Rapid induction of functional and morphological continuity between severed ends of mammalian or earthworm myelinated axons.

The inability to rapidly restore the loss of function that results from severance (cutting or crushing) of PNS and CNS axons is a severe clinical problem. As a novel strategy to help alleviate this problem, we have developed in vitro procedures using Ca2+-free solutions of polyethylene glycol (PEG solutions), which within minutes induce functional and morphological continuity (PEG-induced fusion) between the cut or crushed ends of myelinated sciatic or spinal axons in rats. Using a PEG-based hydrogel that binds to connective tissue to provide mechanical strength at the lesion site and is nontoxic to nerve tissues in earthworms and mammals, we have also developed in vivo procedures that permanently maintain earthworm myelinated medial giant axons whose functional and morphological integrity has been restored by PEG-induced fusion after axonal severance. In all these in vitro or in vivo procedures, the success of PEG-induced fusion of sciatic or spinal axons and myelinated medial giant axons is measured by the restored conduction of action potentials through the lesion site, the presence of intact axonal profiles in electron micrographs taken at the lesion site, and/or the intra-axonal diffusion of fluorescent dyes across the lesion site. These and other data suggest that the application of polymeric fusiogens (such as our PEG solutions), possibly combined with a tissue adherent (such as our PEG hydrogels), could lead to in vivo treatments that rapidly and permanently repair cut or crushed axons in the PNS and CNS of adult mammals, including humans.

Anomalies associated with dye exclusion as a measure of axolemmal repair in invertebrate axons.

After axonal injury, dye exclusion is often used as a measure of the re-establishment of a structural barrier. We now report that this use of dye exclusion is equivocal in two situations. (1) When a negatively-charged hydrophilic fluorescent dye (HFD) was placed in the physiological saline (PS) surrounding a crayfish medial giant axon (CMGA) before transection, this dye did not readily diffuse into the cut ends after transection whereas uncharged or neutralized dyes did do so. (2) When axoplasm flowed out of the cut ends of a transected squid giant axon (SGA), this outflow markedly slowed hydrophilic fluorescent dyes from diffusing into the cut ends. These anomalies suggest that dye exclusion by an injured axon does not always indicate that a structural barrier has formed. Therefore, dye assessments of axonal repair require control experiments that rule out anomalous exclusion due to dye interactions (biochemical and fluid dynamics) with components (axoplasm, axolemma, glial sheath, etc.) of the particular axon under study.

Heat-shock proteins in axoplasm: high constitutive levels and transfer of inducible isoforms from glia.

To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1-3% of the total protein in the axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock.

Endocytotic formation of vesicles and other membranous structures induced by Ca2+ and axolemmal injury.

Vesicles and/or other membranous structures that form after axolemmal damage have recently been shown to repair (seal) the axolemma of various nerve axons. To determine the origin of such membranous structures, (1) we internally dialyzed isolated intact squid giant axons (GAs) and showed that elevation of intracellular Ca2+ >100 microM produced membranous structures similar to those in axons transected in Ca2+-containing physiological saline; (2) we exposed GA axoplasm to Ca2+-containing salines and observed that membranous structures did not form after removing the axolemma and glial sheath but did form in severed GAs after >99% of their axoplasm was removed by internal perfusion; (3) we examined transected GAs and crayfish medial giant axons (MGAs) with time-lapse confocal fluorescence microscopy and showed that many injury-induced vesicles formed by endocytosis of the axolemma; (4) we examined the cut ends of GAs and MGAs with electron microscopy and showed that most membranous structures were single-walled at short (5-15 min) post-transection times, whereas more were double- and multi-walled and of probable glial origin after longer (30-150 min) post-transection times; and (5) we examined differential interference contrast and confocal images and showed that large and small lesions evoked similar injury responses in which barriers to dye diffusion formed amid an accumulation of vesicles and other membranous structures. These and other data suggest that Ca2+ inflow at large or small axolemmal lesions induces various membranous structures (including endocytotic vesicles) of glial or axonal origin to form, accumulate, and interact with each other, preformed vesicles, and/or the axolemma to repair the axolemmal damage.

Delaminating myelin membranes help seal the cut ends of severed earthworm giant axons.

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5-60 min post-transection in physiological salines and Ca2+-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20-100 microm from the cut end at 5-60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15-30 min post-transection. At 30-60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca2+-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca2+-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma.

Calpain activity promotes the sealing of severed giant axons.

A barrier (seal) must form at the cut ends of a severed axon if a neuron is to survive and eventually regenerate. Following severance of crayfish medial giant axons in physiological saline, vesicles accumulate at the cut end and form a barrier (seal) to ion and dye diffusion. In contrast, squid giant axons do not seal, even though injury-induced vesicles form after axonal transection and accumulate at cut axonal ends. Neither axon seals in Ca2+-free salines. The addition of calpain to the bath saline induces the sealing of squid giant axons, whereas the addition of inhibitors of calpain activity inhibits the sealing of crayfish medial giant axons. These complementary effects involving calpain in two different axons suggest that endogenous calpain activity promotes plasmalemmal repair by vesicles or other membranes which form a plug or a continuous membrane barrier to seal cut axonal ends.

Repair of plasmalemmal lesions by vesicles.

Crayfish medial giant axons (MGAs) transected in physiological saline form vesicles which interact with each other, pre-existing vesicles, and/or with the plasmalemma to form an electrical and a physical barrier that seals a cut axonal end within 60 min. The formation of this barrier (seal) was assessed by measuring the decay of injury current at the cut end; its location at the cut end was determined by the exclusion of fluorescent hydrophilic dye at the cut end. When a membrane-incorporating styryl dye was placed in the bath prior to axonal transection and a hydrophilic dye was placed in the bath just after axonal transection, many vesicles near the barrier at the cut axonal end had their limiting membrane labeled with the styryl dye and their contents labeled with the hydrophilic dye, indicating that these vesicles originated from the axolemma by endocytosis. This barrier does not form in Ca2+-free salines. Similar collections of vesicles have been observed at regions of plasmalemmal damage in many cell types. From these and other data, we propose that plasmalemmal lesions in most eukaryotic cells (including axons) are repaired by vesicles, at least some of which arise by endocytosis induced by Ca2+ inflow resulting from the plasmalemmal damage. We describe several models by which vesicles could interact with each other and/or with intact or damaged regions of the plasmalemma to repair small (1-30 microm) plasmalemmal holes or a complete transection of the plasmalemma.