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Axon regeneration in vivo is blocked at boundaries between Schwann cells and astrocytes, such as occur at the dorsal root entry zone and around peripheral nerve or Schwann cell grafts. We have created a tissue culture model of these boundaries in Schwann cell - astrocyte monolayer co-cultures. Axon behaviour resembles that in vivo, with axons showing a strong preference for Schwann cells over astrocytes. At boundaries between the two cell types, axons growing on astrocytes cross readily onto Schwann cells, but only 15% of axons growing on Schwann cells are able to cross onto astrocytes. Treatment with chondroitinase or chlorate to reduce inhibition by proteoglycans did not change this behaviour. The neural adhesion molecule L1 is present on Schwann cells and not astrocytes, and manipulation of L1 by application of an antibody, L1-Fc in solution, or adenoviral transduction of L1 into astrocytes increased the proportion of axons able to cross onto astrocytes to 40-50%. Elevating cAMP levels increased crossing from Schwann cells onto astrocytes in live and fixed cultures, and had a co-operative effect with NT-3 but not with NGF. Inactivation of Rho with a cell-permeant form of C3 exoenzyme also increased crossing from Schwann cells to astrocytes. Our experiments indicate that the preference of axons for Schwann cells is largely mediated by the presence of L1 on Schwann cells but not astrocytes, and that manipulation of growth cone signalling pathways can allow axons to disregard boundaries between the two cell types.

The development of the dendritic tree of a neuron is a complex process which is thought to be regulated strongly by signals from afferent fibers. In particular the synaptic activity of afferent fibers and activity-dependent signaling by neurotrophic factors are thought to affect dendritic growth. We have studied Purkinje cell dendritic arbor development in organotypic cultures under suppression of glutamate-mediated excitatory neurotransmission, achieved with multiple combinations of blockers of glutamate receptors. Despite the presence of either single receptor blockers or combinations of blockers predicted to fully suppress glutamate-mediated excitatory neurotransmission Purkinje cell dendritic arbors developed similar to those of control cultures. Furthermore, Purkinje cell dendritic arbors in organotypic cultures from brain-derived neurotrophic factor (BDNF) knockout mice or after pharmacological blockade of trk-receptors also developed in a way similar to control cultures. Our results demonstrate that during the stage of rapid dendritic arbor growth signals from afferent fibers are of minor importance for Purkinje cell dendritic development because a seemingly normal Purkinje cell dendritic tree developed in the absence of excitatory neurotransmission and BDNF signaling. Our results suggest that many aspects of Purkinje cell dendritic development can be achieved by an intrinsic growth program.

Matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs) are involved in many cell migration phenomena and produced by many cell types, including neurons and glia. To assess their possible roles in brain injury and regeneration, we investigate their production by glial cells, after brain injury and in tissue culture, and we investigate whether they are capable of digesting known axon-inhibitory proteoglycans. To determine the action of MMPs, we incubated astrocyte conditioned medium with activated MMPs, then did western blots for several chondroitin sulphate proteoglycans. MMP-3 digested all five proteoglycans tested, whereas MMP-2 digested only two and MMP-9 none. To determine whether MMPs or TIMPs are produced by astrocytes in vitro, we tested both primary cultures and astrocyte cell lines by western blotting, and compared them with Schwann cells. All cultures produced at least some MMPs and TIMPs, with no obvious correlation with the ability of axons to grow on those cells. Both MMP-9 and TIMP-3 were regulated by various cytokines. To determine which cells produce MMPs and TIMPs after brain injury, we made lesions of adult rat cortex, and did immunohistochemistry. MMP-2 was seen to be induced in activated astrocytes through the whole thickness of the cortex but not deeper, but MMP-3 was not seen in the injured brain. TIMP-2 and TIMP-3 immunoreactivities were induced in activated astrocytes in deep cortex and the underlying white matter. In situ hybridisation confirmed induction of TIMP-2 in glia as well as neurons, but showed no expression of TIMP-4. These results show that both MMPs and TIMPs are produced by some astrocytes, but TIMP production is particularly strong, especially in deep cortex and white matter which is more inhibitory for axon regeneration. Conversely the MMPs produced may not be adequate to promote migration of cells and axons within the glial scar.

