Masuo Yamagishi

Examination and classification of human olfactory mucosa in patients with clinical olfactory disturbances

SummaryTo determine objectively the degree of olfactory disturbance, we biopsied the olfactory mucosa from patients who complained of anosmia. The olfactory disturbances in this study were caused by choanal atresia, chronic sinusitis, viral inflammation, and head trauma, as well as by congenital and idiopathic anosmia. The biopsy specimens were examined by light microscopy and the degree of mucosal degeneration present was classified according to five grades. The clinical courses of the patients studied paralleled the changes found in the olfactory mucosa.

Immunohistochemical analysis of the olfactory mucosa by use of antibodies to brain proteins and cytokeratin.

The present study deals with the immunohistochemical detection of four brain-derived proteins and cytokeratin in the normal olfactory mucosa of humans and guinea pigs. Neurofilament protein immunoreactivity was found in the olfactory vesicles, dendrites, and perikaryon of receptor cells, and in thick nerve bundles located deep in the lamina propria. The antiserum to neuron-specific enolase (NSE) selectively stained olfactory receptor cells throughout the length of the bundles. The NSE immunoreactivity also was recognized in nerve bundles of various sizes throughout the lamina propria. Glia-specific S-100 protein immunoreactivity was present in Bowmans glands as well as in all nerve bundles in the lamina propria, but not in any cellular elements constituting the olfactory epithelium. Immunoreactivity for spot-35 protein, which was considered to be specific for cerebellar Purkinje cells, was found in flasklike cells (microvillar cells) occurring near the free surface of the epithelium. The basal cells in the olfactory and respiratory epithelium were stained positively with a cytokeratin antiserum.

Pathology of Olfactory Mucosa in Patients with Alzheimer's Disease

Characteristic changes that appear in the biopsied olfactory mucosa of patients with Alzheimers disease (AD) were examined with immunohistochemical staining. Specimens were obtained from patients with clinical diagnoses of AD. Patients with vascular dementia and age-matched patients without dementia were used for controls. In most AD cases, neurofibrillary tangle-like abnormal tau protein (Tau) immunoreactivity was seen in the dendrites and perikarya of the olfactory receptor cells and in the nerve bundles. A senile plaquelike extracellular mass was found in the olfactory epithelium, and it reacted strongly to an anti-Tau antiserum and weakly to an antiamyloid-β protein antiserum. Ubiquitin (Ubq) immunoreactivity was also observed in the dendrites. Tau immunoreactivity of the perikarya and extracellular mass, and Ubq immunoreactivity were especially characteristic of the olfactory mucosa of AD patients. From these results, it is clear that the same pathologic changes in the brain are also present in the olfactory mucosa of patients with AD. Not only disruption of the central olfactory pathway, but also an olfactory disturbance of AD patients is caused by peripheral changes. Furthermore, an olfactory mucosal biopsy could be a useful method for a definitive diagnosis of AD.

Microscopic studies of human olfactory epithelia following traumatic anosmia

SummaryWe devised our own instrument for obtaining biopsy specimens of human olfactory epithelium. We obtained specimens from two patients with anosmia caused by head trauma. The morphological changes in the olfactory epithelia were studied by light microscopy and transmission electron microscopy. With little damage or early stage of changes, the characteristic finding involved degeneration of the olfactory vesicles, especially with the disappearance of cilia. In the case of great damage and the final stage of injury, no olfactory vesicles were found at the epithelial surface and the olfactory epithelia presented a disorganized cellular arrangement of cells with an atrophic appearance. Our findings indicate that the epithelial degeneration occurring in traumatic olfactory dysfunction varies according to the degree of damage incurred and the time lapse present from injury.

Ontogenetic Expression of Spot 35 Protein (Calbindin-D28K) in Human Olfactory Receptor Neurons and its Decrease in Alzheimer's Disease Patients

Expression of a calcium-binding protein, spot 35 protein (S-3S, calbindin-D28k), was investigated immunohistochemically in the human olfactory mucosa of patients who ranged in age from 16 weeks of fetal development to 98 years old, including some with Alzheimers disease (AD). S-35 immunoreactivity was observed clearly in olfactory receptor neurons (ORNs) and olfactory nerve bundles that were identified previously with antibodies to olfactory marker protein (OMP) and neuron-specific enolase (NSE). Throughout all ages, the mean number of ORNs immunoreactive for OMP did not change significantly, whereas the mean number of NSE- and S-35-immunoreactive ORNs declined markedly in the postnatal infant, young, and old patients when compared with that of the prenatal fetuses. S-35-immunoreactive ORNs decreased significantly in AD patients when compared with AD control patients. These results indicate that ORNs in humans express S-35 and that there is an age-related trend in the expression of S-35. Furthermore, the marked decrease of S-35 expression in ORNs of AD patients suggests that cell excitability associated with calcium ions and cell protective function against overload of intracellular calcium ions decline in these patients.

