Immunization does not interfere with uptake and transport by motor neurons of the binding fragment of tetanus toxin.

The nontoxic binding domain of tetanus toxin (fragment C or TTC) readily undergoes retrograde axonal transport from an intramuscular injection site. This property has led to investigation of TTC as a possible vector for delivering therapeutic proteins to motor neurons. However, the vast majority of individuals in the developed world have been vaccinated with tetanus toxoid and have circulating antitetanus antibodies that cross-react with TTC and may block the delivery of a TTC-linked therapeutic protein. However, it is uncertain whether the immune response is capable of completely neutralizing an intramuscular depot of protein prior to its internalization by presynaptic nerve terminals, where it is inaccessible to antibody. We have evaluated uptake of rhodamine-labeled TTC following intramuscular injection in normal animals and animals vaccinated with tetanus toxoid prior to injection of fluorescently labeled TTC. All animals demonstrated uptake of TTC, with fluorescence appropriately localized to the hypoglossal nerve and nucleus. The distribution and intensity of fluorescence within neurons and processes were indistinguishable between the two groups and were characteristic of TTC. Vaccinated animals showed levels of uptake of TTC into the brain comparable to those of immunologically naïve animals as measured by quantitative fluorimetry. All vaccinated animals had protective levels of antitetanus antibodies as measured by ELISA. Uptake of TTC by nerve terminals from an intramuscular depot is an avid and rapid process and is not blocked by vaccination associated with protection from tetanus toxin.

Ultrastructural localization of immunoglobulin G and complement C9 in the brain stem and spinal cord following peripheral nerve injury: an immunoelectron microscopic study.

The ultrastructural localization of immunoreactivity for immunoglobulin G (IgG), F(ab')2 and complement C9 was examined with preembedding immunoelectron microscopy in the hypoglossal nucleus and gracile nucleus as well as in the L4 spinal cord dorsal horn 1 week following hypoglossal or sciatic nerve transection, respectively. Only a few scattered immunoreactive profiles were observed on the unoperated side. On the operated side, IgG and F(ab')2 immunoreactivity was present in the membranes of all reactive microglial cells observed. In addition, the cell membrane of some hypoglossal motoneurons showed IgG immunoreactivity. Complement C9 immunoreactivity was present in the cytoplasm of all reactive microglial cells examined. In addition, there was diffuse C9 immunoreactivity in motoneuron perikarya ipsilateral to nerve injury as well as in cell membranes in the neuropil, some of which could be identified as neuronal. Our interpretation of these findings is (1) that peripheral nerve injury results in binding of IgG to reactive microglia, as well as to some axotomized neurons, and (2) that C9 is synthesized by reactive microglia in response to axon injury and is also associated with axotomized motoneurons. These findings suggest that IgG and complement C9 are involved in microglia-neuron interactions after peripheral nerve injury.

Accelerated Nerve Regeneration in Mice by upregulated expression of interleukin (IL) 6 and IL-6 receptor after trauma.

In this study we aimed to examine a role for interleukin 6 (IL-6) and its receptor (IL-6R) in peripheral nerve regeneration in vivo. We first observed that cultured mouse embryonic dorsal root ganglia exhibited dramatic neurite extension by simultaneous addition of IL-6 and soluble IL-6R (sIL-6R), a complex that is known to interact with and activate a signal transducing receptor component, gp130. After injury in the hypoglossal nerve in adult mice by ligation, immunoreactivity to IL-6 was upregulated in Schwann cells at the lesional site as well as in the cell bodies of hypoglossal neurons in the brain stem. In the latter, upregulation of the immunoreactivity to IL-6R was also observed. Regeneration of axotomized hypoglossal nerve in vivo was significantly retarded by the administration of anti-IL-6R antibody. Surprisingly, accelerated regeneration of the axotomized nerve was achieved in transgenic mice constitutively expressing both IL-6 and IL-6R, as compared with nontransgenic controls. These results suggest that the IL-6 signal may play an important role in nerve regeneration after trauma in vivo.

Evidence for activation of the terminal pathway of complement and upregulation of sulfated glycoprotein (SGP)-2 in the hypoglossal nucleus following peripheral nerve injury.

In a previous study, we found immunoreactivity for complement factors C3, C3d, and C4d, as well as endogenous IgG in the hypoglossal nucleus following hypoglossal nerve transection, suggesting that activation of the complement cascade had taken place in the vicinity of the axotomized motorneurons. In the present study, we found increased immunoreactivity for complement factor C1 and C1q in reactive microglia, indicating an increased potential for initiation of the classical pathway by binding of IgG to C1q. Furthermore, we found immunoreactivity for C9, which contributes to the formation of C5b-9, the final lytic product of the complement cascade close to the axotomized neurons and perineuronal glia. In addition, immunoreactivity and mRNA labeling of sulfated glycoprotein (SGP-2), a putative complement inhibitor, was increased in a subpopulation of the axotomized motorneurons. SGP-2 immunoreactivity was also increased in astroglial cells ipsilateral to the nerve injury. The results lend further support to the hypothesis that the complement cascade is activated in the vicinity of axotomized neurons, which in turn may be protected by complement inhibitors. The balance between activation of complement and complement inhibitors might have an impact on the degenerative components of the axon reaction and, in particular, the events leading to nerve cell death.

