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Zusammenfassung Mein Briefträger ist ein intelligenter und findungsreicher Mann. Seit 25 Jahren versorgt er mich pünktlich und zuverlässig mit meiner Briefpost. Von den vielen medizinischen Zeitschriften, die meine Frau und ich bekommen, weiß er, dass wir Ärzte sind. Eines Tages erzählte er mir aufgeregt vom Onkel, der ins Krankenhaus musste, weil er den linken Arm und das linke Bein nicht mehr wie gewohnt bewegen konnte. Die Ärzte hätten ihm aber versichert, dass an den Extremitäten nichts krank sei. Alles käme vom Kopf. Ob das denn wahr sein könne. Natürlich wusste er grob, was ein Schlaganfall war. Konfrontiert aber mit den Lähmungen fehlte ihm dennoch das Verständnis dafür, dass ein Ereignis im Großhirn den Onkel in so eindrucksvoller Weise einseitig seiner Beweglichkeit beraubt hatte. Wenige Krankheitsereignisse beeindrucken uns so sehr wie der Verlust der Motorik, sei es als Halbseitenlähmung nach einem Schlaganfall, als Querschnittslähmung nach einer Rückenmarksverletzung oder als Gesichtslähmung nach einem Schaden am Fazialisnerven. Der allgemeine Sprachgebrauch setzt Lähmung mit Ausfall der Motorik gleich und verbindet damit nicht selten den Verlust anderer oder höherer Funktionen des Nervensystems.

Our knowledge on Neuregulin-1 (Nrg-1) during development of the nervous system is increasing rapidly, but little is known about Nrg-1-ErbB signaling in the adult brain. Nrg-1 is involved in determination, proliferation, differentiation, and migration of neurons and glial cells in the developing brain. In the peripheral nervous system, Nrg-1 signaling is required for Schwann cell differentiation and myelination, and establishment of neuromuscular junctions (NMJs). Multiple alternative splicing of Nrg-1 was shown, but correlation of its structural and functional diversity was rarely addressed. Therefore, we investigated the expression of Nrg-1 isoforms in the rat brain and brain-derived cell types, and their involvement in regeneration of the adult brain, using immunohistochemistry, in situ hybridization, and semiquantitative RT-PCR. We found expression of at least 12 distinct Nrg-1 isoforms in the brain and altered expression of several isoforms in the facial motor nucleus after peripheral transection of the seventh cranial nerve. An upregulation of Nrg-1 type-I mRNA, probably type- I-alpha, was observed in reactive astrocytes of the facial nucleus 1 d postaxotomy. Nrg-1 type-III and the splice variants beta1 and beta5 are dramatically downregulated in axotomized motoneurons, which lack contact to their target tissue. Baseline expression levels were reestablished when the first axons reached the facial muscles and reformed NMJs. Nrg-1-beta1 and -beta5 might act in maintenance of NMJs. The splice variants beta2 and beta4 display an initial downregulation of mRNA levels, followed by an increase during the period of axon remyelination. Thus, Nrg- 1-beta2 and -beta4 might be involved in myelination.

The response of plasminogen activator activity in the CNS to peripheral nerve axotomy was examined in vivo. After transection of the rat facial nerve, a transient increase in plasminogen activator activity was observed in the facial nucleus on the operated side with maximal activity 3-5 days after lesion. This activity was inhibited by the urokinase-specific inhibitor amiloride but not by antibodies against tissue plasminogen activator. The molecular mass of the induced form of plasminogen activator was estimated to be approximately 48 kDa. An in vitro assay of plasminogen hydrolysis also demonstrated an increase in amiloride-sensitive plasminogen activator activity in facial nerve extracts following facial nerve axotomy. These data indicate that the plasminogen activator activity induced in the facial nucleus following axotomy of facial motoneurons is of the urokinase type. It is suggested that the urokinase-type plasminogen activator might play a role in the events accompanying injury and regeneration in the facial nucleus following motoneuron lesion.

The effects of transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) were examined on the accumulation of plasminogen activator inhibitor-1 (PAI-1) mRNA in astrocytes in vitro. Both cytokines stimulated PAI-1 mRNA expression transiently with a maximal fivefold (bFGF) and 30-fold (TGF-beta1) at 4 h, decreasing to basal levels within 32 h. EC50 values were 1.4 nM for bFGF and 6.7 pM for TGF-beta1 on PAI-1 mRNA accumulation. A twofold increase in content of tPA mRNA was observed with bFGF but not with TGF-beta1. The action of TGF-beta1 on PAI-1 mRNA was inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. In contrast, cycloheximide potentiated the action of bFGF. Nuclear run-on assays showed that bFGF, but not TGF-beta1, stimulated astrocytic PAI-1 gene transcription. Thus, TGF-beta1 predominantly uses posttranscriptional mechanisms to raise the level of PAI-1 mRNA in astrocytes, whereas bFGF acts at both the transcriptional and posttranscriptional levels. The data reveal differences in the mechanisms underlying the regulation of PAI-1 mRNA levels by TGF-beta1 in astrocytes compared with other cells. The action of TGF-beta1 and bFGF on the plasminogen activator system in astrocytes might be involved in the cellular events accompanying glial activation following injury of the CNS.

Microglia have long been ignored by neurooncologists. This has changed with the realization that microglial cells not only occur within and around brain tumors but also contribute significantly to the actual tumor mass, notably in astrocytic gliomas. In addition, it has been speculated that microglia could play a role in the defense against neoplasms of the nervous system. However, the biological success of these tumors, i.e., their highly malignant behavior, indicates that natural microglial defense mechanisms do not function properly in astrocytomas. In fact, there is evidence that microglial behavior is controlled by tumor cells, supporting their growth and infiltration. This unexpected "Achilles heel" of microglial immune defense illustrates the risk of generalizing on the basis of a single aspect of microglial biology. Microglia are highly plastic cells, capable of exerting cytotoxic functions under conditions of CNS infections, but not necessarily during glioma progression. Thus, the suggestion that microglial activation through stimulation by cytokines (e.g., interferon-gamma) will benefit patients with brain tumors could prove fatally wrong. Therapeutic recruitment of microglia to treat such diffusely infiltrative brain tumors as astrocytic gliomas must be considered premature.

Gene therapy in the central nervous system (CNS) is hindered by the presence of the blood-brain barrier, which restricts access of serum constituents and peripheral cells to the brain parenchyma. Expression of exogenously administered genes in the CNS has been achieved in vivo using highly invasive routes, or ex vivo relying on the direct implantation of genetically modified cells into the brain. Here we provide evidence for a novel, noninvasive approach for targeting potential therapeutic factors to the CNS. Genetically-modified hematopoietic cells enter the CNS and differentiate into microglia after bone-marrow transplantation. Up to a quarter of the regional microglial population is donor-derived by four months after transplantation. Microglial engraftment is enhanced by neuropathology, and gene-modified myeloid cells are specifically attracted to the sites of neuronal damage. Thus, microglia may serve as vehicles for gene delivery to the nervous system.

