Chem. Senses 27: 521-527,
2002
© Oxford University Press 2002
Cell Death of Olfactory Receptor Neurons in a Rat with Nasosinusitis Infected Artificially with Staphylococcus
1 People's Hospital of Beijing University, Beijing, China 2 Kanazawa University Graduate School of Medical Science, Kanazawa City, Japan
Correspondence to be sent to: Takaki Miwa, Department of Otorhinolaryngology, Kanazawa University Graduate School of Medical Science, Takara Machi 13-1, Kanazawa City, Japan 920-8641. e-mail: miwataka{at}orl.m.kanazawa-u.ac.jp
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Abstract |
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Nasosinusitis is a common cause of acquired hyposmia or anosmia. To study the apoptotic death of olfactory receptor neurons in nasosinusitis, we made an inflammation model in rat infected with Staphylococcus. The histochemical changes in olfactory epithelium were examined using antibodies against protein gene product 9.5 (PGP 9.5), single-strand DNA (ssDNA), Bcl-2 and Bax that might be involved in the apoptosis of olfactory receptor neurons. The thickness of olfactory epithelium and the number of ssDNA-labeled cells were evaluated in each post-treatment group and the results were analyzed by two-way analysis of variance (ANOVA) and post hoc tests. Hematoxylin—eosin staining showed that a severe inflammatory reaction had occurred on the infected side of the nasal cavity and sinus, but not on the non-infected side. However, apoptosis of olfactory receptor neurons occurred on both sides; the apoptosis on the non-infected side started later and behaved like a shadow curve similarly to the infected side. Repeated measures ANOVA showed significant differences of both the thickness of olfactory epithelium (P < 0.0001) and the number of ssDNA-labeled cells (P = 0.0339) in the epithelium between the infected side and non-infected side comparing treatment, time and their interactions. Bcl-2 and Bax were detected only on the infected side in the early stages. Thus, nasosinusitis induced the apoptosis of olfactory receptor neurons. However, the apoptosis occurred not only on the infected side, but also on the non-infected side with no significant inflammation. The Bcl-2/Bax family seems to play an important role in the apoptosis induced by infection, but not in the apoptosis on the non-infected side. The results suggest that mechanisms of apoptosis of olfactory receptor neurons on the infected side may differ from those on the non-infected side.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Apoptosis is a programmed gene-regulated cell death in which tissue architecture can be preserved (Oppenheim, 1991
). Neuronal apoptosis plays an important role not only in the
development of the nerve system, but also in trauma, infection and
neurodegenerative disease (Rink et
al., 1995; Stefanis
et al., 1997). In mammals, olfactory neurons are renewed
through a cyclic process of life and death during the lifespan of the animal.
Since the olfactory epithelium is constantly exposed to airborne environmental
stresses, the ability to adapt to injury from these insults is essential in
maintaining olfactory function. Investigations of apoptosis of olfactory
neurons under pathologic conditions are therefore of considerable
interest.
Nasosinusitis and head injury are common causes of acquired hyposmia or
anosmia. Experiments by Deckner and colleagues
(Deckner et al., 1997)
showed loss of olfactory receptor neurons following olfactory nerve
transection in the rat and showed cell death to be due to apoptosis.
Nasosinusitis can also cause hyposmia or anosmia due to nasal obstruction; it
may also damage olfactory epithelium. Is neuron apoptosis also involved during
inflammation? Does olfactory receptor neuron apoptosis occur only in the
infected area? Is the mechanism of apoptosis in nasal inflammation the same as
in nerve injury?
In this study, we made a nasosinusitis model in rats to answer these
questions about the apoptosis of olfactory receptor neurons. Four kinds of
primary antibody were used to observe apoptotic change of olfactory
neuroepithelium. Protein gene product 9.5 (PGP 9.5) is a neurospecific peptide
(Doran et al., 1983).
Here we used it to show the olfactory receptor neurons
(Johnson et al.,
1995). Single-strand DNA (ssDNA) is the product of apoptosis,
which comes from the effect of endonuclease on DNA. This is a new and
convenient method to detect in situ apoptosis immunohisto-chemically
and has been shown to be effective
(Watanabe et al.
