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.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Wang, Yadong; Committee Member: Philip Santangelo; Committee Member: Ravi Bellamkonda

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Since the discovery of peripheral nerve regeneration nearly a century ago, the mechanisms that guide this regeneration have been elusive. This project aimed to describe how an axon is able to traverse the environment of the body and precisely reinnervate its target cell. Using a novel technique of combining light and electron microscopy, I observed reinnervating axons in transgenic mice to answer the questions of whether Schwann cells are an important guidance cue for the motor neuron and whether the outgrowing axon is fully developed or the process is a step-wise process of activation. The data suggests that Schwann cell contact is important for the tip of the regenerating axon to guide the axon back to its synapse on the muscle fiber. Further, it seems that the tip of the axon is not capable of synaptic transmission as it lacks active zones, suggesting that reinnervation is a step-wise process. / Department of Physiology and Health Science

Peripheral nerve injury in adults often leads to permanent functional loss with or without pain. Traumatic injury or surgery, metabolic injury (diabetic neuropathy), and drug toxicity can lead to neuropathies and all negatively impact the quality of life1-8. Damage to the nervous system is often permanent since neurons in the brain and periphery are post-mitotic and have limited regenerative capacity. Nerve repair involves axon regeneration, a complex and incompletely understood process with repair potential declining with age9-15. The research and design discussed involves the induction of endogenous repair mechanisms of the peripheral nerve using embryonic stem cells, alginate hydrogel, and the guided support of a biomaterial scaffold composed of PGS. Three different populations of cells are discussed: human embryonic stem cells, neural progenitor cells derived from human embryonic stem cells16, and primary rat bone marrow stromal cells. This study was innovative in that it was the first attempt for use of an elastomeric biomaterial scaffold in an injury model for the purpose of clinical application. This research is significant as it has direct clinical relevance in that it incorporates both functional and neuropathic recovery of patients affected by peripheral nerve damage.

Indiana University-Purdue University Indianapolis (IUPUI) / Although peripheral motoneurons are phenotypically endowed with robust regenerative capacity, functional recovery is often suboptimal following peripheral nerve injury (PNI). Research to date indicates that the greatest success in achieving full functional recovery will require the use of a combinatorial approach that can simultaneously target different aspects of the post-injury response. In general, the concept of a combinatorial approach to neural repair has been established in the scientific literature but has yet to be successfully applied in the clinical situation. Emerging evidence from animal studies supports the use of electrical stimulation (ES) and testosterone as one type of combinatorial treatment after crush injury to the facial nerve (CN VII). With the facial nerve injury model, we have previously demonstrated that ES and testosterone target different stages of the regeneration process and enhance functional recovery after facial nerve crush injury. What is currently unknown, but critical to determine, is the impact of a combinatorial treatment strategy of ES and testosterone on functional recovery after crush injury to the sciatic nerve, a mixed sensory and motor spinal nerve which is one of the most serious PNI clinical problems. The results of the present study indicate that either treatment alone or in combination positively impact motor recovery. With regard to molecular effects,single and combinatorial treatments differentially alter the expression of regeneration-associated genes following sciatic nerve crush injury relative to facial nerve injury. Thus, our data indicate that not all injuries equally respond to treatment. Furthermore, the results support the importance of treatment strategy development in an injury-dependent manner and based upon the functional characteristics of spinal vs. cranial nerves.