A major challenge in the assessment of brain injury and its relationship to the ensuing functional deficits is the accurate delineation of the areas of damage. Here, we test the hypothesis that the anatomical distribution pattern of activated microglia, a normally dormant population of resident brain macrophages, can be used as a surrogate marker of neuronal injury not only at the primary lesion site but also in the antero- and retrograde projection areas of the lesioned neurones. Two patients with asymmetrical herpes simplex encephalitis were serially scanned 6 and 12 months after the acute illness using PET with [11C] (R)-PK11195, a marker of activated microglia/brain macrophages. The evolving structural changes in the brain were measured by volumetric MRI and compared with the pattern of [11C](R)-PK11195 binding. Corresponding to the clinically observed cognitive deficits, quantitative [11C](R)-PK11195-PET revealed highly significant signal increases within the affected limbic system and additionally in areas connected to the limbic system by neural pathways, including the lingual gyrus in the occipital lobe and the inferior parietal lobe, which had normal morphology on structural MRI. The increased [11C](R)-PK11195 binding, signifying the presence of activated microglia, persisted many months (>12) after antiviral treatment. Cortical areas that showed early high [11C](R)-PK11195 binding subsequently underwent atrophy. These observations demonstrate that in vivo imaging of activated microglia/brain macrophages provides a dynamic measure of active tissue changes following an acute focal lesion. Importantly, the glial tissue response in the wake of neuronal damage is protracted and widespread within the confines of the affected distributed neural system and can be related to the long-term functional deficits.

Macrophages in the brain can have a triple source. They may originate from recently blood-derived precursors, from the largely resident perivascular cell population (perivascular macrophages and related cells), and from intrinsic parenchymal as well as perivascular microglia. Although continuous exchange of part of the perivascular cell population with bone marrow-derived precursors is now accepted, the turnover of adult parenchymal microglia has remained enigmatic. Using bone-marrow chimeras carrying an unexpressed marker gene and carbon labeling of peripheral monocyte/macrophages in a combined model of facial nerve axotomy and transfer experimental autoimmune encephalitis, we demonstrate for the first time that there is an easy to induce exchange between parenchymal central nervous system (CNS) microglia and the macrophage precursor cell pool of the bone marrow. Furthermore, very low level infiltration of the CNS parenchyma by recently bone marrow-derived microglia could be observed after simple peripheral nerve axotomy that is followed by neuronal regeneration. Thus, microglial cells can be considered wanderers between the peripheral immune system and the CNS where they may act as a "Trojan horse" in infections. The fact that recently bone marrow-derived parenchymal microglia fully integrate into a regenerating brain nucleus' architecture encourages entirely new approaches for delivering genes into the adult CNS.

Motoneurons respond to peripheral nerve transection by either regenerative or degenerative events depending on their state of maturation. Since the expression of c-Jun has been involved in the early signalling of the regenerative process that follows nerve transection in adults, we have investigated c-Jun on rat neonatal axotomized motoneurons during the period in which neuronal death is induced. Changes in levels of c-Jun protein and its mRNA were determined by means of quantitative immunocytochemistry and in situ hybridization. Three hours after nerve transection performed on postnatal day (P)3, c-Jun protein and mRNA is induced in axotomized spinal cord motoneurons, and high levels were reached between 1 and 10 days after. This response is associated with a detectable c-Jun activation by phosphorylation on serine 63. No changes were found in the levels of activating transcription factor -2. Most of dying motoneurons were not labelled by either a specific c-Jun antibody or a c-jun mRNA probe. However, dying motoneurons were specifically stained by a polyclonal anti c-Jun antibody, indicating that some c-Jun antibodies react with unknown epitopes, probably distinct from c-Jun p39, that are specifically associated with apoptosis. J. Neurosci. Res. 63:469–479, 2001. © 2001 Wiley-Liss, Inc.

Direct injury of the brain is followed by inflammatory responses regulated by cytokines and chemoattractants secreted from resident glia and invading cells of the peripheral immune system. In contrast, after remote lesion of the central nervous system, exemplified here by peripheral transection or crush of the facial and hypoglossal nerve, the locally observed inflammatory activation is most likely triggered by the damaged cells themselves, that is, the injured neurons. The authors investigated the expression of the chemoattractants monocyte chemoattractant protein MCP-1, regulation on activation normal T-cell expressed and secreted (RANTES), and interferon-gamma inducible protein IP10 after peripheral nerve lesion of the facial and hypoglossal nuclei. In situ hybridization and immunohistochemistry revealed an induction of neuronal MCP-1 expression within 6 hours postoperation, reaching a peak at 3 days and remaining up-regulated for up to 6 weeks. MCP-1 expression was almost exclusively confined to neurons but was also present on a few scattered glial cells. The authors found no alterations in the level of expression and cellular distribution of RANTES or IP10, which were both confined to neurons. Protein expression of the MCP-1 receptor CCR2 did not change. MCP-1, expressed by astrocytes and activated microglia, has been shown to be crucial for monocytic, or T-cell chemoattraction, or both. Accordingly, expression of MCP-1 by neurons and its corresponding receptor in microglia suggests that this chemokine is involved in neuron and microglia interaction.

The versatility of stem cells has only recently been fully recognized. There is evidence that upon adoptive bone marrow (BM) transplantation (BMT), donor-derived cells can give rise to neuronal phenotypes in the brains of recipient mice. Yet only few cells with the characteristic shape of neurons were detected 1–6 mo post-BMT using transgenic or newborn mutant mice. To evaluate the potential of BM to generate mature neurons in adult C57BL/6 mice, we transferred the enhanced green fluorescent protein (GFP) gene into BM cells using a murine stem cell virus-based retroviral vector. Stable and high level long-term GFP expression was observed in mice transplanted with the transduced BM. Engraftment of GFP-expressing cells in the brain was monitored by intravital microscopy. In a long-term follow up of 15 mo post-BMT, fully developed Purkinje neurons were found to express GFP in both cerebellar hemispheres and in all chimeric mice. GFP-positive Purkinje cells were also detected in BM chimeras from transgenic mice that ubiquitously express GFP. Based on morphologic criteria and the expression of glutamic acid decarboxylase, the newly generated Purkinje cells were functional.

The discovery that missense mutations in the alpha-synuclein gene represent a rare genetic cause of Parkinson's disease (PD) has had significant impact on the development of research into neurodegenerative disorders. It is becoming increasingly clear that alpha-synuclein plays a central role in the pathological process, which causes Lewy body formation and neurodegeneration in PD. Importantly, there is evidence to suggest that mutated alpha-synuclein is toxic to both nerve cells and glia. However, the regulation and function of wild-type alpha-synuclein are as yet ill defined. Using the facial nerve axotomy model, we have addressed the question whether the expression of alpha-synuclein in nerve cells may change in response to injury. We were particularly interested in testing the hypothesis that the severity of neuronal injury had an effect on alpha-synuclein metabolism. Facial nerve cut and crush, respectively, were performed in adult rats where normal facial motoneurones do not express alpha-synuclein. Following axotomy, a subset of facial motoneurones newly expressed high levels of alpha-synuclein immunoreactivity in their cell body and, occasionally, their nucleus. Significantly more nerve cells were labelled following facial nerve transection than following facial nerve crush. Confocal microscopy revealed a granular pattern of alpha-synuclein aggregation in degenerating nerve cells. Interestingly, the observed cell death phenotype was clearly non-apoptotic and developed over days or weeks rather than hours. Thus, axotomy of adult rat facial motoneurones triggers de novo expression of alpha-synuclein and this expression is associated with a non-apoptotic, slow form a neurodegeneration. In addition, the extent of alpha-synuclein expression is related to the severity of neuronal injury.

