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.

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.

Parkin expression in the adult mouse brain.

Mutations in a protein designated Parkin were shown to be involved in the pathogenesis of autosomal recessive juvenile parkinsonism. Nothing is known about its regional and subcellular distribution in the mouse. In order to elucidate the Parkin mRNA and protein distribution in the adult mouse, the mouse cDNA was cloned and polyclonal antisera were generated against the N-terminal part of mouse Parkin. The antibodies were shown to be specific using Western blot analysis, immunostaining of cells transfected with mouse Parkin and pre-absorption tests. The Parkin protein expression profile was studied using immunohistochemistry and Western blot analysis and was compared with that of the mRNA yielded by in situ hybridization and RT-PCR analysis. Parkin protein was widely distributed in all subdivisions of the mouse brain. Low levels were found in the telencephalon and diencephalon, while the brainstem contained a large number of cells heavily expressing Parkin. Ultrastructural analysis and double immunohistochemistry revealed that the majority of Parkin-expressing cells were neurons, while only single glial cells exhibited immunostaining. The protein was distributed nonhomogeneously throughout the entire cytoplasm. A subpopulation of Parkin-immunopositive cells displayed speckled immunodeposits in the nucleus. Dopaminergic cells of the substantia nigra pars compacta exhibited high levels of Parkin mRNA but no Parkin protein, while the striatum contained immunopositive profiles but no mRNA signals. Our data indicate that Parkin is neither restricted to a single functional system nor associated with a particular transmitter system. The speckled nuclear distribution of Parkin immunoreactivity strongly suggests a role for Parkin in gene expression.

Characterization of neuronal migration disorders in neocortical structures: loss or preservation of inhibitory interneurons?

PURPOSE: Neuronal migration disorders (NMD) are often associated with therapy-resistant epilepsy. In human cerebral cortex, this hyperexcitability has been correlated with a loss of inhibitory interneurons. We used a rat model of focal cortical NMD (microgyria) to determine whether the expression of epileptiform activity in this model coincides with a decrease in inhibitory interneurons. METHODS: In 2-to 4-month-old rats, the density of interneurons immunoreactive for gamma-aminobutyric acid (GABA), calbindin, and parvalbumin was determined in fronto-parietal cortex in nine 200-microm-wide sectors located up to 2.5 mm lateral and 2.0 mm medial from the lesion center in primary parietal cortex (Par1). Quantitative measurements in homotopic areas of age-matched sham-operated rats served as controls. RESULTS: The freeze lesion performed in newborn rat cortex resulted in adult rats with a microgyrus extending in a rostro-caudal direction from frontal to occipital cortex. The density of GABA-and parvalbumin-positive neurons in fronto-parietal cortex was not significantly different between lesioned and control animals. Only the density of calbindin-immunoreactive neurons located 1.0 mm lateral and 0.5 mm medial from the lesion was significantly (Student t test, p < 0.05) larger in freeze-lesioned rats (5,817 +/- 562 and 6,400 +/- 795 cells per mm3, respectively; n = 12) compared with measurements in homotopic regions in Par1 cortex of controls (4,507 +/- 281 and 4, 061 +/- 319 cells per mm3, respectively; n = 5). CONCLUSIONS: The previously reported widespread functional changes in this model of cortical NMD are not related to a general loss of inhibitory interneurons. Other factors, such as a decrease in GABA receptor density, modifications in GABAA receptor subunit composition, or alterations in the excitatory network, e.g., an increase in the density of calbindin-immunoreactive pyramidal cells, more likely contribute to the global disinhibition and widespread expression of pathophysiological activity in this model of cortical NMD.

Chondroitin sulfates expressed on oligodendrocyte-derived tenascin-R are involved in neural cell recognition. Functional implications during CNS development and regeneration.

