Ingo Bechmann

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.

Immunity is required to eliminate dangerous or degenerated material and to support regeneration, but also causes significant parenchymal damage. In the eye and the brain, in which cornea and lens poorly regenerate and neurons are hardly replaceable, early transplantation experiments demonstrated remarkable tolerance to various grafts. This “immunologically privileged status” (Billingham and Boswell, 1953) may reflect evolutionary pressure to down-modulate certain actions of immune cells within particularly vulnerable tissues. As an example, tolerating certain “neurotrophic” viruses may often be a more successful strategy for survival than the elimination of all infected neurons. While several constitutive and inducible signals maintaining or re-establishing immune tolerance within the brain have been identified, it has also become evident that the resulting anti-inflammatory environment limits certain beneficial effects of neuroinflammation such as neurotrophin secretion or glutamate buffering by T-cells and the clearance of growth-inhibiting myelin or amyloid. Following spinal cord injury, the costs and benefits of neuroinflammation seem to come close because enhancing as well as suppressing innate or adaptive immunity caused amelioration and aggravation of functional regeneration in similar experiments. Evaluating such balances has also begun in (animal models of) Alzheimer’s disease, central nervous system trauma, and stroke, and the appreciation of the beneficial side of neuroinflammation has caused a rethinking of the ill-defined use of immune suppressants. As dual roles for individual molecules have been recognized (Merrill and Benveniste, 1996), we are uncovering an already fine-tuned system, but the challenge remains to further support beneficial immune cascades without causing additional damage, and vice versa.

This chapter begins with a brief overview of the different brain phagocyte populations in regard to the kinetics of their permanent supplementation by blood-derived precursors. It then provides a timely view of how (astro)glial cells regulate the local adoption of invading antigen-presenting cells (APCs).

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 postlesion, 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 (lpr)‐ 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 NMD A 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.

Virchow–Robin's perivascular spaces lie between the basement membrane around pericytes and the basement membrane at the surface of the glia limitans of the brain vessels. They are directly connected to the subpial space and harbour a population of cells distinct from pericytes, perivascular microglia and other cells within perivascular spaces (e.g. T cells and mast cells) in their ability to quickly phagocytose particles from the cerebrospinal fluid (CSF). Morphology, function, and cell surface proteins of these perivascular cells suggest an origin from the monocyte/macrophage lineage. It is currently unclear to what extent these brain perivascular cells represent a resident population of histiocytes or undergo continuous supplementation from blood monocytes. Using transplants of green‐fluorescent‐protein (GFP)‐transfected bone marrow cells, we therefore investigated the replacement of perivascular cells by blood‐borne macrophages in adult mice. GFP‐positive cells in the perivascular spaces were found as early as 2 weeks post transplantation. The substitution of host perivascular cells by donor‐derived macrophages was then evaluated using immunocytochemistry and intraventricular injection of hydrophilic rhodamine‐fluorescent tracers. Such tracers diffuse along perivascular spaces and are subsequently phagocytosed by perivascular cells leading to stable phagocytosis‐dependent labelling. Thus, the population of newly immigrated macrophages could be related to the total number of perivascular macrophages. This approach revealed a continuous increase of donor‐derived perivascular cells. At 14 weeks post transplantation, all perivascular cells were donor‐derived. These data show that brain perivascular cells are a population of migratory macrophages and not resident histiocytes.