Full text of DOI:10.1097/00005537-200204000-00002

The Laryngoscope
Lippincott Williams & Wilkins, Inc., Philadelphia
© 2002 The American Laryngological,
Rhinological and Otological Society, Inc.
THE 2000 OGURA LECTURE
Olfactory Neural Cells: An Untapped
Diagnostic and Therapeutic Resource
Christopher Perry, MBBS, FRACS; Alan Mackay-Sim, PhD; Francois Feron, PhD;
John McGrath, MD, PhD, FRANZCP
Objective: This is an overview of the cellular biol-
ogy of upper nasal mucosal cells that have special
characteristics that enable them to be used to diag-
nose and study congenital neurological diseases and
to aid neural repair. Study Design: After mapping the
distribution of neural cells in the upper nose, the au-
thors’ investigations moved to the use of olfac-
tory neurones to diagnose neurological diseases of
development, especially schizophrenia. Olfactory-
ensheathing glial cells (OEGs) from the cranial cavity
promote axonal penetration of the central nervous
system and aid spinal cord repair in rodents. The
authors sought to isolate these cells from the more
accessible upper nasal cavity in rats and in hu-
mans and prove they could likewise promote neural
regeneration, making these cells suitable for hu-
man spinal repair investigations. Methods: The
schizophrenia-diagnosis aspect of the study entailed
the biopsy of the olfactory areas of 10 schizophrenic
patients and 10 control subjects. The tissue samples
were sliced and grown in culture medium. The ease of
cell attachment to fibronectin (artificial epithelial
basement membrane), as well as the mitotic and apo-
ptotic indices, was studied in the presence and ab-
sence of dopamine in those cell cultures. The neural
repair part of the study entailed a harvesting and
insertion of first rat olfactory lamina propria rich in
OEGs between cut ends of the spinal cords and then
later the microinjection of an OEG-rich suspension
into rat spinal cords previously transected by open
laminectomy. Further studies were done in which
OEG insertion was performed up to 1 month after rat
cord transection and also in monkeys. Results: Schizo-
phrenic patients’ olfactory tissues do not easily attach
to basement membrane compared with control sub-
jects, adding evidence to the theory that cell wall
anomalies are part of the schizophrenic “lesion” of
neurones. Schizophrenic patient cell cultures had
higher mitotic and apoptotic indices compared with
control subjects. The addition of dopamine altered
these indices enough to allow accurate differentia-
tion of schizophrenics from control patients, leading
to, possibly for the first time, an early objective diag-
nosis of schizophrenia and possible assessment of
preventive strategies. OEGs from the nose were
shown to be as effective as those from the olfactory
bulb in promoting axonal growth across transected
spinal cords even when added 1 month after injury in
the rat. These otherwise paraplegic rats grew motor
and proprioceptive and fine touch fibers with corre-
sponding behavioral improvement. Conclusions: The
tissues of the olfactory mucosa are readily available
to the otolaryngologist. Being surface cells, they must
regenerate (called “neurogenesis”). Biopsy of this
area and amplification of cells in culture gives the
scientist a “window to the developing brain,” includ-
ing early diagnosis of schizophrenia. The “Holy Grail”
of neurological disease is the cure of traumatic para-
plegia and OEGs from the nose promote that repair.
The otolaryngologist may become the necessary part-
ner of the neurophysiologist and spinal surgeon
to take the laboratory potential of paraplegic cure
into the day-to-day realm of clinical reality. Key
Words: Olfactory neurones, olfactory ensheathing
glial cells, paraplegia cure, schizophrenia diagnosis
prevention.
Laryngoscope, 112:603–607, 2002
INTRODUCTION
Thank you very much for the honor and privilege of
presenting the Ogura Lecture for the Triological Society
for the year 2000. Joe Ogura died in 1983 when I was in
my second year of advanced surgical training in otolaryn-
gology. I had been a doctor for 6 years. Although new to
the field of otolaryngology, I knew all about Joe, his influ-
ence as a surgeon and teacher and his role in the devel-
opment of partial laryngeal surgery. This article concerns
nasal physiology and anatomy, the diagnosis of schizo-
phrenia and perhaps other neurological diseases, and the
treatment of nerve injuries and traumatic paraplegia.