This study identifies by microautoradiography activated microglia/macrophages as the main cell type expressing the peripheral benzodiazepine binding site (PBBS) at sites of active CNS pathology. Quantitative measurements of PBBS expression in vivo obtained by PET and [(11)C](R)-PK11195 are shown to correspond to animal experimental and human post-mortem data on the distribution pattern of activated microglia in inflammatory brain disease. Film autoradiography with [(3)H](R)-PK11195, a specific ligand for the PBBS, showed minimal binding in normal control CNS, whereas maximal binding to mononuclear cells was found in multiple sclerosis plaques. However, there was also significantly increased [(3)H](R)-PK11195 binding on activated microglia outside the histopathologically defined borders of multiple sclerosis plaques and in areas, such as the cerebral central grey matter, that are not normally reported as sites of pathology in multiple sclerosis. A similar pattern of [(3)H](R)-PK11195 binding in areas containing activated microglia was seen in the CNS of animals with experimental allergic encephalomyelitis (EAE). In areas without identifiable focal pathology, immunocytochemical staining combined with high-resolution emulsion autoradiography demonstrated that the cellular source of [(3)H](R)-PK11195 binding is activated microglia, which frequently retains a ramified morphology. Furthermore, in vitro radioligand binding studies confirmed that microglial activation leads to a rise in the number of PBBS and not a change in binding affinity. Quantitative [(11)C](R)-PK11195 PET in multiple sclerosis patients demonstrated increased PBBS expression in areas of focal pathology identified by T(1)- and T(2)-weighted MRI and, importantly, also in normal-appearing anatomical structures, including cerebral central grey matter. The additional binding frequently delineated neuronal projection areas, such as the lateral geniculate bodies in patients with a history of optic neuritis. In summary, [(11)C](R)-PK11195 PET provides a cellular marker of disease activity in vivo in the human brain.

Nerve injury leads to the release of a number of cytokines which have been shown to play an important role in cellular activation after peripheral nerve injury. The members of the signal transducer and activator of transcription (STAT) gene family are the main mediators in the signal transduction pathway of cytokines. After phosphorylation, STAT proteins are transported into the nucleus and exhibit transcriptional activity. Following axotomy in rat regenerating facial and hypoglossal neurons, a transient increase of mRNA for JAK2, JAK3, STAT1, STAT3 and STAT5 was detected using in situ hybridization and semi-quantitative polymerase chain reaction (PCR). Of the investigated STAT molecules, only STAT3 protein was significantly increased. In addition, activation of STAT3 by phosphorylation on position Tyr705 and enhanced nuclear translocation was found within 3 h in neurons and after 1 day in astrocytes. Unexpectedly, STAT3 tyrosine phosphorylation was obvious for more than 3 months. In contrast, none of these changes was found in response to axotomy of non-regenerating Clarke's nucleus neurons, although all the investigated models express c-Jun and growth-associated protein-43 (GAP-43) in response to axonal injury. Increased expression of Janus kinase (JAK) and STAT molecules after peripheral nerve transection suggests changes in the responsiveness of the neurons to signalling molecules. STAT3 as a transcription factor, which is expressed early and is activated persistently until the time of reinnervation, might be involved in the switch from the physiological gene expression to an 'alternative program' activated only after peripheral nerve injury.

The interplay between growing axons and the extracellular substrate is pivotal for directing axonal outgrowth during development and regeneration. Here we show an important role for the neuronal cell adhesion molecule alpha7beta1 integrin during peripheral nerve regeneration. Axotomy led to a strong increase of this integrin on regenerating motor and sensory neurons, but not on the normally nonregenerating CNS neurons. alpha7 and beta1 subunits were present on the axons and their growth cones in the regenerating facial nerve. Transgenic deletion of the alpha7 subunit caused a significant reduction of axonal elongation. The associated delay in the reinnervation of the whiskerpad, a peripheral target of the facial motor neurons, points to an important role for this integrin in the successful execution of axonal regeneration.

CD44 is a cell surface glycoprotein involved in cell adhesion during neurite outgrowth, leukocyte homing, and tumor metastasis. In the current study, we examined the regulation of this molecule 4 days after neural trauma in different forms of central and peripheral injury. Transection of the hypoglossal, vagus, or sciatic nerve led to the appearance of CD44-immunoreactivity (CD44-IR) on the surface of the affected motoneurons, their dendrites, and their axons. Fimbria fornix transection led to CD44-IR on a subpopulation of cholinergic neurons in the ipsi- and contralateral medial septum and diagonal band of Broca and colocalized with galanin-IR. Central projections of axotomized sensory neurons to the spinal cord (substantia gelatinosa, Clarke's column) also showed an increase in CD44-IR, which was abolished by spinal root transection. Nonneuronal CD44-IR was mainly restricted to sites of direct injury. In the crushed sciatic nerve, CD44-IR was found on the demyelinating Schwann cells and on infiltrating monocytes and granulocytes. Direct parasagittal transection of the cerebral cortex led to CD44-IR on resident astrocytes and on leukocytes entering the injured forebrain tissue. CD44-IR also increased on reactive retinal astrocytes and microglia after the optic nerve crush. Additional time points in the retina and hypoglossal nucleus (days 1, 2, and 14) and cerebral cortex (day 2) injury models also showed the same cell type pattern for the CD44-IR. Finally, polymerase chain reaction analysis confirmed the posttraumatic expression of CD44 mRNA and detected only the standard haematopoietic CD44 splice isoform both in direct and indirect brain injury models. Overall, the current study shows the widespread, graded appearance of CD44-IR on neurons and on nonneuronal cells, depending on the form of neural injury. Here, the ability of CD44 to bind to a variety of extracellular matrix and cell adhesion proteins and its common presence in different forms of brain pathology could suggest an important role for this cell surface glycoprotein in the neuronal, glial, and leukocyte response to trauma and in the repair of the damaged nervous system.

The growth-associated protein GAP-43 (B-50) and the transcription factor C-Jun are involved in regeneration of the injured nervous system. In this study, we investigated the possibility of the induction of GAP-43 and C-Jun in axotomized neurons of Clarke's nucleus (CN) in adult rats, of which a large population undergoes degeneration several weeks after a low thoracic lateral funiculotomy of the spinal cord. In situ hybridization and immunohistochemistry revealed a transient up-regulation of GAP-43 mRNA, C-Jun protein, and its activated, phosphorylated form, peaking around 7 days after injury in preferentially large diameter CN-neurons ipsilateral and caudal to the lesion. Our results document that some populations of axotomized central nervous system neurons, similar to axotomized regenerating neurons of the peripheral nervous system, can up-regulate GAP-43 and C-Jun, even if they are destined to degenerate. This might reflect a transient regenerative capacity, which fails over time.

