Brain Research 887 2000 53–62 www.elsevier.com locate bres
Research report
Nerve growth factor applied onto the olfactory epithelium alleviates degenerative changes of the olfactory receptor neurons following
axotomy
a,b b
a a
b
Hiroki Yasuno , Keijiro Fukazawa , Tetsuo Fukuoka , Eiji Kondo , Masafumi Sakagami ,
a ,
Koichi Noguchi
a
Department of Anatomy and Neuroscience , Hyogo College of Medicine, Hyogo 663-8501, Japan
b
Department of Otolaryngology , Hyogo College of Medicine, Hyogo 663-8501, Japan
Accepted 12 September 2000
Abstract
The olfactory neuroepithelium of the mammalian nervous system manifests continuous neurogenesis throughout life. Recent studies suggest that neurotrophic factors and their receptors may play a role in the regulation of development and regeneration in the olfactory
system. However, there have been very few in vivo studies investigating the effect of exogenous neurotrophic factors in the olfactory system. In the present study, nerve growth factor NGF and brain-derived neurotrophic factor BDNF were administered into the rat
olfactory mucosa for 5 days just after the transection of the olfactory nerve. We then examined the effect of exogenous neurotrophic factors on the degenerative changes in axotomized olfactory receptor neurons ORNs. Further, we examined the location of their
receptors, Trk A and Trk B. We found that both mature and immature ORNs expressed more intense signals for olfactory marker protein and beta-tubulin mRNAs, respectively, when NGF was applied to the axotomized olfactory neuroepithelium for 5 days, compared to the
ORNs of saline-treated controls. BDNF at a 10 mg total dose did not show this effect. The effect of NGF applied onto the olfactory epithelium is consistent with the immunohistochemical finding that Trk A was present in the dendrites and axon bundles in normal and
axotomized ORNs. These results suggest that NGF may protect the degenerative changes in mature and immature ORNs following axotomy through the binding to the Trk A receptor located on the surface of the olfactory epithelium.
2000 Elsevier Science B.V. All
rights reserved.
Theme : Sensory systems
Topic : Olfactory senses
Keywords : Olfactory nerve; Olfactory marker protein; Beta-tubulin; Protein gene product 9.5; NGF; BDNF
1. Introduction receptor neurons ORNs derived from the observation that
a developmentally heterogenous population of receptor In mammals, the olfactory neuroepithelium OE,
neurons is present in adult animals, and autoradiographic situated within the nasal cavity, has the ability to regener-
studies have shown that immature neurons in the basal cell
3
ate new neurons continually throughout life from a popula- layer of the OE of adult animals incorporate H-thymidine
tion of precursor cells present in the basal cell layer [5,34]. These immature neurons progressively move to
[15,16]. The concept of continued turnover of olfactory more superficial layers of the epithelium to become mature
ORNs. The process of turnover of ORNs can be modified
following the injury of mature ORNs, such as physical or
Corresponding author. Tel.: 181-798-45-6415; fax: 181-798-45-
chemical damage to the OE proper, the axons of the ORNs
6417. E-mail address
: noguchihyo-med.ac.jp K. Noguchi.
olfactory nerve, or the olfactory bulb [8,40]. For exam-
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 6 6 - 8
54 H
ple, changes in ORNs following the transection of the 2. Materials and method
olfactory nerve have been extensively examined. A few days after transection, massive degeneration and neuronal
2.1. Animal procedures programmed cell death of the ORNs occurred and new
ORNs, derived from a stem cell population in the OE, All animal experiments conformed to the regulations of
generate axons which grow along the olfactory nerve to the the Hyogo College of Medicine Committee on Animal
olfactory bulb. Research and were carried out in accordance with the
The normal OE is a ciliated pseudostratified columnar guidelines of the NIH on animal care. A total of 24 male
epithelium. The basal cells are located at the base of the Sprague-Dawley rats weighing 200–250 g were used. All
epithelium and immature ORNs are situated in the lower experimental procedures were performed on rats which
layer of the epithelium. The immature ORNs express were deeply anesthetized with sodium pentobarbital 50
growth associated protein 43 GAP-43 B50 [39,40], mg kg body weight, i.p.. Additional doses of anesthetics
which is expressed during axonal growth and neural were given as needed.