From the Department of Surgery (C.P.), University of Queensland;
the Centre for Molecular Neurobiology (A.M.-S., F.F.), School of Biomolecular
and Biomedical Science, Griffith University; Queensland Centre for
Schizophrenia Research (F.F., J.M.), Wolston Park Hospital; and the Depart-
ment of Psychiatry (J.M.), University of Queensland, Australia.
Editor’s Note: This Manuscript was accepted for publication January
9, 2002.
Send Correspondence to Christopher Perry, MD, Otolaryngology–
Head and Neck Surgery, 4th floor, Watkins Medical Centre, 225 Wickham
Terrace, Brisbane 4000, Australia. E-mail: cpmedical@hotkey.net.au
Laryngoscope 112: April 2002
Perry et al.: Olfactory Neural Cells
603

This, on the surface, does not appear to have much in
common with Dr. Ogura’s academic interests. However, by
the end of this article, I hope you will share the same
wonder and excitement that I have in this research and
that you will understand the critical role of the otolaryn-
gologist in this research and probably in the future diag-
nosis and treatment of a wide array of neurological condi-
tions. I hope this research reflects something of the ideals
of scholarship Dr. Ogura promoted.
The olfactory nerve or bulb is more an outgrowth of
the brain than a cranial nerve. It is totally within the
cranial cavity, receiving 20 bundles or fascicles of nerves
surrounded by dura mater, pia, and arachnoid from the
foramina of the cribriform plate. The periosteum of the
ethmoid is continuous with this dura mater around these
fascicles. The olfactory mucosa is classically described as
occupying the upper 1 cm of the nasal cavity. This mucosa
has Bowman’s glands with olfactory neurones between
them sending a dendrite to the surface and an axon cen-
trally surrounded by electrically insulating olfactory-
ensheathing glial cells. Between the neurones, on the
basement membrane of the olfactory mucosa, sit imma-
ture neurones and stem cells. The axons of the olfactory
neurones synapse centrally in the glomeruli of the olfac-
tory bulb and are therefore, at least partially, within the
brain.
posterior segment has a function associated with spatial
memory. This hippocampal growth occurs in London taxi
drivers. The authors found large hippocampi in these taxi
drivers and the longer a person has been driving taxis, the
bigger the hippocampus becomes.
In the rat, mature neurones may be identified with
antibodies to the olfactory marker protein (OMP), while
immature neurones are identified with an antibody to
growth-associated protein-43. In the rat, if the fascicles of
the olfactory bulb are cut just above the cribriform plate,
the mature neurones on the olfactory area will die. Imma-
ture neurones will send dendrites to the mucosal surface
and axons centrally to the olfactory bulb, surrounded by
olfactory-ensheathing glia. They will cross the cribriform
plate and scar tissue and then these axons will implant
into glomeruli within the olfactory bulb. Although this
mass movement of axons may not result in implantation
into exactly the correct glomerulus, it has recently been
demonstrated that with lesser degrees of axonal regener-
ation, the penetrating reparative axon is highly accurate
in finding the appropriate glomerulus to register the smell
that that neurone is coded to register.3
Thus, the olfactory mucosa contains neurones at least
partially present within the brain. The neurones have
some features and structures normally associated with
the embryonal brain. The axons of olfactory nerve-
ensheathing glia are able to penetrate scar tissue and the
central nervous system. This mucosa is readily available
to the otolaryngologist to biopsy and study. Can this area
be a window to brain disease, especially diseases of neural
development? Can the central nervous system-penetrative
properties of olfactory nerve-ensheathing glia be recruited
to allow repair of central nervous system lesions? Our
studies indicate they can.
Our studies of human olfactory mucosa1 confirm that
olfactory mucosa is present in the upper 1 cm of the nose.