1999). Bcl-2 is a member of a large gene family of encoding
proteins that can either inhibit (e.g. Bcl-2, Bcl-x) or promote (e.g. Bax,
Bcl-X, Bak) apoptosis. Bcl-2 is an intracellular membrane protein and resides
primarily in the nuclear envelope, outer mitochondrial membrane and
endoplasmic reticulum. Bax also belongs to the Bcl-2 family and is involved in
apoptosis under some conditions and can promote cell death. Bax has also been
shown to be directly induced by tumor suppressor p53, which is involved in DNA
damage. In this study, we used these antibodies to investigate the possible
factors involved in apoptosis of olfactory receptor neurons in infection.
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Materials and methods |
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Animal experiment
Eight-week-old, male Sprague—Dawley rats, weighing from 260 to 280g, were used in this study. All animals were treated in accordance with the guidelines of the Institutional Committee for Experimental Animals, Kanazawa University Faculty of Medicine.
Animals were divided into seven groups, each of which contained three rats. Animals were killed 1, 3, 7, 14, 21 or 28 days after the treatment described below, and three other animals which did not receive any treatment were killed as the control group.
Infection procedure
Animals in the treatment group were deeply anesthetized by i.p. injection of sodium pentobarbital (50 mg/kg). An incision was made in the medial side of the left ala to enlarge the nares. A 1 x 2 x 18 mm polyvinyl acetal applicator (Merocel; XOMED Surgical Products, Jacksonville, FL), smeared with Staphylococcus, was then inserted into the left nasal cavity. Staphylococcus aureus strain 209P was obtained from the Japan Collection of Microorganisms and then cultured for 24 h at 37°C prior to administration. The polyvinyl acetal applicator was left in the nasal cavity until perfusion. The wound was sutured and the animal was allowed to recover from anesthesia before being returned to its cage.
Histochemistry
For morphological analysis, animals were deeply anesthetized and thoracotomied, then perfused and fixed via the heart. The perfusion solution was 0.01 M phosphate-buffered saline (PBS, pH 7.0) and the fixation solution was 4% paraformaldehyde (PFA)—0.1 M PBS. The nasal part was then dissected and postfixed in 4% PFA—0.1 M PBS overnight, followed by decalcification in 12% EDTA for >2 weeks.
After paraffin embedding, the fixed and decalcified nasal tissues were cut in 1 µm-thick sections and used for hematoxylin—eosin (H—E) staining and immunohisto-chemical analysis.
In immunohistochemical staining, after deparaffination and rehydration,
specimens were subjected to antigen retrieval by microwave in citrate buffer
(pH 7.0, 80°C, 20 min) for Bax and Bcl-2, or 0.4% trypsin incubation (room
temperature, 10 min) for ssDNA, or both for PGP 9.5. After incubating with 3%
H2O2 and 1% bovine serum albumin/Tris-buffered saline
(BSA/TBS) to block endogenous peroxidase and nonspecific reactions,
respectively, specimens were incubated with the primary antibodies PGP 9.5
(1:400, DAKO code no. Z5116), ssDNA (1:400, DAKO code no. A4506), Bax (1:800,
Pharmingen catalog no. 554106), Bcl-2 (1:800, Pharmingen catalog no. 13456E)
at room temperature for 
60 min. EnVision peroxidase (Rabbit, DAKO code
no. K4002) was used as the second antibody and 3,3'-diaminobenzidine
(DAB, DAKO code no. K3465) was used as the substrate-chromogen system. Color
development was monitored under a microscope and ended by rinsing with
distilled water. A thorough washing with TBS (pH 7.6) followed each step,
except the incubation with 1% BSA/TBS.
Counterstaining with hematoxylin was done for <30 s, followed by rinsing in running water for 15-18 min.
The brain stems of healthy control group rats were used as the positive control for Bcl-2 and Bax according to the manufacturers' instructions and different primary antibodies incubated with consecutive sections were used as the negative control.
Data analysis
The thickness of the olfactory epithelium and the number of ssDNA-labeled cells were evaluated in three randomly selected olfactory epithelial fields on both sides of the nasal cavity. The number of ssDNA-labeled cells was counted in a 0.25 mm2 area of the olfactory epithelium. Average and standard error (SE) were used to make graphs to show the changes. Results were analyzed by two-way analysis of variance (ANOVA) and post hoc tests comparing treatment, time and their interactions.
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Results |
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The animals showed some general symptoms of inflammation, such as weariness and reduced drinking and eating over the first 3 days. Some rats suffered dyspnea to a certain extent. However, after the day 4, nearly all of the animals seemed to return to normal activities and ate normally.
Histochemistry
Control group
In H-E stained tissue sections, the olfactory mucosa showed a structured
epithelium, comprised of olfactory neuron receptor cells, supporting cells,
basal cells and the duct cells of Bowman's glands. Submucosal structures,
including Bowman's glands, olfactory axon bundles and their associated Schwann
cells, blood vessels and connective tissue, were also observed.
