Experimental strategies to promote axonal regeneration after traumatic central nervous system injury.

A damage or pathological process that destroys the continuity of axons in the mature central nervous system (CNS) has devastating consequences and produces lasting functional deficits. One of the major challenges in this field is to stimulate the regrowth of severed axons and reconstruction of pathways. Recent progress in molecular and cell biology has resulted in an explosion of knowledge on factors in the adult CNS being nonsupportive or even actively inhibitory to axonal regrowth. The new findings have a strong impact on the development of new therapeutic concepts directed to stimulate axonal regeneration. They give rise to cautious optimism, showing that under some circumstances repair of a CNS lesion is possible. In this review the authors summarize the current knowledge on the factors and mechanisms involved in regeneration failure and provide an overview of the current therapeutic approaches that may enable effective CNS regeneration in the future.

Quantitative analysis of the choline acetyltransferase-immunoreactive axonal network in the cat primary visual cortex: I. Adult cats.

The laminar distribution of cholinergic axons was analyzed quantitatively in the visual cortex of adult cats by using immunocytochemistry with a monoclonal antibody against choline acetyltransferase (ChAT). ChAT(+) fibers and varicosities were counted at different locations within area 17 and the distribution patterns in various animals were compared. Choline acetyltransferase-immunoreactivity was localized in fine, varicose fibers, which were present in all layers of the visual cortex. The density of labeled fibers was highest in layer I, which contained 14% of all fibers and 19.5% of all varicosities, and decreased toward deeper layers. The number of varicosities decreased more markedly toward deeper layers than the frequency of fibers. These distribution patterns were very consistent, showing only slight intra- and interindividual variability.

Quantitative analysis of the choline acetyltransferase-immunoreactive axonal network in the cat primary visual cortex: II. Pre- and postnatal development.

The pre- and postnatal development of cholinergic projections was investigated in the cat striate cortex by applying immunohistochemical methods based on a monoclonal antibody against choline acetyltransferase (ChAT). The earliest age investigated was gestational day 54. At this stage a sparse network of ChAT(+) fibers was distributed throughout the striate cortex. Subsequent postnatal maturation of ChAT(+) fibers was characterized by an increase in fiber density that started in layer VI and gradually progressed toward more superficial layers. By 4 weeks of age the density of ChAT(+) fibers and varicosities had reached adult levels in layers V and VI but was still subnormal in layers I-IV. The mature pattern of cholinergic innervation was established by 13 weeks of age. There was no evidence for developmental gradients in the anteroposterior and mediolateral directions within area 17. These results indicate that the cholinergic projection to striate cortex develops continuously in an inside-out sequence as is characteristic for most cortical maturation processes. There was no indication that striate cortex receives an especially dense cholinergic input during the critical period.

Light and electron microscopic immunocytochemical localization of parvalbumin in the dorsal lateral geniculate nucleus of the cat: evidence for coexistence with GABA.

The coexistence in individual neurons of parvalbumin and gamma-aminobutyric acid (GABA) was studied in the dorsal lateral geniculate nucleus (dLGN) of the cat using pre- and postembedding immunocytochemical methods. PV(+) cell bodies and processes were found in the perigeniculate nucleus (PGN) and throughout all laminae of the dLGN. PV(+) neurons were relatively small and had circular to fusiform shapes. Electron microscopy revealed PV(+) reaction product within the perikarya, axons, and dendrites of labeled cells. It was associated preferentially with microtubules, postsynaptic densities, and intracellular membranes. PV(+) presynaptic boutons were identified on the basis of their synaptic relations and ultrastructure as retinal terminals (RLP) and as profiles originating from inhibitory interneurons (F1 and F2). Immunopositive somata and dendrites received asymmetric synaptic contacts from labeled RLP and non-identified, non-immunoreactive synaptic boutons. Moreover, PV(+) dendrites were postsynaptic to labeled F profiles. In the PGN all neurons were both PV(+) and GABA-immunoreactive and in the dLGN the vast majority of PV(+) neurons showed GABA-immunoreactivity. It is suggested that the high incidence of PV in GABAergic neurons is related to the particular activation patterns of these neurons and the resulting demand for calcium buffer systems.