Microglial motility was studied in living mammalian brain tissue using infrared gradient contrast microscopy in combination with video contrast enhancement and time lapse video recording. The infrared gradient contrast allows the visualization of living cells up to a depth of 60 μm in brain slices, in regions where cell bodies remain largely uninjured by the tissue preparation and are visible in their natural environment. In contrast to other techniques, including confocal microscopy, this procedure does not require any staining or labeling of cell membranes and thus guarantees the investigation of tissue which has not been altered, apart from during preparation. Microglial cells are activated and increase in number in the facial nucleus following peripheral axotomy. Thus we established the preparation of longitudinal rat brainstem slices containing the axotomized facial nucleus as a source of activated microglial cells. During prolonged video time lapse recordings, two different types of microglial cell motility could be observed. Microglial cells which had accumulated at the surface of the slice remained stationary but showed activity of the cell soma, developing pseudopods of different shape and size which undulated and which were used for phagocytosis of cell debris. Microglial phagocytosis of bacteria could be documented for the first time in situ. In contrast, ameboid microglia which did not display pseudopods but showed migratory capacity, could be observed exclusively in the depth of the tissue. Some of these cells maintained a close contact to neurons and appeared to move along their dendrites, a finding that may be relevant to the role of microglia in “synaptic stripping”, the displacement of synapses following axotomy. This approach provides a valuable opportunity to investigate the interactions between activated microglial cells and the surrounding cellular and extracellular structures in the absence of staining or labeling, thus opening a wide field for the analysis of the cellular mechanisms involved in numerous pathologies of the CNS.

Microglia represent a population of brain macrophage precursor cells which are intrinsic to the CNS parenchyma. Transection of the facial nerve in the newborn rat causes death of the affected motor neurons which is accompanied by massive activation of local microglia. Many of these cells develop into macrophages as can be shown by immunocytochemistry for OX-42 and ED1. Using the new polyclonal microglial marker ionized calcium binding adapter molecule 1, iba1, in combination with immunocytochemical double-labeling for the proliferating cell nuclear antigen (PCNA), or [3H]thymidine autoradiography, and confocal microscopy, qualitative as well as quantitative differences can be demonstrated between the newborn and the adult axotomized rat facial nucleus. While microglial cells are the only cell population which responds to axotomy by cell division in the adult facial nucleus, GFAP positive reactive astrocytes can be shown to undergo mitosis following axotomy in the newborn rat. Furthermore, ED1 immunoreactivity, early expression of MHC class II molecules and morphological transformation of microglia into macrophages can only be observed under conditions of neuronal degeneration, i.e., in the neonatal rat facial nucleus. Thus, the combination of cellular markers described here should be useful for studies employing the neonatal rat facial nucleus as an in vivo assay system to test the efficacy of neurotrophic factors.

The neuropeptides galanin and calcitonin gene-related peptide (CGRP) are strongly up-regulated in motoneurons following axotomy. Earlier reports have suggested that peptides might be released from injured neurons to recruit surrounding glia. In this study, the effects of galanin and CGRP on cultured rat astrocytes were investigated using the expression of immediate early genes as a model for receptor-mediated transcriptional activation. Galanin was found to induce c-fos, junB, and Tis11 mRNA in cultured astrocytes, providing evidence for the presence of functional galanin receptors on neuroglial cells. In contrast, CGRP only led to the induction of c-fos and junB mRNA. Cholecystokinin (CCK-8) and substance P, which are also up-regulated in select motoneuron populations following axotomy, fail to induce immediate early genes in astrocytes, indicating specificity of neuropeptides in their ability to stimulate glial cells. The differential induction of immediate early gene expression by galanin and CGRP in astrocytes points to differences in intracellular signal transduction mechanisms. Whereas CGRP was found to stimulate the accumulation of cyclic AMP by 10- to 20-fold, galanin had no effect on basal cyclic AMP content. The effect of CGRP on cyclic AMP accumulation was completely reversed by the CGRP receptor antagonist, CGRP(8-37). These results suggest roles for galanin and CGRP in the transcriptional activation of astrocytes.

The macrophage colony-stimulating factor (MCSF) is a 40-76-kD glycoprotein that plays an important role in the activation and proliferation of microglia both in vitro and in injured neural tissue. Here, we examined the regulation of MCSF receptor (MCSFR) and MCSF in the normal and injured mouse central nervous system (CNS) by using confocal laser microscopy, quantitative immunofluorescence, and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. Immunohistochemistry on fixed, floating tissue sections demonstrated low to moderate MCSFR immunoreactivity (MCSFR-IR) on microglia in the gray and white matter throughout the mouse CNS in the forebrain, brainstem, cerebellum, and spinal cord. High levels of MCSFR-IR were restricted to the superficial layer of the spinal cord dorsal horn, substantia nigra, and area postrema, a CNS region that lacks the blood-brain barrier. CNS injury led to a strong and specific increase in MCSFR-IR in the directly injured dorsal forebrain, in the cervical spinal cord (C2) after transection of the sensory, minor occipital nerve, and in the axotomized facial motor nucleus. Further investigation at the mRNA level in the facial nucleus model showed that this increase was accompanied by a rapid induction of the transcript for MCSFR, with a peak 1-2 days after injury, but only a constitutive expression of MCSF-mRNA. In summary, although normal levels of MCSF receptor in most microglia are low, microglial activation is accompanied by a rapid and massive increase. In view of the constitutive expression of MCSF, the early upregulation of the MCSF receptor may play a central role in preparing these macrophage-related cells to take part in the cellular response to CNS injury.

During postnatal development of the rodent cerebellum, a transient enzyme activity of ecto-5′-nucleotidase has been shown in the asymmetrical synapses of Purkinje cells. The alterations of the afferent circuitry and microenvironment of the ectopic Purkinje cells present in the cerebellum of the reeler mutant mouse could enlighten parameters that influence the synaptic 5′-nucleotidase activity of these cells. Ecto-enzyme cytochemistry reveals intense 5′-nucleotidase activity in 43% of synapses of the Purkinje cells throughout the cortex and the core of the reeler cerebellar vermis, although the molecular layer displays large areas with less than 1% of labelled synapses. However, enzymatic labelling is found in considerably more Purkinje cells synapses (73%) throughout the granular layer and the subcortical mass. Climbing fiber synapses of monoinnervated Purkinje cells are labelled by 5′-nucleotidase activity in the molecular layer, as well as asymmetrical synapses made on the subjacent ectopic Purkinje cells by the multiple climbing fibers and by the heterologous afferences. The non-innervated dendritic spines of these cells are also labelled, suggesting that 5′-nucleotidase activity at postsynaptic sites of reeler Purkinje cells does not depend on the presynaptic innervation. Rather, 5′-nucleotidase enzyme activity is enhanced at theses sites when the Purkinje cells have not achieved chemodifferentiation but have conserved immature wiring, i.e., low parallel fiber and multiple climbing fiber inputs. Synapse 29:279–292, 1998. © 1998 Wiley-Liss, Inc.

