Abstracts - Association for Chemoreception Sciences

hours later, the levels of proliferating cell nuclear antigen
(PCNA)-labeled proliferation, and TUNEL- and activated
caspase-3-labeled apoptosis in the OE were measured. We found
that, compared to saline animals, intranasal instillation of ATP
significantly increased PCNA+ cells in the OE. ATP also
significantly decreased TUNEL+ but not activated caspase-3+
apoptotic cells even though the level of apoptosis in normal OE is
relative low. Likewise, in OE primary cell culture, we found that
ATP (100 µM) significantly increased BrdU and 5-ethynyl-2-
deoxyuridine incorporation, suggesting an increase in
proliferating cells. ATP also significantly decreased TUNEL+ and
activated caspase-3+ apoptotic cells. Consistent with ATPinduced
decrease of apoptosis, we observed a significant increase
in the number of OMP+ mature OSNs, nestin+ neuronal
progenitor cells, notch 2+ and NPY+ sustentacular cells following
ATP incubation. Taken together, these data indicate that ATP
plays a role in maintaining OE homeostasis via mitogenic and
protective effects. Acknowledgements: NIDCD DC006897
#P73 POSTER SESSION II:
OLFACTORY PHYSIOLOGY & CELL BIOLOGY;
TASTE MOLECULAR GENETICS;
CHEMESTHESIS & TRIGEMINAL
Nickel Sulfate Induces Location-Dependent Atrophy of
Mouse Olfactory Epithelium: Protective and Proliferative
Role of Purinergic Receptor Activation
Colleen C. Hegg, Carlos Roman, Cuihong Jia
Michigan State University East Lansing, MI, USA
Occupational exposure to nickel sulfate (NiSO4) leads to
impaired olfaction and anosmia through an unknown mechanism.
We investigated the mechanism of NiSO4-induced toxicity and
the potential therapeutic role of ATP in olfactory epithelium.
Male Swiss Webster mice were intranasally instilled with NiSO4
or saline followed by ATP, purinergic receptor antagonists or
saline. We assessed the olfactory epithelium for NiSO4-induced
changes 1-7 days post-instillation and compared results to
olfactory bulb ablation-induced toxicity. Intranasal instillation of
NiSO4 produced a dose- and time-dependent reduction in the
thickness of turbinate OE. These reductions were due to
sustentacular cell loss, measured by terminal dUTP nick end
labeling staining at 1 day post-instillation and caspase-3-
dependent apoptosis of olfactory sensory neurons at 3 days
post-instillation. A significant increase in cell proliferation was
observed at 5 and 7 days post-instillation of NiSO4 evidenced
by BrdU incorporation. Treatment with purinergic receptor
antagonists significantly reduced NiSO4-induced cell
proliferation and post-treatment with ATP significantly increased
cell proliferation. Post-treatment with ATP had no effect on
sustentacular cell viability but significantly reduced caspase-3-
dependent neuronal apoptosis. In a bulbectomy-induced model
of apoptosis, exogenous ATP produced a significant increase in
cell proliferation that was not affected by purinergic receptor
antagonists, suggesting ATP is not released during bulbectomyinduced
apoptosis. ATP is released following NiSO4-induced
apoptosis and has neuroproliferative and neuroprotective
functions. These data provide therapeutic strategies to alleviate the
loss of olfactory function associated with occupational exposure
to nickel compounds. Acknowledgements: CR contributed to this
work as part of the Summer Research Opportunity Program of
the Ronald McNair Program at MSU, and was supported by
NINDS 1R25NS065777-01 and the RISE Program at the
University of Puerto Rico-Cayey. This work was supported by
NIDCD DC006897.
#P74 POSTER SESSION II:
OLFACTORY PHYSIOLOGY & CELL BIOLOGY;
TASTE MOLECULAR GENETICS;
CHEMESTHESIS & TRIGEMINAL
Using a 3-D Culture Model to Identify Factors that Regulate
Olfactory Epitheliopoiesis
Woochan Jang, Jesse N. Peterson, Tyler T. Hickman, James E.
Schwob
Tufts University School of Medicine Boston, MA, USA
Many facets of the molecular mechanisms that regulate the
growth, maintenance, and regeneration of the olfactory
epithelium (OE) remain to be discovered. The ability to efficiently
test the regulatory role of candidate molecules or dissect complex
molecular mixtures requires a tissue culture model that closely
mimics the biology of the in vivo OE. When grown in airinterface
cultures surmounting a layer of feeder cells, OE cells
form “spheres” that closely resemble the OE in vivo and retain
the capacity to engraft following transplantation (Jang et al.,
2008). We show here that an immortalized cell line derived from
the lamina propria (LPimm) stimulates sphere formation in 3-D
cultures of mouse OE cells taken after MeBr lesion as 3T3 cells
did in the original demonstration. Furthermore, conditioned
media (CM) from both 3T3 and LPimm cells greatly enhance the
formation of spheres. In order to identify factors released by
LPimm to influence sphere formation, we took two approaches: 1)
individual growth factors or a combination of them were added to
base media; 2) proteins that are synthesized and secreted into CM
by LPimm were metabolically labeled using Click-iT chemistry
and identified by proteomic methods. Some of the candidate
molecules identified by the proteomic approach were tested by
either adding them to base media or by using antibodies to block
their function. Our culture system, by maintaining the capacity
for progenitor cell engraftment and for easy manipulation in vitro,
can provide a rapid “read” on factors that are worth the further
effort required to define their role(s) in the process of
epitheliopoiesis in vivo. In addition, this type of culture offers us
the opportunity to expand neurocompetent stem cells for eventual
therapeutic purposes. Acknowledgements: NIH R01 DC002167
#P75 POSTER SESSION II:
OLFACTORY PHYSIOLOGY & CELL BIOLOGY;
TASTE MOLECULAR GENETICS;
CHEMESTHESIS & TRIGEMINAL
Molecular markers of stem and progenitor cells are the same in
human olfactory mucosa as in mice and rats
Eric H Holbrook 1,2 , Enming Wu 2 , James E Schwob 2
1
Massachusetts Eye and Ear Infirmary, Harvard Medical School
Boston, MA, USA, 2 Tufts University School of Medicine Boston,
MA, USA
Our incomplete understanding of human olfactory
pathophysiology has limited the range of therapeutic options for
most common forms of olfactory dysfunction. We can identify
different categories of progenitor cells as well as more
differentiated cell types in the olfactory epithelium (OE) of mice
and rats using a panel of antibodies to known proteins. Attempts
to carry out a similar analysis of human OE have been limited by
difficulties in obtaining tissue samples of adequate quality via
biopsy, which has contributed to our lack of knowledge
pertaining to olfactory epitheliopoiesis. Whole mount staining of
P O S T E R S
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Abstracts | 51