With recent advances in understanding the development and regeneration of olfactory epithelium in rodents, we are potentially in a better position to assess the pathological changes in the human olfactory epithelium that are responsible for olfactory loss. However, for an accurate translation from experimental models of injury and repair to human pathophysiology we first need to establish the overall similarity of molecular phenotypes between rodents and humans by immunohistochemical or other means.
We approached the analysis of the human olfactory mucosa on two levels. First, we developed a protocol for the macroscopic assessment of the distribution of olfactory mucosa (OM) within the nasal cavity. Second, we applied our panel of cell type-specific markers, developed for use in rodents, to an in-depth analysis of the composition of the human tissue. With regard to the former, the upper nasal mucosa retains considerable areas of neuroepithelium even in the most elderly individuals. The extent of the olfactory epithelium and the borders of the olfactory area were variable across cases, but invariably included the area directly inferior to the cribriform plate, and often extended to the face of the sphenoid. Intervening patches of respiratory epithelium were common as described previously1
. Although olfactory epithelium has been reported as far anterior as the anterior attachment of the middle turbinate27
, that was not the case in our specimens where the OM was more limited; which may reflect the advanced age of our cases since the human OM may be much more extensive at birth1
. The analysis of the distribution of OM by whole mount (WM) may also provide further insight into the relationship of olfaction to age and disease.
Regardless of the full extent of OM in the nasal cavity, the dense concentration of olfactory neurons in the area directly beneath the cribriform plate is a consistent feature even in the elderly. For purposes of OM biopsies, the cleft is a difficult area to access, and the reported suboptimal rate for obtaining true olfactory epithelium likely reflects that inaccessibility4
. A large portion of the mucosa lining the middle and superior turbinates was also analyzed. Substantial parts of the mucosa on the lateral wall contained neuroepithelium, however, the olfactory area of the septum was more extensive and was characterized by a lesser degree of respiratory replacement. With these findings in mind, opportunities for successful harvest of OM are maximal when directing biopsies to the region of septal epithelium directly below the cribriform plate.
Material harvested at autopsy also provides an advantage for immunohistolgical assessment of the cellular composition of the OM. The full-thickness strip harvested from the septum has the advantage of size, which imparts a more integrated view of the mucosa and includes epithelium rich in olfactory neurons, as verified by whole mount staining. In addition, the sample can be oriented properly for sectioning more easily than biopsies, which is crucial for assessing arrangement of the cells in the epithelium and the prominence of the various cell populations, including HBCs, which normally form a monolayer but can accumulate to a greater number and depth after epithelial injury.
The results of our immunohistochemical analysis of cell types and their prevalence in the human OM are essentially identical in broad strokes and in detail to findings in the OM of experimental animals and are mostly consistent with previous analyses in human tissue harvested at autopsy or by biopsy. In particular, antibodies against several key transcription factors and signal cascades involved in the assembly of the OE in rodents are also informative when applied to the human epithelium.
For example, the status of the basal cell population is of key import to the regenerative capacity of the olfactory epithelium in experimental animals. In addition to the stem and progenitor cell activity within the population of GBCs, the HBC population has been recently identified in mice as an additional reserve stem cell population that can be activated by injury28
. Our analysis has clarified the status of these two populations in the human olfactory epithelium. Suggestions that all of the basal cells of the olfactory epithelium are keratin-expressing and HBC-like are not supported by our data7
. We find a distinct monolayer of HBCs, labeled by the usual markers – K5, EGF-R, p63, Pax6, and Sox2. Situated superficial to the layer of HBCs are cells that have the characteristics of GBCs – marked by Mash1, Sox2, Pax6, Notch1, and Ki-67 (the latter indicating a high rate of mitotic activity) – without expressing K5, K17, or K18. We cannot explain the discrepancy with prior reports in full, however, it should be noted that when staining sections for HBCs and other epithelial components, we confirmed the tissue as OE and not respiratory epithelium with double labeling using anti-neuronal antibodies on the same section () or near adjacent sections. Squamous metaplasia with multiple layers of keratin(+) cells has been described in human OE samples and has been associated with chronic rhinosinusits8
. In these conditions the epithelium is abnormal. Based on our observations using sections of OE with confirmed olfactory neurons, a basal monolayer of CK5(+)/p63(+) HBCs is the norm with a more superficial, separate population of GBCs.
In addition to clarifying the basal cell population, the use of these reagents has also suggested that the various cell types differentiate from their progenitors in a manner similar to that revealed in experimental animals. Thus, the expression of the basic helix-loop-helix transcription factor Hes1 (the canonical downstream effector of Notch signaling) in sustentacular (Sus) cells, and the expression of Notch1 by GBCs is precisely the same as in rodents. In them, manipulating gene expression has shown that Notch1 activation diverts GBCs toward the formation of Sus cells during the regeneration of the injured olfactory epithelium (unpublished results), presumably by blocking the expression of Mash1, which is required for progenitors to advance to the generation of neurons. Likewise, the selective expression of Sox9 by duct/gland cells and Sox2 by Sus cells in partnership with Pax6 in both cell types, is seen in human olfactory epithelium as in rodents, where the switch from Sox9 to Sox2 accompanies the differentiation of Sus cells from residual duct/gland cells during epithelial regeneration.