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a widely expressed glycoprotein, which plays an important role in leukocyte extravasation and in the interaction of lymphocytes with antigen-presenting cells. In the current study we examined the regulation of ICAM-1 in the mouse facial motor nucleus after facial nerve transection, using immunohistochemistry, confocal laser microscopy and electron microscopy. In the normal facial nucleus ICAM-1 immunoreactivity was restricted to vascular endothelium. Transection of the facial nerve led to a strong and selective upregulation of ICAM-1 on activated microglia. Quantitation of microglial ICAM-1 immunoreactivity revealed a biphasic increase. The first peak 1-2 days post operation paralleling the early stage of microglial activation was followed by a decline at 4-7 days. The second induction of ICAM-1 occurred at day 14 accompanying the period of neuronal cell death and microglial phagocytosis of neuronal debris. Immunoelectron microscopy showed strong ICAM-1 reactivity on the cell membrane of activated microglia at day 2. During the second peak (day 14), ICAM-1 was also observed on lymphocytes adhering to phagocytotic microglia forming aggregates around neuronal debris. No immunolabelling was observed on neurons, astrocytes or oligodendroglia. These data suggest the involvement of ICAM-1 in the adhesion of activated microglia, in their phagocytosis of neuronal debris, and also in the interaction with infiltrating lymphocytes following this injury.

CD44 is a cell adhesion molecule which plays an important role in cell movement and adhesion, e.g. in lymphocyte homing and tumour metastasis. Here we studied the expression of CD44 mRNA and protein immunoreactivity in the facial nucleus after nerve injury and during the ensuing regeneration. Transection of the facial nerve led to a strong up-regulation of CD44, peaking 4 days after injury on the motoneurons of the axotomized facial nucleus. Use of the polymerase chain reaction confirmed the de novo expression of CD44 and detected only the standard haematopoietic CD44 isoform. Western blotting also detected the 76 kDa protein subtype, in line with the predicted size of the haematopoietic CD44 variant. At the ultrastructural level, CD44 immunoreactivity was restricted to the surface of the neuronal perikarya, their dendrites and axons. It was not seen in the adjacent activated astrocytes, microglia or vascular endothelia. This study shows strong up-regulation of the cell adhesion molecule CD44 on the regenerating motoneurons in the axotomized facial nucleus. These data suggest that CD44 may play a role in neurite outgrowth, in synaptic stripping or in the adhesion of activated glial cells to the perikaryal surface of the axotomized motoneurons.

Proliferation of ramified microglia is a common phenomenon in brain pathology, but little is known about how this is regulated. In the current study, we examined the effect of different cytokines on the proliferation of ramified microglia in vitro using a combination of autoradiography for [3H]-thymidine and immunocytochemical techniques. Ramified microglia were obtained using a 10-day co-culture on top of a confluent astrocyte monolayer. Addition of macrophage colony-stimulating factor (MCSF), granulocyte-macrophage colony-stimulating factor (GMCSF), and interleukin-3 (IL3), stimulated the proliferation of ramified microglia, with a 7.2-fold, 3.5-fold, and 1.5-fold increase, respectively. Of all the other cytokines tested (IL1, IL2, IL4, IL6, IL10, interferon-gamma (IFNgamma), leukemia inhibitory factor (LIF), and tumor necrosis factor-alpha (TNFalpha) only IL1 strongly enhanced proliferation. However, this effect of IL1 was indirect and could be neutralized by antibodies against MCSF and GMCSF. IL2, IL4, IL10, TNFalpha, and IFNgamma inhibited microglial proliferation. The great number of inhibitory cytokines could point to the importance of containing microglial proliferation in the central nervous system.

Nerve injuries lead to metabolic and morphological changes in the cell bodies of the neurons of origin. Increases in glucose turnover in axotomized facial and hypoglossal motor nuclei have been described. Glucose-regulated protein 78 kDa (GRP78) is implicated in cellular protein folding and subunit assembly and responds to glucose deficiency. We performed Western blot and immunohistochemistry to determine the effect of axotomy on the expression and regulation of GRP78 in the facial nucleus (FN). Facial nerve axotomy caused a larger and longer increase of GRP78 in the ipsilateral FN than in the contralateral FN. In right ipsilateral FN, axotomy resulted in elevation of GRP78 protein levels, first detected at 12 h and which reached significant, maximal induction at 24 h (75 +/- 27% increase). GRP78 protein levels decreased at later time points, but remained elevated over sham-operated controls. In contrast, no significant increase in GRP78 concentrations was found in contralateral left FN. Immunocytochemically, positive GRP78 staining was found mainly in the cytoplasm of motoneurons; there was no nuclear staining. Prominent GRP78-immunostaining appeared in axotomized motoneurons at 24 h postaxotomy as compared with the contralateral, unoperated controls. This augmentation was also observed at 4 and 7 days postaxotomy. The possibility that glucose metabolism and GRP78 levels are two parallel events in the injured facial nucleus is discussed.

To test the effects of insulin-like growth factor-I (IGF-I) on clinical deficits, lesion severity, and immune cell response in acute, non-demyelinative experimental autoimmune encephalomyelitis (EAE), we induced EAE in Lewis rats by passive transfer of an MBP-reactive T lymphocyte line. Four days after receiving 5 x 10(5) MBPL-1 T cells intravenously, ten pairs of rats had the same mild degree of tail and hind limb weakness. Ten were given 300 micrograms IGF-I i.v. twice daily for 6 days, and the other 10 received the same volume of 0.89% NaCl. Pairs of rats were sacrificed after 4 days and 6 days of IGF-I and placebo treatment and spinal cord sections were processed for immunostaining, in situ hybridization, and morphological examination. IGF-I treatment decreased clinical deficits, lesion numbers, and lesion areas significantly. Numbers of CD4-positive T cells, alpha/beta TCR-positive cells, and ED-1-positive macrophages were also significantly reduced by IGF-I treatment. Similar reductions were found in our second trial, when 11 days of placebo and IGF-I injections began the day after transfer. No demyelination was observed in either toluidine blue-stained semithin sections or sections immunostained with an antibody raised against myelin basic protein (MBP). We conclude that IGF-I-induced reductions in immune cell responses can occur in the absence of demyelination and are of major importance in decreasing clinical deficits and lesion severity in EAE. If IGF-I has similar effects in multiple sclerosis, we think that it will be useful therapeutically.

Astrocyte activation is a ubiquitous hallmark of the damaged brain and has been suggested to play an important regulatory role in the activation, survival, and regeneration of adjacent neurons, microglia, and oligodendrocytes. Little is known, however, about the endogenous signals that lead to this activation of astrocytes. Here we examined the regulation of interleukin 6 (IL6), a proinflammatory cytokine, its receptors, and the effects of IL6-deficiency in a model of traumatic central nervous system injury in the axotomized mouse facial motor nucleus. Facial nerve transection led to a massive but transient upregulation of IL6 mRNA in the disconnected motor nucleus, while IL6-receptor subunits were constitutively expressed on motoneurons and astrocytes. Absence of IL6 in genetically IL6-deficient mice led to massive reduction in the number of activated GFAP-positive astrocytes, a more moderate decrease in microglial activation and proliferation, and an increase in the late neuronal response to axotomy. These results emphasize the role of IL6 in the global regulation of neurons, astrocytes, and microglia and their activation in the injured nervous system. GLIA 19:227–233, 1997. © 1997 Wiley-Liss, Inc.

