Lack of galectin‐3 speeds Wallerian degeneration by altering TLR and pro‐inflammatory cytokine expressions in injured sciatic nerve

Wallerian degeneration (WD) comprises a series of events that includes activation of non‐neuronal cells and recruitment of immune cells, creating an inflammatory milieu that leads to extensive nerve fragmentation and subsequent clearance of the myelin debris, both of which are necessary prerequisites for effective nerve regeneration. Previously, we documented accelerated axon regeneration in animals lacking galectin‐3 (Gal‐3), a molecule associated with myelin clearance. To clarify the mechanisms underlying this enhanced regeneration, we focus here on the early steps of WD following sciatic nerve crush in Gal‐3^?/? mice. Using an in vivo model of nerve degeneration, we observed that removal of myelin debris is more efficient in Gal‐3^?/? than in wild‐type (WT) mice; we next used an in vitro phagocytosis assay to document that the phagocytic potential of macrophages and Schwann cells was enhanced in the Gal‐3^?/? mice. Moreover, both RNA and protein levels for the pro‐inflammatory cytokines IL‐1β and TNF‐α, as well as for Toll‐like receptor (TLR)‐2 and ‐4, show robust increases in injured nerves from Gal‐3^?/?mice compared to those from WT mice. Collectively, these data indicate that the lack of Gal‐3 results in an augmented inflammatory profile that involves the TLR–cytokine pathway, and increases the phagocytic capacity of Schwann cells and macrophages, which ultimately contributes to speeding the course of WD.     

Characterization of osteopontin expression and function after status epilepticus

Purpose: Osteopontin is a cytokine found in many tissues and plays a role in tissue injury and repair. This study had two goals: to characterize osteopontin expression after status epilepticus (SE), and to test the hypotheses that osteopontin affects the susceptibility to seizures or alters cell death and inflammation after SE. Methods: Pilocarpine was used to induce SE in OPN^?/? and OPN^+/+ mice to compare seizure susceptibility, neuropathological markers including real time PCR for inflammatory genes, and osteopontin immunohistochemistry. The effect of added osteopontin on excitotoxicity by N‐methyl‐d ‐aspartate in neuronal cultures of ONP^?/? mice was determined. Results: Neurons undergoing degeneration showed osteopontin immunoreactivity 2–3  days after SE. After 10 to 31 days degenerating axons in the thalamus were osteopontin‐positive. The susceptibility to seizures of OPN^?/? and OPN^+/+ mice in the pilocarpine, fluorothyl, and maximal electroshock models was similar. There were no significant differences in the extent of neuronal damage after pilocarpine‐induced SE, the expression of several neuropathological markers or the RNA levels of selected inflammatory genes. Recombinant and natural bovine osteopontin did not affect the extent of NMDA‐induced cell death in OPN^?/? mouse neuronal cultures. Conclusion: We demonstrated that osteopontin is up‐regulated in response to SE in distinct temporal sequences in the hippocampus, specifically in dege‐nerating neurons and axons. However, osteopontin did not appear to regulate neurodegeneration or inflammation within the first 3  days after SE.     

Differences in peripheral nerve degeneration/regeneration between wild-type and neuronal nitric oxide synthase knockout mice

Nitric oxide (NO), a unique biological messenger molecule, is synthesized by three isoforms of the enzyme NO synthase (NOS) and diffuses from the site of production across cellular membranes. A postulated role for NO in degeneration and regeneration of peripheral nerves has been explored in a sciatic nerve model comparing wild-type mice and mice lacking neuronal NOS after transection and microsurgical repair. In NOS knockout mice, regenerative delay was observed, preceded by a decelerated Wallerian degeneration (WD). In the regenerated nerve, pruning of uncontrolled sprouts was disturbed, leading to an enhanced number of axons, whereas remyelination seemed to be less affected. Delayed regeneration was associated with a delayed recovery of sensor and motor function. In such a context, possible NO targets are neurofilaments and myelin sheaths of the interrupted axon, filopodia of the growth cone, newly formed neuromuscular endplates, and Schwann cells in the distal nerve stump. The results presented suggest that 1) local release of NO following peripheral nerve injury is a crucial factor in degeneration/regeneration, 2) success of fiber regeneration in the peripheral nervous system depends on a regular WD, and 3) manipulation of NO supply may offer interesting therapeutic options for treatment of peripheral nerve lesions.     