A re-evaluation of the classification of olfactory epithelia in patients with olfactory disorders

SummaryWe have previously demonstrated that human olfactory epithelia can be classified into five grades according to the degree of degeneration present in patients with various kinds of olfactory disorders. In practice, however, the occurrence of additional types of cell changes in other kinds of olfactory disorders and findings with immunohistochemical techniques have led us to re-evaluate our previous classification. In the present study, changes in olfactory epithelia from ten patients with various kinds of olfactory disorders are discussed and a revised classification is proposed. Microvillar and differentiating cells were also evaluated in the epithelium studied.

Olfactory Mucosa of Patients with Olfactory Disturbance following Head Trauma

The olfactory mucosa in 7 patients with olfactory disturbance following head trauma were sampled for biopsy with special biopsy forceps and examined by immunohistochemical staining with anti—neuron-specific enolase (NSE) and S-100 protein (S-100) antibodies. The residual olfactory receptor cells and nerve bundles were counted, and the degree of degeneration was determined. In 5 patients, olfactory receptor cells that reacted with anti-NSE antiserum remained, although the number varied with the patient, and in 2 patients the receptor cells disappeared. In the lamina propria, the S-100–immunoreactive olfactory nerves were retained in 6 patients. The outcome was poor in all cases regardless of the number of residual receptor cells and nerve bundles. These results indicate that the degree of impairment of the peripheral olfactory region after head trauma differs from case to case, and that even if the receptor cells and nerve bundles remain, it is difficult to improve the condition, although some cases of malingering may be contained.

Increased Density of Olfactory Receptor Neurons Immunoreactive for Apolipoprotein E in Patients with Alzheimer's Disease

Immunolocalization of apolipoprotein E (apoE) was investigated in human olfactory mucosa in which olfactory receptor neurons (ORNs) were identified with antiserum to protein gene product (PGP) 9.5. Tissue was obtained at autopsy from 10 nondemented middle-aged or elderly subjects and 9 patients with Alzheimers disease (AD). Double-labeling immunofluorescence established that apoE immunoreactivity was colocalized in a subpopulation of PGP 9.5-immunoreactive ORNs. The mean number of apoE-immunoreactive ORNs per unit epithelial length in AD patients was about 3.5 times greater than that in nondemented patients, although the mean number of PGP 9.5-immunoreactive ORNs was similar. The apoE-immunopositive Schwann cells in olfactory nerve bundles were the probable source of apoE in the ORNs. The increased numbers of apoE-immunoreactive ORNs in AD patients compared to nondemented subjects demonstrates another manifestation of AD-related neuropathology, in addition to cytoskeletal changes, β-amyloid deposition, and changes in immunoreactivity for other neuroproteins, that parallels changes in neurons in the AD brain.

Immunohistochemical Study of the Fourth Cell Type in the Olfactory Epithelium in Guinea Pigs and in a Patient

The mammalian olfactory epithelium consists of supporting cells, olfactory receptor cells, basal cells and a fourth cell type, which has recently been discovered. In this study we examine this fourth cell type using immunohistochemical techniques. Anti-Purkinje-specific spot-35 protein (S-35), anti-S-100 protein (S-100), anti-neuron-specific enolase (NSE), antichromogranin A and antisynaptophysin antisera were used for the immunostaining. The fourth cell type immunoreacted only to anti-S-35 antiserum and did not react to other antisera in guinea pigs. On the other hand, in the human S-100 immunoreactivity was seen in the fourth cell type as well as weak reactivity to S-35. NSE immunoreactivity was found only in the olfactory receptor cells of guinea pigs and the human. From these results it is assumed that this fourth cell type is a second chemoreceptor different from the olfactory receptor cells because S-35 and S-100 are Ca(++)-binding proteins.

Immunohistochemical Method for the Diagnosis of Olfactory Disturbance

To confirm the diagnosis and determine the cause of the olfactory disturbance, we used an immunohistochemical method to examine biopsy specimens of the olfactory mucosa from a patient who complained of anosmia after head injury. Neuron-specific enolase immunoreactivity was found in the olfactory vesicles and dendrites of the receptor cells in the olfactory epithelium. S-100 protein immunoreactivity was found in the ductal cells of Bowmans gland in the olfactory epithelium and in the acinar cells of Bowmans gland in the lamina propria. This suggests that the olfactory receptor cells and Bowmans gland were normal. The olfactory disturbance in this patient was not caused by nerve transection due to the head injury, but by already existing chronic sinusitis. Immunohistochemical methods are useful for diagnosing olfactory disturbance when used in combination with biopsy of olfactory mucosa.