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Phospholipase A2 plays an important role in myelin breakdown and phagocytosis during wallerian degeneration

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Abstract

Phospholipase A2 (PLA2) hydrolyzes phosphatidylcholine to lysophosphatidylcholine and arachidonic acid. The former can induce myelin breakdown and the latter, via eicosanoids, can stimulate inflammatory responses. Immunohistochemical analysis of secreted (sPLA2) and cytosolic (cPLA2) forms of the enzyme was assessed in the injured adult rat sciatic and optic nerves. sPLA2 and cPLA2 are expressed in the first 2 weeks in the injured sciatic nerve, which correlates with rapid Wallerian degeneration in peripheral nerves. In contrast, both forms of PLA2 were not expressed in the optic nerve for the first 3 weeks after crush injury, which correlates with slow Wallerian degeneration in the central nervous system (CNS). In addition, PLA2 is not expressed in the lesioned sciatic nerve of C57BL/Wlds mutant mice in which Wallerian degeneration is severely retarded. Blocking cPLA2 in the transected sciatic nerve of C57BL/6 mice, which have a naturally occurring null mutation for the major from of sPLA2, resulted in a marked slowing of myelin and axonal degradation and phagocytosis in the distal nerve segment. These results provide direct evidence of an important role for cPLA2 in Wallerian degeneration.

Introduction

Wallerian degeneration consists of the breakdown and phagocytosis of myelin and axons distal to the site of an injury Waller, 1850, George and Griffin, 1994. Axonal degeneration after injury requires Ca2+ influx and activation of calpain George et al., 1995, Wang et al., 2000, while the breakdown and phagocytosis of myelin requires the influx and activation of hematogenous macrophages Beuche and Friede, 1984, Beuche and Friede, 1986, Brück et al., 1996, Crang and Blakemore, 1986, Müller and Minwegen, 1987 and possibly also contributions from Schwann cells Bigbee et al., 1987, Stoll et al., 1989a, Fernandez-Valle et al., 1995, Rutkowski et al., 1999. A number of molecules, such as adhesion molecules (ICAM1, VCAM1), complement proteins (C3), the complement receptor CR3, and cytokines Castano et al., 1996, Brück and Friede, 1990, Brück and Friede, 1991, Vougioukas et al., 1998, Rotshenker, 2001, have been implicated in mediating the immune responses in Wallerian degeneration. However, the key molecular signal(s) that triggers these cellular responses is not fully known.

Understanding the molecular control of myelin phagocytosis during Wallerian degeneration has acquired increasing importance for central nervous system (CNS) regeneration, since axon growth inhibitors associated with myelin contribute to the failure of regeneration in the adult mammalian CNS Bandtlow and Schwab, 2000, David and Lacroix, 2003. In contrast to the CNS, axon regeneration occurs readily in injured peripheral nerves despite the presence of axon growth-inhibitory activity in peripheral nerve myelin David et al., 1995, Bähr and Przyrembel, 1995. One plausible reason for this might be the rate at which myelin is cleared after peripheral nerve injury. Wallerian degeneration proceeds rapidly in injured peripheral nerves (3 to 14 days) Griffin et al., 1992, Stoll et al., 1989a, Brück, 1997, but is much slower in the adult mammalian CNS (weeks to months) Perry et al., 1987, George and Griffin, 1994, Lawson et al., 1994, Stoll et al., 1989b.

One likely candidate that can trigger myelin breakdown and inflammatory cell responses during Wallerian degeneration is the enzyme phospholipase A2 (PLA2). This enzyme hydrolyzes phospholipids to produce lysophospholipids and a free fatty acid (Glaser, 1995). If the phospholipid is phosphatidylcholine, PLA2 can produce lysophosphatidylcholine (LPC) and arachidonic acid. LPC acts as a detergent to cause myelin breakdown Hall, 1972, Hall and Gregson, 1971, Jeffery and Blakemore, 1995, Ousman and David, 2000, and can induce the expression of inflammatory chemokines and cytokines David, and Ousman, 2001, Ousman and David, 2001. Arachidonic acid gives rise to eicosanoids that are potent mediators of inflammation (Glaser, 1995). PLA2 and its metabolites could, therefore, be key mediators of the immune cell responses during Wallerian degeneration. Differences in the expression of PLA2 may contribute to the differences in the rate of Wallerian degeneration in the CNS and peripheral nervous system (PNS). Various forms of PLA2 exist that can be broadly grouped under secreted (sPLA2) and cytosolic (cPLA2) forms (Dennis, 1994). Of these forms we are particularly interested in PLA2 enzymes that can release arachidonic acid and are expressed in neural cells and cells of the immune system. PLA2 enzymes that meet these criteria include group IIA sPLA2 and group IV cPLA2 (Dennis, 1994).

Group IIA sPLA2 was shown by Paul and Gregson (1992) to be rapidly expressed after peripheral nerve injury. In this article; we report our findings on the expression of both sPLA2 and cPLA2 during Wallerian degeneration after crush injury of PNS (sciatic nerve) and CNS (optic nerve) in adult rats. We also report the expression of PLA2 in the injured sciatic nerve of the C57BL/Wlds mutant mice in which Wallerian degeneration is markedly slow after peripheral nerve injury. In addition, we have carried out experiments to obtain direct evidence for the involvement of cPLA2 in Wallerian degeneration.

Section snippets

Expression of cPLA2 and sPLA2 during Wallerian degeneration in the PNS and CNS

To assess if there is a correlation between the rate of Wallerian degeneration and the expression of cPLA2 and sPLA2 we carried out immunohistochemistry on adult rat sciatic and optic nerves at various survival times after crush injury.

Expression of PLA2 correlates with the rate of Wallerian degeneration

We show that both sPLA2 and cPLA2 are expressed rapidly in the sciatic nerve within hours of crush injury, and continue to be expressed throughout the entire degenerating distal nerve segment for the next 2 weeks. The time course of this expression correlates with the rapid rate of Wallerian degeneration in peripheral nerves Stoll et al., 1989a, Griffin et al., 1992, Griffin and Hoffman, 1993, Brück, 1997. In contrast, immunoreactivity for both forms of PLA2 was not detected in the optic nerve

Sciatic and optic nerve crush injuries

Female Sprague–Dawley rats (200–240 g; Charles River, Wilmington, MA, USA), C57BL/6 mice (18–20 g; Harlan Olac, Indianapolis, IN, USA) and C57BL/Wlds mice (18–20 g; Harlan Olac) were used. Under deep anesthesia the left sciatic nerve was crushed at the level of the midthigh in C57BL/6 mice, C57BL/Wlds mice, and Sprague–Dawley rats using a fine jeweler's forceps. The rat optic nerve was exposed intraorbitally and crushed in a similar manner 1–2 mm behind the eye. All procedures were approved by

Acknowledgements

This work was supported by a grant from the Canadian Institutes of Health Research to S.D. The authors thank Margaret Attiwell for her expertise with the illustrations.

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    1

    Current address: Instituto de Bioingeniería, Facultad de Medicina, Universidad Miguel Hernández de Elche, Apartado de Correos 18, 03550 San Juan de Alicante (Alicante), Spain.

    2

    These authors contributed equally.

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