Injuries to the peripheral nervous system rank among the most common causes of chronic neuropathic pain. Afflicting millions of people for months or even years, symptoms of this condition have proven difficult to treat clinically. A thorough understanding of the pathophysiological changes induced by such nerve lesions is essential to the development of more efficient therapeutic options.
Peripheral nerve injury induces a robust and tightly regulated innate immune response in the dorsal horn of the spinal cord. The precise molecular mechanisms regulating the spatiotemporal dynamics and functional impact of the response remain incompletely understood. Preclinical evidence suggests mitigating this immune response can have a significant therapeutic benefit in the treatment of neuropathic pain, however these findings have yet to be clinically validated.
To elucidate the mechanisms regulating the spinal immune response, we used a mouse model of partial sciatic nerve injury exclusively in male adult (2-3-month-old) mice. The spared nerve injury (SNI) model employed throughout our studies induces robust, persistent neuropathic pain-like behavior.
We established a time course for the spinal immune response to SNI and used mRNA extracted from the ipsilateral dorsal horn of lumbar spinal cord segments L4 and L5 to analyze changes in the transcriptome at the peak of the immune reaction 7 days after nerve lesion. We discovered upregulation of multiple elements of the triggering receptor expressed on myeloid cells 2 (Trem2) pathway. Trem2 is considered a regulator of toll-like receptor signaling in innate immune cells. It also promotes microglia-mediated phagocytosis in the central nervous system. Recent work from our lab has established neuronal apoptosis in the ipsilateral dorsal horn after SNI as an essential mechanism leading to the development of chronic neuropathic pain-like behavior. We used TUNEL staining of L4 spinal cord sections to compare the clearance of apoptotic cell profiles in Trem2-/- mice to wild-type littermates and discovered a key role for Trem2 in the clearance of apoptotic cells after SNI.
We further used genetic deletion of Trem2 as well as administration of a Trem2 agonist in C57Bl/6 mice to assess the impact of Trem2 signaling on both the spinal immune response and neuropathic pain-like behavior after SNI. Neither removal nor augmentation of Trem2 signaling significantly affected the development of neuropathic pain-like behavior.
Utilizing flow cytometry, we also evaluated the cellular composition of the spinal immune response. We found no evidence that monocytes from the peripheral circulation invade the spinal cord after SNI, as has been previously suggested. These findings were corroborated by immunohistochemical analysis of spinal cord sections from transgenic mice that express distinct fluorescent proteins in their monocyte and microglia cell populations.
To better understand the different mechanisms modulating the spinal immune response, we further examined several transcriptionally regulated signaling pathways. We achieved the greatest reduction of mechanical allodynia in nerve-lesioned mice treated with a P2x4r antagonist. Surprisingly, the removal of fractalkine (Cx3cl1) signaling, another prominent chemokine signaling pathway in microglia, had no significant impact on either the spinal immune response or mechanical allodynia after SNI. Reducing the number of spinal microglia by blocking Csf1r activation did not prevent the development of mechanical allodynia after SNI either.
Our findings reveal a more nuanced concept of microglial activation after nerve injury. The impact on neuropathic pain-like behavior and phagocytosis appear to be regulated by pathways that differ from those controlling immune cell recruitment and global activation. These findings provide a greater understanding of the complex mechanisms governing microglial function and offer new insight into molecular targets essential to the development of more efficient treatment options for neuropathic pain.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D87Q0GDM |
| Date | January 2019 |
| Creators | Smith, Brendan M. |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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