MICROSCOPY RESEARCH AND TECHNIQUE 23:103-110 (1992)
Ultrastructural Histopathology of Human Olfactory Dysfunction DAVID T. MORAN, BRUCE W. JAFEK, PAMELA M. ELLER, AND J. CARTER ROWLEY 111 Rocky Mountain Taste and Smell Center, Department of Otolaryngology and Head and Neck Surgery, University of Colorado School of Medicine, Denver, Colorado 80262
KEY WORDS
Olfaction, Anosmia, Pathology, Nose, Smell
This paper presents electron-microscopic observations on biopsies of the olfactory ABSTRACT mucosae of several classes of patients with smell disorders: 1) patients with loss of smell function following head injury (post-traumatic anosmics or hyposmics); 2) patients with loss of smell function following severe head colds and/or sinus infections (post-viral olfactory dysfunction, or PVOD); and 3) patients that have lacked smell function since birth (congenital anosmics). Of these, the traumatic anosmics’ olfactory epithelia were quite disorganized; the orderly arrangement of supporting cells, ciliated olfactory receptor neurons, microvillar cells, and basal cells was disrupted. Although many somata of ciliated olfactory receptors were present, few of their dendrites reached the epithelial surface. The few olfactory vesicles present usually lacked olfactory cilia. The postviral anosmics, too, had a greatly reduced number of intact ciliated olfactory receptor neurons, and most of those present were aciliate. The post-viral hyposmics had a larger population of intact, ciliated olfactory receptor cells. In the seven cases of congenital anosmia studied, no biopsies of olfactory epithelium were obtained, indicating the olfactory epithelium is either absent-or greatly reduced in area-in these individuals. o 1992 Wiley-Liss, Inc. cluding a detailed examination of the nose, paranasal INTRODUCTION This paper reviews some of the ultrastructural inves- sinuses, and head and neck region. The airways were tigations of the histopathology of olfactory dysfunction normal and unobstructed. Medical histories showed performed in our laboratory during the past 6 years that 16 patients complained of loss of smell function (1984-1990). During that time, more than two hun- following head trauma; 16 reported loss of smell followdred patients have presented at the ENT clinic of the ing a n upper respiratory infection; and 7 had never Rocky Mountain Taste and Smell Center with specific experienced proper smell function. All 39 patients were given several tests of smell funccomplaints of olfactory dysfunction. Although these tion, including the University of Pennsylvania Smell cases encompass a broad range of olfactory deficits, many of them can be categorized into one of three ma- Identification Test (the “UPSIT” test; Doty et al., 19841, a seven-item discrmination test, and a bilateral jor classes: 1. Patients who have lost all-or part-of their smell threshold test (Cain et al., 1983). Patients were classifunction following a head injury. These individuals are fied as normosmic (normal sense of smell), hyposmic called “traumatic anosmics” if they have lost all of (partial loss of smell function), or anosmic (complete their smell function, and “traumatic hyposmics” if they loss of smell function). have lost part of it. Biopsy Procedure 2. Patients who have lost all (or part) of their smell In all cases, the tool and technique described by Lovfunction following a severe head cold or sinus infection. These individuals are called “post-viral anosmics” or ell et al. (1982), recently reviewed by Strahan et al. “post-viral hyposmics,” respectively; the syndrome, (19911, were used, with which a small (1 mm’), thin called “post-viral olfactory dysfunction,” is abbreviated piece of nasal epithelium can safely be removed while the patient is under local anesthesia. First, the interior as PVOD. 3. Patients who have never been aware of any sense of the nasal cavity was sprayed with 4% aqueous cocaine to achieve both local anesthesia and vasoconstricof smell have been classified a s congenital anosmics. The present paper reviews ultrastructural investiga- tion. Next, the interior of the nasal cavity was intions of the nasal mucosae of 39 patients; 16 with trau- spected with a small-caliber endoscope (the Olympus matic anosmia or hyposmia, 16 with post-viral olfactory Selfoscope SED-11). Following that, a specially made dysfunction (PVOD), and 7 with congenital anosmia.
MATERIALS AND METHODS Olfactory Testing Thirty-nine patients with chemosensory complaints were each given a complete physical examination, in-
0 1992 WILEY-LISS, INC
Received July 9, 1991; accepted in revised form July 15, 1991. Address reprint requests to Dr. David T. Moran, 1415 Cherryvale, Boulder, CO 80303.
104
D.T. MORAN ET AL. TABLE 1. Thirty-nine patients with olfactory disorders and descriptions of biopsies
F’ost-traumatic anosmic F’ost-traumatic hyposmic Post-viral anosmic Post-viral hyposmic Congenital anosmic
of
their nasal mucosae
No. of patients tested and biopsied
Abnormal olfactory epithelium
Normal olfactory epithelium
Respiratory epithelium’
Unidentified epithelium’
14 2
9 1 2 7 0
0 0 0 0 0
3 1 1 5 7
2 0 1 0 0
4 12
7
~
‘In 7.69%of these cases the biopsy specimens were distorted in such a way that the tissue obtained could not be identified. ‘In 43.5%of these cases the biopsy specimens did not contain olfactory epithelium; only respiratory epithelium was observed. With the exception of the specimens from congenital anosmic patients (see text), the failure to obtain olfactory epithelium is thought to be a result of the biopsy technique and the variability of the epithelium in human subjects.
