N. H. Chi

Glial fibrillary acidic (GFA) protein in Schwann cells: fact or artifact?

Antisera to the glial fibrillary acidic (GFA) protein stained a subpopulation of Schwann cells in cryostat sections of rat sciatic nerve by indirect immunofluorescence and by the peroxidase-antiperoxidase (PAP) procedure. The staining pattern was entirely different from that obtained with vimentin antisera, which uniformly decorated endoneurial tubes. Electron microscopic examination of sciatic nerve provided a possible explanation for the relatively small number of Schwann cells decorated by GFA antisera: 10 nm filaments were mainly confined to Schwann cell processes surrounding nonmyelinated axons. A marked increase in GFA-positive Schwann cells and in Schwann cells containing filaments by electron microscopy was observed in sciatic nerves undergoing Wallerian degeneration. Conversely, immunochemical procedures failed to demonstrate the presence of antigen reacting with GFA antisera in extracts of sciatic nerve, both normal and degenerated. These include absorption experiments, double immunodiffusion, immunoaffinity chromatography, and immunoradiometric assay. Two explanations may be considered for these findings: i) Schwann cell intermediate filaments and GFA protein share common antigenic determinants, the immunohistological methods being more sensitive to detect cross-reactivity as compared to immunochemical procedures on tissue extracts; and ii) the binding of anti-GFA to Schwann cell 10 nm filaments is not due to immunological cross-reactivity.

Laminin in rat sciatic nerve undergoing Wallerian degeneration: immunofluorescence study with laminin and neurofilament antisera

Immunofluorescence with laminin antisera revealed a striking change in the localization of this basal membrane glycoprotein in rat sciatic nerve as a result of Wallerian degeneration. The staining was confined to the endoneurium in normal sciatic nerve and during the first days of degeneration. On day 11 endoneurial tubes were no longer identified in the distal stump of crushed nerves or of nerves that had been transected and tightly ligated to prevent regeneration. In both crushed and ligated nerves proliferating Schwann cells forming the cell-bands of Bünger were intensely laminin positive. With double-labeling experiments, laminin and neurofilament antisera revealed similar but not identical staining patterns in crushed nerves, which suggests a close relation between laminin and regenerating axons. Crushed nerves had recovered their normal appearance 18 days after operation while anti-laminin reactivity was decreased in parts of ligated nerves undergoing fibrosis. The localization of laminin in reactive Schwann cells was confirmed by electron microscopy using the indirect immunoperoxidase procedure. Axons did not contain reaction product.

Neurofilament phosphorylation in peripheral nerve regeneration

A monoclonal antibody to the 200 kdalton neurofilament (NF) polypeptide selectively decorated axons in tissue sections. Neuronal perikarya and dendrites, including motor and sensory neurons reacting to axotomy, were not stained. Axonal staining was abolished by dilution of the monoclonal supernatants with phosphate buffer and by digestion of tissue sections with phosphatase, thus suggesting that the antibody reacted with a phosphorylated epitope. Conventional monoclonal and polyclonal antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites) were not affected by these procedures. Compared to conventional NF antibodies, staining with the axon-specific monoclonal antibody was a late event in peripheral nerve regeneration. One week after operation, the whole distal stump of crushed rat sciatic nerve was invaded by bundles of axons strongly reacting with conventional NF antibodies. Axon-specific NF immunoreactivity was confined to the proximal segment of the stump at this time and progressively extended distally in the following week. Furthermore, NF phosphorylation appeared to coincide with the return of a normal nerve structure as evidenced by the distribution of laminin immunoreactivity. Bundles of axons growing within columns of laminin-positive Schwann cells did not stain with the axon-specific NF antibody. Immunoreactivity with this antibody coincided with the return of a normal laminin pattern, i.e. selective decoration of the endoneurial basal membranes surrounding the axons.

Autologous sciatic nerve grafts to the rat spinal cord: Immunofluorescence studies with neurofilament and gliofilament (GFA) antisera

Abstract Autologous sciatic nerve grafts to the spinal cord contained many regenerating nerve fibers intensely immunofluorescent with neurofilament antisera. Axonal growth was not confined to the graft but also occurred in the surrounding spinal cord. In this location regenerating nerve fibers were invariably associated with Schwann-like cells. The reverse situation, that is, invasion of the graft by GFA-positive astroglia, also occurred but was a more limited phenomenon involving only a few cells.

Plasminogen activators in rat neural tissues during development and in Wallerian degeneration.

SummaryThe fibrinolytic activity of blood is caused by plasminogen activators (PA) converting plasminogen to plasmin, the active fibrinolytic protease. PA activity in rat neural tissues was studied by Todds fibrin slide technique. Cryostat sections overlayed with a film of plasminogen and fibrin were incubated for 60–90 min. PA activity was related to the size of the zone of fibrinolysis surrounding the sections. No lysis occurred with fibrin alone. In rats perfused with saline prior to decapitation the size of the zone of lysis was approximately the same as in non-perfused animals. PA activity was compared in the following tissues: adult (2–3 month) cerebellum and 6–14-day postnatal cerebellum; normal sciatic nerve and transected sciatic nerve 1–9 weeks after operation (in these experiments the sciatic nerve was crushed on the left side, on the right side it was transected and the stumps were tightly ligated to prevent regeneration); normal optic nerves and optic nerves undergoing Wallerian degeneration 1–2 weeks after enucleation of the eye. As compared to normal cerebellum PA activity was increased in 6–14-day cerebellum. PA activity was also markedly increased in both crushed and ligated sciatic nerves 1–4 weeks after operation while no differences were observed between normal sciatic nerves and sciatic nerves 9 weeks after ligation. The zone of fibrinolysis surrounding normal optic nerves and the optic nerves of blinded rats was approximately the same. It is proposed that the fibrinolytic system may be relevant to the problem of CNS regeneration.

First appearance of laminin in peripheral nerve, cerebral blood vessels and skeletal muscle of the rat embryo: Immunofluorescence study with laminin and neurofilament antisera

The formation of basal laminae in peripheral nerve was studied by immunofluorescence with laminin antisera in the rat embryo. Peripheral nerves were identified with neurofilament antisera in double labeled sections. In the adult rat perineurium and endoneurium were uniformly decorated by the antisera. Sensory neurons in posterior root ganglia were surrounded by a laminin positive basal lamina. Laminin immunoreactivity was first observed in posterior spinal roots on day 14. Anterior spinal roots and peripheral nerves remained laminin negative until day 17. The adult pattern (uniform decoration of endoneurium in large and small nerve trunks) was only observed on day 21. The formation of a basal lamina surrounding posterior root ganglion neurons was still not completed in 3‐day‐old rats. The only laminin positive structures in the brain and spinal cord were the external basal laminae and the blood vessels. The external basal lamina was present at all stages of development. In the spinal cord and brain stem vascular basal laminae were first identified with laminin antisera on day 14, in the diencephalon and telencephalon on day 15. Laminin immunoreactivity in the basal laminae surrounding myotubes was first observed on day 16.

Immunohistochemical characterization of neurofibrillary tangles induced by mitotic spindle inhibitors

SummaryNeurofibrillary tangles were induced in the motor neurons of the rabbit spinal cord by the intrathecal injection of colchicine, vinblastine, and vincristine. The tangles stained intensely by immunofluorescence and by the peroxidase-anti-peroxidase procedure using neurofilament antisera raised against chicken brain antigen, as previously reported for aluminum-induced neurofibrillary tangles. No immunohistochemical reactivity could be demonstrated between the tangles and a 150,000 dalton bovine neurofilament antiserum, although the adjacent axons were intensely stained in cryostat sections of the spinal cord.