However, there is no clear line of demarcation between
respiratory and olfactory mucosa. Rather, there is a fine
patchwork of intermingling microscopic areas of respira-
tory and olfactory mucosa that are mostly olfactory in the
upper zones but become respiratory the further down the
nose you biopsy. There is a concentration of olfactory
mucosal zones in the front of the middle turbinate but the
higher and further back the biopsy is taken, the more
likely one will find larger areas of olfactory mucosa
present. Remarkably, olfactory mucosa is also found more
anteriorly and inferiorly than previously appreciated, sug-
gesting that it is present as a patchwork of microscopic
sites over large areas of the septum and lateral wall.
Olfactory neural cells are the only surface neural
cells of the body and must therefore withstand wear and
tear and be able to replicate and regenerate. As said
previously, part of these neurones is within the meninges
and cranial cavity. At least some of the ultrastructural
elements of the olfactory neurones, for example,
␤-tubulin3 and microtubule-associated protein-5, are oth-
erwise only found in embryonal brain cells.
The growth and replication of neural cells is termed
“neurogenesis.” It is of great interest that after the age of
51/2 years, the areas of the mammalian brain known to
undergo neurogenesis are areas associated with olfaction,
the olfactory bulb, the hippocampus, and areas adjacent to
the upper lateral ventricle. Cells that are produced near
the lateral ventricle stream into the olfactory bulb, at least
in laboratory animals. The flow of these cells in humans
has not been established, but a recent study published in
The Proceedings of the National Academy of Sciences of the
United States of America2 has demonstrated volume
changes of the adult human hippocampus, which in its
Diagnostic Resources in the Upper Nose
Schizophrenia is the most common psychosis, affect-
ing approximately 1% of the population. There is undoubt-
edly a genetic component to the onset of this condition,
although inheritance is probably polygenetic. The risk of
disease is higher in affected families with a risk of approx-
imately 50% if a monozygotic twin has the disease. Risk
factors include several perinatal environmental variables
such as mother’s nutrition, winter birth, and city versus
country childhood. Schizophrenics often have physical
characteristics of a high-arched palate and other minor
physical anomalies, suggesting factors operate during pre-
natal development. They are frequently strangely behav-
ing young teenagers with few friends and then develop
hallucinations and full-blown schizophrenia in the 18- to
25-year age group. It is now generally accepted that the
brain lesion of schizophrenia starts early and is therefore
probably a lesion involving the brain during the prenatal
period. If olfactory neurones are similar to brain tissue
and indeed have characteristics of a developing brain, can
cellular biology techniques differentiate the olfactory neu-
rones of the patient with schizophrenia from the normal
control?
Our group reported on a small study of schizophren-
ics and normal control subjects whose olfactory neurones
were grown in the laboratory from upper nasal biopsies
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Perry et al.: Olfactory Neural Cells

taken under local anesthetic as an office procedure.4 Bi-
opsy samples were sliced into 200-␮g thicknesses and
grown on fibronectin-coated glass slides immersed in a
serum-free medium containing antibiotics and growth
factors.
The biopsies of schizophrenic patients do not attach
to fibronectin, a component of the normal epithelial base-
ment membrane. Normal control biopsies in culture at-
tached within 1 to 2 days; however, biopsies from schizo-
phrenic patients would often not attach even after
multiple attempts over 14 days after being taken. These
differences in attachment were statistically significant.
Additionally, there were statistically significantly more
A study of the literature to date highlights the pio-
neering work of Ramon-Cueto. Her 1994 experiment8 in-
volved the division of the dorsal sensory nerve root close to
the spinal cord of the laboratory rat. The dorsal nerve root
was rejoined to the spinal cord using microsurgical tech-
niques. In some of these anastomoses, a suspension of
OEG cells taken from allograft donor rats was injected
into multiple sites on either side of the cut. In those
animals, OEG implantation facilitated the regrowth of the
sensory axons into the dorsal horn from the severed dorsal
root. Similar regrowth was absent in the controls. How-
ever, there was no behavioral improvement in the dener-
vated areas of these rats.
mitoses in the schizophrenics’ cultures and
a
non-
Ramon-Cueto’s 1998 study7 entailed a laminectomy
in 300-g Wistar white rats and excision of 4 mm of spinal
cord in the T8–T10 segments. A 6-mm plastic tube con-
taining a medium rich in Schwann cells was placed as a
bridge between the cut ends and a suspension of OEGs
microinjected into the cut ends of the cord. In this study,
OEG grafts facilitated axonal penetration across the plas-
tic tube and gel bridge and into the distal spinal cord
stump. The regenerating axons tended to cross peripher-
ally and probably did not join appropriate tracts on the
opposite side. Once again, there was no behavioral im-
provement in the OEG injected rats; they remained
paraplegic.