Long-term consequences of impaired regeneration on facial motoneurons in the C57BL/Ola mouse

Peripheral nerves of the C57BL/Ola mouse mutant undergo markedly slowed Wallerian degeneration following injury. This is associated with impaired regeneration of both sensory and motor axons. Following a crush lesion of the facial nerve, there was no cell loss in facial nuclei of normal (C57BL/6J) adult mice, but 40% cell loss occurred in Ola mice and the survivors increased in size during the period when functional reinnervation was established. These results are interpreted as a result, first, of prolonged deprivation of target-derived trophic factor in the slowly regenerating Ola motoneurons and second, increased peripheral field size of the survivors. Within the regenerated facial nerve, there was marked heterogeneity of myelinated fibre size in Ola mice. Some Ola axons, both proximal and distal to the lesion site, had areas over twice as great as the largest 6J axons when measured 1 year following injury. A population of small diameter fibres, not observed in 6J nerves, persisted distal to the crush site in Ola nerves, and this was associated with an increase in the total number of myelinated axons in the distal nerve: on average, each parent Ola axon retained three persistent daughter axons. The delayed Wallerian degeneration in Ola mice not only impairs immediate axon regrowth, but also results in a breakdown of the normal mechanisms which regulate axon number and size in regenerating nerve.     

Electrophysiological investigation of motor axonal excitability in a mouse model of nerve constriction injury

Peripheral nerve injuries caused by focal constriction are characterised by local nerve ischaemia, axonal degeneration, demyelination, and neuroinflammation. The aim of this study was to understand temporal changes in the excitability properties of injured motor axons in a mouse model of nerve constriction injury (NCI). The excitability of motor axons following unilateral sciatic NCI was studied in male C57BL/6J mice distal to the site of injury at the acute (6 hours‐1 week) and chronic (up to 20 weeks) phases of injury, using threshold tracking. Multiple measures of nerve excitability, including strength‐duration properties, threshold electrotonus, current‐threshold relationship, and recovery cycle were examined using the automated nerve excitability protocol (TRONDNF). Acutely, injured motor axons developed a pattern of excitability characteristic of ischemic depolarisation. In most cases, the sciatic nerve became transiently inexcitable. When a liminal compound muscle action potential could again be recorded, it had an increase in threshold and latency, compared to both pre‐injury baseline and sham‐injured groups. These axons showed a greater threshold change in response to hyperpolarising threshold electrotonus and a significant upward shift in the recovery cycle. Mathematical modelling suggested that the changes seen in chronically injured axons involve shortened internodes, reduced myelination, and exposed juxtaparanodal fast K⁺ conductances. The findings of this study demonstrate long‐term changes in motor excitability following NCI (involving alterations in axonal properties and ion channel activity) and are important for understanding the mechanisms of neurapraxic injuries and traumatic mononeuropathies.     

Loss of neural recognition molecule NB-3 delays the normal projection and terminal branching of developing corticospinal tract axons in the mouse

Neural recognition molecule NB‐3 is involved in neural development and synapse formation. However, its role in axon tract formation is unclear. In this study, we found that the temporal expression of NB‐3 in the deep layers of the motor cortex in mice was coincident with the development of the corticospinal tract (CST). Clear NB‐3 immunoreactivity in the CST trajectory strongly suggested that NB‐3 was expressed specifically in projecting CST axons. By tracing CST axons in NB‐3^?/? mice at different developmental stages, we found that these axons were capable of projecting and forming a normal trajectory. However, the projection was greatly delayed in NB‐3^?/? mice compared with wild‐type (WT) mice from the embryonic to postnatal stages, a period that is coincident with the completion of the CST projection in mice. Subsequently, although their projection was delayed, CST axons in NB‐3^?/? mice gradually completed a normal projection. By stage P21, the characteristics of CST projections in NB‐3^?/? mice were not statistically different from those in WT mice. In addition, we found that the branching of CST axons into spinal gray matter also was delayed in NB‐3^?/? mice. The CST innervation area in the spinal gray matter of NB‐3^?/? mice was greatly reduced in comparison with WT mice until P30 and gradually became normal by P45. These data suggest that NB‐3 is involved in the normal projection and terminal branching of developing CST axons. J. Comp. Neurol. 520:1227–1245, 2012. © 2011 Wiley Periodicals, Inc.     

