Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice

Beirowski, Bogdan; Berek, Livia; Adalbert, Róbert; Wagner, Diana; Grumme, Daniela; Addicks, Klaus; Ribchester, Richard R.; Coleman, Michael P.

doi:10.1016/j.jneumeth.2003.10.016

Cited by 104 publications

(109 citation statements)

References 42 publications

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“…25 We previously showed that in the absence of Wld S all labelled axons fragment within 52 h of nerve transection, 26 whereas Wld S /YFP-H axons remain continuous after 20 days, showing only localised atrophy. 23 Homozygotes of highly expressing …”

Section: Resultsmentioning

confidence: 97%

“…YFP-H mice 25 were obtained from the Jackson Laboratories and genotyped as described previously. 26 Western blotting. Sagittally-divided half brains were homogenised in five volumes of RIPA buffer (phosphate-buffered saline (PBS) containing 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecylsulphate).…”

Section: Methodsmentioning

confidence: 99%

“…Nerves were fixed for at least 24 h in 0.1 M phosphate buffer containing 4% paraformaldehyde and 2.5% glutardialdehyde, embedded in Durcupan resin (Fluka) and processed for light and electron microscopy as described previously. 26 Analysis of YFP-labelled nerves. Tibial nerves were quickly removed from humanely killed mice and processed as described previously 26 Primary culture for transgenic experiments (Coleman group).…”

Section: Methodsmentioning

confidence: 99%

“…26 Analysis of YFP-labelled nerves. Tibial nerves were quickly removed from humanely killed mice and processed as described previously 26 Primary culture for transgenic experiments (Coleman group). DRGs were dissected from E15.5 mouse embryos using sterile technique, initially into L15 medium (Invitrogen) for removal of other tissue, and then plated ca.…”

Section: Methodsmentioning

confidence: 99%

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NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

et al. 2006

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The slow Wallerian degeneration protein (Wld S ), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of Wld S mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to Wld S mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for Wld S , and lentiviral overexpressed enzyme-dead Wld S still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than Wld S at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of Wld S requires more N-terminal sequences of the protein.

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Section: Resultsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

et al. 2006

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“…Finally, the extent to which degenerating PNS myelin is inhibitory to regenerating axons has been controversial. To determine whether the absence of mature B-cells delays axon regeneration, we crossed the JHD mice to YFP-H mice in which 3% of myelinated axons in sciatic nerve are YFP positive (18,19). These mice enable us to examine the regenerative rates of a specific subset of myelinated axons with high spatial resolution.…”

Section: Passive Transfer Rescues Delayed Macrophage Entry Injured Scmentioning

confidence: 99%

Endogenous antibodies promote rapid myelin clearance and effective axon regeneration after nerve injury

Vargas¹,

Watanabe²,

Singh³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

144

129

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Degenerating myelin inhibits axon regeneration and is rapidly cleared after peripheral (PNS) but not central nervous system (CNS) injury. To better understand mechanisms underlying rapid PNS myelin clearance, we tested the potential role of the humoral immune system. Here, we show that endogenous antibodies are required for rapid and robust PNS myelin clearance and axon regeneration. B-cell knockout JHD mice display a significant delay in macrophage influx, myelin clearance, and axon regeneration. Rapid clearance of myelin debris is restored in mutant JHD mice by passive transfer of antibodies from naïve WT mice or by an anti-PNS myelin antibody, but not by delivery of nonneural antibodies. We demonstrate that degenerating nerve tissue is targeted by preexisting endogenous antibodies that control myelin clearance by promoting macrophage entrance and phagocytic activity. These results demonstrate a role for immunoglobulin (Ig) in clearing damaged self during healing and suggest that the immune-privileged status of the CNS may contribute to failure of CNS myelin clearance and axon regeneration after injury.humoral immunity | nerve regeneration D uring the process of wound healing, rapid and efficient clearance of cellular debris is necessary for tissue regeneration (1). Myelin debris remains in white matter tracks years after an injury to the CNS in humans and primates (2, 3). The prolonged presence of myelin-associated inhibitors of axon regeneration is thought to contribute to the lack of recovery following CNS injury. Although peripheral myelin also contains inhibitors of axon regeneration, PNS myelin is rapidly cleared after injury, thereby permitting rapid axon regeneration (4-6). It is not known why the rates of clearance of PNS and CNS are so different (7).Antibodies, like other opsonins, coat exogenous debris and pathogens, thereby targeting them for clearance by phagocytes. The recognition of self antigens by natural antibodies produced by B1 cells is well documented (8). Although it is hypothesized that these antibodies may have a physiological role other than immune defense, their role in clearing necrotic cellular debris is not known (8). Therefore, we tested whether endogenous antibodies contribute to rapid removal of degenerating myelin after PNS injury, thereby promoting axon regeneration. ResultsAntibodies Accumulate in Sciatic Nerve After Injury. To investigate whether endogenous antibodies aid in removal of myelin debris, we first examined whether antibodies accumulate in peripheral nerves following injury. We compared nerve injury responses between WT and JHD mice, which have a targeted deletion of the JH locus that prevents VDJ recombination and the formation of mature B-cells and antibodies (9). Control and crushed sciatic nerves were obtained from WT and JHD mice and stained with anti-mouse Ig antibodies. Six days after crush injury, we observed a strong increase in immunoreactivity for Ig on degenerating myelin sheathes distal to the site of injury in WT but not in JHD nerves (Fig. S1A). To ...

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Confocal Microendoscopy of Neuromuscular Synapses in Living Mice

Blanco

Ribchester

2012

CP Mouse Biology

Self Cite

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Here we describe a step-by-step method for vital imaging of neuromuscular junctions (NMJ) and axons using fiber-optic confocal microendoscopy (CME). A commercially available system, the Cellvizio Lab, can be applied to transgenic mouse lines expressing yellow fluorescent protein in all or pseudorandom sub-subsets of motor neurons. Microscopic imaging in vivo is achieved by means of a flexible optical fiber probe that excites and collects the emitted light from fluorescently labeled structures. The hand-held probe is introduced through small skin incisions to visualize nerves and neuromuscular junctions from superficial muscles. Interpolation software then reconstructs the images in real time. The images are of sufficient quality to permit screening of axonal and neuromuscular synaptic integrity and other aspects of their phenotype in live animals. Curr. Protoc. Mouse Biol. 2:1-8 © 2012 by John Wiley & Sons, Inc.

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Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice

Cited by 104 publications

References 42 publications

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

Endogenous antibodies promote rapid myelin clearance and effective axon regeneration after nerve injury

Confocal Microendoscopy of Neuromuscular Synapses in Living Mice

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