development, and tubulin [25,32], which participates in Rats received local anesthetic 1 carbocaine, 200 m1
spindle formation in dividing cells. A third type of cell, the to the site of the skin incision on the middle of the head.
mature ORNs, are localized in the middle layer of the The left skull, rostral to the bone suture bregma, was
epithelium and express olfactory marker protein OMP opened using a micro drill and the left olfactory bulb was
[4,6,10,27,30,43], a 19 kDa cytoplasmic protein. Immature exposed. A microsurgery scalpel was inserted to a depth of
and mature neurons express neural cell adhesion molecules 4 mm against the olfactory bulb side of the cribriform plate
[28], which are important during all stages of neurode- and the fila olfactoria were transected according to the
velopment and are especially important for synaptic struc- method reported by Harding et al. [19]. In a preliminary
turing and protein gene product 9.5 PGP 9.5 [23,36], experiment, we confirmed that our method could success-
which is a neurospecific peptide. A fourth type of cell in fully transect the olfactory nerves by checking the thick-
the OE are supporting cells, which are located in the upper ness of the olfactory epithelium. Next, NGF 20 mg ml,
layer of the epithelium. Injury of ORNs also affects the recombinant rat NGF, RD Systems, Minneapolis, MN,
neurochemical expression pattern and axotomized ORNs USA, or BDNF 20 mg ml, recombinant human BDNF,
lose almost all OMP immunoreactive profiles after about a Sumitomo Pharmaceutical, Osaka, Japan was adminis-
week [31]. tered twice a day for 5 days. Rats received NGF or BDNF
It is well known that several growth factors play a solution using a Hamilton syringe intermittently for 15 min
critical role in the regulation of neurogenesis in the until a total volume of 50 ml of solution had been injected
developing central nervous system. The typical neuro- into the left nasal cavity. In control rats, saline was
trophic factors, nerve growth factor NGF and brain- administered. The total dose of 10 mg of NGF or BDNF
derived neurotrophic factor BDNF, are known to have was administered over a 5 day period. This dose was
crucial roles in the differentiation, survival, morphogenesis determined according to the doses of neurotrophins, topical
and regeneration of sensory neurons [2,20,24,26,37]. Re- injections of which were reported previously 6 mg day
cently, high-affinity receptors of the neurotrophic factor NGF [11,41], 5 mg day BDNF [35], 12 mg day BDNF
tyrosine kinases Trks have been cloned; NGF is the [12], and considering the very limited area of the olfac-
preferred ligand of Trk A [7] and BDNF is the preferred tory epithelium.
ligand of Trk B [13]. A number of studies have suggested that endogenous neurotrophins play a role in the regulation
2.2. In situ hybridization histochemistry of neurogenesis within normal development of the OE and
altered turnover of OE following injury to the ORNs. For Five days after surgery, rats were deeply anesthetized
example, NGF injected into the olfactory bulb is trans- with sodium pentobarbital and perfused transcardially with
ported to the OE for 18 h in the mouse [29]. Antibodies to 4 paraformaldehyde in 0.1 M phosphate buffer pH 7.4.