Cell body response to injury in motoneurons and primary sensory neurons of a mutant mouse,ola (Wld), in which wallerian degeneration is delayed

We examined the response to axon injury in the facial motoneurons and dorsal root ganglion (DRG) neurons of C57BL/Ola (Wld) mice, compared with the responses of C57BL/6J mice. The peripheral nerves of Ola mutants undergo remarkably slowed and muted Wallerian degeneration after injury. The increase in GAP-43 mRNA levels in facial motoneurons and DRG neurons was similar in both strains of mice, as was the initial decrease in medium-weight neurofilament (NFM) mRNA in facial motoneurons, and the increase in JUN immunoreactivity in both types of neurons. However, the subsequent recovery to normal low levels of JUN and GAP-43 mRNA expression and high levels of NFM mRNA was delayed in Ola motoneurons. We ascribe this delay to the slow regeneration and target reinnervation of facial axons in the Ola mice. These results show that absence of rapid Wallerian degeneration does not affect the initial cell body response to axonal injury. They also provide further evidence that restoration of normal levels of expression of GAP-43 and NFM mRNAs is dependent on target reinnervation and/or trophic factors provided by the distal nerve, Impaired regeneration in the Ola mouse does not seem to be a consequence of a defective cell body response to injury, and our results illustrate the general principle that, even if there is a vigorous cell body response to injury, normal axonal regeneration requires the additional provision of a favorable environment for growth. © 1995 Wiley-Liss, Inc.     

Delayed peripheral nerve repair: methods,including surgical ‘cross-bridging' to promote nerve regeneration

Despite the capacity of Schwann cells to support peripheral nerve regeneration, functional recovery after nerve injuries is frequently poor, especially for proximal injuries that require regenerating axons to grow over long distances to reinnervate distal targets. Nerve transfers, where small fascicles from an adjacent intact nerve are coapted to the nerve stump of a nearby denervated muscle, allow for functional return but at the expense of reduced numbers of innervating nerves. A 1-hour period of 20 Hz electrical nerve stimulation via electrodes proximal to an injury site accelerates axon outgrowth to hasten target reinnervation in rats and humans, even after delayed surgery. A novel strategy of enticing donor axons from an otherwise intact nerve to grow through small nerve grafts(cross-bridges) into a denervated nerve stump, promotes improved axon regeneration after delayed nerve repair. The efficacy of this technique has been demonstrated in a rat model and is now in clinical use in patients undergoing cross-face nerve grafting for facial paralysis. In conclusion, brief electrical stimulation, combined with the surgical technique of promoting the regeneration of some donor axons to ‘protect' chronically denervated Schwa nn cells, improves nerve regeneration and, in turn, functional outcomes in the management of peripheral nerve injuries.     

The delayed depolarization in rat cutaneous afferent axons is reduced following nerve transection and ligation,but not crush: Implications for injury-induced axonal NA + channel reorganization

Two distinct populations of Na⁺ channels (kinetically fast and slow) are present on the cell bodies and axons of cutaneous afferent neurons; the fast current is increased and the slow current reduced in amplitude following nerve injury. The present study was undertaken to determine if similar changes occur on the axons of these neurons following peripheral nerve injury. The compound action potentials from rat sural nerves were recorded in a sucrose gap chamber. Following application of 4-aminopyridine, a prominent and well-characterized depolarization (the delayed depolarization) followed the action potential. This potential, only present on cutaneous afferent axons, has been correlated with activation of a slow Na⁺ current. The delayed depolarization was reduced after nerve transection. The refractory period of transmission of the action potential was shortened in the transected nerves, but that of the delayed depolarization was prolonged. The changes were largest when the sural nerve was cut and ligated [control: 38.1 ± 1.7% (n = 5); injury: 24.5 ± 2.8% (n = 5), P < 0.05], which prevented reconnection to its peripheral target. When the nerve was crushed and allowed to reestablish peripheral target connections, the delayed depolarization was minimally effected. These results indicate that the changes in Na⁺ channel organization following peripheral target disconnection observed on cutaneous afferent cell bodies also occur on their axons. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:1040–1047, 1988.     