man olfactory epithelium, described elsewhere in detail (Jafek, 1983; Moran et al., 1982a, 19911, is briefly summarized below. Supporting cells, thought to perform both absorptive and secretory functions, comprise the bulk of the epithelium. They surround the ciliated olfactory recepTissue Preparation for Electron Microscopy tors-bipolar neurons that each send a dendrite to the Immediately following their removal from the nose, site of stimulus reception at the epithelial surface and biopsies of nasal epithelium were fixed in a freshly a n axon to the olfactory bulb of the brain. The dendrite made mixture of 2% glutaraldehyde and 0.6% parafor- tip, illustrated in Figure 2, forms a n olfactory vesicle maldehyde buffered to pH 7.2 with 0.06M sodium ca- (often called a n “olfactory knob”) that bears 10-30 sencodylate (Monti-Graziadei and Graziadei, 1979), post- sory cilia whose membranes contain the receptor macfixed in buffered 2% osmium tetroxide, dehydrated in a romolecules that participate in odorant binding and graded series of acetones, and embedded in Spurr’s chemosensory transduction (see Bruch, 1990, for re(1969) low-viscosity epoxy resin. Thin sections were cut view). The microvillar cells, fewer in number than the on a Porter-Blum MT-2B ultramicrotome, mounted on ciliated olfactory receptors (Moran et al., 1982b1, have a Formvar-coated slot grid using a Domino Rack flask-shaped cell bodies located near the epithelial sur(Moran and Rowley, 1987), and doubly-contrasted with face. The precise functions of these microvillar cells, uranyl acetate and lead citrate. Specimens were exam- whose ultrastructure resembles that of “brush cells” ined and photographed with a Philips CM-10 transmis- occasionally found in the conductive airways of the ression electron microscope operated a t a n accelerating piratory tract (see Moran and Rowley, 1988, Fig. 112B), remain to be determined. The basal cells, small voltage of 80 kV. cells located near the basement membrane, are mitotRESULTS ically-active stem cells that replace lost, worn-out, or Table 1 summarizes data from 39 patients with ol- damaged cells of the olfactory epithelium-including factory dysfunctions who were tested and biopsied a t ciliated olfactory receptor neurons (see Graziadei, the Otorhinolaryngology clinic of the Rocky Mountain 1973, for review). Taste and Smell Center at the University of Colorado Ultrastructure of Olfactory Mucosae From Health Sciences Center. One striking feature of the Patients With Traumatic Anosmia data is this: of the 39 patients from whom biopsies were The ultrastructure of olfactory epithelium from paobtained, all samples of olfactory epithelia showed evidence of histopathologic changes when examined by tients with traumatic anosmia (Figs. 3-4) is quite difelectron microscopy. Seven patients, diagnosed as con- ferent from that of normosmic subjects (Figs. 1-21. The genital anosmics, were believed to have no olfactory orderly arrangement of supporting, receptor, and basal epithelium at all. All of their biopsies were of respira- cells-and the relative positions of their nuclei-is dratory epithelium. Of the 32 patients in whom olfactory matically disrupted in olfactory epithelium from trauepithelium was presumed present, biopsies of olfactory matic anosmics (Jafek et al., 1989; Moran e t al., 1985). mucosa were obtained from 19, indicating a “success As shown in Figure 3, the number of degenerating rate” of 59.5% for the biopsy procedure. cells-evidenced by condensed, electron-dense cytoplasm and pyknotic nuclei-has increased. The nuclei Ultrastructure of Normal Olfactory Mucosa and cell bodies of ciliated olfactory receptor neurons, When a biopsy of olfactory epithelium is taken from normally located between those of supporting and a subject with a normal sense of smell and viewed by basal cells, are scattered throughout the epithelium; transmission electron microscopy, it appears as shown many have assumed a superficial location not seen in in Figure 1. Here, the tissue is seen to be a pseudo- normal olfactory epithelia. The dendrites of the bipolar stratified columnar epithelium consisting of four mor- receptor neurons are fewer, shorter, and thicker, and phologically distinct cell types: supporting (or susten- very few of them reach the free surface of the olfactory tacular) cells, ciliated olfactory receptors, microvillar epithelium. Those few that do are usually topped by a cells, and basal cells. The ultrastructure of normal hu- rounded olfactory vesicle that is devoid of sensory cilia
biopsy instrument was placed through the nostril, advanced along the nasal septum to a depth of approximately 6-7 cm, and a small piece of olfactory mucosa was removed from the superior region of the nasal septum.
OLFACTORY ULTRASTRUCTURAL HISTOPATHOLOGY
Fig. 1. Low-magnification TEM of olfactory epithelium from a human subject with a normal sense of smell. B, basal cell; C , ciliated olfactory receptor cell; D, dendrite of ciliated olfactory receptor cell; M, microvillar cell; NC, nasal cavity; NS, nucleus of supporting cell; arrows, olfactory vesicles. x 1,300.
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Fig. 2. This TEM illustrates the dendrite (D) of a ciliated olfactory receptor tipped by a n olfactory vesicle (OW, from which several olfactory cilia (arrow) project. S, supporting cell. x 2,050.
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D.T. MORAN ET AL.
Fig. 3. TEM of abnormal olfactory epithelium from a patient with traumatic anosmia. Nuclei of ciliated olfactory receptors (C) are located more superficially than those in normal olfactory epithelia. Dendrites (D) are short, thick, and tortuous; few reach the epithelial surface. Degenerating cells (DC) abound. S , supporting cell. x 3,700.
