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Contributions of Monoclonal Antibodies With Anti-Neural Cell Adhesion Molecule Like Activity to Peripheral Nerve Regeneration.

L. G. Remsen

Published 1993 in Unknown venue

ABSTRACT

Tubes containing monoclonal antibodies with anti-neural cell adhesion molecule (N-CAM)-like activity were applied to transected sciatic nerves to attempt to perturb the recovery of muscle function. Physiological recordings were used to estimate the return of function. The decline of implanted antibody over 28 days was estimated. No significant immune responses were detected in response to the implanted material. Electron microscopic and immunohistological analyses evaluated particular cellular disruptions in nerves due to the presence of these antibodies with anti-NCAM like activity. Histological sections of fixed experimental nerves consistently revealed abnormal gaps between Schwann cells of regenerating nerves. This specific Schwann cell abnormality was not present in nerves of control animals and was no longer observed in experimental nerves after 60 days of survival. This time course was associated with antibody clearance and restoration of muscle function. We proposed that perturbed Schwann cell adhesive interactions disrupted the advance of neurites across nerve gaps and resulted in delayed regeneration. The data implicated N-CAM as a potential contributor to nerve regeneration. INTROD UCTIO N To regenerate an injured axon, a neuron must undergo a series of physiological rearrangements. It must reorganize its terminal membrane and cytoplasm to generate a motile structure called the growth cone, that will move in a directed manner to appropriate target cells and eventually form a synaptic terminal. The neuron must also produce the membrane, cytoplasm and cytoskeleton required by the elongating and expanding sprout. In the peripheral nervous system of adult mammals, damaged axons can regenerate for many centimeters from the site of injury (Ramon y Cajal, 1928a,b). These regenerating axons are typically found within conduits of basement membrane and are often in contact with Schwann cells (Hillarp and Olivecrona, 1946). The basement membrane in the peripheral nervous system, like many other basement membranes, is comprised of laminin, fibronectin and other extracellular matrix materials. Purified fibronectin, collagen and laminin have been shown to stimulate neural outgrowth and to guide nerve processes under in vitro conditions (Woolley et al., 1990). This growth requires binding of cell-surface receptors to extracellular matrix adhesive proteins and requires binding of regenerating axonal sprouts to Schwann cells (Hillarp and Olivecrona, 1946). In the regenerating neurite, there are multiple adhesive systems for both cell-cell and cell-matrix receptors that are present on neurons, Schwann cells and fibroblasts (Dellon, 1990). The neural cell adhesion molecule (N-CAM) is a membrane glycoprotein that serves as a homophilic ligand in the formation of adhesions between cells (Hoffman and Edelman, 1933). It is expressed by several types of cells during embryogenesis including nerve, muscle and glial cells of the developing nervous system (Edelman, 1984). N-CAM-mediated adhesion is involved in many developmental events including axon guidance, segregation of cells into discreet layers, and the formation and innervation of muscles. Molecular interactions at the axon-Schwann cell interface that initiate the formation of myelin sheaths involve N-CAM and LI adhesion molecule. Both N-CAM and LI are present on the membrane of axons and Schwann cells before the onset of myelination, but are reduced and weakly detectable after myelination commences (Seilheimer and Schachner, 1988). N-CAM is on Schwann cells and their basal laminae and may mediate Schwann cell adhesion (Daniloff et al., 1986b). These cells adhere to one another during regeneration to form cords that guide regenerating nerves through an injured area. These proliferating Schwann cells align longitudinally within the confines of the basal lamina or endoneurial tube, creating a continuous column of cells called the bands of Biingner (Sunderland and Bradley, 1952; Thomas, 1963; Komiyama et al., 1991). Schwann cells also adhere to the basal lamina as they grow toward the site of injury (Nathaniel and Pease, 1963; Ide et al., 1983). N-CAM can be detected in the basement membrane of Schwann cells and 3 collagen fibrils of the endoneurium (Daniloff et al., 1986a). It has been found that axon-Schwann cell interactions are characterized by the sequential appearance of cell adhesion molecules (CAMs) and myelin basic protein coordinated in time and space. It was deduced that N-CAM was involved in fasciculation, initial axon-Schwann cell interactions and the onset of myelination (Daniloff et al., 1986b). Two to six days following transection, small diameter regenerating axons were found to be positive for N-CAM in regions where they made contact with one another or with Schwann cells. Large diameter axons showed negligible amounts of N-CAM. Fourteen days after transection, when