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

Abstract

We investigated the usefulness of YFP-H transgenic mice [Neuron 28 (2000) 41] which express yellow fluorescent protein (YFP) in a restricted subset of neurons to study Wallerian degeneration in the PNS. Quantification of YFP positive axons and myelin basic protein (MBP) immunocytochemistry revealed that YFP was randomly distributed to approximately 3% of myelinated motor and sensory fibres. Axotomy-induced Wallerian degeneration appeared as fragmentation of fluorescent signals in individual YFP positive axons with a morphology and timing similar to Wallerian degeneration observed by more traditional methods. In YFP-H transgenic mice co-expressing a high dosage of Wld^S, a chimeric gene that protects from Wallerian degeneration [Nat Neurosci. 4 (2001) 1199], axonal fragmentation in distal tibial nerves after sciatic nerve axotomy was approximately 10 times delayed. Considerable retardations of Wallerian degeneration using the same transgenic expression system were also observed in cultures of nerve explants, enabling in vitro real-time imaging of axonal fragmentation. Remarkably, single YFP-labelled axons could be traced in peripheral nerves for unusually long distances of up to 2.9 cm exploiting confocal fluorescence imaging. Altogether transgenic YFP-H mice prove to be a valuable tool to study mechanisms of Wallerian degeneration in vivo and in vitro.

Introduction

Wallerian degeneration is a spontaneous degenerative process of the distal portions of peripheral or central nerve axons that are separated from the parent cell body (Waller, 1850). Not only do traumatic disorders such as spinal cord injury result in this form of degeneration but it is now broadly accepted that Wallerian degeneration is mechanistically related to axon loss in many neurodegenerative disorders such as amyotrophic lateral sclerosis, multiple sclerosis and toxic neuropathy (Bjartmar et al., 1999, Coleman and Perry, 2002, Griffin et al., 1996). Wallerian degeneration has usually been described in common neuropathology textbooks as a focal phenomenon just focusing on a microscopically small part of the degenerating fibre, often neglecting the fact that axons can reach lengths of many centimetres to meters and that topographic factors of Wallerian degeneration along axons play an important role in many axon disorders (Cavanagh, 1979, King, 1999a, Spencer and Schaumburg, 1976). There is relatively little and contradictory published information regarding the behaviour of the axon in its entirety after disconnection from its supporting neuronal cell body mainly due to limits of axon visualisation over longer distances. To the best of our knowledge, only very few and moreover exceedingly complicated classical histological techniques are available to date for reliable visualisation of pathological processes in single axons over substantial nerve distances, and these are often particularly labour-intensive and susceptible to artefacts due to preparation and processing (King, 1999b, Somogyi et al., 1979). Against this background, the availability of a method for long-range tracing of axons undergoing pathological processes would improve our understanding of axodegenerative mechanisms.

Yellow fluorescent protein (YFP) is a derivative of green fluorescent protein (GFP), a versatile reporter molecule which has found use in many neuroscience applications (Chalfie et al., 1994). Transgenic mice are now available that express YFP or related fluorophores such as cyan fluorescent protein (CFP) at high levels selectively in various neuronal subsets under the control of regulatory elements derived from the mouse Thy1 gene (Feng et al., 2000). In contrast to mice expressing the CFP fluorophore in a majority of PNS neurites, transgenic YFP-H mice obtained from the Jackson Laboratories (Bar Harbor, USA) show only a few axons in peripheral nerves that are brightly and homogeneously fluorescent all the way to the terminals. No expression is detectable in non-neuronal cells such as Schwann cells. Thus YFP, as a strong and specific vital marker for a small percentage of axons, should provide an innovative and convenient opportunity for axon tracing over long distances since YFP-labelled axons can be observed among the bulk of unlabelled fibres in a nerve applying confocal fluorescence microscopy. Because YFP is localized in neuronal cytoplasm, intrinsic morphological changes in axoplasm due to various insults can be visualised in labelled axons by fluorescence microscopy techniques (Brendza et al., 2003, Gillingwater et al., 2002). Importantly, it has been shown that expression of YFP in neurons results in no apparent toxic effect (Feng et al., 2000), so that even sensitive physiological processes such as synapse elimination during early postnatal development are not disturbed (Keller-Peck et al., 2001). Therefore, morphological events such as axon disintegration that are known to appear in the course of Wallerian degeneration should correlate with YFP signal changes in fluorescent axons.

In this study, we performed a detailed morphological analysis of the restricted subset of YFP positive axons in peripheral nerves of transgenic YFP-H mice and then addressed the question whether these fibres are suitable to visualise and evaluate Wallerian degeneration by objective, sensitive and valid criteria in vivo and in vitro. Further, we asked whether selective neuronal YFP expression in transgenic YFP-H mice permits continuous long-range and long-term tracing of individual axon courses in peripheral nerve wholemount preparations in conjunction with the optical sectioning capability of the confocal laser scanning microscope.