Takao Omura

Accelerating axonal growth promotes motor recovery after peripheral nerve injury in mice

Although peripheral nerves can regenerate after injury, proximal nerve injury in humans results in minimal restoration of motor function. One possible explanation for this is that injury-induced axonal growth is too slow. Heat shock protein 27 (Hsp27) is a regeneration-associated protein that accelerates axonal growth in vitro. Here, we have shown that it can also do this in mice after peripheral nerve injury. While rapid motor and sensory recovery occurred in mice after a sciatic nerve crush injury, there was little return of motor function after sciatic nerve transection, because of the delay in motor axons reaching their target. This was not due to a failure of axonal growth, because injured motor axons eventually fully re-extended into muscles and sensory function returned; rather, it resulted from a lack of motor end plate reinnervation. Tg mice expressing high levels of Hsp27 demonstrated enhanced restoration of motor function after nerve transection/resuture by enabling motor synapse reinnervation, but only within 5 weeks of injury. In humans with peripheral nerve injuries, shorter wait times to decompression surgery led to improved functional recovery, and, while a return of sensation occurred in all patients, motor recovery was limited. Thus, absence of motor recovery after nerve damage may result from a failure of synapse reformation after prolonged denervation rather than a failure of axonal growth.

Diminished Schwann Cell Repair Responses Underlie Age-Associated Impaired Axonal Regeneration

The regenerative capacity of the peripheral nervous system declines with age. Why this occurs, however, is unknown. We demonstrate that 24-month-old mice exhibit an impairment of functional recovery after nerve injury compared to 2-month-old animals. We find no difference in the intrinsic growth capacity between aged and young sensory neurons in vitro or in their ability to activate growth-associated transcriptional programs after injury. Instead, using age-mismatched nerve transplants in vivo, we show that the extent of functional recovery depends on the age of the nerve graft, and not the age of the host. Molecular interrogation of the sciatic nerve reveals that aged Schwann cells (SCs) fail to rapidly activate a transcriptional repair program after injury. Functionally, aged SCs exhibit impaired dedifferentiation, myelin clearance, and macrophage recruitment. These results suggest that the age-associated decline in axonal regeneration results from diminished Schwann cell plasticity, leading to slower myelin clearance.

A Systems-Level Analysis of the Peripheral Nerve Intrinsic Axonal Growth Program

The regenerative capacity of the injured CNS in adult mammals is severely limited, yet axons in the peripheral nervous system (PNS) regrow, albeit to a limited extent, after injury. We reasoned that coordinate regulation of gene expression in injured neurons involving multiple pathways was central to PNS regenerative capacity. To provide a framework for revealing pathways involved in PNS axon regrowth after injury, we applied a comprehensive systems biology approach, starting with gene expression profiling of dorsal root ganglia (DRGs) combined with multi-level bioinformatic analyses and experimental validation of network predictions. We used this rubric to identify a drug that accelerates DRG neurite outgrowth in vitro and optic nerve outgrowth in vivo by inducing elements of the identified network. The work provides a functional genomics foundation for understanding neural repair and proof of the power of such approaches in tackling complex problems in nervous system biology.

A MILD ACUTE COMPRESSION INDUCES NEURAPRAXIA IN RAT SCIATIC NERVE

The pressure that induces neurapraxia in rat remains unrevealed. To determine the appropriate force to induce neurapraxia, two types of clips were applied to the sciatic nerve and were evaluated with functional, electrophysiological, and histological examinations. With a compression of 60 g/mm2, walking track analysis showed complete sciatic nerve paralysis one day postoperatively, but became normal in 14 days. Electrophysiologically, complete conduction block occurred one day post operatively, whereas the motor conduction velocity (MCV) below the compression site remained normal. Histologically, only limited signs of Wallerian degeneration were seen. The model in this study exhibited the features of neurapraxia.

