Release of membrane-associated growth factors during neural injury.

The release of membrane-associated growth factors after neural injury may influence the outcome of the recovery. For example, for remyelination to occur after neural injury it is critical for the glial cell to proliferate prior to remyelination in both the PNS and CNS. In the CNS, the relative response of the oligodendrocytes and astroglia to growth factors mobilized during neural injury may play a role in the cellular dynamics of repair of neural injury or scarring and subsequent failure to repair neural injury. In support of this view, we have studied the mitotic potential and cell cycle kinetics of cultured adult oligodendrocytes and found that these adult cells respond only weakly to factors such as FGF which are known to be potent mitogens for neonatal cells. However, given the same dose of FGF, adult astrocytes are mitotically stimulated to a much greater degree than are the adult oligodendrocytes (Vick and De Vries, unpublished observations). Given the pathways which may be operative in the release of growth factors after injury, it has not escaped our attention that, provided the released factors are in equilibrium with easily accessible and peripheral body fluids, these released factors may serve as new markers for neural injury. Further experiments are in progress to explore this possibility.

A mitogen for Schwann cells is derived from myelin basic protein.

Evidence is presented that a mitogen can be produced from myelin basic protein (MBP) which may be related to the Schwann cell proliferation characteristic of Wallerian degeneration. Myelin derived from the shiverer mutant which is devoid of MBP is also devoid of mitogenic activity. Absorption of the mitogen with a polyclonal antisera to MBP abolishes the mitogenic effect. In addition, only liposomes containing MBP are mitogenic to cultured Schwann cells; liposomes containing other myelin-specific proteins do not stimulate Schwann cell division. These results directly demonstrate the MBP origin of a mitogen for Schwann cells.

Macrophage-mediated myelin-related mitogenic factor for cultured Schwann cells.

Conditioned medium from cultured peritoneal macrophages that have phagocytosed a myelin membrane fraction is mitogenic for cultured Schwann cells. Production of the mitogenic supernatant was time- and dose-dependent with a maximal Schwann cell-proliferative response from supernatants after 48-hr incubation of cultured macrophages with myelin-enriched fraction (200 micrograms of protein per ml). The response was specific for myelin membrane: supernatants derived from macrophages incubated with axolemma, liver microsomes, polystyrene beads, or lipopolysaccharide were not mitogenic. Lysosomal processing of the myelin membrane was necessary for the production of the mitogenic factor, which was shown to be heat labile and trypsin sensitive. There was no species specificity because myelin membranes isolated from the central and peripheral nervous systems of rat, bovine, and human were equally potent in eliciting mitogenic supernatant. However, supernatants derived from central nervous system myelin membranes were two to three times more mitogenic than those obtained from peripheral nervous system fractions of the same species. Previous observations that myelin is mitogenic for cultured Schwann cells may, in part, involve the intermediate processing of myelin by macrophages that are present in Schwann cell cultures. These results suggest that macrophages play a crucial role in Schwann cell proliferation during Wallerian degeneration.

An automated method for autoradiographic analysis of cultured Schwann cells.

A semi-automated analysis system based on video image analysis was developed to count labelled and unlabelled nuclei of Schwann cells which had been exposed to tritiated thymidine followed by processing for autoradiography. A Model 3000 Image Analysis system (Image Technology Corporation, Deer Park, NY) was used to acquire and process the images and provide quantitative measurements based on the distinctive size and shape of the Schwann cell nucleus. The maximum and minimum dimensions for the labelled and unlabelled nuclei were determined. These stored dimensional parameters were then compared with the dimensions of a given field of cell nuclei by the image analysis system. The counts from various fields were collected until a total of 1000 labelled and unlabelled nuclei had been analyzed. A labelling index (LI = ratio of labelled cells to total cells X 100) was then calculated and printed by the system. LIs of autoradiographs determined by automated analysis correlated well with those determined by visual cell counting. The principle of the image analysis program as described here is applicable to other systems for the measurement of LIs of a particular cell type in a mixed population. This automated process eliminates both the subjectivity and fatigue of visual counting and facilitates the rapid measurement of the LI of large numbers of autoradiographs with precision.