Immunohistochemical localization of calcium-binding proteins, parvalbumin and calbindin-D 28k, in the adult and developing visual cortex of cats: a light and electron microscopic study.

In the cat primary visual cortex, we investigated with immunohistochemical techniques the developmental changes in the cellular and subcellular localization of the Ca2+-binding proteins parvalbumin (PV) and calbindin-D 28K (CBP), in order to determine whether there is a correlation between the expression of Ca2+-dependent processes and the time course of the critical period for use-dependent plasticity. On the 54th day of gestation and at 1 week postnatally, both calcium-binding proteins were present only in a subpopulation of neurons in layers V and VI. During subsequent maturation, the number of PV(+) and CBP(+) neurons increased significantly and labeled cells were detected in more superficial layers. Moreover, the homogeneous labeling of some CBP(+) neurons in layers IV to VI decreased and changed to a punctate pattern. In adult cats PV(+) neurons were evenly distributed throughout layers II to VI, whereas CBP(+) neurons were concentrated in layers II/III. Only a few immunoreactive cells had morphological features characteristic of pyramidal cells; the large majority were nonpyramidal. Electron microscopy confirmed the presence of PV- and CBP-reaction product within the perikarya, axons, and dendrites of labeled cells. It was associated preferentially with microtubules, postsynaptic densities, and intracellular membranes. Immunoreactive neurons received immunonegative asymmetric synapses on their dendritic shafts and made symmetric synaptic contacts with labeled and unlabeled somata and with unlabeled dendritic shafts. The large number and widespread distribution of immunoreactive neurons implies that PV and CBP play an important role in the regulation of calcium-dependent processes in the visual cortex. Furthermore, the developmental redistribution of PV and CBP points to changes in the organization of Ca2+-dependent processes during maturation.

Basal membrane-depleted scar in lesioned CNS: characteristics and relationships with regenerating axons.

The lesion scar formed after CNS injury is an impediment to axonal regeneration and leads to growth arrest or misrouting of sprouting axons. Our previous study showed that pharmacological reduction of basal membrane formation within the scar can overcome this scar impermeability [Stichel C. C. et al. (1999) Eur. J. Neurosci. 11, 632-646]. The aim of the present study was to characterize the basal membrane-depleted scar and to analyse its relationships with penetrating axons. The experiments comprised two groups of animals in which the left postcommissural fornix was transected; in addition, one group received a local immediate injection of the collagen IV-reducing agent dipyridyl, while the other group received an injection of phosphate-buffered saline. Immunohistochemical methods were used to characterize scar formation and scar-axon relationships. Animals receiving dipyridyl showed reduction of collagen IV-immunopositive basal membrane in the lesion center, which was accompanied by: (i) a decrease in laminin, as well as chondroitin and heparan sulfate proteoglycan, deposition in the lesion center; (ii) an increase in chondroitin and keratan sulfate proteoglycan expression in the perilesional area; (iii) a typical activation pattern of astrocytes and microglia/macrophages; (iv) axons regenerating through this modified scar were closely associated with various glial cell types and crossed a prominent chondroitin/keratan sulfate proteoglycan matrix. Our results suggest that neither the formation of a reactive astroglial network nor the accumulation of microglia/macrophages or the deposition of chondroitin and keratan sulfate proteoglycans in the perilesional area represent a barrier to regrowing axons. The present approach demonstrates that the lesion-induced basal membrane itself is the primary determinant of scar impermeability.

A search for choline acetyltransferase-like immunoreactivity in neurons of cat striate cortex.

The goal of this study was to determine whether cat striate cortex contains neurons with choline acetyltransferase-like immunoreactivity. Two different monoclonal antibodies were applied with various fixation protocols. As controls served selected regions of the cat brain and the rat neocortex where cholinergic neurons had been demonstrated previously with independent methods. All experimental protocols labelled the presumptive cholinergic neurons in the central regions but revealed only a few weakly stained neurons in cat striate cortex. These had a non-pyramidal morphology and were scattered throughout all layers.

Developmental regulation of decorin expression in postnatal rat brain.