The isoquinoline PK 11195 has been suggested as a marker of glial pathology in the lesioned brain. The aim of the present study is to clarify the precise cellular location of its binding site in the central nervous system. Here, we report that in the facial nucleus after facial nerve axotomy–a lesion causing a retrograde neuronal reaction without nerve cell death while keeping the blood–brain barrier intact–activated microglia are the predominant source of lesion-induced increases of PK 11195 binding. Likewise, increased PK 11195 binding is seen in the gracile nucleus after anterograde neuronal injury following sciatic nerve transection. The peak of PK 11195 binding, using the single isomer R-PK 11195, was observed 4 days after the peripheral nerve lesion, consistent with the well-known time course of microglial activation. Photoemulsion microautoradiography confirmed the restriction of PK 11195 binding to activated microglia. The increase of PK 11195 binding in the facial nucleus seen after selective cell death of facial motoneurons by retrograde suicide transport of toxic ricin, a lesion that is accompanied by the rapid transformation of microglia into phagocytes, was no higher than that seen following axotomy. This suggests that the full transformation of microglia into parenchymal phagocytes is not necessary to reach maximal levels of PK 11195 binding. PK 11195, therefore, is a well-suited marker to detect microglial activation in areas of subtle brain pathology, where neither a disturbance of the blood–brain barrier function nor the presence of macrophages and inflammatory cells indicate an on-going disease process.

Brain lesions, even of the most subtle type, are accompanied by the activation of microglia, the main immune cells of the brain. Microglial cells dramatically increase in number through proliferation and adhere to the injured neurons, where they displace the synaptic input. After proliferation, microglia gradually migrate into the nearby parenchyma and appear to decrease in number. Here we examined the possible involvement of apoptosis in the regulation of the microglial cell number using Terminal transferase mediated d-UTP Nick End-Labelling (TUNEL). In vitro, cell death is a common phenomenon in microglial cell cultures, and is enhanced by the withdrawal of the mitogen, granulocyte-macrophage colony stimulating factor. In vivo, application of the TUNEL-reaction revealed TUNEL-positive microglia beginning at day 4, with a peak 7 days after transection of the facial nerve. Surprisingly, TUNEL-labelling in vivo was localized on the outer side of the nuclear membrane and in the microglial cytoplasm, with very little staining within the nucleus itself. These TUNEL-labelled cells also lacked other classic morphological signs of apoptosis, like membrane blebbing, chromatin condensation and apoptotic bodies. These data suggest that the regulation of post-mitotic microglia is not mediated by the classic pathway of apoptosis.

Thrombospondin (TSP) is a multifunctional extracellular matrix protein that plays a role in neuronal migration and axonal outgrowth in the developing central nervous system. In the current study we have examined the localization and regulation of TSP immunoreactivity (TSP-IR) during neuronal regeneration in the axotomized facial motor nucleus using Western blotting and light and electron microscopy. Transection of the facial nerve led to a gradual increase in TSP-IR in the regenerating motoneurons, peaking 4-7 days after injury (DAI). In addition to regenerating neurons, axotomy also caused a rapid upregulation of TSP-IR on activated microglia throughout the facial nucleus, with a maximum of 2-3 DAI, and a second increase at 14-21 DAI on microglial aggregates surrounding degenerating motoneurons and in neuronophagic microglia. In summary, injury leads to the induction of thrombospondin on axotomized neurons and activated microglia, peaking at the times of maximal posttraumatic microglial proliferation and during neuronal phagocytosis. Since thrombospondin is a multimodal extracellular matrix protein with a variety of cell attachment sites, thrombospondin might serve to link microglia and injured neurons, followed by microglial proliferation and removal of the neuronal debris.

Light microscopic enzyme histochemistry was employed to study the alterations of acetylcholinesterase (AChE) within lumbosacral ventral horn neurons at survival times of 1, 4, 7, 14, 28, 60, and 90 days after low thoracic spinal cord hemisection in adult rats. The intensity of histochemical staining was quantified using densitometric techniques. Virtually all ventral horn neurons of sham-operated and unoperated animals, which served as controls, displayed intense AChE staining. Hemisection of the spinal cord induced a transient ipsilateral decrease of AChE staining in most neuronal cell bodies and in the neuropil of lamina IX at all segmental levels caudal to the lesion. Quantitative analysis of representative segments revealed a reduction of AChE in the ventral horn during a postoperative (p.o.) period of 1 to 28 days followed by a phase of recovery over the next two months. AChE activity still remained slightly reduced, even at 90 days p.o. The transient decrease in AChE is a well-known metabolic response of axotomized motoneurons. However, the observed changes of AChE reactivity in intact motoneurons ipsilateral and caudal to the hemisection are presumably induced by the interruption of supraspinal descending pathways. These metabolic changes may functionally affect the whole motor unit and be involved in the disturbances of motor function following spinal cord injury.

When scientists hear about scientific fraud, they quickly denounce the culprits as not being ‘true’ scientists. The true scientist, they argue, is only interested in unveiling step by step the countless enigmas of nature. He or she labours long hours and weekends at a desk or in the laboratory to find the truth, not to invent it. When describing her attitude to science, Nobel Prize‐winning cytogeneticist Barbara McClintock once said, “I was so interested in what I was doing I could hardly wait to get up early in the morning and get at it. One of my friends said I was a child, because only children can't wait to get up in the morning to get at what they want to do” (National Academy of Sciences, 1995). It is probably the scientist's greatest motivation and satisfaction to understand or observe what has never been understood or described before. But is the ensuing ‘eureka’ experience really the greatest award for all that hard work? Is this the only reason for doing science? Or is this too idealistic and naive a view of scientists, one that ignores the fact that our profession may be driven by other ambitions, such as glory, recognition or even money? > Science and scientists have been entrusted to set up their own rules, based on trust, respect and the welfare of society We scientists think that we enjoy the highest degree of freedom in our work. Many societies have also accepted the notion that research is done best when unhindered, and have included in their constitutions the freedom of science as a basic human right. Science and scientists have been entrusted to set up their own rules, based on trust, respect and the welfare of society. The general public shares this idealistic view of how research is done …

Factors affecting gene expression in microglial cells were investigated using the induction of immediate early genes in cultured microglia as a model. In particular, the actions of calcitonin gene-related peptide (CGRP) and ATP, both of which have been proposed as signalling molecules in the activation of glial cells, were evaluated using Northern blotting and in situ hybridization methods. In the presence of CGRP, c-fos and junB mRNAs accumulated in microglial cultures, whereas no significant change in c-jun and TIS11 mRNAs occurred. A similar pattern of immediate early gene activation was obtained when adenylate cyclase was stimulated with forskolin. CGRP also stimulated cyclic AMP accumulation with a half-maximal effect in the range 2–5 nM, suggesting a possible role for cyclic AMP as a mediator of the effects of CGRP on gene expression. In contrast to the selective induction of c-fos and junB by CGRP and forskolin, ATP led to the accumulation of all four immediate early genes studied, i.e., c-fos, junB, c-jun, and TIS11. Similar results were obtained when protein kinase C was stimulated with phorbol ester indicating that the induction of immediate early gene expression by ATP and CGRP involves different intracellular mechanisms. The action of ATP was mimicked by ADP and the poorly hydrolyzable analogues, ADPβS and 2-methylthio ATP, but not by β,γ-methylene ATP, AMP, or adenosine, indicating that the receptor mediating the actions of ATP on microglial gene expression is probably of the P2Y-purinoreceptor type. The results suggest roles for CGRP and ATP as transcriptional activators in microglial cells. © 1995 Wiley-Liss, Inc.