regrowing axons were seen in the distal part of the transected nerve, N-CAM was observed where regrowing axons made contact with Schwann cells. Most Schwann cells that were associated with degenerating myelin also expressed N-CAM. During myelination, N-CAM expression is reduced and disappears in compacted myelin (Mirsky et al., 1986; Martini and Schachner, 1988). N-CAM is also involved in the initial stages of nerve-muscle contact (Rieger et al., 1985) but is not essential for the formation of electrophysiologically active synapses. Overview of Nerve Structure Peripheral nerve trunks are composed of bundles of neurons, axons and connective tissue elements. Most nerves contain motor and sensory fibers; the latter conduct electrical impulses at a faster rate than do motor fibers. The presence of myelin profoundly enhances the velocity of impulse transmission. Each myelinated fiber has a compact myelin sheath, which is composed of a lipid and protein bilayer. This sheath is formed by a Schwann cell wrapping spirally around the axon. Individual Schwann cells meet at the nodes o f Ranvier, where small gaps in the myelin exist. At these nodes of Ranvier, the axon is surrounded only by the Schwann cell basal lamina. Small bundles of nonmyelinated axons can be encircled by a single Schwann cell, but no myelin is present (Kuczynski, 1980). There are three layers of connective tissue in a nerve: endoneurium, perineurium, and epineurium. All individual axons are covered by endoneurium. This covering gives tensile strength to the nerve and promotes resistance to internal axonal pressure. Bundles of nerve fibers form fasciculi and are encircled by perineurium. The perineurium can be sutured in order to anastomose severed nerves (Kline and Kahn, 1982). Thick, outer connective tissue, called epineurium, covers the nerve trunk; epineurium has extensions that separate fasciculi and blend with the perineurium (Kucynski, 1980). This epineurium can be used to manipulate the nerve during repair and is a frequent site of suture placement in anastomosis of transected nerves (Sunderland, 1980; Braun, 1982). Injuries to Nerves Mammalian peripheral nerve fibers are capable of repair by regeneration after injury. Conditions for successful regeneration will be best after nerve crush; the Schwann cell basement membrane tubes (endoneurial tubes) remain intact and this gives the injured axons a measure of protection from extracellular fluid and products of tissue damage at the injury site. The tubes also serve to contain the Schwann cells needed to support the regenerating axon sprouts and to guide them into the distal stump and then on to reinnervate peripheral targets (Horch, 1979; Horch and Lisney, 1981). Recovery is more likely to occur if axons are simply crushed (Nicholson and Seddon, 1957) or have a very short (less than 5 mm) interstump gap to cross (McQuarrie, 1986). This regeneration distal to the cell body is more likely to fail if the interstump gap is greater than 1 cm and associated with soft tissue damage. Even though reactive axonal sprouting is an intrinsic neuronal response to injury, the subsequent reorganization of these axonal sprouts does not occur unless Schwann cells are present (Aguayo and Bray, 1980; Lisney, 1989). In normal, young adult rats sustaining experimental nerve gaps of 10mm, there are significant attempts to regenerate and some recovery occurs (Gibson and Daniloff, 1989b; Madison et al., 1987). Transection of peripheral nerves causes a breakdown of myelin in the distal stump. This results in macrophage recruitment to remove myelin debris from this area. It has been suggested that these macrophages interact with Schwann cells and may be a source of Schwann cell mitogens (Scheidt and Friede, 1987). During the injury response, Schwann cells proliferate. If the distal stump is separated from the proximal stump, Schwann cells co-migrate with regrowing axons. Schwann cells also respond to axonal cues by transient upregulation or re-expression of molecules which provide a favorable environment for axonal extension. They also attract bundles of regrowing axons and their associated Schwann cells across interstump gaps up to 1 cm in length (Ramon y Cajal, 1928b). Schwann cell basal lamina is similar to basal laminae elsewhere, in that it contains molecules such as laminin and fibronectin, which are potent promoters of neurite growth in culture (Rogers et al., 1983; Bozyczko and Horwitz, 1986). The behavior of Schwann cells inside the basal lamina depends on the presence of an axon. The initial breakdown products of axons after axotomy stimulate Schwann cell multiplication in preparation for phagocytosis o f debris (Aguayo et al., 1976). Subsequently, a regenerating axon is required for differentiation of the Schwann cell and production of myelin for the remyelination of the axon by the Schwann cell. The degree of remyelination is determined by the type of axon regenerating into the basal lamina (Hillarp and Olivecrona, 1946; Weinberg and Spencer, 1975). The environment through which axons regene

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