Different expressions of BDNF, NT3, and NT4 in muscle and nerve after various types of peripheral nerve injuries

Abstract  The changes in the expression of brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4 (NT‐4) in the rat neuromuscular system as a result of three different types of sciatic nerve injuries have been evaluated. The changes in mRNA and protein levels for BDNF, NT‐3, and NT‐4 in the soleus muscle and sciatic nerve were assessed 4–28 days after sciatic nerve transection (neurotmesis), sciatic nerve crush (axonotmesis), and mild acute compression (neurapraxia). BDNF mRNA levels increased dramatically with nerve transection in the soleus muscle and the sciatic nerve 7–14 days after injury, whereas the changes were low in other types of injury. The changes of protein levels for BDNF were also similar. The mRNA and the protein levels of NT‐3 in the soleus muscle did not show any significant difference. The mRNA for NT‐4 in the soleus muscle decreased from 4 to 14 days after sciatic nerve transection, and the protein level was also minimum 14 days after sciatic nerve transection. Our results indicate that the neurotrophic factors in the neuromuscular system could play a role in differentiating peripheral nerve injury.

The BMP Coreceptor RGMb Promotes While the Endogenous BMP Antagonist Noggin Reduces Neurite Outgrowth and Peripheral Nerve Regeneration by Modulating BMP Signaling

Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb−/− mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb−/− mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin.

The recovery of blood-nerve barrier in crush nerve injury: a quantitative analysis utilizing immunohistochemistry

The purpose of this study is to reveal whether the application of immunohistochemical examinations to the peripheral nervous system (PNS) can be a reliable method for the quantitative analysis of the blood-nerve barrier (BNB) and the relationship between restoration of BNB and nerve regeneration. Sciatic nerves in rats were examined after nerve crush. Immunohistochemical staining with anti-rat endothelial cell antigen-1 (anti-RECA-1) that recognizes endothelial cells and anti-endothelial barrier antigen (anti-EBA) for the detection of barrier-type endothelial cells were used. Neurofilament for staining axons was also performed. A quantitative analysis of the BNB was assessed using the ratio of EBA positive cells and RECA-1 positive cells. The ratio of EBA/RECA-1 decreased significantly 3 days postoperatively and reached its lowest level at day 7 in the segment 5 mm proximal and the entire distal stump. The ratio gradually recovered from the proximal and the regeneration of axons started a week earlier than BNB. The ratio of EBA/RECA-1 applied to the PNS can be a reliable method for the quantitative analysis of BNB. In crush injuries, the breakdown of BNB occurred simultaneously in the segment 5 mm proximal and the entire distal stump; restoration began from the proximal to distal and followed a week later to nerve regeneration.

Spatiotemporal quantification of recruit and resident macrophages after crush nerve injury utilizing immunohistochemistry

The purpose of this study was to investigate quantitatively the temporal and spatial regulation and the morphological changes of the recruit and resident macrophages in the sciatic nerve during Wallerian degeneration and the following regeneration using immunohistochemistry. Sciatic nerves in Sprague-Dawley (SD) rats were examined after nerve crush. The rats were anesthetized with 100 mg of ketamine and 20 mg of xylazine in a dose of 1 ml/kg by intraperitoneal injection. Anti-ED-1 antibody was used to detect phagocytic macrophage and anti-OX-6 antibody was used to detect MHC class II cells. Few ED-1-immunopositive cells were seen within the normal sciatic nerve. After crush injury the number and the size of ED-1-immunopositive cells started to increase in all the segments distal to the crush site 3 days after injury and the number and size reached its peak on day 14 when the population of macrophage was 150 times higher in all the segments compared to controls. However, the number of ED-1-immunopositive cells and the size of the cells remains significantly high even after day 56 when functional recovery and axonal regeneration were complete. OX-6-immunopositive cells were observed within the control sciatic nerves. The number decreases significantly 3 days after injury in all the segments distal to the crush site but showed no significant difference thereafter. There were also no significant differences in the cell areas. ED-1-immunopositive phagocytic macrophages show significant differences temporally in both the cell number and the size even after axonal regeneration.