Chondroitin sulfate proteoglycan (CS-PG) expression is increased in response to CNS injury and limits the capacity for axonal regeneration. Previously we have shown that neurocan is one of the CS-PGs that is upregulated (Asher et al., 2000). Here we show that another member of the aggrecan family, versican, is also upregulated in response to CNS injury. Labeling of frozen sections 7 d after a unilateral knife lesion to the cerebral cortex revealed a clear increase in versican immunoreactivity around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed considerably more versican in the injured tissue extract. In vitro studies revealed versican to be a product of oligodendrocyte lineage cells (OLCs). Labeling was seen between the late A2B5-positive stage and the O1-positive pre-oligodendrocyte stage. Neither immature, bipolar A2B5-positive cells, nor differentiated, myelin-forming oligodendrocytes were labeled. The amount of versican in conditioned medium increased as these cells differentiated. Versican and tenascin-R colocalized in OLCs, and coimmunoprecipitation indicated that the two exist as a complex in oligodendrocyte-conditioned medium. Treatment of pre-oligodendrocytes with hyaluronidase led to the release of versican, indicating that its retention at the cell surface is dependent on hyaluronate (HA). In rat brain, approximately half of the versican is bound to hyaluronate. We also provide evidence of a role for CS-PGs in the axon growth-inhibitory properties of oligodendrocytes. Because large numbers of OLCs are recruited to CNS lesions, these results suggest that OLC-derived versican contributes to the inhospitable environment of the injured CNS.

We hypothesized that creatine (Cr) supplementation would preserve energy metabolism and thus ameliorate the energy failure and the extent of brain edema seen after severe but transient cerebral hypoxia-ischemia (HI) in the neonatal rat model. Six-day-old (P6) rats received subcutaneous Cr monohydrate injections for 3 consecutive days (3 g/kg body weight/day), followed by 31P-magnetic resonance spectroscopy (MRS) at P9. In a second group, P4 rats received the same Cr dose as above for 3 days prior to unilateral common carotid artery ligation followed 1 h later by 100 min of hypoxia (8% O2) at P7. Rats were maintained at 37 degrees C rectal temperature until magnetic resonance imaging was performed 24 h after HI. Cr supplementation for 3 days significantly increased the energy potential, i.e. the ratio of phosphocreatine to beta-nucleotide triphosphate (PCr/betaNTP) and PCr/inorganic phosphate (PCr/Pi) as measured by 31P-MRS. Rats with hemispheric cerebral hypoxic-ischemic insult that had received Cr showed a significant reduction (25%) of the volume of edemic brain tissue compared with controls as calculated from diffusion-weighted images (DWI). Thus, prophylactic Cr supplementation demonstrated a significant neuroprotective effect 24 h after transient cerebral HI. We hypothesize that neuroprotection is probably due to the availability of a larger metabolic substrate pool leading to a reduction of the secondary energy failure because DWI has been reported to correlate with the PCr/Pi ratio in the acute phase of injury. Additional protection by Cr may be related to prevention of calcium overload, prevention of mitochondrial permeability transition pore opening and direct antioxidant effects., (Copyright 2002 S. Karger AG, Basel)

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.

We have developed a novel Schwann cell line, SCTM41, derived from postnatal sciatic nerve cultures and have stably transfected a clone with a rat glial cell line-derived neurotrophic factor (GDNF) construct. Coculture with this GDNF-secreting clone enhances in vitro survival and fiber growth of embryonic dopaminergic neurons. In the rat unilateral 6-OHDA lesion model of Parkinson's disease, we have therefore made cografts of these cells with embryonic day 14 ventral mesencephalic grafts and assayed for effects on dopaminergic cell survival and process outgrowth. We show that cografts of GDNF-secreting Schwann cell lines improve the survival of intrastriatal embryonic dopaminergic neuronal grafts and improve neurite outgrowth into the host neuropil but have no additional effect on amphetamine-induced rotation. We next looked to see whether bridge grafts of GDNF-secreting SCTM41 cells would promote the growth of axons to their striatal targets from dopaminergic neurons implanted orthotopically into the 6-OHDA-lesioned substantia nigra. We show that such bridge grafts increase the survival of implanted embryonic dopaminergic neurons and promote the growth of axons through the grafts to the striatum.