The β-amyloid precursor protein (APP) bears characteristics of an acute-phase protein and therefore is likely to be involved in the glial response to brain injury. In the brain, APP is rapidly synthesized by activated glial cells in response to comparatively mild neuronal lesions, e.g., a remote peripheral nerve injury. Perfusion deficits in the brain result largely in neuronal necrosis and are a common condition in elderly patients. This neuronal necrosis is accompanied by a pronounced reaction of astrocytes and microglia, which can also be observed in animal models. We have therefore studied in the rat, immunocytochemically, the induction of APP after 30 min of global ischemia caused by four-vessel occlusion. The postishemic brain injuries were examined at survival times from 12 h to 7 days. From day 3 onward, APP immunoreactivity was strongly induced in the CA1and CA4regions of the rat dorsal hippocampus as well as in the dorsolateral striatum. In these areas, the majority of APP-immunoreactive cells were reactive glial fibrillary acidic protein (GFAP)-positive astrocytes, as shown by double-immunofluorescence labeling for GFAP and APP. Additionally, small ramified cells, most likely activated microglia, expressed APP immunoreactivity. In contrast, in the parietal cortex, APP immunoreactivity occurred focally in clusters of activated microglia rather than in astrocytes, as demonstrated by double-immunofluorescence labeling for APP and the microglia-binding lectin Griffonia simplicifolia isolectin B4. In conclusion, following global ischemia, APP is induced in reactive glial cells with spatial differences in the distribution pattern of APP induction in astrocytes and microglia.

The controversial hypothesis that intraspinal sprouting by dorsal root axons promotes reinnervation of partially denervated neurons caudal to a low thoracic cord hemisection was re-investigated in rats using quantitative immunohistochemical analysis of the neural specific growth-associated protein B-50 (GAP-43) at postoperative survival times of 3, 10, 21, 42, and 90 days. The lack of increase in B-50-immunoreactivity in all segments below the hemisection at all survival times does not support the concept of intraspinal sprouting following the removal of supraspinal descending pathways.

Lesions to the nervous system are nearly universally accompanied by a glial response involving both microglia and astrocytes. The growth and immunoregulatory cytokine transforming growth factor-beta 1 (TGF-beta 1) has potent effects on glial cells in vitro and may play a role in regulating glial activation in vivo. Though present only at very low levels in the normal brain, TGF-beta 1 mRNA is strongly upregulated in a number of different experimental models suitable to study glial responses. Following axotomy of the facial nerve of the rat, about a three-fold increase of TGF-beta 1 mRNA in the regenerating nucleus was observed with a time-course closely matching that of glial activation. Putative activated microglial cells are the major cellular source as revealed by in-situ hybridization. TGF-beta 1 was also found to be upregulated around brain tumors, in the spinal cord in response to peripheral nerve inflammation and in the postischemic hippocampus. In all systems investigated, TGF-beta 1 mRNA could be localized predominantly to cells with the typical nuclear morphology of microglia. In the peripheral nervous system, nerve transection leads to a massive increase in TGF-beta mRNA expression both proximal and distal to the cut site. However, whereas TGF-beta 1 mRNA is restricted to the nerve stump in the proximal segment, expression is diffuse and widespread throughout the denervated distal segment where it was localized mainly to cells with macrophage morphology.(ABSTRACT TRUNCATED AT 250 WORDS)

Lesions of the optic nerve in young animals lead to rapid retrograde degeneration of the axon stumps and to death of retinal ganglion cells. We injected different neurotrophic factors into the eye at the time of an intracranial freeze-crush lesion of the optic nerve in 8 day old rats. Optic nerve axons were visualized by anterograde tracing with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and by electron microscopy. The lesion induced a rapid die-back of the axons, which could be prevented by BDNF and to a lesser extent by neurotrophin-3 (NT-3) or ciliary neurotrophic factor (CNTF). No effect was seen in animals injected with nerve growth factor (NGF) or a mixture of acidic and basic fibroblast growth factor (FGF). In contrast to this effect on the axons, none of these factors was able to counteract the rapidly progressing degeneration of the retinal ganglion cells. These results suggest a selective influence of BDNF on the mechanisms responsible for the maintenance of optic nerve axons.

The adenosine-producing ecto-enzyme 5′-nucleotidase has recently been assigned to malleable axon terminals in both the developing and regenerating adult nervous system, but is otherwise only glia-bound. Using a cytochemical lead method, we now show that 5′-nucleotidase activity is localized predominantly at glomerular and mitral synapses within the main olfactory bulb of normal, adult rats. As these terminals are prone to synaptic turnover even at maturity, the present findings favour the view that this enzyme constitutes a marker molecule for plastic synapses. It is suggested that functions of 5′-nucleotidase in purinergic neuromodulation and cell adhesion are unique to the olfactory bulb, and implied in synaptic arrangements and information processing.

Clinical and experimental data have indicated an up-regulation of amyloid precursor protein (APP) after various types of CNS injury. In the present study the cellular source of lesion-induced APP has been investigated in a neurotoxic CNS model. Quinolinic acid injection into the striatum results in neuronal degeneration, while glial cells survive. APP immunoreactivity was detected in glial cells starting at postoperative day 3 and persisted until day 21, the last time point studied. Double immunocytochemistry identified the majority of APP-immunoreactive cells as glial fibrillary acidic protein-immunoreactive astrocytes. There was no evidence of amyloid fibril deposition during this time. It is concluded that following excitotoxic neuronal degneration APP is mainly produced by reactive astrocytes in the lesioned area.

Ciliary neurotrophic factor (CNTF) was first identified and partially purified from embryonic chick eye tissues. Subsequently, it was shown that CNTF is also present in large amounts in sciatic nerves of adult rats and rabbits, which led to its final purification and cloning. CNTF is not secreted by the classical secretory pathway involving the endoplasmatic reticulum and Golgi complex, but can be detected in high quantities within the cytoplasm of myelinating Schwann cells and astrocytes using immunohistochemistry. CNTF supports survival and/or differentiation of a variety of neuronal cell types including sensory, sympathetic, and motoneurons. Also, nonneuronal cells, such as oligodendrocytes, microglial cells, liver cells, and skeletal muscle cells, respond to exogenously administered CNTF, both in vitro and in vivo. During development, expression of CNTF is very low, if indeed it is expressed at all, and the phenotype of mice lacking endogenous CNTF after inactivation of the CNTF gene by homologous recombination suggests that CNTF does not play a crucial role for responsive cells during embryonic development. However, motoneurons are lost postnatally in mice lacking endogenous CNTF, suggesting that CNTF acts physiologically on the maintenance of these cells. The ability of exogenous CNTF to protect against motoneuron loss following lesion or in other animal models indicates that CNTF might be useful in the treatment of human motoneuron disorders, provided appropriate means of administration can be found.

