Ulrike Gimsa

Turnover of Rat Brain Perivascular Cells

Brain perivascular spaces harbor a population of cells which exhibit high phagocytic capacity. Therefore, these cells can be labeled by intraventricular injection of tracers. Such perivascular cells at the interface between blood and brain are believed to belong to the monocyte/macrophage lineage and to be involved in antigen presentation. Currently, it is unclear whether these cells undergo a continuous turnover by entering and leaving the bloodstream. Using bone-marrow-chimeric animals, migration of donor macrophages into brain perivascular spaces has been reported. On the other hand, following intracerebral injection of india ink into nontransplanted animals, ink-labeled perivascular cells were still found 2 years after injection, suggesting a high stability of this cell pool. Thus, the turnover of perivascular cells observed in chimeras might be a result of bone marrow transplantation rather than a physiological occurrence. To address this issue, we monitored de novo invasion of macrophages into perivascular spaces of apparently healthy adult rats by applying techniques other than bone marrow transplantation, (i) consecutive injections of different tracers and (ii) ex vivo isolation of macrophages from the blood, cell labeling, and reinjection into the same animal to avoid MHC mismatch. Both approaches revealed vivid de novo invasion of macrophages into perivascular spaces, but not into brain parenchyma, rendering untenable the concept of perivascular cells forming a stable population of macrophages in the brain. Thus, brain perivascular spaces are under permanent immune surveillance of blood borne macrophages in normal adult rats.

Choosing electrodes for deep brain stimulation experiments--electrochemical considerations.

Deep brain stimulation (DBS) is a therapy of movement disorders including Parkinsons disease (PD). Commercially available electrodes for animal models of Parkinsons disease vary in geometry and material. We characterized such electrodes and found a drift in their properties within minutes and up to about 60 h after immersion in cell culture medium, both with and without a stimulation signal. Electrode properties could largely be restored by proteolytic treatment for platinum/iridium electrodes but not for stainless steel ones. Short-term drift and irreversible aging could be followed by impedance measurements. Aging was accompanied by metal corrosion and erosion of the plastic insulation. For both materials, the degradation rates depended on the current density at the electrode surfaces. Fourier analysis of the DBS pulse (60 micros, repetition rate 130 Hz) revealed harmonic frequencies spanning a band of more than three decades, with significant harmonics up to the MHz range. The band is located in a window imposed by electrode processes and capacitive cell membrane bridging at the low and high frequency ends, respectively. Even though electrode processes are reduced at higher frequencies they only vanish above 1 MHz and cannot be avoided. Therefore, the use of inert electrode materials is of special importance. The neurotoxicity of iron makes avoiding stainless steel electrodes imperative. Future developments need to avoid the use of corrosive materials and current density hot spots at the electrode surface, and to reduce low frequency components in the DBS pulses in order to diminish electrode processes.

Dissecting the effects of mtDNA variations on complex traits using mouse conplastic strains

Previous reports have demonstrated that the mtDNA of mouse common inbred strains (CIS) originated from a single female ancestor and that mtDNA mutations occurred during CIS establishment. This situation provides a unique opportunity to investigate the impact of individual mtDNA variations on complex traits in mammals. In this study, we compiled the complete mtDNA sequences of 52 mouse CIS. Phylogenetic analysis demonstrated that 50 of the 52 CIS descended from a single female Mus musculus domesticus mouse, and mtDNA mutations have accumulated in 26 of the CIS. We then generated conplastic strains on the C57BL/6J background for 12 mtDNA variants with one to three functional mtDNA mutations. We also generated conplastic strains for mtDNA variants of the four M. musculus subspecies, each of which contains hundreds of mtDNA variations. In total, a panel of conplastic strains was generated for 16 mtDNA variants. Phenotypic analysis of the conplastic strains demonstrated that mtDNA variations affect susceptibility to experimental autoimmune encephalomyelitis and anxiety-related behavior, which confirms that mtDNA variations affect complex traits. Thus, we have developed a unique genetic resource that will facilitate exploration of the biochemical and physiological roles of mitochondria in complex traits.

Astrocyte-induced T cell elimination is CD95 ligand dependent

The brain has an intrinsic capacity to remove infiltrating T cells by inducing apoptosis. However, the pathways and cellular components driving this process are still under debate. Astrocytes seem to play an important role because they colocalize with apoptotic lymphocytes in vivo and induce apoptosis of transformed T cells in vitro. Since we previously demonstrated the expression of the death ligand CD95L (APO-1L/FasL) on astrocytes in the brain, we wanted to know whether nontransformed astrocytes induce cell death in nontransformed T cells, reflecting the in vivo situation and, if so, whether CD95/CD95 ligand interaction is important. T cell apoptosis measured by Annexin V binding and DNA fragmentation was significantly lower using CD95 ligand-deficient (gld) astrocytes compared to nondeficient controls. Moreover, neutralizing anti-CD95 ligand antibody reduced astrocyte-induced T cell apoptosis. Thus, adult astrocytes are capable of inducing the apoptotic death of T cells by involving the CD95/CD95 ligand pathway without undergoing cell death in vitro. Since astrocytic end-feet contribute to the formation of the blood-brain barrier, this depletion mechanism may play an important role as the first line of defense in the brain.

Reactive astrocytes upregulate Fas (CD95) and Fas ligand (CD95L) expression but do not undergo programmed cell death during the course of anterograde degeneration.

