How to regenerate peripheral nerves
Controlling the timing of immune response to nerve damage may be key to promoting nerve cell repair.
Our peripheral nervous system is a vast network that exists outside of our brain and spinal cord, connecting the far reaches of our body. This great expanse is highly vulnerable to injury and diseases such as Charcot-Marie-Tooth, Guillain-Barre syndrome as well as diabetes.
Unlike the brain and spinal cord of the central nervous system, peripheral nerves have the capacity to regenerate. Inflammatory immune responses play a key role in that regeneration.
Case Western Reserve University School of Medicine scientists now demonstrates in lab animals the dynamics of regeneration as initiated by a specific signaling protein, C-C class chemokine 2 (CCL2).
CCL2 sends inflammatory immune cells (macrophages) to peripheral nerve cell clusters promoting repair and triggering gene expression leading to new growth in nerve cells. Their findings were posted online in mid-November and appear in the January 2016 published issue of Experimental Neurology.
"We are excited about our findings because we had no reason to expect that just expressing the chemokine CCL2 would be enough to stimulate nerve regeneration. It is remarkable that CCL2 should be so powerful."
Richard E. Zigmond PhD, Professor of Neurosciences and Pathology, Case Western Reserve University School of Medicine, and senior author.
Post-injury, CCL2 affects peripheral nerve cell clusters known as ganglia. Each peripheral nerve cell has a main body and a tail-like extension called an axon. After leaving the cell body, axons of sensory nerve cells split: one part projects from the nerve cell body to capture sensations, while the other returns that information to the spinal cord and brain.
After an injury to a region of peripheral nerves, CCL2 signals macrophages to move into the area of damaged axons and remove cell debris. This clearing of debris is also seen to promote nerve sprouting.
Regeneration occurs partly due to macrophages triggering genes to promote new axon growth.
Zigmond's most recent work revealed the response of CCL2 in lab animals. In one experiment wild-type mice were injected with a virus designed to trigger CCL2 to be expressed. The boost in CCL2 led to a greater accumulation of macrophages three weeks later in their dorsal root ganglia — the cluster of sensory nerve cells projecting into both the peripheral and central nervous systems. More macrophage build-up produced greater neuron sprouting — or beginnings of nerve regeneration.
However, results were entirely different in mice without the CCR2 receptor which enables CCL2. Without the CCR2 receptor, there was no spike in macrophage accumulation. These mice had scant nerve regrowth — not matching nerve regeneration seen in wild-type mice.
"We did the same experiments in another type of mouse and found the same correlation. If macrophages don't come into the ganglia, then regeneration was substantially impeded. We found this true of sensory and sympathetic neurons and conclude there is a correlation between macrophage entry into ganglia and nerve regeneration."
Richard E. Zigmond PhD
Researchers also saw how CCL2 overexpression altered gene response. Investigators screened for mRNA molecules found with nerve regeneration in mice with CCL2 overexpression and saw changes in one mRNA and one protein — leukemia-inhibitory factor (LIF) mRNA and neural pSTAT3 (Signal Transducers and Activators of Transcription 3).
STAT3 initiates the response of a variety of genes to stimuli, playing a key role in cell growth and cell death (apoptosis). By causing macrophages to increase, CCL2 increased levels of LIF mRNA and pSTAT3, which led to an increase in the regenerative capacity of dorsal root ganglia neurons.
To test their hypothesis, they then blocked LIF activation using inhibitors of STAT3. Inhibiting STAT3 did not result in an increase in neuron growth, despite the increase in CCL2 - which previously had increased growth of dorsal root ganglia.
The findings by Zigmond and fellow investigators shed a new light on inflammation. Rather than fight inflammation at the very outset of a peripheral nerve injury, perhaps allowing limited inflammation post-injury may be therapeutic to stimulating neuron regeneration. These findings about CCL2 could also have implications for illnesses that affect peripheral nerves.
Zigmond: "Our conclusion is that the immune system and the nervous system interact in a beneficial way to create macrophage-induced inflammation and promote nerve regeneration. This is occurring around the cell bodies. There is something happening within the cell in response to macrophage activity, in addition to macrophage action on to the damaged distal nerve axon."
• Intrathecal injection of AAV5-CCL2 leads to overexpression of the chemokine in DRGs.
• CCL2 overexpression causes macrophage accumulation in uninjured DRGs.
• A conditioning-like increase in neurite outgrowth in CCL2 overexpression.
• LIF mRNA and pSTAT3 are significantly increased in DRGs overexpressing CCL2.
• Overexpression-induced neurite outgrowth occurs via a STAT3-dependent mechanism.
Neuroinflammation plays a critical role in the regeneration of peripheral nerves following axotomy. An injury to the sciatic nerve leads to significant macrophage accumulation in the L5 DRG, an effect not seen when the dorsal root is injured. We recently demonstrated that this accumulation around axotomized cell bodies is necessary for a peripheral conditioning lesion response to occur. Here we asked whether overexpression of the monocyte chemokine CCL2 specifically in DRG neurons of uninjured mice is sufficient to cause macrophage accumulation and to enhance regeneration or whether other injury-derived signals are required. AAV5-EF1α-CCL2 was injected intrathecally, and this injection led to a time-dependent increase in CCL2 mRNA expression and macrophage accumulation in L5 DRG, with a maximal response at 3 weeks post-injection. These changes led to a conditioning-like increase in neurite outgrowth in DRG explant and dissociated cell cultures. This increase in regeneration was dependent upon CCL2 acting through its primary receptor CCR2. When CCL2 was overexpressed in CCR2 −/− mice, macrophage accumulation and enhanced regeneration were not observed. To address the mechanism by which CCL2 overexpression enhances regeneration, we tested for elevated expression of regeneration-associated genes in these animals. Surprisingly, we found that CCL2 overexpression led to a selective increase in LIF mRNA and neuronal phosphorylated STAT3 (pSTAT3) in L5 DRGs, with no change in expression seen in other RAGs such as GAP-43. Blockade of STAT3 phosphorylation by each of two different inhibitors prevented the increase in neurite outgrowth. Thus, CCL2 overexpression is sufficient to induce macrophage accumulation in uninjured L5 DRGs and increase the regenerative capacity of DRG neurons via a STAT3-dependent mechanism.
Contributing authors: Alicia DeFrancesco-Lisowitz, Jared Cregg, and Madeline Howarth, all of the Department of Neurosciences, Case Western Reserve University School of Medicine.
This research was supported by National Institutes of Health grant DK097223 and training grant NS077888.
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