ST8SIA2 promotes oligodendrocyte differentiation and the integrity of myelin and axons

ST8SIA2 is a polysialyltransferase that attaches polysialic acid to the glycoproteins NCAM1 and CADM1. Polysialylation is involved in brain development and plasticity. ST8SIA2 is a schizophrenia candidate gene, and St8sia2 ^?/? mice exhibit schizophrenia‐like behavior. We sought to identify new pathological consequences of ST8SIA2 deficiency. Our proteomic analysis suggested myelin impairment in St8sia2 ^?/? mice. Histological and immune staining together with Western blot revealed that the onset of myelination was not delayed in St8sia2 ^?/? mice, but the content of myelin was lower. Ultrastructure analysis of the corpus callosum showed thinner myelin sheaths, smaller and irregularly shaped axons, and white matter lesions in adult St8sia2 ^?/? mice. Then we evaluated oligodendrocyte differentiation in vivo and in vitro . Fewer OLIG2+ cells in the cortex and corpus callosum, together with the higher percentage of undifferentiated oligodenroglia in St8sia2 ^?/? mice suggested an impairment in oligodendrocyte generation. Experiment on primary cultures of oligodendrocyte precursor cells (OPCs) confirmed a cell‐autonomous effect of ST8SIA2 in oligodendroglia, and demonstrated that OPC to oligodendrocyte transition is inhibited in St8sia2 ^?/? mice. Concluding, ST8SIA2‐mediated polysialylation influences on oligodendrocyte differentiation, and oligodendrocyte deficits in St8sia2 mice are a possible cause of the demyelination and degeneration of axons, resembling nerve fiber alterations in schizophrenia. GLIA 2016;65:34–49     

Corticospinal tract regeneration after spinal cord injury in receptor protein tyrosine phosphatase sigma deficient mice

Receptor protein tyrosine phosphatase sigma (RPTPσ) plays a role in inhibiting axon growth during development. It has also been shown to slow axon regeneration after peripheral nerve injury and inhibit axon regeneration in the optic nerve. Here, we assessed the ability of the corticospinal tract (CST) axons to regenerate after spinal hemisection and contusion injury in RPTPσ deficient (RPTPσ^−/−) mice. We show that damaged CST fibers in RPTPσ^−/− mice regenerate and appear to extend for long distances after a dorsal hemisection or contusion injury of the thoracic spinal cord. In contrast, no long distance axon regeneration of CST fibers is seen after similar lesions in wild‐type mice. In vitro experiments indicate that cerebellar granule neurons from RPTPσ^−/− mice have reduced sensitivity to the inhibitory effects of chondroitin sulfate proteoglycan (CSPG) substrate, but not myelin, which may contribute to the growth of CST axons across the CSPG‐rich glial scar. Our data suggest that RPTPσ may function to prevent axonal growth after injury in the adult mammalian spinal cord and could be a target for promoting long distance regeneration after spinal cord injury. © 2009 Wiley‐Liss, Inc.     

Schwann cell‐derived exosomes enhance axonal regeneration in the peripheral nervous system

Axonal regeneration in the peripheral nervous system is greatly supported by Schwann cells (SCs). After nerve injury, SCs dedifferentiate to a progenitor‐like state and efficiently guide axons to their original target tissues. Contact and soluble factors participate in the crosstalk between SCs and axons during axonal regeneration. Here we show that dedifferentiated SCs secrete nano‐vesicles known as exosomes which are specifically internalized by axons. Surprisingly, SC‐derived exosomes markedly increase axonal regeneration in vitro and enhance regeneration after sciatic nerve injury in vivo. Exosomes shift the growth cone morphology to a pro‐regenerating phenotype and decrease the activity of the GTPase RhoA, involved in growth cone collapse and axon retraction. Altogether, our work identifies a novel mechanism by which SCs communicate with neighboring axons during regenerative processes. We propose that SC exosomes represent an important mechanism by which these cells locally support axonal maintenance and regeneration after nerve damage. GLIA 2013;61:1795–1806     

Diabetic Schwann cells suffer from nerve growth factor and neurotrophin‐3 underproduction and poor associability with axons

