Interaction of transplanted olfactory-ensheathing cells and host astrocytic processes provides a bridge for axons to regenerate across the dorsal root entry zone.

A single fourth lumbar dorsal rootlet was transected at the entry point into the spinal cord. The nerve fibres were labelled with biotin dextran injected into the rootlet. An endogenous matrix containing olfactory-ensheathing cells (OECs) labelled with green fluorescent protein was applied to the opposing cut surfaces of the rootlet and the spinal cord, which were then brought into apposition and held in place by fibrin glue. Two weeks later, a ladderlike bridging structure has been formed by astrocytic processes growing out for about 200-300 microm from the spinal cord. The transplanted cells remained largely confined to this area. They were elongated along the nerve axis but did not enter the spinal cord itself. Labelled dorsal root axons crossed the repaired dorsal root entry zone in alignment with the bridging astrocytic processes and the transplanted cells and then proceeded beyond the transplant to enter the grey matter of the dorsal horn and send axons both rostrally and caudally for at least 10 mm in the white matter of the ascending dorsal columns.

Absence of glial fibrillary acidic protein and vimentin prevents hypertrophy of astrocytic processes and improves post-traumatic regeneration.

The regenerative capacity of the CNS is extremely limited. The reason for this is unclear, but glial cell involvement has been suspected, and oligodendrocytes have been implicated as inhibitors of neuroregeneration (Chen et al., 2000, GrandPre et al., 2000; Fournier et al., 2001). The role of astrocytes in this process was proposed but remains incompletely understood (Silver and Miller, 2004). Astrocyte activation (reactive gliosis) accompanies neurotrauma, stroke, neurodegenerative diseases, or tumors. Two prominent hallmarks of reactive gliosis are hypertrophy of astrocytic processes and upregulation of intermediate filaments. Using the entorhinal cortex lesion model in mice, we found that reactive astrocytes devoid of the intermediate filament proteins glial fibrillary acidic protein and vimentin (GFAP-/-Vim-/-), and consequently lacking intermediate filaments (Colucci-Guyon et al., 1994; Pekny et al., 1995; Eliasson et al., 1999), showed only a limited hypertrophy of cell processes. Instead, many processes were shorter and not straight, albeit the volume of neuropil reached by a single astrocyte was the same as in wild-type mice. This was accompanied by remarkable synaptic regeneration in the hippocampus. On a molecular level, GFAP-/-Vim-/- reactive astrocytes could not upregulate endothelin B receptors, suggesting that the upregulation is intermediate filament dependent. These findings show a novel role for intermediate filaments in astrocytes and implicate reactive astrocytes as potent inhibitors of neuroregeneration.

Early onset of degenerative changes at nodes of Ranvier in alpha-motor axons of Cntf null (-/-) mutant mice.

The nodes of Ranvier are sites of specific interaction between Schwann cells and axons. Besides their crucial role in transmission of action potentials, the nodes of Ranvier and in particular the paranodal axon-Schwann cell networks (ASNs) are thought to function as local centers in large motor axons for removal, degradation, and disposal of organelles. In order to test whether ciliary neurotrophic factor (CNTF), which is present at high levels in the Schwann cell cytoplasm, is involved in the maintenance of these structures, we have examined lumbar ventral root nerve fibers of alpha-motor neurons by electron microscopy in 3- and 9-month-old Cntf null ((-/-)) mutant mice. Nerve fibers and nodes of Ranvier in 3-month-old Cntf(-/-) mutants appeared morphologically normal, except that ASNs were more voluminous in the mutants than in wild-type control animals at this age. In 9-month-old Cntf(-/-) animals, morphological changes, such as reduction in nerve fiber and axon diameter, myelin sheath disruption, and loss of ASNs at nodes of Ranvier, were observed. These findings suggest that endogenous CNTF, in addition to its role in promoting motor neuron survival and regeneration, is needed for long-term maintenance of alpha-motor nerve fibers. The premature loss of paranodal ASNs in animals lacking CNTF, which seems to be a defect related to a disturbed interaction in the nodal region between the axon and its myelinating Schwann cells, could impede the maintenance of a normal milieu in the motor axon, preceding more general neuronal damage.

Expression of the myelin and oligodendrocyte progenitor marker sulfatide in neurons and astrocytes of adult rat brain.

Sulfatide is a myelin component of the central (CNS) and peripheral nervous system (PNS) and is used extensively to identify oligodendrocyte progenitor cells. We have explored sulfatide expression in CNS gray matter (cerebellum, cerebral cortex, and hippocampus) and the PNS in adult rats using an anti-sulfatide antibody (Sulph I) and confocal microscopy. Biochemical analyses revealed two Sulph I antigens, sulfatide and seminolipid; sulfatide was present at about five times higher concentration, and the affinity of Sulph I for sulfatide was 2.5 times higher than that for seminolipid. Thus sulfatide was considered the dominant antigen. We found Sulph I immunostaining, in addition to that in myelinated areas in subpopulations of astrocytes and neurons. Astrocyte Sulph I staining was localized to the cell bodies and in some cases also to the processes. In the cerebellum, some Sulph I-positive astrocytes corresponded to Golgi epithelial cell bodies. We also found Sulph I staining in neuronal cell bodies, which in some neurons was clearly localized to the cytoplasm and in others to the nuclear membrane. Sulph I immunostaining in the PNS was located in the myelin sheath and paranodal end segments. These results demonstrate the expression of sulfatide in cell types other than oligodendrocytes and Schwann cells, showing that sulfatide is not a selective marker for adult oligodendrocyte progenitor cells. Moreover, these findings show that sulfatide is localized also to intracellular compartments and indicate that other roles of sulfatide in astrocytes and neurons, compared to myelin, might be considered.

Computer-assisted simulation of high-voltage electron microscopy using serial images recorded by conventional transmission electron microscopy.

Here we describe a computer-assisted method which, based on conventional transmission electron microscopy, renders simulated high-voltage electron micrographs. We perform arithmetic minimum filtering on stacks of aligned serial transmission electron microscopic images. In this way, the structural information of the separate images is fused into one compound image that highlights organization patterns otherwise easily overlooked or impossible to comprehend. Our method, like high-voltage electron microscopy, offers the possibility to build stereo-pairs for high-resolution three-dimensional analysis of tissue layers 1-2 microm thick. The use of background elimination and the development of a depth enhancement routine improved the three-dimensional effect and facilitated the analysis of the interior of objects. As an example, we use the method to display the distribution of axonal organelles at nodes of Ranvier and the shape and contents of a highly branched hippocampal dendritic spine.