significant trend to more cell death or apoptoses. How-
ever, when dopamine was added to the culture, and most
schizophrenic drugs are dopamine receptor blockers, there
were marked differences between mitoses and apoptoses
in the schizophrenic and control populations. Dopamine
reduced the number of mitoses in both groups but de-
creased apoptoses in schizophrenics, whereas it increased
apoptoses in control subjects. These were constant fea-
tures and allow differentiation of the schizophrenic group
from the control group.
Thus, a nasal biopsy has the potential to provide an
objective and additional sign for the differential diagnosis
of schizophrenia, possibly long before other firm diagnos-
tic criteria become evident. Obviously the study, being
only recently published in December 1999,4 needs to be
confirmed, and we are currently doing a second study.
Early diagnosis of schizophrenia is important because
early intervention with medication leads to a better prog-
nosis and milder continuing symptoms.
Two further studies from 1998 are worth reviewing.
Studies of Li et al.9 from London and Imaizumi from
Yale10 showed that OEG transplants could facilitate re-
myelination after localized lesions in the corticospinal
tracts and dorsal columns.
The most exciting study to date on the axonal regener-
ation properties of OEGs was once again performed by
Ramon-Cueto and published in February 2000.11 Here, rats
were made paraplegic by laminectomy and open, complete
transection of the spinal cord at T8. A suspension of OEGs
was made from olfactory bulbs and injected into the cut ends
of the spinal cord. OEG grafts stimulated regrowth of axons
across the scar and in some rats significant behavioral im-
provement occurred, including coordinated locomotion,
weight bearing, touch, and proprioceptive functions. This
study was hailed as a milestone in paraplegia cure.
In 1999, our collaborators similarly cut the spinal
cords of 18 rats by open laminectomy at T10. Half of these
animals had pieces of donor rat olfactory mucosal lamina
propria inserted between the cut ends and half had respi-
ratory mucosa lamina propria inserted. The lamina pro-
pria of the olfactory area is rich in OEGs. Another group of
six similarly transected rats received injections into the
injured cord of OEGs purified from olfactory lamina pro-
pria. A further six received saline injections. These rats
were killed at 8 to 10 weeks after surgery, compared with
7 months in Ramon-Cueto’s series. Even after only 10
weeks recovery, there was significant behavioral improve-
ment in the nasal OEG-grafted rats, whereas the controls
were left completely paraplegic.
With the olfactory area being a “window to the brain,”
other neurological diseases such as Alzheimer’s disease or
multiple sclerosis may, like schizophrenia, manifest them-
selves in olfactory tissues readily available for study in the
upper nose.
Therapeutic Resources in the Upper Nose
The “Holy Grail” of neurobiology is the cure of trau-
matic paraplegia, and the olfactory mucosa may have a
vital role to play.
Our group, and our collaborators at the Spinal Injury
Research Unit at the University of New South Wales
Department of Anatomy and at the Fourth Military Med-
ical University, Xian, China, have been studying spinal
cord regeneration using olfactory-ensheathing glial cells
(OEGs) from the upper nasal cavity.5 OEGs have been
postulated to promote axon penetration of the central
nervous system and even scar tissue within it. OEGs have
both Schwann cell and glial cell characteristics. They are
able to form myelin sheaths and promote axonal growth
like Schwann cells. They can reside in both the central
and peripheral nervous systems and penetrate an
astrocyte-rich and oligodendroglial-rich central nervous
system, which normally secretes compounds inhibitory to
axonal penetration. For example, oligodendroglial cells
secrete “Nogo,” a 1200 amino acid protein that is inhibi-
tory to axon growth, whereas astrocytes have been shown
to secrete a number of similarly inhibitory compounds.6,7
Histologic cross-sections of the sectioned spinal cord
showed the formation of a syrinx commonly found in hu-
man paraplegia. The OEGs entered the spinal cord ros-
trally and caudally and descending motor axons pene-
trated the distal spinal cord. Fluororuby placed caudally
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Perry et al.: Olfactory Neural Cells
605

was identified in brainstem neuronal cell bodies, and se-
rotonin projections from the brainstem were identified
distal to the transection site. Therefore, it appears that
OEGs could be harvested from a patient’s own upper nasal
mucosa and be used to promote healing and axonal pene-
tration of spinal cord lesions, at least in rats. This exciting
breakthrough needs to be extended toward humans.