Tenascin-R (TN-R), an extracellular matrix constituent of the central nervous system (CNS), has been implicated in a variety of cell-matrix interactions underlying axon growth inhibition/guidance, myelination and neural cell migration during development and regeneration. Although most of the functional analyses have concentrated exclusively on the role of the core protein, the contribution of TN-R glycoconjugates present on many potential sites for N- and O-glycosylation is presently unknown. Here we provide first evidence that TN-R derived from whole rat brain or cultured oligodendrocytes expresses chondroitin sulfate (CS) glycosaminoglycans (GAGs), i.e., C-4S and C-6S, that are recognized by CS-56, a CS/dermatan sulfate-specific monoclonal antibody. Based on different in vitro approaches utilizing substrate-bound glycoprotein, we found that TN-R-linked CS GAGs (1) promote oligodendrocyte migration from white matter microexplants and increase the motility of oligodendrocyte lineage cells; (2) similar to soluble CS GAGs, induce the formation of glial scar-like structures by cultured cerebral astrocytes; and (3) contribute to the antiadhesive properties of TN-R for neuronal cell adhesion in an F3/F11-independent manner, but not to neurite outgrowth inhibition, by mechanism(s) sensitive to chondroitinase or CS-56 treatments. Furthermore, after transection of the postcommissural fornix in adult rat, CS-bearing TN-R was found to be stably upregulated at the lesion site. Our findings suggest the functional impact of TN-R-linked CS on neural cell adhesion and migration during brain morphogenesis and the contribution of TN-R to astroglial scar formation (CS-dependent) and axon growth inhibition (CS-independent), i.e., suppression of axon regeneration after CNS injury.

Effects of schwann cell suspension grafts on axon regeneration in subacute and chronic CNS traumatic injuries.

In a previous study, we have shown that microtransplanted Schwann cell suspensions foster structural recovery of the acutely transected postcommissural fornix. The emphasis of the present study was to examine whether subacutely and chronically injured axons also demonstrate significant responsiveness to implanted Schwann cells. Microinjected suspensions of cultured Schwann cells i) elicited a growth response and attracted axons in a subacute and chronic traumatic lesion but ii) failed to stimulate regrowth of the postcommissural fornix projection at any nonacute postlesion stage. In conclusion, the single intervention strategy of Schwann cell microimplantation is not sufficient to ensure regeneration of the subacutally or chronically transected postcommissural fornix. The use of Schwann cells as stimulators of axon regrowth depends on the neuronal cell type and the appropriate postinjury time point.

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.

Inhibition of collagen IV deposition promotes regeneration of injured CNS axons.

Scarring impedes axon regrowth across the lesion site and is one major extrinsic constraint to effective regeneration in the adult mammalian central nervous system. In the present study we determined whether specific biochemical or immunochemical modulation of one major component of the scar, the basal membrane (BM), would provide a means to stimulate axon regeneration in the mechanically transected postcommissural fornix of the adult rat. Basal membrane developed within the first 2 weeks after transection in spatiotemporal coincidence with the abrupt growth arrest of spontaneously regrowing axons. Local injection of anticollagen IV antibodies or alpha, alpha'-dipyridyl, an inhibitor of collagen triple helix formation and synthesis, significantly reduced lesion-induced BM deposition. This treatment allowed massive axon elongation across the lesion site. Anterograde tracing provided unequivocal evidence that regenerating axons follow their original pathway, reinnervate the appropriate target, the mammillary body, and become remyelinated with compact myelin. Presynaptic electrophysiological recordings of regenerated fibre tracts showed recovery to nearly normal conduction properties. Our results indicate that lesion-induced BM is an impediment for successful axonal regeneration and its reduction is a prerequisite and sufficient condition for regrowing axons to cross the lesion site.

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.

The CNS lesion scar: new vistas on an old regeneration barrier.

Scar formation represents a reaction of nervous tissue to any form of physical injury. Research over the past decade has demonstrated that the scar composed of glial cells and several extracellular matrix molecules constitutes an obstacle to axon regeneration in the CNS. This review briefly summarizes the current knowledge on (a) the structural and functional features of the lesion scar and (b) the development of therapeutic interventions to override this regeneration barrier.

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.

Lack of immune responses to immediate or delayed implanted allogeneic and xenogeneic Schwann cell suspensions.