Fig. 4. Traumatic anosmics have few olfactory vesicles. When present, most, like this one (OW, have no sensory cilia. Basal bodies (B) are often evident. NC, nasal cavity; D, dendrite of ciliated olfactory receptor. x 21,600.
OLFACTORY ULTRASTRUCTURAL HISTOPATHOLOGY
(Fig. 4). The disorganized olfactory epithelium, reduced number of dendrites, and aciliate olfactory vesicles are distinct manifestations of ultrastructural histopathology consistently observed in the olfactory epithelia of all traumatic anosmics biopsied to date. In addition, increased numbers of axons are present in the olfactory mucosa both within the epithelium itself and just beneath the basement membrane.
Ultrastructure of Olfactory Mucosae From Patients With Post-Viral Anosmia and Hyposmia The olfactory epithelium of patients with post-viral anosmia (Figs. 5-6) presents a n ultrastructure similar to that of patients with post-traumatic anosmia. Although the overall organization of the olfactory epithelium in PVOD patients is closer to normal than it is in post-traumatic anosmics, the number of ciliated olfactory receptors that have dendrites which reach the epithelial surface is quite limited. Few olfactory vesicles are present a t the epithelial surface in post-viral anosmics, and those that are present usually lack olfactory cilia. Figure 5, a longitudinal section through the junction between the respiratory and olfactory epithelium of a post-viral anosmic, shows the respiratory epithelium to the left of the field, and the olfactory epithelium, to the right. In the latter, only one olfactory vesicle is seen at the epithelial surface, and it is aciliate. Figure 6 shows another olfactory vesicle from the same patient. Although basal bodies are present, no cilia are evident. The olfactory epithelia of patients with post-viral hyposmia (Figs. 7,8) appear more normal than do those from post-viral anosmics. More dendrites display olfactory vesicles at the epithelial surface, and those olfactory vesicles often have sensory cilia. In Figure 7, a n electron micrograph that shows the junction between respiratory and olfactory epithelia from a patient with post-viral hyposmia, a n olfactory vesicle is present that bears several sensory cilia. Another olfactory vesicle equipped with sensory cilia is seen in Figure 8. Although more olfactory vesicles are present in olfactory epithelia from post-viral hyposmic patients than postviral anosmic patients, their number is still reduced from normal.
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DISCUSSION The investigations reviewed in this paper were designed to answer two questions: 1) Do the olfactory mucosae of dysosmic patients exhibit ultrastructural abnormalities? 2) If so, are there any correlations between ultrastructural changes in the olfactory mucosa and functional deficits? The answer to both questions is yes: dysosmic patients examined in these investigations do possess a n olfactory epithelium that appears abnormal when examined by electron microscopy, and in several cases the ultrastructural changes can be correlated with degrees of loss of smell function.
Patients With Traumatic Anosmia It has been known for some time that severe head injuries are often accompanied by partial or complete loss of smell function (Douek, 1974; Leigh, 1943). Often, the degree of olfactory dysfunction is correlated with the severity of head injury (Caruso e t al., 1969; Douek, 1974; Hasegawa et al., 1986). Recovery of smell function in head-injured patients can occur; Zusho (19821, for example, reports that 14% of the cases he studied showed substantial recovery of smell function. Traumatic anosmia is thought to result from shearing of the delicate fila olfactoria a s they pass through tiny holes in the cribriform plate on their path from the olfactory epithelium to the olfactory bulb in the brain. The shearing of nerves occurs as the brain moves suddenly within the calvarium in response to direct or contrecoup forces (Doty, 1979; Douek, 1974; Sumner, 1964). Theoretically, the ciliated olfactory receptor neurons, destroyed by having their axons thus severed, can be replaced by new olfactory neurons derived from basal cells (see Graziadei, 1973, for review). This phenomenon probably accounts for observed cases of recovery of smell function. In those traumatic anosmic patients in whom functional recovery does not occur, it seems likely that the tiny holes in the cribriform plate scar over following injury, blocking the path of regenerating axons attempting to find their way to the brain (Jafek et al., 1989; Moran et al., 1985). The ultrastructural data from traumatic anosmics support the scheme presented above. In these patients, the epithelium is disorganized, the number of dendrites Absence of Olfactory Epithelia in Patients With and olfactory vesicles is reduced, and the few olfactory Congenital Anosmia vesicles present are usually aciliate. These observations are consistent with experiments done with roAs seen in Table 1, seven patients with congenital anosmia were examined in this study. Their case his- dents, in which developing olfactory epithelia placed in tories have been described elsewhere in detail (Jafek et organ culture with pieces of olfactory bulbs develop far al., 1990). Although biopsies were taken in the same more olfactory vesicles and cilia after synaptic contact way a s for other patients in the study, not a single piece is made between receptor axons and the bulb (Chuah et of olfactory epithelium was found. Instead, all seven al., 1985; Farbman, 1977, 1986). patients yielded samples of respiratory epithelium. Patients With Post-Viral Anosmia These results suggest the olfactory epithelium is not These data are more difficult to interpret, since present in patients with congenial anosmia. Recently Paik e t al. (1991) have observed olfactory epithelium in PVOD is a general syndrome describing loss of olfacseveral patients with congenital anosmia; conse- tory function due to a variety of causes involving viral quently, the “negative” biopsies in the present study and/or bacterial rhinologic infections. The one consismay result from the presence of a very small area of tent, significant observation is this: the populationolfactory epithelium, decreasing the probability of cap- density of olfactory receptors reflects the degree of loss of smell function (Jafek et al., 1990). That is, post-viral turing it with a small biopsy instrument.