Here, we report on the expression of the small chondroitin/dermatan sulfate proteoglycan decorin in the developing postnatal rat brain. Northern analysis of brain RNA demonstrated decorin transcripts with peak expression on postnatal day 3 followed by a slow decline to the lower adult level. In situ hybridization and immunohistochemistry revealed postnatal decorin expression in the grey matter of neocortex, hippocampus and thalamus, in myelinated fibre tracts and in several mesenchymal tissues (blood vessels, pia mater and the choroid plexus). In the neocortex, decorin is expressed in a specific laminar pattern with intense staining of the cortical plate and its derivatives, which differs remarkably from the distributions observed for other proteoglycans [B. Miller, A.M. Sheppard, A.R. Bicknese, A.L. Pearlman, Chondroitin sulfate proteoglycans in the developing cerebral cortex: the distribution of neurocan distinguishes forming afferent and efferent axonal pathways, J. Comp. Neurol. 355 (1995) 615-28]. Thus, decorin seems to serve yet unknown functions in the developing rat brain parenchyma in addition to its well-established role as a constituent of the mesenchymal extracellular matrix.

Differential expression of the small chondroitin/dermatan sulfate proteoglycans decorin and biglycan after injury of the adult rat brain.

Chondroitin sulfate proteoglycans are widespread extracellular matrix proteins and are specifically upregulated after CNS injury at the lesion site. Many proteoglycan core proteins have been described in the rat brain, but detailed analysis of individual proteoglycans expressed after injury are missing. The present study represents an initial attempt to assess the diversity and timing of lesion-induced expression of proteoglycans in order to elucidate their functional role in CNS injury and repair. Using immunocytochemical methods we analysed the expression of decorin and biglycan in the transected postcommissural fornix of the adult rat. Transection of the fornix induced the upregulation of both decorin and biglycan. However, their expression differed with respect to time course, regional extent and cellular localization. The rapid upregulation of decorin within a wide area around the lesion was followed by a massive appearance of biglycan that remained restricted to the transection site. Three months after lesion, differences of the area size of decorin- and biglycan-immunoreactivities were no longer detectable. Both proteoglycans were restricted to the lesion site and the fornix stumps. While decorin was primarily expressed by astrocytes, biglycan was deposited extracellularly in sheet-like structures. The upregulation of both proteoglycans persisted for at least up to 6 months after lesion. These strong but divergent lesion-induced expression patterns indicate important but different roles of decorin and biglycan in CNS injury.

Expression of MUPP1 protein in mouse brain.

Localizing cell surface receptors to specific subcellular sites can be crucial for proper functioning. PDZ proteins apparently play central roles in such protein localizations. 5-HT(2C) receptors have previously been shown to interact with MUPP1, a multi PDZ domain protein, in heterologous systems and in rat choroid plexus. We now report the generation and characterization of two independent MUPP1 antisera, which recognise distinct areas of the mouse brain in agreement with previous in-situ hybridization studies. Our results indicate that MUPP1 immunoreactivity co-localizes with 5-HT(2A) or 5-HT(2C) receptor expression in all regions of the mouse brain, including the choroid plexus where 5-HT(2C) receptors are highly enriched.

Ontogenetic changes in the level and subcellular distribution of protein kinase C in cat visual cortex.

Ontogenetic changes of the total activity and the subcellular distribution of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) were investigated in the cat visual cortex. Following homogenization of the tissue in Ca2+-free buffer and separation of membrane-bound and soluble protein kinase C, the enzyme was partially purified by diethylaminoethyl-cellulose chromatography. Total, membrane-bound and soluble enzyme activity were determined by measuring the phosphate incorporation into lysine-rich histone, a substrate for protein kinase C. It was found that the total activity level, while being low in the first 4 weeks, increased rapidly to peak values at 5 weeks of age and declined slightly thereafter. Although at all developmental stages most of the enzyme was recovered in the soluble fraction, the membrane-bound activity exhibited a considerable increase at 5 weeks of age, reflecting a relative shift of protein kinase C from the cytosol to the membranes. The increase and redistribution of protein kinase C activity coincide in time with the developmental phase during which the visual cortex is particularly susceptible to undergo use-dependent modifications. This finding is compatible with the hypothesis that protein kinase C-mediated phosphorylation processes are involved in activity-dependent modifications of neuronal transmission.