In PGP 9.5 staining, olfactory neurons and submucosa axon bundles were strongly stained. These neurons showed in an orderly arrangement in about four to eight layers and their dendrites can also be seen clearly between supporting cells. ssDNA staining showed only a few positive cells dispersed in olfactory epithelium in the normal 8-week-old rats. There was no Bcl-2 or Bax staining detected in the olfactory epithelium in this group (data not shown).
Nasosinusitis model
In this model, H-E stained tissue clearly showed sinusitis-related changes
on the infected side only (Figure
1A,B), such as inflammatory cell infiltration, vascular dilation
and severe luminal exudation. These changes were most prominent on day 1 in
both the nasal cavity and the sinus and subsequently declined gradually. The
thickness of the olfactory epithelium decreased, beginning on day 1 and
reached its thinnest level at about day 14. Nearly no supporting cells were
left by day 3, which may have been due to necrosis, and did not begin to
recover until day 14. On the other side in the same rat, there was nearly no
inflammatory reaction in the olfactory epithelium, but the thickness of the
olfactory epithelium was also decreased on day 7
(Figure 1C) compared to control
animals and reached a minimum on day 21. The changes of epithelium thickness
on both sides are shown in Figure
2. On the infected side, the decrease in thickness of the
olfactory epithelium was significant in all the post-treatment days
(P < 0.0001), while on the non-infected side, the thickness
decreased significantly from day 7 (P < 0.0001). Repeated measures
ANOVA showed significant differences between the infected side and the
non-infected side (P < 0.0001). Moreover, supporting cells did not
decrease until day 14.
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), it decreased on day 7 and
reached the minimum on day 21. *P < 0.0001.
With the anti-PGP 9.5 immunohistochemical stain, the layers of positive
olfactory neurons decreased from day 1 on the infected side. By day 3, some
parts had only the basal cells remaining. Dendrites and cilia of olfactory
neurons were also lost in some portions, especially at 1 week
(Figure 3A). By day 28,
although the PGP 9.5 positive cells were smaller than normal olfactory cells,
some of these small olfactory neurons had regained their dendrites. Supporting
cells also diminished in most of the rats and olfactory neurons were then
directly exposed to the nasal cavity. The olfactory epithelium on the
non-infected side showed nearly normal structure until day 3, while from day
7, the olfactory epithelium became thinner and unstained olfactory neurons,
which were located mainly in the deep layers, could be seen clearly on day 14.
However, the ciliary structure and orderly arrangement was maintained and most
of the supporting cells were still intact
(Figure 3B).
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Anti-ssDNA positive cells increased from day 1 on the infected side and reached a maximum at day 7 (Figure 4A). By day 21, the number of positive cells was nearly normal. On the other side of nasal cavity, apoptosis began to increase in some areas from day 3 (Figure 4B) and reached a maximum at day 14. On day 28, there were still many positive cells on the non-infected side. So the change on the non-infected side looked like a shadow curve of that on the infected side. Figure 5 shows the changes in both sides. On the infected side, the number of ssDNA-labeled cells increased significantly on days 1, 3, 7 and 14 (P < 0.0001) compared with control animals. On the non-infected side, ssDNA-labeled cells increased from day 3 (P < 0.01), reached a maximum on day 14 and were still not back to normal by day 28 (P < 0.0001). Repeated measures ANOVA showed significant differences between the two sides (P = 0.0339) comparing treatment, time and their interaction. The curve patterns were also significantly different between the infected and non-infected sides (P < 0.0001). The positive cells on the infected side were mainly scattered in the superficial part in the early stage and turned to reach the whole epithelium by day 7 (Figure 4A). On the non-infected side, olfactory neuron apoptosis appeared mainly in the deep part of the olfactory epithelium (Figure 4B).
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Bcl-2 expression can be clearly detected in some of the olfactory receptor neurons on days 1 and 3 on the infected side. Some of the positive cells were concentrated on the superficial part of the olfactory epithelium, adjacent to where necrotic changes were detected (Figure 6A). On day 7, there was nearly no expression of Bcl-2. On the other side of the nasal cavity, no positive cells were detected in any groups.