Spatiotemporal quantification of tumor necrosis factor-alpha and interleukin-10 after crush injury in rat sciatic nerve utilizing immunohistochemistry

The purpose of this study was to investigate quantitatively the longitudinal temporal, spatial changes of the tumor necrosis factor-alpha (TNF) and interleukin-10 (IL-10) immunopositive cells during Wallerian degeneration and the following regeneration after crush injury in rat sciatic nerve using immunohistochemistry and enzyme linked immunosorbent assay (ELISA). The number of TNF-immunopositive cells reached its peak and increased significantly in all the segments distal to the crush site 3 days after injury. On Day 7, TNF-immunopositive cells decreased in all the segments distal to the crush site, and a significant decrease was observed 14 days after injury. From Day 21 to Day 56, there were no significant differences in the numbers of TNF-immunopositive cells. The average size of TNF immunopositive cells became significantly larger with degeneration. The number of IL-10-immunopositive cells decreases significantly 1 day after crush injury. IL-10-immunopositive cells increased on Day 3, returning to control levels. Seven days after injury, a significant increase in the number of IL-10-immunopositive cells was observed. There was also no significant difference in the number of IL-10-immunopositive cells beyond Day 14 except for a part of distal segments. The number of IL-10-immunopositive cells showed no significant differences in all the segments on Day 56. The protein levels of IL-10 measured by ELISA were similar to the result of immunohistochemistry. These results suggest that the significant change in IL-10 occurred prior to the significant change in TNF and that IL-10 may be the key to the change in TNF.

Inhibition of the kinase WNK1/HSN2 ameliorates neuropathic pain by restoring GABA inhibition

Mice lacking the HSN2 form of the kinase WNK1 are protected from neuropathic pain due to nerve injury. “WNK”ing out pain Mutations in the HSN2 exon present in the nervous system–specific isoform of the kinase WNK1 cause an ulcerating neuropathy disorder called hereditary sensory and autonomic neuropathy type IIA (HSANII). HSANII affects the peripheral and spinal nerves and results in loss of touch, temperature, and pain sensation. Kahle et al. generated transgenic mice specifically lacking this alternatively spliced variant of WNK1, which is present in the spinal cord’s dorsal horn, the gateway for pain processing from the periphery to the brainstem. These mice exhibited no gross neurological defects and did not exhibit symptoms of HSANII, likely because mutations in HSANII patients generate a truncated form of the kinase that has an intact catalytic domain. The HSN2-deficient mice were protected from pain hypersensitivity in a model of neuropathic pain resulting from peripheral nerve injury, but not in an inflammatory pain model. Mechanistically, HSN2-deficient mice had less phosphorylation of the K+-Cl− cotransporter KCC2 in the nerves, which resulted in an increase in KCC2 activity, a decrease in the amount of Cl− in the nerves, and restoration of the inhibitory response to GABA. Thus, by alleviating GABA “disinhibition,” a known major contributor to neuropathic pain, drugs that inhibit HSN2 might reduce injury-induced neuropathic pain. HSN2 is a nervous system predominant exon of the gene encoding the kinase WNK1 and is mutated in an autosomal recessive, inherited form of congenital pain insensitivity. The HSN2-containing splice variant is referred to as WNK1/HSN2. We created a knockout mouse specifically lacking the Hsn2 exon of Wnk1. Although these mice had normal spinal neuron and peripheral sensory neuron morphology and distribution, the mice were less susceptible to hypersensitivity to cold and mechanical stimuli after peripheral nerve injury. In contrast, thermal and mechanical nociceptive responses were similar to control mice in an inflammation-induced pain model. In the nerve injury model of neuropathic pain, WNK1/HSN2 contributed to a maladaptive decrease in the activity of the K+-Cl− cotransporter KCC2 by increasing its inhibitory phosphorylation at Thr906 and Thr1007, resulting in an associated loss of GABA (γ-aminobutyric acid)–mediated inhibition of spinal pain-transmitting nerves. Electrophysiological analysis showed that WNK1/HSN2 shifted the concentration of Cl− such that GABA signaling resulted in a less hyperpolarized state (increased neuronal activity) rather than a more hyperpolarized state (decreased neuronal activity) in mouse spinal nerves. Pharmacologically antagonizing WNK activity reduced cold allodynia and mechanical hyperalgesia, decreased KCC2 Thr906 and Thr1007 phosphorylation, and restored GABA-mediated inhibition (hyperpolarization) of injured spinal cord lamina II neurons. These data provide mechanistic insight into, and a compelling therapeutic target for treating, neuropathic pain after nerve injury.