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Fig. 5. Low-magnification electron micrograph of the junction between respiratory (RE) and olfactory epithelium (OE) in a patient with post-viral anosmia. A lone olfactory vesicle (arrowhead) is seen Note non-sensory, at upper right in a field of supporting cells 6). motile cilia (arrow) extending from cells in respiratory epithelium. x 3.600.
Fig. 6. This olfactory vesicle (OV), found in the olfactory epithelium of a patient with post-viral anosmia, has no olfactory cilia. S, supporting cell; NC, nasal cavity. x 39,000.
OLFACTORY ULTRASTRUCTURAL HISTOPATHOLOGY
Fig. 7. This electron micrograph illustrates the interface of respiratory epithelium (RE), with its motile cilia (arrowhead), and olfactory epithelium (OE) of a patient with post-viral hyposmia. The olfactory vesicle (OV), which is ciliated (arrows), projects above the supporting cell (S) that surrounds the dendrite (D). X 15,600.
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Fig. 8. Ciliated olfactory vesicle (OW from a patient with postviral hyposmia. s, supporting cell; arrows, sensory cilia. X 17,800.
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D.T. MORAN ET AL.
anosmics have very few olfactory vesicles, and those that have been observed are largely aciliate. Post-viral hyposmics, on the other hand, have fewer olfactory vesicles than normal subjects (but more than post-viral anosmics), and most of their olfactory vesicles are equipped with olfactory cilia. Consequently, studies of patients with PVOD indicate that the number of intact ciliated olfactory receptors within the olfactory epithelium is directly related to the patient’s olfactory acuity.
ACKNOWLEDGMENTS These investigations were supported by NIH Program Project Grant NIDCDBPOl DC 00244 (to the Rocky Mountain Taste and Smell Center). We thank Drs. Glen Morangie and Edward W. Johnson for reviewing the manuscript and providing useful comments. REFERENCES Bruch, R.C. (1990) G proteins in olfactory neurons. In: G Proteins and Calcium Signaling, F.H. Naccache, ed. CRC Press, Boca Raton, Florida, pp. 123-134. Gain, W.S., Gent, G., and Catalanotto, F.A. (1983) Clinical evaluation of olfaction. Am. J. Otolaryngol., 4252-256. Caruso, V., Hagan, J., and Manning, H. (1969) Quantitative olfactometry in the measurement of post-traumatic anosmia. Arch. Otolaryngol. Head Neck Surg., 90:500-503. Chuah, M.I., Farbman, A.I., and Menco, B.P.M. (1985) Influence of olfactory bulb on dendritic knob density of rat olfactory receptor neurons in uitro. Brain Res., 338:259-266. Qoty, R.L. (1979) A review of olfactory dysfunction in man. Am. J . Otolaryngol., 1:57-79. Qoty, R.L., Shaman, P., and Dann, M. (1984) Developments of the University of Pennsylvania smell identification test: A standard microencapsulated test of olfactory function. Physiol. Behav., 32: 489-502. Douek, E. (1974) The Sense of Smell and Its Abnormalities. Churchill Livingstone, London. Farbman, A.I. (1977) Differentiation of olfactory receptor cells in organ culture. Anat. Rec., 189:187-200. Farbman, A.I. (1986) Prenatal development of mammalian olfactory receptor cells. Chem. Senses, 11:3-18. Graziadei, P.P.C. (1973) The ultrastructure of vertebrate olfactory mucosa. In: The Ultrastructure of Sensory Organs, I. Friedman, ed. North Holland Publ. Co., Amsterdam, pp. 267-305. Hasegawa, S., Yamagichi, M., and Nakano, Y. (1986) Microscopic
studies of human olfactory epithelia following traumatic anosmia. Arch. Otorhinolaryngol., 243:112-116. Jafek, B.W. (1983) Ultrastructure of human nasal mucosa. Laryngoscope 93:1576-1599. Jafek, B.W., Eller, P.M., Esses, B.A., and Moran, D.T. (1989) Posttraumatic anosmia: Ultrastructural correlates. Arch. Neurol., 46: 300-304. Jafek, B.W., Gordon, A.S.D., Eller, P.M., Esses, B.A., Moran, D.T., Johnson, E.W., and Straham, R.C. (1990) Congenital anosmia. ENT Journal, 69:331-337. Jafek, B.W., Hartman, D., Eller, P.M., Johnson, E.W., Straham, R.C., and Moran, D.T. (1990)Post-viral olfactory dysfunction. Am. J. Rhinol. 491-100. Leigh, A.D. (1943) Defect of smell after head injury. Lancet, 11:3840. h v e l l , M.A., Jafek, B.W., Moran, D.T., and Rowley, J.C. I11 (1982) Biopsy of human olfactory mucosa: An instrument and a technique. Arch. Otolaryngol., 108:247-249. Monti-Graziadei, M.A., and Graziadei, P.P.C. (1979) Neurogenesis and neuron regeneration in the olfactory system of mammals: 11. Degeneration and reconstitution of the olfactory sensory neurons after axotomy. J. Neurocytol., 8:197-213. Moran, D.T., Rowley, J.C. 111, Jafek, B.W., and Lovell, M.A. (1982a) The fine structure of the olfactory mucosa in man. J . Neurocytol., 11:72 1-746. Moran. D.T.. Rowlev. J.C. 111. and Jafek. B.W. (1982b) Electron microscopy of h u m a i olfactoryepithelium’reveals a new cell type: The microvillar cell. Brain Res., 253:39-46. Moran, D.T., Jafek. B.W., Rowley, J.C. 111, and Eller, P.M. (1985) Electron microscopy of olfactory epithelia in 2 patients with anosmia. Arch. Otolaryngol., 111:122-126. Moran, D.T., and Rowley, J.C. 111 (1987) Biological specimen preparation for correlative light and electron microscopy. In: Correlative Microscopy, M.A. Hayat, ed. Academic Press, New York, pp. 1-22. Moran, D.T,. and Rowley, J.C. 111 (1988) Visual Histology. Lea & Febiger, Philadelphia, PA. Moran, D.T., Jafek, B.W., and Rowley, J.C. 111 (1991) The ultrastructure of the human olfactory mucosa. In: The Human Sense of Smell. D.G. Laing, R.I. Doty, and W. Breipohl, eds. Springer-Verlag, Berlin, pp. 1-25. Paik, E.I., Seiden, A.M., Duncan, H.J., Smith, D.V. (1991) Olfactory mucosal biopsy in patients with congenital anosmia. Proc. AChemS XIII, Sarasota, FL: Abstr. 