Schwann cells (SCs) are integral to peripheral nerve biology, contributing to saltatory conduction along axons, nerve and axon development, and axonal regeneration. SCs also provide a microenvironment favoring neural regeneration partially due to production of several neurotrophic factors. Dysfunction of SCs may also play an important role in the pathogenesis of peripheral nerve diseases such as diabetic peripheral neuropathy where hyperglycemia is often considered pathogenic. In order to study the impact of diabetes mellitus (DM) upon the regenerative capacity of adult SCs, we investigated the differential production of the neurotrophic factors nerve growth factor (NGF) and neurotrophin‐3 (NT3) by SCs harvested from the sciatic nerves of murine models of type 1 DM (streptozotocin treated C57BL/6J mice) and type 2 DM (LepR^?/? or db/db mice) or non‐diabetic cohorts. In vitro, SCs from diabetic and control mice were maintained under similar hyperglycemic and euglycemic conditions respectively. Mature SCs from diabetic mice produced lower levels of NGF and NT3 under hyperglycemic conditions when compared to SCs in euglycemia. In addition, SCs from both DM and non‐DM mice appear to be incapable of insulin production, but responded to exogenous insulin with greater proliferation and heightened myelination potentiation. Moreover, SCs from diabetic animals showed poorer association with co‐cultured axons. Hyperglycemia had significant impact upon SCs, potentially contributing to the pathogenesis of diabetic peripheral neuropathy. GLIA 2013;61:1990–1999     

Functional recovery following peripheral nerve injury in the transgenic Thy1‐GFP rat

Transgenic mice have been previously used to assess nerve regeneration following peripheral nerve injury. However, mouse models are limited by their small caliber nerves, short nerve lengths, and their inability to fully participate during behavioral assessments. The transgenic Thy1 GFP rat is a novel transgenic rat model designed to assess regeneration following peripheral nerve injury. However, return of functional and behavioral recovery following nerve injury has not yet been evaluated in these rats. In this study, we ask whether differences in anatomy, recovery of locomotion, myological, and histomorphological measures exist between transgenic Thy1 GFP rats when compared to wild type (WT) Sprague Dawley rats following unilateral sciatic nerve injury. We found that both motor and sensory neuronal architecture, overground and skilled locomotion, muscle force, motor unit number estimation (MUNE) and wet muscle weights, and histomorphometric assessments are similar between both genetic phenotypes. Overall, these data support the use of the transgenic Thy1‐GFP rat in experiments assessing functional and behavioral recovery following nerve injury and repair.     

The effects of pyronin on sprouting and regeneration of mouse motor nerves

Administration to mice of a 0.1% solution of pyronin G in their drinking water caused an acceleration both of axonal sprouting from nodes of Ranvier in partly denervated gluteus maximus muscles, and of motor nerve regeneration following a crush to the soleus nerve. Sprouting from soleus motor nerve terminals in response to botulinum toxin-induced paralysis was, however, unaffected. Removal of degenerating axons following nerve section was also accelerated by pyronin treatment. Pyronin is therefore likely to act upon the process of Wallerian degeneration, rather than upon intact motor nerves directly.     

Impaired peripheral nerve regeneration in type‐2 diabetic mouse model

Peripheral neuropathy is one of the most common and serious complications of type‐2 diabetes. Diabetic neuropathy is characterized by a distal symmetrical sensorimotor polyneuropathy, and its incidence increases in patients 40 years of age or older. In spite of extensive research over decades, there are few effective treatments for diabetic neuropathy besides glucose control and improved lifestyle. The earliest changes in diabetic neuropathy occur in sensory nerve fibers, with initial degeneration and regeneration resulting in pain. To seek its effective treatment, here we prepared a type‐2 diabetic mouse model by giving mice 2 injections of streptozotocin and nicotinamide and examining the ability for nerve regeneration by using a sciatic nerve transection‐regeneration model previously established by us. Seventeen weeks after the last injection, the mice exhibited symptoms of type‐2 diabetes, that is, impaired glucose tolerance, decreased insulin level, mechanical hyperalgesia, and impaired sensory nerve fibers in the plantar skin. These mice showed delayed functional recovery and nerve regeneration by 2 weeks compared with young healthy mice and by 1 week compared with age‐matched non‐diabetic mice after axotomy. Furthermore, type‐2 diabetic mice displayed increased expression of PTEN in their DRG neurons. Administration of a PTEN inhibitor at the cutting site of the nerve for 4 weeks promoted the axonal transport and functional recovery remarkably. This study demonstrates that peripheral nerve regeneration was impaired in type‐2 diabetic model and that its combination with sciatic nerve transection is suitable for the study of the pathogenesis and treatment of early diabetic neuropathy.     