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Bax-positive cells were also detected in the early stage of inflammation on the infected side. The positive cells were scattered over some portions and the reaction was not as prominent as Bcl-2 staining (Figure 6B). No positive reaction showed on the non-infected side.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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There are several experimental manipulations used to study apoptosis in olfactory receptor neurons: olfactory bulbectomy (Michel et al., 1994
);
olfactory nerve transection (Simmons
et al., 1981); and sensory deprivation by naris occlusion
(Farbman et al.,
1988). Although previous studies have shown that infection can
cause apoptosis of hippocampal neurons
(Inja et al., 1997),
little is known about apoptosis in olfactory receptor neurons due to
nasosinusitis. In this study, we successfully used a delivery system smeared
with Staphylococcus to make a nasosinusitis model in rat. The results
showed that inflammation and substantive olfactory receptor neuron apoptosis
occurred during the infection. Farbman's experiment
(Farbman et al., 1988)
showed that reduction of olfactory neurons started 20 days after naris
occlusion for sensory deprivation. In our nasosinusitis model, neuron
apoptosis and reduction started within the first 2 weeks. Therefore, we can
conclude that the apoptosis reaction in our model may not due to the temporal
sensory deprivation effect, but to inflammation.
Infection can damage the structure of the olfactory epithelium and cause
olfactory receptor neurons loss. Part of the cell loss is due to necrosis,
including both olfactory receptor neurons and supporting cells, and the
remaining neurons may also lose part of the dendrites or ciliary structures,
as shown in the present experiment. The impairment of neuron structure or
intercellular connection may be part of the reason for neuron apoptosis in
this model. Inflammation can also produce inflammatory chemokines, such as
RANTES, MIP-1
, MCP-1, MCP-3 and IP-10, and cytokines such as
TNF-, IL-1ß, IL-6, IL-18 and IFN-/ß, etc. These
factors can exaggerate the inflammatory reaction and some of them are ligands
that can induce apoptosis by themselves. Earlier experiments
(Farbman et al., 1999)
have shown that both sets of ligands and receptors, Fas/FasL and
TNF-/TNFR1, were present in olfactory epithelium. So the apoptosis of
olfactory receptor neurons during infection may be either a host defense
mechanism or may reflect the pathogenesis of the infection. On the one hand,
the host is influenced by the inflammatory insult passively and, on the other
hand, the organism can eliminate those injured cells actively to maintain
normal function.
There are many kinds of mechanisms involved in apoptosis. During the
commitment or induction phase of apoptosis, a number of signals cause the cell
to enter the apoptotic pathway by altering the balance of proapoptotic and
antiapoptotic proteins that determine either susceptibility or resistance to
apoptosis. The Bcl-2 family is just one of those modulation proteins. Bcl-2
inhibits apoptosis by preventing the release of apoptosis inducing factor
(AIF) and cytochrome C from mitochondria. Bax is a type of factor that can
promote cell death. It has been suggested that the tumor suppressor p53 and
exposure to DNA-damaging agents could induce Bax release directly. In our
experiment, although Bcl-2 was expressed clearly on days 1 and 3 on the
infected epithelium, the expression of Bax was scattered and massive olfactory
neuron apoptosis followed subsequently and was evident by day 7. The results
suggested that apoptosis caused by inflammation might be mediated through a
pathway regulated by the Bcl-2 family. However, according to earlier workers
(Calof et al. 1996),
the apoptosis of mature olfactory neurons following bulbectomy was p53
independent and not regulated by Bcl-2. The different expressions of Bcl-2 and
Bax suggest that different apoptotic mechanisms are involved in these two
situations.
Another interesting finding is the apoptosis of olfactory neurons observed on the non-infected side of the nasal cavity during infection. In our experiment, the sinusitis occurred mainly on the infected side, while the non-infected side showed little inflammatory reaction. However, olfactory receptor neuron apoptosis appeared not only on the infected side, but also on the non-infected side which, though delayed a little compared with the infected side, was followed by a decrease in the thickness of the olfactory epithelium. In contrast to the infected side, the structure of the epithelium on the non-infected side maintained well and there was no positive reaction with either Bcl-2 or Bax. Thus, these responses might indicate active regulation by the organism itself through a different apoptotic pathway from the directly infected area. The physiological and pathological significance of this kind of effect is still not clear, but it seems to be the clinical manifestation of nasosinusitis. It is still unclear if the information or signals are transmitted to other areas by inflammatory media, endotoxins, or the nervous system. Further work should be carried out for a clear understanding of this matter.
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Acknowledgments |
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We thank Dr T. Tatsutomi for help with the animal experiments. This research was partially supported by Japan China Medical Association.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Accepted April 11, 2002
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