Dissociated cell culture of rat cerebral cortical neurons in serum-free, conditioned media: GABA-immunopositive neurons.

The gamma-aminobutyric acid (GABA)ergic properties of embryonic (E15d) rat cortical neurons were studied in dissociated serum-free culture by immunohistochemical methods. GABA-like immunoreactivity was found in a subpopulation of neurons from the first day onwards. The number of GABA-positive neurons reached mature values (10.5-12.6%) within the first week, while their morphological differentiation was not found to be fully completed until the 11th day of culture and was characterized by several discrete developmental stages. First, GABA-positive neurons gained their mature complement of neurites at 3 days in vitro (DIV). Three days later somal maturation became evident, followed at least by the maturation of the neuritic arbor. Double-labelling studies revealed the coexpression of GABA and tyrosine hydroxylase within the same cells. The similarities of relative number, morphology, time course of development and biochemistry of cultured GABAergic neurons compared with those in situ suggest that the applied culture system is a useful model to investigate several aspects of GABAergic neurotransmission at the cellular level.

Expression of inherent neuronal shape characteristics after transient sensitivity to epigenetic factors.

We investigated effects of different substrates and culture media on the early morphological differentiation of rat neocortical neurons in culture. In particular, we examined the effects of homotypic astrocytes, the adhesive glycoprotein laminin and the polycationic substrate poly-L-lysine, as well as diffusible astrocyte-derived conditioned medium factors and serum on (1) soma area, (2) total neuron area and (3) primary neurite number. To assess variations in morphological reactions of neurons with a defined neurotransmitter phenotype, we analyzed the differentiation of GABAergic neurons. The morphology of young neocortical neurons was dramatically affected by both substrate and culture medium. Replacement of the astrocytic monolayer or the astrocyte-conditioned medium by other substrates or non-conditioned medium, respectively, was accompanied by (1) spreading and flattening of neuronal somata, (2) a marked decrease in total neuron area and (3) an increase in the number of primary neurites. The various morphological parameters studied exhibited different sensitivities to changes of these external factors. Moreover, the influences of epigenetic factors on the generation of primary neurites depended on the transmitter phenotype of the neuron. The induced morphological alterations were transient. At the end of the first week in culture, the surviving neurons underwent substantial remodeling of their morphology leading to an expression of in vivo shape characteristics. These observations suggest that despite an early, transient sensitivity to environmental influences, the neuronal differentiation with respect to the morphological parameters studied in culture is to a large degree determined by intrinsic factors.

Organization and morphological characteristics of choline acetyltransferase-containing fibers in the visual thalamus and striate cortex of the cat.

The morphological characteristics and the distribution of cholinergic fibers in the cat visual system were studied with immunohistochemical methods employing monoclonal antibodies directed against choline acetyltransferase (ChAT). ChAT-positive fibers form dense meshworks within the main laminae of the dorsal lateral geniculate nucleus (LGNd), the perigeniculate nucleus (PGN) and the visual cortex. The fibers are of fine caliber, branch extensively and possess numerous varicosities in close contact with cellular elements. The density of ChAT + fibers is not uniform and is particularly high in cortical laminae I-III, the PGN complex and laminae A and A1 of the LGNd.

Inflammatory processes in the aging mouse brain: participation of dendritic cells and T-cells.

Increased inflammatory activity accompanies normal brain aging. Whereas local glial cell activation, upregulation of cytokines and transcriptional alterations of inflammatory factors are well-documented components of this complex process, it is unclear whether blood-derived leukocytes also contribute to the age-related changes. The present study of normal mouse brain applied single and double immunohistochemistry to reveal for the first time that dendritic cells (DCs) and T-cells are important components of the general increased inflammatory state, which was documented by upregulation of reactive astrocytes and microglia. B-cells and mast cells do not contribute to this inflammatory response. Dendritic cells and T-cells appeared at about 12 months of age and their number increased further during aging. In 24-month-old animals a dense network of DCs interspersed with T-cells pervaded brain areas where substantial histopathological changes and a volumetric decrease have been reported. All CD11c(+)-DCs displayed the typical dendritic shape and expressed the myeloid specific integrin CD11b. Some of the DCs were also CD205- or MIDC8-immunoreactive and expressed the cathepsins S and X. The emergence and prolonged presence of leukocytes might indicate a crucial role of these cells in local, age-related immune responses in the brain.

