Alleviation of neuropathic pain by over-expressing a soluble colony-stimulating factor 1 receptor to suppress microgliosis and macrophage accumulation.

Microglial proliferation and activation and macrophage accumulation are implicated in neuropathic pain development. In this study, we aim to suppress microgliosis and macrophage accumulation by over-expressing a non-functional soluble colony stimulating factor-1 receptor (sCSF1R) using an adeno-associated virus 9 vector (AAV9). AAV9/sCSF1R and the control vector AAV9/GFP were intrathecally administered into the lumbar spine of adult C57BL/6 mice. Two weeks later, these mice underwent partial sciatic nerve ligation to induce neuropathic pain. GFP and sCSF1R were highly expressed in lumbar dorsal root ganglia (DRG) and spinal cord of AAV9-injected mice. A significant increase in microglia densities in the dorsal and ventral horns of lumbar spinal cords and macrophage densities in DRG and sciatic nerves were observed in the mice with either ligation alone or pre-treated with AAV9/GFP. In nerve-ligated mice pre-treated with AAV9/sCSF1R the microglia densities in the dorsal and ventral horns and macrophage densities in DRG and sciatic nerves were significantly lower compared to nerve-ligated mice pre-treated with AAV9/GFP. Behavioral tests showed that nerve-ligated mice pre-treated with AAV9/sCSF1R had a significantly higher paw withdrawal threshold, indicating the alleviation of neuropathic pain. The results implicate that viral vector-mediated expression of sCSF1R may represent a novel strategy in the alleviation of neuropathic pain.

Increased expression of colony-stimulating factor-1 in mouse spinal cord with experimental autoimmune encephalomyelitis correlates with microglial activation and neuronal loss.

Microglia contribute to pathophysiology at all stages of multiple sclerosis. Colony-stimulating factor-1 (CSF1) is crucial for microglial proliferation and activation. In this study we measured the CSF1 levels and studied its cellular expression in the mouse spinal cords with experimental autoimmune encephalomyelitis (EAE) to explore the potential contribution of CSF1 in neuronal death. ELISA data showed that CSF1 levels were significantly higher in the spinal cords with acute and chronic EAE than those of normal and adjuvant-injected mice. Immunohistochemical studies demonstrated that CSF1 was expressed in astrocytes and neurons in normal mouse spinal cord. In acute EAE, CSF1 expression was significantly increased, especially in astrocytes in peripheral white matter and large motoneurons. High density of activated microglia was observed in the gray matter where motoneurons expressed high-level CSF1 in acute EAE. Significant large motoneuron loss was seen in chronic EAE and the remaining motoneurons with high-level CSF1 were enwrapped by microglia. Viral vector mediated over-expression of CSF1 in spinal neurons induced profound proliferation and activation of microglia at the injection site and microglia enwrapped CSF1-transduced neurons and their neurites. Significant loss of large CSF1-transduced neurons was seen at 2 and 3 weeks post-viral injection. Demyelination in the CSF1-transduced areas was also significant. These results implicate that CSF1 upregulation in CNS may play an important role in the proliferation and activation of microglia in EAE, contributing to neuroinflammation and neurodegeneration. © 2018 Wiley Periodicals, Inc.

Intraneural Injection of ATP Stimulates Regeneration of Primary Sensory Axons in the Spinal Cord.

Injury to the peripheral axons of sensory neurons strongly enhances the regeneration of their central axons in the spinal cord. It remains unclear on what molecules that initiate such conditioning effect. Because ATP is released extracellularly by nerve and other tissue injury, we hypothesize that injection of ATP into a peripheral nerve might mimic the stimulatory effect of nerve injury on the regenerative state of the primary sensory neurons. We found that a single injection of 6 µl of 150 µm ATP into female rat sciatic nerve quadrupled the number of axons growing into a lesion epicenter in spinal cord after a concomitant dorsal column transection. A second boost ATP injection 1 week after the first one markedly reinforced the stimulatory effect of a single injection. Single ATP injection increased expression of phospho-STAT3 and GAP43, two markers of regenerative activity, in sensory neurons. Double ATP injections sustained the activation of phospho-STAT3 and GAP43, which may account for the marked axonal growth across the lesion epicenter. Similar studies performed on P2X7 or P2Y2 receptor knock-out mice indicate P2Y2 receptors are involved in the activation of STAT3 after ATP injection or conditioning lesion, whereas P2X7 receptors are not. Injection of ATP at 150 µm caused little Wallerian degeneration and behavioral tests showed no significant long-term adverse effects on sciatic nerve functions. The results in this study reveal possible mechanisms underlying the stimulation of regenerative programs and suggest a practical strategy for stimulating axonal regeneration following spinal cord injury.SIGNIFICANCE STATEMENT Injury of peripheral axons of sensory neurons has been known to strongly enhance the regeneration of their central axons in the spinal cord. In this study, we found that injection of ATP into a peripheral nerve can mimic the effect of peripheral nerve injury and significantly increase the number of sensory axons growing across lesion epicenter in the spinal cord. ATP injection increased expression of several markers for regenerative activity in sensory neurons, including phospho-STAT3 and GAP43. ATP injection did not cause significant long-term adverse effects on the functions of the injected nerve. These results may lead to clinically applicable strategies for enhancing neuronal responses that support regeneration of injured axons.

Observations on the function of nuclear factor kappa B (NF-kappaB) in the survival of adult primary sensory neurons after nerve injury.

Peripheral nerve transections cause much more neuronal death in embryonic and neonatal dorsal root ganglia (DRG) than in adult DRG. Here we used transgenic approaches to examine the hypothesis that NF-kappaB is an important intrinsic factor of adult DRG neurons for their in vivo capacity to survive after nerve injury. We generated transgenic mice expressing the NF-kappaB super-inhibitor (IkappaBalpha-SI), a multi-mutant form of IkappaBalpha, specifically in adult neurons. Adult DRG neurons in these transgenic animals are not abnormally susceptible to apoptosis after peripheral nerve injury, although there is a significant inhibition in the ability of NF-kappaB to translocate into their nucleus. We investigated the observed lack of NF-kappaB neuroprotective function at the level of NF-kappaB transcriptional activity using transgenic NF-kappaB/LacZ reporter mice. We show that the expression of the NF-kappaB reporter transgene is restricted in naïve and injured DRG neurons. However, NF-kappaB transcriptional activity in adult DRG neurons is evident upon exposure to Trichostatin A (TSA) which is a specific inhibitor of histone deacetylases. Taken together our results illustrate that the functions of NF-kappaB are limited in adult primary sensory neurons due to a transcriptional repression mechanism mediated by histone deacetylases, and that intrinsic neuroprotective factors other than NF-kappaB are responsible for the resistance of adult DRG neurons to apoptosis in response to nerve injury.