Previous studies have shown that Schwann cell implantation offers a potential therapeutic approach to a variety of neurodegenerative disorders and traumatic injuries. In a clinically relevant paradigm, however, the implantation of autologous Schwann cells is problematic and the use of heterogenetic Schwann cells will be required. In the present study we addressed this important issue and analysed the immunogenicity and survival of allogeneic and xenogeneic Schwann cell suspension grafts in a prelesioned CNS fiber tract, the transected postcommissural fornix of the adult Wistar rat. Cultured Schwann cells from Wistar rat or human peripheral nerve were injected either immediately or after a delay into the transection site and the spatio-temporal pattern of leukocyte infiltration and of major histocompatibility antigen expression was characterized and semiquantified with immunocytochemical methods. Our main findings are that (1) invasive cerebral lesions induce the expression of MHC class I and II antigens, but only sparse infiltration of T-lymphocytes, (2) both allogeneic and xenogeneic discordant Schwann cell suspension grafts, from either neonatal or adult peripheral nerve, survive without any overt signs of rejection for up to 10 weeks after implantation; and (3) delayed implantation procedures have no effect on immune responses to allogeneic Schwann cell grafts. These results demonstrate that there is no marked ongoing immune reactions to heterogenetic Schwann cell suspension grafts and that long-term survival of cross-species Schwann cell grafts can be achieved in the absence of any immunsuppressive treatment. Thus the conditions for functional transplantation of Schwann cells across immunological barriers seem to be favourable and will have implications for future cross-species studies, and possibly also for clinical application.

Chondroitin/dermatan sulphate promotes the survival of neurons from rat embryonic neocortex.

Recently we have shown that biglycan, a small chondroitin sulphate proteoglycan of the extracellular matrix, supports the survival of cultured neurons from the developing neocortex of embryonic day 15 rats. Here we investigate the structure-function relationship of this neurotrophic proteoglycan and show that chondroitin/dermatan sulphate chains are the active moieties supporting survival. Heparin, a highly sulphated glucosaminoglycan, is less active than the galactosaminoglycans (chondroitin-4-sulphate, chondroitin-6-sulphate and dermatan sulphate), whereas hyaluronic acid, an unsulphated glucosaminoglycan, does not support neuron survival. Galactosaminoglycans must be in direct contact with neurons to cause survival. Experiments with elevated potassium concentrations and antagonists of voltage-gated calcium channels exclude the involvement of membrane depolarization. However, genistein and an erbstatin analogue, which are inhibitors of tyrosine kinases with low specificity, abolished neuron survival in the presence of chondroitin/dermatan sulphate, whereas a selective inhibitor of neurotrophin receptor kinases (K252a) had no suppressive effect. Thus, yet unidentified tyrosine kinases are involved in the chondroitin/dermatan sulphate-dependent survival of neocortical neurons. In the embryonic stages of rat neocortical development chondroitin sulphate is mainly located in layers I, V and VI and the subplate. Chondroitin sulphate expression is maintained after birth, extends up to cortical layer IV on postnatal day 7, and is down-regulated until postnatal day 21 concomitant with the period of naturally occurring cell death. The latter observation is consistent with a putative role of chondroitin sulphate in the control of neuron survival during cortical histogenesis.

Reconstruction of transected postcommissural fornix in adult rat by Schwann cell suspension grafts.