Relationship between injury-induced astrogliosis, laminin expression and axonal sprouting in the adult rat brain.

Lesion-induced regenerative sprouting of CNS axons is accompanied by structural and metabolic changes of astrocytes. In order to evaluate the effects of these astrocytic changes on axonal regeneration, we investigated the spatio-temporal relationship of gliosis, laminin expression and axonal sprouting in the postcommissural fornix of the adult rat. Using immunocytochemical methods we observed (1) a perilesional area with a transient lack of astrocytes and axons, (2) the reappearance of reactive astrocytes followed by the ingrowth of sprouting fibres and finally an increase in laminin-immunoreactivity, (3) the absence of lesion-induced laminin-expression in reactive astrocytes and (4) the formation and long-lasting (at least 28 months) persistence of a dense plexus of laminin-immunopositive blood vessels at the site of transection and in the proximal and distal stumps. These data indicate that astrogliosis is permeable for regrowing axons and that injury-induced axonal sprouting in the transected postcommissural fornix may be mediated by laminin-independent mechanisms.

Extensive and long-lasting changes of glial cells following transection of the postcommissural fornix in the adult rat.

Injury to the adult brain results in the formation of a glial scar that involves both activated astroglia and microglia/macrophages. Although reactive gliosis has been linked to failure of axonal regeneration in the adult mammalian CNS, the spatio-temporal pattern of the postlesion responses in morphology and distribution of the major cellular constituents of the gliosis has attracted little attention. In an attempt to define these relationships, we have undertaken a systematic study of astrocytic and microglial/macrophagic responses after stereotactic transection of the postcommissural fornix in rat. To visualize astrocytes, microglia, and macrophages, antibodies against glial fibrillary acidic protein (GFAP), vimentin (VIM), complement receptor type 3 (OX42), and ED1 were used. The cellular responses occurring between 2 h and 1 year postlesion (PL) at various distances distal and proximal to the lesion site were studied. Transection of the postcommissural fornix leads to: 1) a very early microglial reaction both distal and proximal to the lesion, as indicated by OX42-immunoreactivity, followed by a massive astrocytic response; 2) a transient GFAP(-) but VIM(+) region around the lesion center; 3) the appearance of numerous ED1(+) macrophages at PL3d; and 4) long-lasting (at least 1 year) persistence of both astrocytes and microglia/macrophages. The timing and extension of the sequential glial reactions after postcommissural fornix transection are discussed in relation to the myelin degradation and spontaneous sprouting of injured axons that have previously been observed in this lesion model (Wunderlich et al: Glia 10:49-58, 1994).

Localization of isoenzymes II/III of protein kinase C in the rat visual cortex (area 17), hippocampus and dentate gyrus.

Monoclonal antibodies against type II and type III subspecies of protein kinase C PkC(II/III) were used to map the distribution of these isoenzymes in the visual cortex (area 17), hippocampus and dentate gyrus of the rat. PkC(II/III)-immunocytochemistry resulted in a specific staining of neuropil and of neuronal somata with their proximal dendrites. The majority of immunopositive cells exhibited a punctate distribution of reaction product, while only a few neurons were homogeneously labeled. In the visual cortex stained neurons were distributed throughout all laminae and reached a particularly high density in layers II/III. Moreover, PkC(II/III)-positive neurons were found within the strata pyramidale and radiatum of the hippocampus proper and in the stratum granulosum, the subgranular zone and the hilar region of the dentate gyrus. The present results suggest that PkC(II/III)-positive neurons constitute a distinct population of both projection and local circuit neurons that are not exclusively associated with any one neurotransmitter system.