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Abstract

The exceptional capacity for regeneration of olfactory sensory neurons in the olfactory epithelium
and their axonal projections into the central nervous system (CNS) has led to extensive
investigation of olfactory ensheathing cell (OEC) transplantation as a therapy to promote repair
within the injured CNS. In those studies that report enhanced anatomical and functional recovery,
the beneficial effects of OECs have been variously attributed to OECs re-myelinating axons,
forming a supportive tract for axon growth across the boundary between the peripheral and
central nervous systems or producing growth factors, cell adhesion molecules and extracellular
matrix proteins that promote axon growth. This thesis investigates the possibility that OECs
modulate the inflammatory activation of astrocytes and could thereby contribute to CNS injury
repair after therapeutic transplantation.
Following CNS injury, factors released by damaged cells, activated microglia and invading
peripheral immune cells activate surviving astrocytes in the lesion penumbra. The activated
astrocytes undergo reactive astrogliosis and become another important source of inflammatory
mediators. These inflammatory responses often lead to delayed secondary neuronal loss and
tissue damage that can exceed the initial traumatic damage. In addition, astrogliosis leads to the
development of a glial scar around the lesion that is a barrier to axonal regeneration. Therefore,
moderation of astrocyte activation by transplanted OECs could potentially both protect neurons
by moderating inflammation and promote axon regeneration by moderating astrogliosis and glial
scarring.
The transcription factor nuclear factor-kappaB (NF-κB) is a key regulator of inflammatory
responses, including many of the protein expression changes that characterize astrogliosis. NF-κB
exists as inactive dimers in the cytoplasm of most cells and must translocate to the nucleus in
order to regulate gene transcription. Phorbol myristate acetate (PMA) and calcium ionophore
(PMA/ionophore) stimulation was found to induce rapid robust translocation of NF-κB to
astrocyte nuclei (p < 0.001), providing an in vitro model of astrocyte inflammatory activation.
NF-κB translocation was readily detectable by immunocytochemistry, providing a relatively
simple, direct, unambiguous measure of this early key event in astrocyte activation. Subsequently,
soluble factors released by microglia were found to similarly induce NF-κB translocation in
astrocytes (p < 0.001), illustrating that this detection method can be utilised to investigate other
inflammatory stimuli.Most importantly, soluble factors released by OECs moderated the NF-κB translocation induced
in astrocytes by either PMA/calcium ionophore or the microglia-derived factors (p < 0.001).
Immunostaining confirmed that insulin-like growth factor-1 (IGF-1) was expressed by the
cultured OECs and may have contributed to the moderation of astrocyte activation, since IGF-1
also significantly moderated (p < 0.05) the NF-κB translocation induced by either stimulus, albeit
insufficiently (p < 0.01) to account in full for the OEC-induced moderation. OECs also
significantly moderated the increased transcription of the NF-κB-regulated pro-inflammatory
cytokine, granulocyte macrophage-colony stimulating factor (GM-CSF) in the activated
astrocytes (p < 0.01). High levels of GM-CSF released by astrocytes can stimulate microglia to
produce cytotoxic levels of pro-inflammatory cytokines that may further amplify and prolong
inflammation. More severe inflammation leads to more severe astrogliosis, increased secondary
neuronal damage and more extensive glial scarring, accompanied by increased deposition of axon
growth-inhibitory extracellular matrix molecules. Hence, the identified moderation of astrocyte
activation by OECs represents a plausible mechanism whereby transplanted OECs could facilitate
neural repair after CNS injury. Furthermore, PMA/ionophore and the microglia-derived factors
did not induce NF-κB translocation in OECs, suggesting that OECs transplanted into CNS injury
sites could be resistant to pro-inflammatory stimuli and may be able to maintain the in vitro
phenotype associated here with the amelioration of astrocyte activation.
CNS injury and the associated ischemia induces increases in intracellular calcium levels that can
initiate inflammatory responses via NF-κB or, when excessive, cell death by necrosis or apoptosis.
Calcium ionophore alone activated significantly more astrocytes than PMA alone (p < 0.001),
with only a relatively small additive effect attributable to PMA, when astrocytes were treated
simultaneously with PMA and ionophore. TUNEL assay demonstrated that at higher doses
(250nM-1μM), calcium ionophore dose-dependently (R2 = 0.9996) induced significant (p < 0.001)
apoptosis of astrocytes. OECs did not appear to protect astrocytes against the ionophore-induced
apoptosis and they became apoptotic more rapidly and at lower ionophore doses (125nM) than
astrocytes. Consequently, OECs could become apoptotic due to excessive calcium influx if
transplanted into ischemic regions of CNS lesions where there are high levels of extracellular
calcium ions, glutamate and ATP. Disruption of the surviving vasculature during OEC
transplantation could re-initiate ischemia and in vivo research may be required to determine the
optimal timing and location for transplanting OECs. Both the OEC moderation of astrocyte
activation and the absence of OEC inflammatory activation imply that if transplanted OECs
survive, they are likely to enhance neuronal survival by moderating inflammation.In summary, this thesis provides evidence that OECs moderate the inflammatory activation of
astrocytes, thus providing a plausible mechanism that could contribute to improved
neuroprotection and tissue regeneration in response to therapeutic OEC transplantation after CNS
injury. IGF-1 was identified as soluble factor that may have contributed to the anti-inflammatory
effects of the cultured OECs. Further research to identify and isolate the responsible soluble
factors released by the OECs could lead to more precisely targeted molecular therapies. The in
vitro model of astrocyte activation established during the thesis could be further utilised to
investigate other CNS injury therapies.

Item Type:	Thesis - PhD
Authors/Creators:	Hale, DM
Keywords:	OEC, astrocyte, NFkappaB,inflammation, CNS injury, GM-CSF microglia
Additional Information:	Copyright 2011 the Author
Item Statistics:	View statistics for this item

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