Introduction: The glial scar has been considered as an impediment to axonal regeneration, albeit studies also show that astrocytes are necessary for axonal re-growth following spinal cord injury (SCI). The principal cell type that form the glial scar are proliferating and migrating reactive astrocytes, which secrete both helpful and harmful effectors. Modulation of astroglial proliferation and migration can alter the properties of the glial scar and thereby, influence the outcomes of SCI. Evidence indicates that Toll like receptors (TLRs), which are expressed by SC neurons, glia and infiltrating immune cells, play important roles in SCI. Our laboratory has previously shown that a TLR9 antagonist, CpG ODN 2088, administered intrathecally, improves the functional and histopathological outcomes of SCI, and attenuates the pro-inflammatory phenotype of SC astrocytes, in vitro, through direct actions. The current studies were undertaken to determine whether the TLR9 antagonist modulates astroglial functions pertinent to glial scar formation such as proliferation and migration.
Methods: Mixed glial cultures, derived from the SC of postnatal day 2-3 mouse pups were used to isolate astrocytes, which were passaged three times for 99% purification. Astrocyte cultures were incubated in Minimum Essential Medium containing 1% heat inactivated fetal bovine serum, in the presence or absence of CpG ODN 2088. EdU, a thymidine analogue was used to label proliferating cells whereas a scratch-wound assay and live cell imaging were utilized to assess the astroglial migration.
Results: CpG ODN 2088 significantly reduced by 40% the number of proliferating astrocytes (p<0.001; n=4). The antagonist also significantly decreased the astroglial migration into the gap formed by the scratch. These effects necessitated TLR9 since CpG ODN 2088 did not affect the proliferation or migration TLR9-/- astrocytes.
Conclusions: Astroglial TLR9 antagonism inhibits both proliferation and migration, in vitro. Thus, CpG ODN 2088 has the potential of targeting astrocyte functions pertinent to glial scar formation.
Patient Care: Our laboratory has previously shown that intrathecal administration of a TLR9 antagonist improves the functional and histopathological outcomes of SCI in mice. We now expanded these findings and show that the antagonist has the potential of modulating astrocyte function pertinent to glial scar formation. Since the glial scar plays a critical role in axonal re-growth, the antagonist could target mechanisms that have relevance to regeneration.
Learning Objectives: By the conclusion of this session, participants should be able to: 1) Describe the importance of TLR9 in astroglial function especially as it relates to glial scar formation following SCI, 2) Discuss, in small groups, whether inhibition of TLR9 in the spinal cord following injury is beneficial or detrimental to the injury outcomes, 3) Identify a potential therapeutic approach to modulate glial scar formation following SCI.