Neurocan is upregulated in injured brain and in cytokine-treated astrocytes.

Injury to the CNS results in the formation of the glial scar, a primarily astrocytic structure that represents an obstacle to regrowing axons. Chondroitin sulfate proteoglycans (CSPG) are greatly upregulated in the glial scar, and a large body of evidence suggests that these molecules are inhibitory to axon regeneration. We show that the CSPG neurocan, which is expressed in the CNS, exerts a repulsive effect on growing cerebellar axons. Expression of neurocan was examined in the normal and damaged CNS. Frozen sections labeled with anti-neurocan monoclonal antibodies 7 d after a unilateral knife lesion to the cerebral cortex revealed an upregulation of neurocan around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed substantially more neurocan in the injured CNS. Western blot analysis revealed neurocan and the processed forms neurocan-C and neurocan-130 to be present in the conditioned medium of highly purified rat astrocytes. The amount detected was increased by transforming growth factor beta and to a greater extent by epidermal growth factor and was decreased by platelet-derived growth factor and, to a lesser extent, by interferon gamma. O-2A lineage cells were also capable of synthesizing and processing neurocan. Immunocytochemistry revealed neurocan to be deposited on the substrate around and under astrocytes but not on the cells. Astrocytes therefore lack the means to retain neurocan at the cell surface. These findings raise the possibility that neurocan interferes with axonal regeneration after CNS injury.

Comparing astrocytic cell lines that are inhibitory or permissive for axon growth: the major axon-inhibitory proteoglycan is NG2.

Astrocytes, oligodendrocytes, and oligodendrocyte/type 2 astrocyte progenitors (O2A cells) can all produce molecules that inhibit axon regeneration. We have shown previously that inhibition of axon growth by astrocytes involves proteoglycans. To identify inhibitory mechanisms, we created astrocyte cell lines that are permissive or nonpermissive and showed that nonpermissive cells produce inhibitory chondroitin sulfate proteoglycans (CS-PGs). We have now tested these cell lines for the production and inhibitory function of known large CS-PGs. The most inhibitory line, Neu7, produces three CS-PGs in much greater amounts than the other cell lines: NG2, versican, and the CS-56 antigen. The contribution of NG2 to inhibition by the cells was tested using a function-blocking antibody. This allowed increased growth of dorsal root ganglion (DRG) axons over Neu7 cells and matrix and greatly increased the proportion of cortical axons able to cross from permissive A7 cells onto inhibitory Neu7 cells; CS-56 antibody had a similar effect. Inhibitory fractions of conditioned medium contained NG2 coupled to CS glycosaminoglycan chains, whereas noninhibitory fractions contained NG2 without CS chains. Enzyme preparations that facilitated axon growth in Neu7 cultures were shown to either degrade the NG2 core protein or remove CS chains. Versican is present as patches on Neu7 monolayers, but DRG axons do not avoid these patches. Therefore, NG2 appears to be the major axon-inhibitory factor made by Neu7 astrocytes. In the CNS, NG2 is expressed by O2A cells, which react rapidly after injury to produce a dense NG2-rich network, and by some reactive astrocytes. Our results suggest that NG2 may be a major obstacle to axon regeneration.

Bridge grafts of fibroblast growth factor-4-secreting schwannoma cells promote functional axonal regeneration in the nigrostriatal pathway of the adult rat.

Axons damaged in the adult mammalian central nervous system are able to regenerate when their inhibitory glial environment is replaced with a more permissive substrate. Here, we have used long oblique "bridge" grafts of fibroblast growth factor-4-transfected RN-22 schwannoma cells to allow mechanically lesioned nigrostriatal axons to regenerate back to their original target in the adult rat brain. Regenerated axons were able to leave the bridge graft to form terminal arborizations and increase the density of tyrosine hydroxylase-immunoreactive fibres within the striatum. Bridge grafting also resulted in an increase in the number of neurons within the substantia nigra pars compacta taking up the fluorescent retrograde tracer Fluoro-Gold from the striatum. Animals which had received RN-22 bridge grafts showed lower rates of amphetamine-induced rotation 10 weeks after a mechanical lesion of the nigrostriatal tract compared to lesioned controls, the magnitude of the behavioural effect being related to the number of regenerated axons, and this comparative reduction was reversed by mechanical section of the bridge graft. It is concluded that our bridge grafting strategy allowed the partial anatomical and functional regeneration of the mechanically lesioned nigrostriatal tract, an unmyelinated central axon bundle, and that bridge grafting therefore represents a realistic approach to the repair of central nervous system lesions involving axon tract damage.

Functional and anatomical reconstruction of the 6-hydroxydopamine lesioned nigrostriatal system of the adult rat.

In an attempt to reconstruct the 6-hydroxydopamine lesioned nigrostriatal system of the adult rat we have combined homotopic grafting of embryonic ventral mesencephalon suspensions with the implantation of long oblique "bridge" grafts of fibroblast growth factor-4-transfected RN-22 schwannoma cells stretching from the site of the neuronal grafts to the striatum. At seven weeks after receiving both grafts, animals were killed and processed for immunohistochemistry against tyrosine hydroxylase. Tyrosine hydroxylase-immunoreactive axons were seen to extend from the nigral grafts, along the bridge graft to the striatum where terminal arborizations could be seen. The retrograde tracer Fluoro-gold was injected intrastriatally in some of the experimental animals and was taken up by grafted neurons confirming their projection to the striatum. In parallel to the anatomical reconstruction of the system, a decrease in amphetamine-induced rotation was demonstrated in those animals receiving both grafts which had received > 98% complete lesions. This decrease was greatest in those animals with the most tyrosine hydroxylase-immunoreactive axons in their bridge grafts. The presence of the bridge graft also led to an increase in neuronal graft survival with twice as many tyrosine hydroxylase-immunoreactive neurons being found in the grafts of those animals that had received both grafts compared to those that had received a neuronal graft but no bridge graft.

The effects of stereotyped behaviour on orienting and tonic cardiac activity.

The relationship of cardiac activity to stereotyped behaviour was studied in profoundly mentally retarded persons, using both inter-group comparisons. The first intra-group comparison involved examining the relative magnitude of an orienting response to an auditory stimulus during the occurrence or nonoccurrence of stereotyped body rocking. While no differences were found between periods of rocking and non-rocking when behavioural measures of orienting were used, smaller changes in post-tone heart rate were observed during periods of stereotyped rocking. A second intra-group comparison found that stereotyped behaviour was associated with a significant increase in heart rate but no change in heart rate variability when compared to periods of no stereotyped activity. Finally, using an inter-group comparison, individuals who displayed stereotyped body rocking exhibited higher tonic heart rates and lower heart rate variability during periods of no stereotyped behaviour than was observed in matched control subjects. This result suggests that stereotyped behaviour may be correlated with reduced vagal tone.

Time-series analysis of stereotyped movements: relationship of body-rocking to cardiac activity.

The pathological stereotypies frequently observed among severely mentally retarded and autistic persons are highly rhythmical in nature. Most attempts to quantify such behavior, however, have not analyzed stereotypy in terms of its cyclical properties. In the present paper we have detailed a method for electronically transducing stereotyped body-rocking and analyzing its frequency and amplitude characteristics with a standard polygraph and microprocessor. The relationship between stereotyped body-rocking and cardiac activity was also described using time-series analysis. The method described should provide a sensitive index of various experimental manipulations and treatment effects.