control monkey was hemisected but received no graft. This
animal also walked normally. The two experimental ani-
mals were re-anesthetized and a second hemisection was
made at T9 either contralaterally or ipsilaterally to the
first cut. Two months later, both animals had recovered.
Histologic and immunochemical assessment of all four
lesions in these animals showed nerve fibers crossing the
lesion sites, those with and those without lamina propria
grafts.
From Rats to Humans
Previous studies in rats have universally used OEGs
harvested from the embryonic or adult olfactory bulb,
which is a rich source of these cells, but the harvesting of
OEGs from the olfactory bulb is not practical in humans.
It requires an open craniotomy with all the attendant
morbidity and mortality. It would involve significant re-
duction or loss of the sense of smell in a human patient
who is already paralyzed from the waist down from pre-
vious multi-trauma, which may have already affected the
sense of smell. With loss of mobility and possibly sexual
function associated with paraplegia, the sense of smell
enhances the enjoyment of food and beverages and is a
major part of the enjoyment of life for the paraplegic
patient. Endangerment of life with a craniotomy and en-
dangerment of the sense of smell by the harvesting of
olfactory bulb tissues makes OEGs harvested from olfac-
tory bulbs difficult to study in the human clinical experi-
mental situation. The harvesting of the same cells from a
regenerating upper nose under local anesthetic by biopsy
is a more feasible and justifiable proposition for further
experiments.
We have been studying the upper nasal OEGs in
normal subjects undergoing routine nasal surgery. As ex-
pected, OEGs become more numerous the higher up one
goes in the nasal submucosa. Human OEGs are more
densely intertwined and more difficult to separate than
those of rats. Neurones and mucosal epithelial cells can be
separated off the lamina propria containing OEGs by en-
zymatic dissolution of the basement membrane. Numer-
ous cocktails of collagenases and proteases have been used
to dissociate the OEG cells into a suspension. Techniques
of immuno-panning and binding magnetic beads with
anti-p75 receptor antibody, which selectively bind to OEGs,
have allowed the separation and purification of these cells.
We have been able to grow OEGs in culture, and by using a
serum-free medium we have been able to kill fibroblasts
and macrophages while keeping an OEG culture alive and
amplifying.
Thus to Humans
The microinjection of a pure OEG autograft suspen-
sion in the paraplegic human is feasible without excessive
risk of harm. Experiments on the usefulness of these cells
should be able to begin shortly. Because crush and devi-
talization lesions are much more common in humans,
OEGs would probably be needed to be instilled over a wide
area of the spinal lesion. The remyelination properties of
OEGs in spinal injuries raise interesting possibilities. The
property of the promotion of axonal penetration of OEGs is
ready for human study, especially autograft OEGs har-
vested by a minimally invasive technique in the con-
stantly regenerating upper nose.
CONCLUSION
The olfactory mucosal cells, the neurones, and the
olfactory-ensheathing glia offer remarkable possibilities
in the diagnosis of neurologic and psychiatric disorders
and in the healing of central nervous system lesions.
Within the near future, the otolaryngologist and neuro-
physiologist may be the necessary partners of the psychi-
atrist and spinal surgeon to bring this experimental evi-
dence for neurologic diagnosis and paraplegia cure into
the realm of clinical, everyday reality.
Acknowledgments
The studies referred to in this paper, performed by
members of our research group, have been largely funded
by the Stanley Foundation and The Garnett Passe and
Rodney Williams Memorial Foundation.
The authors thank Jike Lu and Phil Waite of the
Spinal Injury Research Unit of the University of New
South Wales and the Fourth Military Medical University,
Xian, China.
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