NGF injected in the olfactory bulb induce degeneration of The left olfactory mucosa was dissected, postfixed over-
ORNs [38]. ORNs can be matured in vitro by treatment night, followed by immersion in 20 sucrose in phosphate
with neurotrophins, especially treatment with a combina- buffer for cryoprotection. After 1 or 2 days, olfactory
tion of NGF and BDNF [33]. However, there has been no mucosae were frozen with powdered dry ice, cut trans-
in vivo experiments in which exogenous neurotrophins versely with a cryostat into 10 mm thick sections, thaw-
were applied into the nasal cavity. In this study, we mounted onto Vectabond-coated slides Vector Laborator-
administered NGF or BDNF into rats’ olfactory mucosa, ies, Burlingame, CA, USA and stored at 2808C until use.
and examined in vivo the effect on axotomized ORNs. The An olfactory marker protein-specific probe, a mature ORN
dependent measures examined were altered mRNA expres- marker, and a beta-tubulin-specific probe, an immature
sion of neurochemical markers using in situ hybridization ORN marker, were used. The probes consisted of 48-base
histochemistry, and we also examined their receptors, Trk oligonucleotides and the OMP probe was complementary
A and Trk B, using immunohistochemistry. to bases 439–486 and beta-tubulin probe to bases 18–65 of
H . Yasuno et al. Brain Research 887 2000 53 –62
55
the respective cDNAs. These probes were labeled with drated in a graded ethanol series, cleared in xylene, and
35
S-deoxyadenosine triphosphate NEN, Boston, MA, coverslipped.
USA and terminal deoxynucleotidyl transferase Amer- sham, Buckinghamshire, UK, giving a specific activity of
2.4. Quantitative analysis
9 9
1.0310 –1.5310 cpm mg. Tissue sections were hybrid- ized after thawing, without any pretreatment, overnight at
Each experimental group consisted of six rats. For the in
5
428C in humidified boxes with 5310 c.p.m. of labeled situ hybridization histochemistry, measurements of the
probe per 100 ml of a mixture containing 43 SSC, 50 relative density of the areas of silver grains over selected
formamide, 0.12 M phosphate buffer, 13 Denhardt’s tissue profiles were performed. OMP mRNA and beta-
solution, 0.2 sodium dodecyl sulfate, 250 mg ml yeast tubulin mRNA levels in the OE were quantified using a
tRNA, 10 dextran sulfate, and 100 mM dithiothreitol. computerized image analysis system IBAS, Zeiss, Ger-
After hybridization, the sections were rinsed four times for many. At a magnification of 320 with bright-field
15 min each at 558C in 13 SSC, dipped into distilled illumination, OE were set such that only silver grains were
water, transferred through 60, 80, and 95 ethanol, and accurately discriminated from the background in the
then air dried. For autoradiography, the sections were lamina propria and read pixel-by-pixel by the computer.
coated with NTB-3 emulsion Kodak, Rochester, NY, We selected randomly 18 fields of view from each rat and
USA; diluted 1:1 with distilled water at 458C and exposed quantified the signal density. The signals for OMP and
for 2–3 weeks in light-tight boxes at 48C. After develop- beta-tubulin were expressed as a percentage of the signal
ment in D19 Kodak and fixation in 24 sodium thiosul- intensity of naive rats. Unpaired comparisons t-test were
fate, the sections were rinsed in distilled water, counter- used to determine if there were significant differences
stained with neutral red, dehydrated in a graded ethanol between groups. P , 0.05 two tail was considered to be
series, cleared in xylene, and coverslipped. statistically significant. All data points are expressed as the
mean6S.E.M. For immunohistochemistry, the number of PGP 9.5-
2.3. Immunohistochemistry labeled profiles per each field of view was counted and
averaged. Eighteen fields of view were selected randomly The sections were immunostained using the avidin–
from each rat. Unpaired comparisons t-test were used to biotin complex ABC with nickel ammonium sulfate
determine if there were significant differences between intensification. The primary antibody for PGP 9.5 poly-
groups. P , 0.05 two tail was considered to be statistical- clonal antibody Ultra Clone Limited, UK was diluted
ly significant. All data points are expressed as the 1:1500 with 0.1 M Tris–HCl buffered saline TBS, pH
mean6S.E.M. 7.5 containing 2 normal goat serum NGS. Anti-Trk A
polyclonal antibody Chemicon International, Temecula, CA, USA was diluted 1:250 and anti-Trk B polyclonal
3. Results
