Increased spinal adenosine after subacute cervical injury correlates with sustained upregulation of CD39 and CD73 in microglia.

Cervical spinal cord injuries (cSCI) are associated with decreased breathing ability. Although no treatment options are currently available, moderate acute intermittent hypoxia (mAIH) is a promising therapeutic modality to improve breathing function after cSCI. Moderate AIH elicits phrenic motor plasticity via distinct, competing serotonin- or adenosine-driven mechanisms that interact via powerful crosstalk inhibition that constrains or even abolishes plasticity. The dominant mechanism driving plasticity depends on the spinal serotonin/adenosine balance. Shortly after cSCI, repeated AIH exposure elicits plasticity via an adenosine-dependent mechanism but reverts to serotonin-dominance with chronic cSCI. In healthy CNS, microglia regulate AIH-induced phrenic motor plasticity via enzymatic activities of ectonucleotidases (CD39, CD73) by converting extracellular ATP to adenosine. We hypothesized that cSCI increases microglial ectonucleotidase expression, elevating adenosine levels that may alter therapeutic responses to mAIH post-cSCI. We assessed microglial CD39 and CD73 expression at the subacute (1 & 2 weeks) and chronic (8 weeks) stages post C2-hemisection, both at the injury site (C1-C3) and in spinal segments containing phrenic motor neurons below the injury (C3-C6). Both enzymes were upregulated (mRNA & protein) 1- and 2-weeks post injury but returned to baseline by 8 weeks. In association, spinal adenosine increased significantly at 2, but not 8 weeks post-injury. Further, microglial CD39 and CD73 expression strongly correlate with P2Y12 receptor expression. Thus, shifting adenosine levels between subacute and early chronic cSCI may impact mechanism regulating mAIH-induced respiratory motor plasticity and breathing recovery at different times post-cSCI.