Olfactory ensheathing cells moderate astrocyte inflammatory activation

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-˜í‚à´B) is a key regulator of inflammatory responses, including many of the protein expression changes that characterize astrogliosis. NF-˜í‚à´B 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-˜í‚à´B to astrocyte nuclei (p < 0.001), providing an in vitro model of astrocyte inflammatory activation. NF-˜í‚à´B 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-˜í‚à´B 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-˜í‚à´B 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-˜í‚à´B 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-˜í‚à´B-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-˜í‚à´B 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-˜í‚à´B 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-1˜í¬¿M), 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.