Characterization of retrograde axonal transport of antibodies in central and peripheral neurons.

Retrograde axonal transport of antibodies against synaptic membrane glycoproteins was studied in the hypoglossal nerve and several CNS pathways of the rat. Injection into the tongue of polyclonal antibodies against synaptic membrane glycoproteins produced immunocytochemically labeled cells in the hypoglossal nucleus 4-5 hr later. Immunoreactive staining increased through 48 hr after injection and then declined. Injections of Fab preparations of the antibody gave labeling patterns indistinguishable from those of the whole antibody. The specificity of this method is shown by control studies in which antibodies against antigens that are not known to be present on the surface of presynaptic membranes were injected and gave no retrograde labeling. Retrograde labeling was also demonstrated in CNS pathways. However, labeling was never as intense as that seen in the hypoglossal nucleus, and some CNS pathways failed to show any retrograde labeling. Furthermore, retrograde labeling after control injections could be demonstrated in some cases. To determine if antibodies were also transported anterogradely, injections were made into the vitreous body of the eye, and the superior colliculus was processed for immunocytochemistry. Unlike wheat-germ agglutinin and several other tracers, antibodies were not found to be anterogradely transported in the optic nerve.

Differential expression of Thy-1 on the various components of connective tissue of rat nerve during postnatal development.

The glycoprotein Thy-1 is found on the surface of both neurons, and most fibroblasts, in tissue culture of embryonic or neonatal rat nervous tissue. In adult rat nerves, however, we find the antigen restricted in vivo to neurons and their axons (R. Morris, P. Barber, J. Beech, and G. Raisman, 1983, J. Neurocytol., 12, 1017-1039). We show here that this discrepancy is due to a loss of Thy-1 from neural connective tissue during postnatal development. Moreover, the different elements of connective tissue loose Thy-1 at different times. Epineurial fibroblasts, for instance, express Thy-1 for at least 2 weeks after endoneurial fibroblasts lack detectable antigen. As judged by their loss of Thy-1 antigen, therefore, the different components of neural connective tissue mature at different rates. One important practical implication of this is that Thy-1 cannot be used as a cell surface "marker" for all classes of fibroblasts in vitro. Unusual problems encountered in the immunohistochemical staining of Thy-1 on perineurium, but not on the other elements of the nerve, are also described.

The distribution of Thy-1 antigen in the P.N.S. of the adult rat.

The distribution of the cell surface glycoprotein Thy-1 in the P.N.S. of adult rats was examined using immunohistochemical and experimental techniques. In the hypoglossal nerve the pattern of Thy-1 labelling suggested the antigen was on the plasma membrane of all axons, not only in their major myelinated course but also on their fine terminal branches and at the motor end plate itself. Similarly in other peripheral nerves examined [phrenic and vagus nerves, dorsal and ventral roots, and both the preganglionic and postganglionic trunks of the superior cervical ganglion (SCG) and the submandibular ganglion] Thy-1 was always associated with axons, but the resolution obtained with immunohistochemical techniques was not in itself sufficient to exclude the possibility that the antigen was on the surface of the ensheathing Schwann cell where it apposed the axons. However, in the hypoglossal nerve the antigen was found to accumulate proximal to a ligation of the nerve, suggesting it was made by the neurons and transported down the nerve by axoplasmic flow. This impression was supported by examining neuronal cell bodies in the SCG, dorsal root ganglia and submandibular ganglion, all of which contain readily detectable cytoplasmic Thy-1. In the SCG this cytoplasmic antigen was shown to include the pool of newly synthesized Thy-1. It was increased by treatment of the ganglion with colchicine, and decreased by cycloheximide. Conversely, treatment of hypoglossal nerve trunk with colchicine did not lead to the appearance of the antigen around the non-neuronal perikarya. It is therefore concluded that in those parts of the adult rat P.N.S. examined, Thy-1 is made by neurons and occurs generally on the plasma membrane of axons.

Density of Thy-1 on axonal membrane of different rat nerves.

The density of the cell surface antigen, Thy-1, has been measured on the axonal plasma membrane of the optic and hypoglossal nerves, and on the sympathetic (preganglionic) chain of the superior cervical ganglion in the rat. For each, the amount of Thy-1 in a standard length of nerve was determined. The amount of axonal plasma membrane in a similar length was then measured by use of a computer graphics system which determined the total length of membrane in electron micrographs of cross sections of nerve. The axons of the two peripheral nerves were found to have a similar density (1100-1500 molecules/microns2) of Thy-1 on their surface, and this was two- to threefold higher than the density on axons of optic nerve (500 molecules/microns2). These figures indicate that the density of Thy-1 on the surface of these axons is somewhat lower than that found on the cells of the lymphoid system. Moreover, contrary to the impression gained from previous determinations of Thy-1 levels, axons in the peripheral nervous system do not necessarily have a lower density of Thy-1 on their surface than do those of the central nervous system.