Of course, the other feature that defines the overall "health" of the mucosa is the status of the neuronal population of the epithelium, which has been assessed in the past (by ourselves and others) by the extent of staining with various neuronal markers. The olfactory epithelium can be aneuronal (but distinguished from respiratory by labeling of the latter with beta-tubulin, type IV) or the neuronal population can vary in size and the relative proportion of immature vs. mature olfactory neurons (as determined by the number of neurons that are marked by TuJ-1 alone or by both TuJ-1 and OMP, respectively). The presence of any immature neurons is an indirect, but powerful indication that neurogenesis is carrying on, even in patients as old as these. However, the population of mature neurons should be larger than that of immature ones, and is generally found to be so when the epithelium is thick and neurons are numerous. A high ratio of immature to mature neurons suggests either a recent insult to the epithelium requiring neuronal replacement, or an inability of mature neurons to connect with the olfactory bulb for whatever reason--known (for example, due to head trauma or other forms of damage to the bulb itself) or unknown. On the other hand, scant numbers of both mature and immature neurons may be due to a dwindling of the neurogenic capacity as stem and progenitor cells become exhausted by ongoing damage to the epithelium itself or permanent interruption of the axonal pathway to the bulb. A fuller view of the neurogenic capacity of the epithelium must take the status of the GBC population into account. We show here that labeling for Mash1 (the neurogenic basic helix-loop-helix transcription factor) and Ki-67 (characteristic of proliferating cells) provides an estimate, albeit incomplete, of that capacity. Mash1, which is labeling only GBCs and not marker-identified neurons or HBCs, marks cells in clusters or layered groups that are mitotically active. Given that neurogenesis cannot proceed in the absence of functional Mash1 in rodents20,21,29
, and that the phenotype of the Mash1-labeled cells in the human is similar, the status of the Mash1 population may provide a way of distinguishing between progenitor cell exhaustion vs. ongoing regeneration in those case where the neurons are few and immature ones predominant in the setting of clinical olfactory loss.
The potential usefulness of our immunohistochemical panel and approach is also exemplified by the analysis of olfactory epithelial-derived tissue such as esthesioneuroblastomas. These tumors are thought to be of neuroectodermal origin and their derivation from olfactory epithelium is assumed on the basis of their common association with the cribriform area, staining characteristics, and finding of Mash1 mRNA expression within the tumor25,26
. These tumors may present with varying degrees of differentiation and are sometimes difficult to diagnose from other undifferentiated sinonasal tumors such as sinonasal undifferentiated carcinoma and lymphoma. In these settings, definitive antibody labeling characteristics may be helpful in determining the pathology.
Overall the immunostaining in our case confirmed the analysis performed by the clinical pathologists – keratins and p63 were absent, but neurogenic markers stained strongly. Labeling of presumed esthesioneuroblastomas with p63 antibodies has been reported in the literature30
, however the absence of this marker remains a common criterion for identifying lesions as esthesioneuroblastomas – a criterion satisfied by the case presented here. The controversy regarding the expression of p63 in esthesioneuroblastomas may derive, in part, from difficulties in determining what cells are or aren’t part of the tumor, particularly since we see p63(+) HBCs in the remnants of normal epithelium that overrides the invasive mass. That the tumor labeled with Golf
antibodies also provides convincing evidence of its olfactory epithelial origin. Although this G-protein has been found in other areas of the body including the testis, retina, basal ganglia, liver, and pancreatic islet cells31
, it is the G-protein associated with signal transduction in olfactory neurons32
and is not found in other nasal tissue. In addition to Golf
staining, our findings of extensive antibody labeling with Mash1 are a honed extension of prior mRNA studies25,26
and further support the association with olfactory epithelium. The combined use of these markers may prove beneficial in the diagnosis of undifferentiated masses of the nasal cavity.
Most strikingly, we were able to subdivide the tumor into two major cell types and regions using other markers from our panel. One type of tumor cell is strongly labeled with K18 and Sox2 antibodies reminiscent of Sus cells or other non-neuronal epithelia, and the other is labeled with neuronal markers such as Tuj-1 and NCAM, as well as Sox9. The latter was a surprising and intriguing finding, since Sox9 is limited to the Bowman’s gland and duct cells of the OE (which also do not express Sox2), and does not cross-over to neurons in normal tissue. E-Cadherin staining was common to both regions of the tumor and is expressed by both duct/gland cells and supporting cells in the normal epithelium. These findings suggest a glandular component to the Sox9/neuronal portion of the tumor and a distinct pathway toward differentiation of the surrounding K18/Sox2(+) sustentacular-like cells. It is worth noting that both GBCs and HBCs can give rise to duct/gland cells during the regeneration of injured epithelium10,28
. Indeed, transplanted GBCs give rise to only neurons and duct/gland cells on occasion10
. Thus, the Sox9/neuronal tumor cells may resemble that set of GBCs responsible for that result. Alternatively, the cellular phenotype may reflect a capacity of Bowman’s gland and/or duct cells to serve as stem or progenitor cells during epithelial regeneration, as has been suggested previously33
. Cells expressing Sox9 in other epithelia such as the epidermis have been associated with stemness; there, the Sox9-expressing cells occupy a specific niche of the hair follicle and give rise to all epidermal lineages34
. Sox9 expressing cells within the Bowman’s glands may be playing a similar role in the OE, and also may be a potential source for mutagenesis and development of esthesioneuroblastomas.
We are now in a position to analyze the human olfactory epithelium in depth and in detail by application of a panel of immunohistochemical reagents with additional emphasis on cell lineage markers. By this approach we can discern the dynamic state of the epithelium and correlate pathological findings with clinical olfactory ability with more assurance. Further studies of human olfactory biopsies will be needed to elucidate the pathophysiology of olfactory disorders further. Moreover, additional investigations of olfactory pathology, for example as shown by the study of esthesioneuroblastoma, may also lend insight into the regenerative processes of the olfactory epithelium, as well as tumorigenesis.