Circulating monocytic cells infiltrate layers of anterograde axonal degeneration where they transform into microglia.

In this study, we demonstrate the infiltration of blood-derived monocytic cells and their morphologic transformation into microglia in zones of acute, anterograde (Wallerian) axonal degeneration induced by entorhinal cortex lesion (ECL). ECL was performed in mice which had received green fluorescent protein (GFP)-transduced bone marrow grafts allowing identification of blood-derived elements within the brain. While in the unlesioned hemisphere GFP+ cells were restricted to perivascular and leptomeningeal sites, many round fluorescent cells appeared in hippocampal zones of axonal degeneration at 24 h post lesion (hpl). Within 72 hpl, these GFP+ cells acquired ramified, microglia-like morphologies, which persisted for at least 7 days post ECL. Differentiation of GFP+ cells into glial fibrillary acidic protein (GFAP)+ astrocytes was never observed. To exclude that this recruitment is an artifact of irradiation or bone marrow transplantation, the fluorescent cell tracker 6-carboxylfluorescein diacetate (CFDA) was injected into spleens of normal mice 1 day before ECL. Again, fluorescent cells appeared at the lesion site and along the layers of axonal degeneration at 48 hpl and CFDA+/MAC-1+, cells exhibited amoeboid and ramified morphologies. Thus, blood-derived cells infiltrate not only the site of mechanical lesion, but also the layers of anterograde axonal degeneration, where they readily transform into microglia-like elements. A role for infiltrating leukocytes in facilitating or modulating postlesional plasticity, e.g., by phagocytosis of growth-inhibiting myelin should now be considered. Moreover, monocytic cells may serve as vehicles to transport therapeutic substances such as neurotrophic factors or caspase inhibitors to zones of axonal degeneration.

CXCR3-dependent microglial recruitment is essential for dendrite loss after brain lesion.

Microglia are the resident macrophage population of the CNS and are considered its major immunocompetent elements. They are activated by any type of brain pathology and can migrate to the lesion site. The chemokine CXCL10 is expressed in neurons in response to brain injury and is a signaling candidate for activating microglia and directing them to the lesion site. We recently identified CXCR3, the corresponding receptor for CXCL10, in microglia and demonstrated that this receptor system controls microglial migration. We have now tested the impact of CXCR3 signaling on cellular responses after entorhinal cortex lesion. In wild-type mice, microglia migrate within the first 3 d after lesion into the zone of axonal degeneration, where 8 d after lesion denervated dendrites of interneurons are subsequently lost. In contrast, the recruitment of microglia was impaired in CXCR3 knock-out mice, and, strikingly, denervated distal dendrites were maintained in zones of axonal degeneration. No differences between wild-type and knock-out mice were observed after facial nerve axotomy, as a lesion model for assessing microglial proliferation. This shows that CXCR3 signaling is crucial in microglia recruitment but not proliferation, and this recruitment is an essential element for neuronal reorganization.

Entorhinal cortex lesion in the mouse induces transsynaptic death of perforant path target neurons.

Entorhinal cortex lesion (ECL) is a well described model of anterograde axonal degeneration, subsequent sprouting and reactive synaptogenesis in the hippocampus. Here, we show that such lesions induce transsynaptic degeneration of the target cells of the lesions pathway in the dentate gyrus. Peaking between 24 and 36 hours post-lesion, dying neurons were labeled with DeOlmos silver-staining and antisera against activated caspase 3 (CCP32), a downstream inductor of programmed cell death. Within caspase 3-positive neurons, fragmented nuclei were co-localized using Hoechst 33342 staining. Chromatin condensation and nuclear fragmentation were also evident in semithin sections and at the ultrastructural level, where virtually all caspase 3-positive neurons showed these hallmarks of apoptosis. There is a well-described upregulation of the apoptosis-inducing CD95/L system within the CNS after trauma, yet a comparison of caspase 3-staining patterns between CD95 (Ipr)- and CD95L (gld)-deficient with non-deficient mice (C57/bl6) provided no evidence for CD95L-mediated neuronal cell death in this setting. However, inhibition of NMDA receptors with MK-801 completely suppressed caspase 3 activation, pointing to glutamate neurotoxicity as the upstream inducer of the observed cell death. Thus, these data show that axonal injury in the CNS does not only damage the axotomized neurons themselves, but can also lethally affect their target cells, apparently by activating glutamate-mediated intracellular pathways of programmed cell death.