NF-kappa B is an inducible transcription factor involved in the induction of multiple genes during inflammatory processes. So far the information pertaining to the role of NF-kappa B in autoimmune processes has been restricted to in vitro analysis. To further characterize the role of NF-kappa B in vivo, the involvement of NF-kappa B has been studied by immunocytochemistry in T cell-mediated autoimmune encephalomyelitis (EAE) of the Lewis rat. In non-diseased animals, immunoreactivity for the DNA-binding subunit p50 and for the DNA-binding and transactivating subunit p65 was low and restricted to the surface of small to medium-sized blood vessels. Strong immunoreactivities for p50 and p65 were detected at the peak of clinical disease. At the recovery stage of EAE, p50 and p65 immunoreactivities had declined to base line levels. Within the resident glial cell population, p50 and p65-immunoreactive cells were identified as OX-42-positive microglia. GFAP-positive astrocytes did not show significant p50 or p65 immunoreactivity. In the core and the vicinity of perivascular inflammatory lesions, both ED-1-positive macrophages and W3/13-positive T lymphocytes and monocytes were strongly immunoreactive for NF-kappa B. Our data suggest a crucial involvement of the transcription factor NF-kappa B in autoimmune diseases of the central nervous system. Furthermore, NF-kappa B appears as a useful marker for inflammatory processes in vivo.

There is increasing evidence that deprivation of the retrogradely transported neurotrophic protein nerve growth factor (NGF) accounts for some functional deficits known to occur in experimental diabetic neuropathy. Here we have studied changes in the axonal transport of endogenous NGF, NGF receptor (NGFR), and NGFR saturation (NGF/NGFR ratio) in the rat sciatic nerve after 2 months of streptozotocin (STZ)-induced diabetes mellitus. Compared with vehicle-treated control rats (blood glucose: 6-12 mM), there was a very clear reduction in the retrograde transport of NGF by 50% (P < 0.001) in STZ-treated, diabetic animals (blood glucose: 33-62 mM). No significant reduction in NGF axonal transport was observed in a subpopulation of STZ-treated rats (poor responders) with nearly normal glucose levels (range: 9-12 mM). No change was observed in any group in the retrograde transport of NGFR. Compared with control rats, however, the apparent NGFR saturation was reduced by 45% (P < 0.002) in STZ diabetics, whereas no change in NGFR saturation was observed in the STZ-poor responders. Moreover, the NGFR saturation and amount of retrogradely transported NGF were negatively correlated to the individual glucose concentration in diabetics (r2 = 0.47 and 0.55, respectively; P < 0.0001). These findings indicate that, while NGFR expression is normal in the STZ-diabetic neuropathy model, the marked decrease in receptor saturation observed in diabetics may reflect low peripheral NGF levels, which in consequence leads to the apparent deprivation of neuronal NGF in diabetic rats.

Activation and proliferation of microglia are common cellular hallmarks in many different pathological processes of the central nervous system. Although a number of colony-stimulating factors enhance microglial proliferation in vitro, little is known about the endogenous mitogens. In the present study we show a strong and selective inhibition of microglial proliferation in the facial nucleus of osteopetrotic (op/op) mice, with a genetic deficiency in biologically active macrophage colony-stimulating factor (MCSF). Posttraumatic activation of adjacent axotomized neurons and reactive astrocytes was not affected, emphasizing the specificity of MCSF as a microglial growth factor.

In development, the ectoenzyme 5′-nucleotidase marks maturing cerebellar and cortical synapses, but it is localized in glial cells in the normal, adult nervous system. With a histochemical lead technique, we have now investigated its localization during reactive synaptogenesis in the dentate gyrus of adult rats deprived of entorhinal afferents. A band of enhanced 5′-nucleotidase reaction product was present in the outer portions of the dentate molecular layer between 5 and 75 days after destruction of the ipsilateral entorhinal cortex. At the ultrastructural level, 5′-nucleotidase-positive microglia and degenerating axon terminals were numerous within this band during the first postoperative week. Between Days 7 and 75, intact synapses were found that exhibited 5′-nucleotidase reaction product in their clefts. Astrocytic labeling was abundant. No enzyme-positive synapses and few labeled glial elements were present in the control molecular layer. Conspicuous 5′-nucleotidase activity within synaptic clefts of mossy, fiber terminals was present between Postoperative Days 10 and 40 on the operated side, but the staining was sporadic on the control side. We conclude that 5′-nucleotidase is associated with lesion-induced synaptic remodeling in the dentate gyrus. The band of 5′-nucleotidase reaction product within the outer molecular layer corresponds to the zone where the lesioned entorhinal fibers degenerate and where other afferents sprout. Here, the transient appearance of 5′-nucleotidase within synaptic clefts parallels the time course of synaptic reinnervation. The enzyme is also indicative of the sprouting response of mossy fiber terminals. Functional properties of 5′-nucleotidase in purinergic neuromodulation and cellular adhesion may be relevant for the generation and plasticity of synaptic contracts.

We have investigated the T cell receptor (TCR) repertoire in the inflammatory infiltrates of T line-transferred experimental autoimmune encephalomyelitis (EAE) of the Lewis rats. Using a panel of TCR Vβ-specific monoclonal antibodies (mAbs) and immunocytochemistry, we studied the nature of the T cells entering the central nervous system (CNS) after transfer of either myelin basic protein (MBP)-reactive, or MBP-reactive but non-encephalitogenic T cell lines. All the MBP-specific T cell lines predominantely used the Vβ8.2 TCR chain. T cell lines specific for the tuberculin purified protein derivative (PPD), using TCR V genes different from Vβ8.2, served as controls. We first studied the time course of T cells entering the CNS. In all recipient rats, small, but significant numbers of αβ-TCR-expressing infiltrate cells appeared in the CNS within the first 24 h after T cell transfer. In animals injected with either type of MBP-reactive T cells, the early infiltrate cells were preferentially located within the parenchyma of the spinal cord, while in PPD T lineinjected rats, the lymphocytes were mostly found in the meninges. TCR Vβ gene usage was examined on the peak of clinical disease. Six days after T cell transfer, the TCR repertoire used by infiltrating lymphocytes in general seemed to be highly diverse. None of the Vβ isotypes examined (i.e. Vβ8.2, Vβ8.5 or Vβ10) was used by a major population of the αβ-TCR-positive T cells. A more detailed, quantitative analysis of individual infiltrate compartments revealed, however, a preferential accumulation of Vβ8.2-positive T cells within the parenchyma. In contrast, perivascular infiltrating cells used Vβ genes randomly. Our results confirm first that activated T lymphocytes enter the brain rapidly irrespective of their antigen specificity. Second, the data show that most of the perivascular infiltrate T cells in the acute EAE lesion are host-derived, recruited presumably from the recirculating T cell pool, while the encephalitogenic, Vβ8.2-positive T cells preferentially persist within the parenchyma.