Schwann cell (SC) transplantation has emerged as a powerful tool to promote regeneration in the lesioned central nervous system (CNS). Most studies focused on the use of guidance channels to introduce the SCs into CNS neuropil; a technique that itself causes extensive damage to host tissue and is only applicable in superficial brain areas. The present study examines the efficacy of microinjected SC suspensions to promote structural reconstruction of the transected postcommissural fornix in the adult rat. Stereotactic, unilateral fornix transection was performed using a tungsten wire knife. Immediately after transection lesion cavities received either a Dulbecco's modified Eagle's medium injection or a pure SC suspension graft derived from a highly purified SC culture that was prepared from syngeneic rat (P1) sciatic nerves. After 4 days to 8 months, the implant characteristics as well as the structural reconstruction of the tract were analyzed using immunocytochemical methods and anterograde tracing techniques. Numerous SCs of the graft could be identified for up to 8 months. They rapidly dispersed from the injection site and migrated freely for considerable distances into the host tissue. The SCs exhibited a low proliferation activity that ceased within 2 weeks after transplantation. They did not prevent retrograde axonal degeneration of the fornix tract for a short distance (600 micron) but promoted structural reconstruction of the transected fornix tract. Regenerating fibers traversed the lesion site and extended along their former pathway up to the mammillary body, their proper target. Moreover, the applied transplantation technique allowed remyelination of the regenerating fibers by host oligodendrocytes. In conclusion, microtransplantation of SC suspensions represents a promising strategy for promoting structural reconstruction of lesioned CNS projections.

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.

Restricted appearance of tenascin and chondroitin sulphate proteoglycans after transection and sprouting of adult rat postcommissural fornix.

Transected fibres of the adult rat postcommissural fornix sprout over short distances but fail to traverse the lesion site and terminate in close vicinity to the wound. As a step in defining the molecular environment responsible for regeneration failure at the lesion site, we have used immunocytochemistry to analyse the spatio-temporal expression pattern of two putative growth-inhibitory extracellular matrix components, tenascin and chondroitin sulphate proteoglycans and their topographical relationship to the sprouting axons. Both tenascin and chondroitin sulphate proteoglycan labelling appeared after fornix transection and were confined to the immediate vicinity of the lesion site. While tenascin-labelling was associated with astrocytes and microglia/macrophages, which accumulate preferentially at the tract borders, chondroitin sulphate proteoglycan labelling appeared as a homogeneous meshwork around the wound. Tenascin-like immunoreactivity disappeared between 17 days and 4 weeks, but chondroitin sulphate proteoglycan staining persisted at least up to 14 months after transection. Regrowing fornix fibres invaded and elongated within the chondroitin sulphate proteoglycan-immunopositive region up to the lesion site, where they terminated. This zone of axonal growth inhibition was neither characterized by an increase of chondroitin sulphate proteoglycan immunoreactivity nor by the presence of tenascin-immunopositive structures. The spatio-temporal distribution patterns of tenascin and chondroitin sulphate proteoglycan and the permeability of the chondroitin sulphate proteoglycan-immunopositive region for sprouting axons do not support the hypothesis that chondroitin sulphate proteoglycan alone and/or tenascin inhibit the advance of sprouting fornix fibres.

Clearance of myelin constituents and axonal sprouting in the transected postcommissural fornix of the adult rat.

Following transection of the postcommissural fornix in the adult rat, fibres retract from the lesion zone but then start to regrow within their former pathway up to the lesion site, where they terminate. The fibres neither penetrate nor bypass this region. In order to define the molecular mechanisms that cause regenerative failure at the lesion site, we analysed the spatiotemporal relationship between clearance/re-expression of myelin constituents and axon sprouting. Using immunocytochemical methods, we investigated the distribution of myelin-associated growth inhibitor (NI-35/250) and myelin basic protein after transection of the postcommissural fornix. In the studies described here we demonstrate the sequential removal of neurofilaments and myelin constituents in a perilesion zone and at the lesion site. The removal of myelin constituents was followed by the extensive regrowth of fornix fibres in the proximal segment. However, these fibres stopped at the lesion site, an area that lacked immunostaining for NI-35/250 and. In the distal stump we observed the disappearance of neurofilament along the entire fornix segment but spatial differences in the removal of myelin constituents. While both NI-35/250 and myelin basic protein disappeared in the perilesion zone, they persisted in the more distal segment for at least 28 months after lesion. In conclusion, our study indicates that the onset of axon sprouting is correlated with the removal of myelin basic protein and NI-35/250. Furthermore, we suggest that it seems unlikely that the myelin growth inhibitor NI-35/250 constitutes the stop signal of the axon growth barrier in the transected formix.

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.