The correlation between severity of paraparesis and reduced density of resident antigen-presenting cells implicates an unknown role for the spinal perivascular macrophages in experimental autoimmune encephalomyelitis in rats.

To study alterations in the morphology of spinal perivascular macrophages (SPM) during experimental allergic encephalomyelitis (EAE), we labelled SPM by intracerebroventricular (i.c.v.) injection of horseradish peroxidase (HRP). As earlier electron microscopical analysis had shown severely damaged SPM, we suspected that each inflammatory process is accompanied by the death of SPM. To prove this hypothesis, we compared the numerical density of resident SPM (i.c.v. labelled in red by Fluoro-Ruby) with that of monocytes/macrophages recruited to the perivascular space (i.c.v. labelled in green by Fluoro-Emerald). At the peak of paraparesis, the density of resident SPM was reduced by 33%. Since this reduction contrasted sharply with earlier data indicating a massive increase in the density of SPM during EAE, we checked our findings after general or selective suppression of the immune response to myelin autoantigens with the drugs dexamethasone and copaxone, respectively. Dexamethasone treatment commenced after evident paraparesis accelerated recovery, but did not influence SPM density. Immunisation with copaxone completely prevented the occurrence of EAE (monitored by video-based motion analysis of tail motility); the subsequent histological analysis revealed no reduction in SPM density. Based on this inverse correlation between the severity of EAE and the density of resident macrophages, we conclude that SPM plays an important role in the pathogenesis of EAE.

IDO (indolamine 2,3-dioxygenase) expression and function in the CNS.

From an immunological perspective the placenta is an allograft and therefore requires a special immune suppressive status termed immune privilege. Other organs of the body, which possess poor regenerative capacity share this special status, e.g. the brain, the eye and the gonads. The biological function of immune privilege in all these tissues is to protect them from inflammation-mediated injury. The mechanism maintaining immune privilege are poorly understood and are apparently site-specific. In the placenta, inhibition of IDO leads to spontaneous abortion, showing the crucial role of this enzyme for the maintenance of immune privilege. By catabolizing extracellular tryptophan IDO inhibits local T cell proliferation thereby preventing placental rejection. Here, we show that this mechanism can also be active in suppressing inflammatory responses in the CNS, where inflammations must be tightly regulated to prevent the loss of irreplaceable neurons. Employing RT-PCR and Western blot analysis we could show that, upon activation with the pro-inflammatory cytokine interferon-gamma, astrocytes and microglia are capable of expressing IDO in vitro and in vivo. To test the functional capacity of IDO in the CNS, we performed blockade experiments using actively induced experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease which correlates to the human disease multiple sclerosis (MS). Inhibition of IDO activity by daily subcutaneous administration of the specific IDO inhibitor 1-methyl-DL-tryptophan during EAE significantly exacerbates EAE, shown by comparing clinical disease scores. Thus, local expression of IDO during inflammation is apparently a self-protection mechanism which limits antigen-specific immune responses in the CNS.

Comparison of neuronal density and subfield sizes in the hippocampus of CD95L-deficient (gld), CD95-deficient (lpr) and nondeficient mice.

During brain development, the majority of neurons undergo programmed cell death. It is now clear that caspases are involved in this process of selective induction of neuronal apoptosis, yet the signals for this caspase activation remain undefined. As an upstream activator of these enzymes, the death receptor CD95 (Fas, APO1) was recently shown on neurons in the cornu ammonis (CA)2 and CA3 hippocampal subfields of early postnatal mice and rats. In vitro, cortical neuroblast cells are susceptible to CD95 ligand (CD95L, FasL, APO-1 L)-induced apoptosis. It was therefore suggested that the CD95/CD95L system is involved in neuronal apoptosis during hippocampal development. We therefore performed a blinded study comparing field size and neuronal density in the hippocampi of p20 CD95-deficient (lpr), CD95L-deficient (gld) and C57 mice. Whereas field sizes did not differ significantly between these strains, paired Mann-Whitney analyses revealed an increased number of neurons in the CA2 regions of CD95-deficient mice (P = 0.008), and minor, yet at 1% nonsignificant, differences between gld, lpr and C57 strains in the CA1 and CA3 regions. However, joint comparison of the three strains using the Kruskal-Wallis test rendered all differences insignificant. We conclude that the CD95/CD95L system is either not involved, or can be replaced by alternate mechanisms in the control of neuronal populations during hippocampal development.