70. Spurr, A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J . Ultrastruct. Res., 26:31-43. Strahan, R.C., Jafek, B.W., and Moran, D.T. (1991) Biopsy of the olfactory neuroepithelium. In: Smell and Taste in Health and Disease. T.V. Getchell, R.L. Doty, L.M. Bartoshuk, and J.B. Snow, eds. Raven Press, New York (in press). Sumner, D. (1964) Post-traumatic anosmia. Brain 87:107-120. Zusho, H. (1982) Posttraumatic anosmia. Arch. Otolaryngol. Head Neck Surg., 108:90-92.
Ultrastructural Histopathology of Human Olfactory Dysfunction DAVID T. MORAN, BRUCE W. JAFEK, PAMELA M. ELLER, AND J. CARTER ROWLEY 111 Rocky Mountain Taste and Smell Center, Department of Otolaryngology and Head and Neck Surgery, University of Colorado School of Medicine, Denver, Colorado 80262
KEY WORDS
Olfaction, Anosmia, Pathology, Nose, Smell
This paper presents electron-microscopic observations on biopsies of the olfactory ABSTRACT mucosae of several classes of patients with smell disorders: 1) patients with loss of smell function following head injury (post-traumatic anosmics or hyposmics); 2) patients with loss of smell function following severe head colds and/or sinus infections (post-viral olfactory dysfunction, or PVOD); and 3) patients that have lacked smell function since birth (congenital anosmics). Of these, the traumatic anosmics’ olfactory epithelia were quite disorganized; the orderly arrangement of supporting cells, ciliated olfactory receptor neurons, microvillar cells, and basal cells was disrupted. Although many somata of ciliated olfactory receptors were present, few of their dendrites reached the epithelial surface. The few olfactory vesicles present usually lacked olfactory cilia. The postviral anosmics, too, had a greatly reduced number of intact ciliated olfactory receptor neurons, and most of those present were aciliate. The post-viral hyposmics had a larger population of intact, ciliated olfactory receptor cells. In the seven cases of congenital anosmia studied, no biopsies of olfactory epithelium were obtained, indicating the olfactory epithelium is either absent-or greatly reduced in area-in these individuals. o 1992 Wiley-Liss, Inc. cluding a detailed examination of the nose, paranasal INTRODUCTION This paper reviews some of the ultrastructural inves- sinuses, and head and neck region. The airways were tigations of the histopathology of olfactory dysfunction normal and unobstructed. Medical histories showed performed in our laboratory during the past 6 years that 16 patients complained of loss of smell function (1984-1990). During that time, more than two hun- following head trauma; 16 reported loss of smell followdred patients have presented at the ENT clinic of the ing a n upper respiratory infection; and 7 had never Rocky Mountain Taste and Smell Center with specific experienced proper smell function. All 39 patients were given several tests of smell funccomplaints of olfactory dysfunction. Although these tion, including the University of Pennsylvania Smell cases encompass a broad range of olfactory deficits, many of them can be categorized into one of three ma- Identification Test (the “UPSIT” test; Doty et al., 19841, a seven-item discrmination test, and a bilateral jor classes: 1. Patients who have lost all-or part-of their smell threshold test (Cain et al., 1983). Patients were classifunction following a head injury. These individuals are fied as normosmic (normal sense of smell), hyposmic called “traumatic anosmics” if they have lost all of (partial loss of smell function), or anosmic (complete their smell function, and “traumatic hyposmics” if they loss of smell function). have lost part of it. Biopsy Procedure 2. Patients who have lost all (or part) of their smell In all cases, the tool and technique described by Lovfunction following a severe head cold or sinus infection. These individuals are called “post-viral anosmics” or ell et al. (1982), recently reviewed by Strahan et al. “post-viral hyposmics,” respectively; the syndrome, (19911, were used, with which a small (1 mm’), thin called “post-viral olfactory dysfunction,” is abbreviated piece of nasal epithelium can safely be removed while the patient is under local anesthesia. First, the interior as PVOD. 3. Patients who have never been aware of any sense of the nasal cavity was sprayed with 4% aqueous cocaine to achieve both local anesthesia and vasoconstricof smell have been classified a s congenital anosmics. The present paper reviews ultrastructural investiga- tion. Next, the interior of the nasal cavity was intions of the nasal mucosae of 39 patients; 16 with trau- spected with a small-caliber endoscope (the Olympus matic anosmia or hyposmia, 16 with post-viral olfactory Selfoscope SED-11). Following that, a specially made dysfunction (PVOD), and 7 with congenital anosmia.