NADPH oxidase and aging drive microglial activation,oxidative stress,and dopaminergic neurodegeneration following systemic LPS administration

Parkinson's disease is characterized by a progressive degeneration of substantia nigra (SN) dopaminergic neurons with age. We previously found that a single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) injection caused a slow progressive loss of tyrosine hydroxylase immunoreactive (TH+IR) neurons in SN associated with increasing motor dysfunction. In this study, we investigated the role of NADPH oxidase (NOX) in inflammation‐mediated SN neurotoxicity. A comparison of control (NOX2^+/+) mice with NOX subunit gp91^phox‐deficient (NOX2^?/?) mice 10 months after LPS administration (5 mg/kg, i.p.) resulted in a 39% (P < 0.01) loss of TH+IR neurons in NOX2^+/+ mice, whereas NOX2^?/? mice did not show a significant decrease. Microglia (Iba1+IR) showed morphological activation in NOX2^+/+ mice, but not in NOX2^?/? mice at 1 hr. Treatment of NOX2^+/+ mice with LPS resulted in a 12‐fold increase in NOX2 mRNA in midbrain and 5.5–6.5‐fold increases in NOX2 protein (+IR) in SN compared with the saline controls. Brain reactive oxygen species (ROS), determined using diphenyliodonium histochemistry, was increased by LPS in SN between 1 hr and 20 months. Diphenyliodonium (DPI), an NOX inhibitor, blocked LPS‐induced activation of microglia and production of ROS, TNFα, IL‐1β, and MCP‐1. Although LPS increased microglial activation and ROS at all ages studied, saline control NOX2^+/+ mice showed age‐related increases in microglial activation, NOX, and ROS levels at 12 and 22 months of age. Together, these results suggest that NOX contributes to persistent microglial activation, ROS production, and dopaminergic neurodegeneration that persist and continue to increase with age. © 147.     

Heat shock protein is a key therapeutic target for nerve repair in autoimmune peripheral neuropathy and severe peripheral nerve injury

Guillain-Barré syndrome (GBS) is an autoimmune peripheral neuropathy and a common cause of neuromuscular paralysis. Preceding infection induces the production of anti-ganglioside (GD) antibodies attacking its own peripheral nerves. In severe proximal peripheral nerve injuries that require long-distance axon regeneration, motor functional recovery is virtually nonexistent. Damaged axons fail to regrow and reinnervate target muscles. In mice, regenerating axons must reach the target muscle within 35 days (critical period) to reform functional neuromuscular junctions and regain motor function. Successful functional recovery depends on the rate of axon regeneration and debris removal (Wallerian degeneration) after nerve injury. The innate-immune response of the peripheral nervous system to nerve injury such as timing and magnitude of cytokine production is crucial for Wallerian degeneration. In the current study, forced expression of human heat shock protein (hHsp) 27 completely reversed anti-GD-induced inhibitory effects on nerve repair assessed by animal behavioral assays, electrophysiology and histology studies, and the beneficial effect was validated in a second mouse line of hHsp27. The protective effect of hHsp27 on prolonged muscle denervation was examined by performing repeated sciatic nerve crushes to delay regenerating axons from reaching distal muscle from 37 days up to 55 days. Strikingly, hHsp27 was able to extend the critical period of motor functional recovery for up to 55 days and preserve the integrity of axons and mitochondria in distal nerves. Cytokine array analysis demonstrated that a number of key cytokines which are heavily involved in the early phase of innate-immune response of Wallerian degeneration, were found to be upregulated in the sciatic nerve lysates of hHsp27 Tg mice at 1 day postinjury. However, persistent hyperinflammatory mediator changes were found after chronic denervation in sciatic nerves of littermate mice, but remained unchanged in hHsp27 Tg mice. Taken together, the current study provides insight into the development of therapeutic strategies to enhance muscle receptiveness (reinnervation) by accelerating axon regeneration and Wallerian degeneration.