Motor nerve terminal destruction and regeneration following anti-ganglioside antibody and complement-mediated injury: An in and ex vivo imaging study in the mouse

Abstract

Both the neural and glial components of the neuromuscular junction (NMJ) have been identified as potential sites for anti-ganglioside antibody (Ab) binding and complement-mediated injury in murine models for the human peripheral nerve disorder Guillain-Barré syndrome (GBS). Some patients suffering from the acute motor axonal neuropathy (AMAN) forms of GBS recover very rapidly from paralysis; it has been proposed that in these cases the injury was restricted to the distal motor axons and nerve terminals (NTs) which are able to regenerate over a very short time-frame.

To test this hypothesis, the ventral neck muscles of mice (n = 45) expressing cytosolic fluorescent proteins in their axons (CFP) and Schwann cells (GFP) were subjected to a single topical application of anti-ganglioside Ab followed by a source of complement. Group A (n = 15) received Ab that selectively bound to the NTs, group B (n = 15) received Abs that bound both to the NTs and the perisynaptic Schwann cells (pSCs) and group C (control animals; n = 15) only received complement. Evolution of the injury was documented by in vivo imaging, and following euthanasia the muscles were reimaged ex vivo both quantitatively and qualitatively, either immediately, or after 1, 2, 3 or 5 days of regeneration (each n = 3 per group).

Within 15 minutes of complement application, a rapid loss of CFP overlying the NMJ could be seen; in group A, the GFP signal remained unchanged, whereas in group B the GFP signal was also lost. In group C no changes to either CFP or GFP were observed. At 24 h, 6% of the superficial NMJs in group A and 12% of the NMJs in group B exhibited CFP. In both groups, CFP returned within the next five days (group A: 93.5%, group B: 94%; p = 0.739), with the recovery of CFP being preceded by a return of GFP-positive cells overlying the NMJ in group B. Auxiliary investigations revealed that the loss of CFP at the NMJ correlated with a loss of NT neurofilament immuno-reactivity and a return of CFP at the NMJ was accompanied by a return of neurofilament. In ultrastructural investigations, injured NTs were electron lucent and exhibited damaged mitochondria, a loss of filaments and a loss of synaptic vesicles. The examination of muscles after five days of regeneration revealed physiological NT-profiles.

The results described above indicate that following a single anti-ganglioside Ab-mediated and complement-mediated attack, independent of whether there are healthy and mature perisynaptic Schwann cells overlying the NMJ, the murine NT is capable of recovering both its architectural and axolemmal integrity very rapidly. This data supports the notion that an equivalent mechanism may account for the rapid recovery seen in some clinical cases of AMAN.

Introduction

Anti-ganglioside antibodies (Abs) are considered important mediators of the disease in the acute human peripheral nerve disorder Guillain-Barré syndrome (GBS) and its variant forms (Willison, 2005). Gangliosides are glycosphingolipids found on plasma membranes throughout the body, but are enriched on neural tissue. They are distinguished from one another by the number and location of sialic acid residues attached to a neutral sugar backbone, and their distribution varies widely between the structures which they are located on (Hughes et al., 1999); this is likely to be of clinical relevance (Willison, 2005, Willison, 2007). Once anti-ganglioside Abs have bound their target membrane, one mechanism by which they induce injury is through the activation of the complement system, which leads to the formation of a membrane attack complex (MAC) on the structures bound (Halstead et al., 2005a). The MAC pore allows an uncontrolled influx of ions and water and ultimately results in pathological changes and dysfunction of the structures targeted (Halstead et al., 2005b, McGonigal et al., 2010, O'Hanlon et al., 2001).

Anti-GM1 and anti-GD1a Abs are the circulating Abs predominantly found in patients suffering from the motor axonal forms of GBS (acute motor axonal neuropathy, AMAN) (Kaida et al., 2000, Press et al., 2001, Willison, 2005). Clinically, AMAN-patients exhibit paralysis; this may be so widespread that the patients are required to be ventilated artificially (McKhann et al., 1993). Necropsies of AMAN-patients reveal Wallerian-like degeneration of the motor nerve fibres more pronounced in the ventral roots than the peripheral nerves, indicating that the initial lesion is to be found in the spinal roots. Occasionally, however, the pathological changes to these areas are not sufficient to be commensurate with the degree of paralysis observed (Griffin et al., 1995, McKhann et al., 1993). At the same time, the very extensive motor fibre degeneration observed in some necropsies is not compatible with a rapid recovery (Griffin et al., 1995, Ho et al., 1997b), which may begin within the first three weeks of disease onset (McKhann et al., 1991). Other investigators have documented the convalescence of AMAN-patients to occur in two subgroups: one which exhibits a rapid improvement within the first two to four weeks after onset of disease, and another, in which recovery is prolonged and the patients are unable to walk independently at six months after disease onset (Hiraga et al., 2005, Kuwabara et al., 1998). Explanations for a rapid recovery include a) distal demyelination, b) a reversible conduction-block along motor axons and c) degeneration restricted to the very distal motor nerve (Griffin et al., 1995, Ho et al., 1997b, Kuwabara et al., 1998) with the investigation of muscle-nerve biopsies obtained from a rapidly recovering AMAN-patient providing evidence for the third hypothesis (Ho et al., 1997a). Since the neuromuscular junction (NMJ) lies outside the blood-nerve-barrier (Olsson, 1968), the motor nerve terminals (NTs) are readily available for Ab-binding (Ho et al., 1997a, Plomp and Willison, 2009).

Perisynaptic Schwann cells (pSCs) are glial cells of the NMJ. These non-myelinating SCs cap the neuromuscular junction (NMJ), completely covering the NTs with their cell bodies and processes (Griffin and Thompson, 2008). In mammals, little is known about the regular functions of these glial cells (Griffin and Thompson, 2008); in frogs, however, pSCs have been shown to be important for the maintenance of the physiological structure and function of the NMJ (Reddy et al., 2003). Traumatic denervation of the NMJ, both in mammals and frogs, leads to reactive expansion and extension of pSC processes (Astrow et al., 1998). Once these processes have reached an innervated NMJ, they entice the resident NT to sprout and guide it back to their parent NMJ, thus regaining innervation (Son and Thompson, 1995a, Son and Thompson, 1995b). Kranocytes, which are fibroblast-like cells capping the NMJ, have been shown to contribute to this process (Court et al., 2008).

Studies conducted in ex vivo muscle-nerve preparations and in vivo murine models of GBS have revealed both the neural and glial components of the NMJ to be targets for anti-ganglioside Ab binding and complement-mediated injury (Halstead et al., 2005a, Halstead et al., 2005b, O'Hanlon et al., 2003). By combining aspects of the murine model of GBS with in and ex vivo imaging of mice expressing fluorescent proteins in the neural and glial structures of their peripheral nervous system (Feng et al., 2000, Zuo et al., 2004), we are able to monitor anti-ganglioside Ab-mediated injury and recovery of the structures affected in vivo and in real-time. Here we exploited this system to determine the rate of NT regeneration following a single anti-ganglioside Ab and complement-mediated injury both with and without a concomitant injury of the pSCs.