MATERIALS AND METHODS Olfactory Testing Thirty-nine patients with chemosensory complaints were each given a complete physical examination, in-
0 1992 WILEY-LISS, INC
Received July 9, 1991; accepted in revised form July 15, 1991. Address reprint requests to Dr. David T. Moran, 1415 Cherryvale, Boulder, CO 80303.
104
D.T. MORAN ET AL. TABLE 1. Thirty-nine patients with olfactory disorders and descriptions of biopsies
F’ost-traumatic anosmic F’ost-traumatic hyposmic Post-viral anosmic Post-viral hyposmic Congenital anosmic
of
their nasal mucosae
No. of patients tested and biopsied
Abnormal olfactory epithelium
Normal olfactory epithelium
Respiratory epithelium’
Unidentified epithelium’
14 2
9 1 2 7 0
0 0 0 0 0
3 1 1 5 7
2 0 1 0 0
4 12
7
~
‘In 7.69%of these cases the biopsy specimens were distorted in such a way that the tissue obtained could not be identified. ‘In 43.5%of these cases the biopsy specimens did not contain olfactory epithelium; only respiratory epithelium was observed. With the exception of the specimens from congenital anosmic patients (see text), the failure to obtain olfactory epithelium is thought to be a result of the biopsy technique and the variability of the epithelium in human subjects.
man olfactory epithelium, described elsewhere in detail (Jafek, 1983; Moran et al., 1982a, 19911, is briefly summarized below. Supporting cells, thought to perform both absorptive and secretory functions, comprise the bulk of the epithelium. They surround the ciliated olfactory recepTissue Preparation for Electron Microscopy tors-bipolar neurons that each send a dendrite to the Immediately following their removal from the nose, site of stimulus reception at the epithelial surface and biopsies of nasal epithelium were fixed in a freshly a n axon to the olfactory bulb of the brain. The dendrite made mixture of 2% glutaraldehyde and 0.6% parafor- tip, illustrated in Figure 2, forms a n olfactory vesicle maldehyde buffered to pH 7.2 with 0.06M sodium ca- (often called a n “olfactory knob”) that bears 10-30 sencodylate (Monti-Graziadei and Graziadei, 1979), post- sory cilia whose membranes contain the receptor macfixed in buffered 2% osmium tetroxide, dehydrated in a romolecules that participate in odorant binding and graded series of acetones, and embedded in Spurr’s chemosensory transduction (see Bruch, 1990, for re(1969) low-viscosity epoxy resin. Thin sections were cut view). The microvillar cells, fewer in number than the on a Porter-Blum MT-2B ultramicrotome, mounted on ciliated olfactory receptors (Moran et al., 1982b1, have a Formvar-coated slot grid using a Domino Rack flask-shaped cell bodies located near the epithelial sur(Moran and Rowley, 1987), and doubly-contrasted with face. The precise functions of these microvillar cells, uranyl acetate and lead citrate. Specimens were exam- whose ultrastructure resembles that of “brush cells” ined and photographed with a Philips CM-10 transmis- occasionally found in the conductive airways of the ression electron microscope operated a t a n accelerating piratory tract (see Moran and Rowley, 1988, Fig. 112B), remain to be determined. The basal cells, small voltage of 80 kV. cells located near the basement membrane, are mitotRESULTS ically-active stem cells that replace lost, worn-out, or Table 1 summarizes data from 39 patients with ol- damaged cells of the olfactory epithelium-including factory dysfunctions who were tested and biopsied a t ciliated olfactory receptor neurons (see Graziadei, the Otorhinolaryngology clinic of the Rocky Mountain 1973, for review). Taste and Smell Center at the University of Colorado Ultrastructure of Olfactory Mucosae From Health Sciences Center. One striking feature of the Patients With Traumatic Anosmia data is this: of the 39 patients from whom biopsies were The ultrastructure of olfactory epithelium from paobtained, all samples of olfactory epithelia showed evidence of histopathologic changes when examined by tients with traumatic anosmia (Figs. 3-4) is quite difelectron microscopy. Seven patients, diagnosed as con- ferent from that of normosmic subjects (Figs. 1-21. The genital anosmics, were believed to have no olfactory orderly arrangement of supporting, receptor, and basal epithelium at all. All of their biopsies were of respira- cells-and the relative positions of their nuclei-is dratory epithelium. Of the 32 patients in whom olfactory matically disrupted in olfactory epithelium from trauepithelium was presumed present, biopsies of olfactory matic anosmics (Jafek et al., 1989; Moran e t al., 1985). mucosa were obtained from 19, indicating a “success As shown in Figure 3, the number of degenerating rate” of 59.5% for the biopsy procedure. cells-evidenced by condensed, electron-dense cytoplasm and pyknotic nuclei-has increased. The nuclei Ultrastructure of Normal Olfactory Mucosa and cell bodies of ciliated olfactory receptor neurons, When a biopsy of olfactory epithelium is taken from normally located between those of supporting and a subject with a normal sense of smell and viewed by basal cells, are scattered throughout the epithelium; transmission electron microscopy, it appears as shown many have assumed a superficial location not seen in in Figure 1. Here, the tissue is seen to be a pseudo- normal olfactory epithelia. The dendrites of the bipolar stratified columnar epithelium consisting of four mor- receptor neurons are fewer, shorter, and thicker, and phologically distinct cell types: supporting (or susten- very few of them reach the free surface of the olfactory tacular) cells, ciliated olfactory receptors, microvillar epithelium. Those few that do are usually topped by a cells, and basal cells. The ultrastructure of normal hu- rounded olfactory vesicle that is devoid of sensory cilia
biopsy instrument was placed through the nostril, advanced along the nasal septum to a depth of approximately 6-7 cm, and a small piece of olfactory mucosa was removed from the superior region of the nasal septum.
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Fig. 1. Low-magnification TEM of olfactory epithelium from a human subject with a normal sense of smell. B, basal cell; C , ciliated olfactory receptor cell; D, dendrite of ciliated olfactory receptor cell; M, microvillar cell; NC, nasal cavity; NS, nucleus of supporting cell; arrows, olfactory vesicles. x 1,300.
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Fig. 2. This TEM illustrates the dendrite (D) of a ciliated olfactory receptor tipped by a n olfactory vesicle (OW, from which several olfactory cilia (arrow) project. S, supporting cell. x 2,050.
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Fig. 3. TEM of abnormal olfactory epithelium from a patient with traumatic anosmia. Nuclei of ciliated olfactory receptors (C) are located more superficially than those in normal olfactory epithelia. Dendrites (D) are short, thick, and tortuous; few reach the epithelial surface. Degenerating cells (DC) abound. S , supporting cell. x 3,700.
Fig. 4. Traumatic anosmics have few olfactory vesicles. When present, most, like this one (OW, have no sensory cilia. Basal bodies (B) are often evident. NC, nasal cavity; D, dendrite of ciliated olfactory receptor. x 21,600.
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(Fig. 4). The disorganized olfactory epithelium, reduced number of dendrites, and aciliate olfactory vesicles are distinct manifestations of ultrastructural histopathology consistently observed in the olfactory epithelia of all traumatic anosmics biopsied to date. In addition, increased numbers of axons are present in the olfactory mucosa both within the epithelium itself and just beneath the basement membrane.
Ultrastructure of Olfactory Mucosae From Patients With Post-Viral Anosmia and Hyposmia The olfactory epithelium of patients with post-viral anosmia (Figs. 5-6) presents a n ultrastructure similar to that of patients with post-traumatic anosmia. Although the overall organization of the olfactory epithelium in PVOD patients is closer to normal than it is in post-traumatic anosmics, the number of ciliated olfactory receptors that have dendrites which reach the epithelial surface is quite limited. Few olfactory vesicles are present a t the epithelial surface in post-viral anosmics, and those that are present usually lack olfactory cilia. Figure 5, a longitudinal section through the junction between the respiratory and olfactory epithelium of a post-viral anosmic, shows the respiratory epithelium to the left of the field, and the olfactory epithelium, to the right. In the latter, only one olfactory vesicle is seen at the epithelial surface, and it is aciliate. Figure 6 shows another olfactory vesicle from the same patient. Although basal bodies are present, no cilia are evident. The olfactory epithelia of patients with post-viral hyposmia (Figs. 7,8) appear more normal than do those from post-viral anosmics. More dendrites display olfactory vesicles at the epithelial surface, and those olfactory vesicles often have sensory cilia. In Figure 7, a n electron micrograph that shows the junction between respiratory and olfactory epithelia from a patient with post-viral hyposmia, a n olfactory vesicle is present that bears several sensory cilia. Another olfactory vesicle equipped with sensory cilia is seen in Figure 8. Although more olfactory vesicles are present in olfactory epithelia from post-viral hyposmic patients than postviral anosmic patients, their number is still reduced from normal.
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DISCUSSION The investigations reviewed in this paper were designed to answer two questions: 1) Do the olfactory mucosae of dysosmic patients exhibit ultrastructural abnormalities? 2) If so, are there any correlations between ultrastructural changes in the olfactory mucosa and functional deficits? The answer to both questions is yes: dysosmic patients examined in these investigations do possess a n olfactory epithelium that appears abnormal when examined by electron microscopy, and in several cases the ultrastructural changes can be correlated with degrees of loss of smell function.
Patients With Traumatic Anosmia It has been known for some time that severe head injuries are often accompanied by partial or complete loss of smell function (Douek, 1974; Leigh, 1943). Often, the degree of olfactory dysfunction is correlated with the severity of head injury (Caruso e t al., 1969; Douek, 1974; Hasegawa et al., 1986). Recovery of smell function in head-injured patients can occur; Zusho (19821, for example, reports that 14% of the cases he studied showed substantial recovery of smell function. Traumatic anosmia is thought to result from shearing of the delicate fila olfactoria a s they pass through tiny holes in the cribriform plate on their path from the olfactory epithelium to the olfactory bulb in the brain. The shearing of nerves occurs as the brain moves suddenly within the calvarium in response to direct or contrecoup forces (Doty, 1979; Douek, 1974; Sumner, 1964). Theoretically, the ciliated olfactory receptor neurons, destroyed by having their axons thus severed, can be replaced by new olfactory neurons derived from basal cells (see Graziadei, 1973, for review). This phenomenon probably accounts for observed cases of recovery of smell function. In those traumatic anosmic patients in whom functional recovery does not occur, it seems likely that the tiny holes in the cribriform plate scar over following injury, blocking the path of regenerating axons attempting to find their way to the brain (Jafek et al., 1989; Moran et al., 1985). The ultrastructural data from traumatic anosmics support the scheme presented above. In these patients, the epithelium is disorganized, the number of dendrites Absence of Olfactory Epithelia in Patients With and olfactory vesicles is reduced, and the few olfactory Congenital Anosmia vesicles present are usually aciliate. These observations are consistent with experiments done with roAs seen in Table 1, seven patients with congenital anosmia were examined in this study. Their case his- dents, in which developing olfactory epithelia placed in tories have been described elsewhere in detail (Jafek et organ culture with pieces of olfactory bulbs develop far al., 1990). Although biopsies were taken in the same more olfactory vesicles and cilia after synaptic contact way a s for other patients in the study, not a single piece is made between receptor axons and the bulb (Chuah et of olfactory epithelium was found. Instead, all seven al., 1985; Farbman, 1977, 1986). patients yielded samples of respiratory epithelium. Patients With Post-Viral Anosmia These results suggest the olfactory epithelium is not These data are more difficult to interpret, since present in patients with congenial anosmia. Recently Paik e t al. (1991) have observed olfactory epithelium in PVOD is a general syndrome describing loss of olfacseveral patients with congenital anosmia; conse- tory function due to a variety of causes involving viral quently, the “negative” biopsies in the present study and/or bacterial rhinologic infections. The one consismay result from the presence of a very small area of tent, significant observation is this: the populationolfactory epithelium, decreasing the probability of cap- density of olfactory receptors reflects the degree of loss of smell function (Jafek et al., 1990). That is, post-viral turing it with a small biopsy instrument.
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Fig. 5. Low-magnification electron micrograph of the junction between respiratory (RE) and olfactory epithelium (OE) in a patient with post-viral anosmia. A lone olfactory vesicle (arrowhead) is seen Note non-sensory, at upper right in a field of supporting cells 6). motile cilia (arrow) extending from cells in respiratory epithelium. x 3.600.
Fig. 6. This olfactory vesicle (OV), found in the olfactory epithelium of a patient with post-viral anosmia, has no olfactory cilia. S, supporting cell; NC, nasal cavity. x 39,000.
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Fig. 7. This electron micrograph illustrates the interface of respiratory epithelium (RE), with its motile cilia (arrowhead), and olfactory epithelium (OE) of a patient with post-viral hyposmia. The olfactory vesicle (OV), which is ciliated (arrows), projects above the supporting cell (S) that surrounds the dendrite (D). X 15,600.
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Fig. 8. Ciliated olfactory vesicle (OW from a patient with postviral hyposmia. s, supporting cell; arrows, sensory cilia. X 17,800.
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anosmics have very few olfactory vesicles, and those that have been observed are largely aciliate. Post-viral hyposmics, on the other hand, have fewer olfactory vesicles than normal subjects (but more than post-viral anosmics), and most of their olfactory vesicles are equipped with olfactory cilia. Consequently, studies of patients with PVOD indicate that the number of intact ciliated olfactory receptors within the olfactory epithelium is directly related to the patient’s olfactory acuity.
ACKNOWLEDGMENTS These investigations were supported by NIH Program Project Grant NIDCDBPOl DC 00244 (to the Rocky Mountain Taste and Smell Center). We thank Drs. Glen Morangie and Edward W. Johnson for reviewing the manuscript and providing useful comments. REFERENCES Bruch, R.C. (1990) G proteins in olfactory neurons. In: G Proteins and Calcium Signaling, F.H. Naccache, ed. CRC Press, Boca Raton, Florida, pp. 123-134. Gain, W.S., Gent, G., and Catalanotto, F.A. (1983) Clinical evaluation of olfaction. Am. J. Otolaryngol., 4252-256. Caruso, V., Hagan, J., and Manning, H. (1969) Quantitative olfactometry in the measurement of post-traumatic anosmia. Arch. Otolaryngol. Head Neck Surg., 90:500-503. Chuah, M.I., Farbman, A.I., and Menco, B.P.M. (1985) Influence of olfactory bulb on dendritic knob density of rat olfactory receptor neurons in uitro. Brain Res., 338:259-266. Qoty, R.L. (1979) A review of olfactory dysfunction in man. Am. J . Otolaryngol., 1:57-79. Qoty, R.L., Shaman, P., and Dann, M. (1984) Developments of the University of Pennsylvania smell identification test: A standard microencapsulated test of olfactory function. Physiol. Behav., 32: 489-502. Douek, E. (1974) The Sense of Smell and Its Abnormalities. Churchill Livingstone, London. Farbman, A.I. (1977) Differentiation of olfactory receptor cells in organ culture. Anat. Rec., 189:187-200. Farbman, A.I. (1986) Prenatal development of mammalian olfactory receptor cells. Chem. Senses, 11:3-18. Graziadei, P.P.C. (1973) The ultrastructure of vertebrate olfactory mucosa. In: The Ultrastructure of Sensory Organs, I. Friedman, ed. North Holland Publ. Co., Amsterdam, pp. 267-305. Hasegawa, S., Yamagichi, M., and Nakano, Y. (1986) Microscopic
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