Tissue homeostasis is determined by a balance between proliferation and apoptosis. Various lesions in the brain are accompanied by proliferation and subsequent death of glial cells, but the mechanisms that limit this expansion of glial populations remains unknown. One possible candidate is the death ligand, FasL, and its receptor Fas, because the expression of both proteins was reported on glial cells. To elucidate the expression and putative function of Fas and FasL on proliferative glial cells, we performed stereotactic lesion of the entorhinal cortex of adult rats. Such lesions induce proliferation of astrocytes and microglial cells in the hippocampal fields of anterograde degeneration. Subsequently, the total number of both cell types returns to pre‐lesion counts. We found that Fas and FasL is strongly upregulated on astrocytes in the zone of anterograde degeneration with a peak 5 days postlesion (dpl) and a return to control levels at 10 dpl. However, evidence for astrocytic cell death was neither detected by TUNEL staining, immunocytochemistry for c‐Jun, and apoptosis‐specific protein (ASP), nor by staining for morphologic hallmarks of apoptotic or necrotic cell death at the light and electron microscopic level. Thus, increased expression of Fas and FasL is not accompanied by cell death of reactive astrocytes during anterograde degeneration. GLIA 32:25–41, 2000.

Axonal Damage Induced by Invading T Cells in Organotypic Central Nervous System Tissue in vitro: Involvement of Microglial Cells

Neuroinflammation in the course of multiple sclerosis and experimental autoimmune encephalomyelitis results in demyelination and, recently demonstrated, axonal loss. Invading neuroantigen specific T cells are the crucial cellular elements in these processes. Here we demonstrate that invasion of activated T cells induces a massive microglial attack on myelinated axons in entorhinal‐hippocampal slice cultures. Flow cytometry analysis of activation markers revealed that the activation state of invading MBP‐specific T cells was significantly lower in comparison to PMA‐activated T cells. Moreover, MBP‐specific T cells showed a significantly lower secretion of IFN‐γ. Conversely, MBP‐specific T cells displayed a significantly higher potential to trigger activation of microglial cells, i.e. upregulation of MHC class II and ICAM‐1 expression, and, most importantly, microglial phagocytosis of pre‐traced axons. Our data suggest that this was mediated via specific cellular interactions of T cells and microglial cells since IFN‐γ alone was not sufficient to induce axonal damage while such damage was apparent in response to TNF‐α which is released by activated microglial cells. TNF‐α secretion by both T cell populations was negligible. Thus, MBP‐specific T cells which invade nervous tissue in the course of neuroinflammation are more effective in axon‐damaging recruiting microglial cells than activated T cells of other specificities.

CD25 regulatory T cells determine secondary but not primary remission in EAE: Impact on long-term disease progression☆

Multiple sclerosis (MS) is often characterized by several relapses and remissions during long-term disease, but neither the responsible cells nor the mechanisms are known to date. Using an animal model of multiple sclerosis, relapsing experimental autoimmune encephalomyelitis (R-EAE) CD4+CD25+ Treg cells expressing Foxp3 and CTLA-4 intracellularly and T lymphocytes expressing surface CTLA-4 were identified in the CNS. The first remission occurred even after depletion of Treg cells, but secondary remissions from EAE were ablated. Despite the unaltered first remission autoantigen rechallenge revealed already an amplified cytokine response during acute phase. These results indicate that the cellular composition during first attack of MS predicts long-term disease progression.

Differential astroglial activation in 6-hydroxydopamine models of Parkinson’s disease

In rat models of Parkinsons disease, injections of 6-hydroxydopamine (6-OHDA) into different areas of the basal ganglia result in dopaminergic neurodegeneration in the substantia nigra. The extent and time course of the dopaminergic lesions varies between the models. While the effects on neurons have been extensively studied, little is known about the effects on astrocytes. We compared astrocytic activation (i.e. increase in number and staining intensity of glial fibrillary acidic protein immunoreactive cells) at the injection site and in downstream structures of the motor loop, i.e. the globus pallidus (GP) and the subthalamic nucleus (STN) following 6-OHDA lesion of the medial forebrain bundle (MFB) or the striatum. Lesions in both regions resulted in astrocytic activation at the lesion site, but their remote effects varied. MFB injections caused astrocytic activation in the ipsi- and contralateral striatum, whereas striatal injections resulted in astrocytic activation in the GP and STN. Since 6-OHDA injections into the MFB and the striatum result in complete and partial SNc lesions, respectively, we hypothesize that communication links exist between astrocytes, or between neurons and astrocytes, along neuronal pathways that transmit activating signals in response to neuronal damage-but only if the neuronal pathways are at least partially intact.

Immune Privilege as an Intrinsic CNS Property: Astrocytes Protect the CNS against T-Cell-Mediated Neuroinflammation

Astrocytes have many functions in the central nervous system (CNS). They support differentiation and homeostasis of neurons and influence synaptic activity. They are responsible for formation of the blood-brain barrier (BBB) and make up the glia limitans. Here, we review their contribution to neuroimmune interactions and in particular to those induced by the invasion of activated T cells. We discuss the mechanisms by which astrocytes regulate pro- and anti-inflammatory aspects of T-cell responses within the CNS. Depending on the microenvironment, they may become potent antigen-presenting cells for T cells and they may contribute to inflammatory processes. They are also able to abrogate or reprogram T-cell responses by inducing apoptosis or secreting inhibitory mediators. We consider apparently contradictory functions of astrocytes in health and disease, particularly in their interaction with lymphocytes, which may either aggravate or suppress neuroinflammation.

Astrocytes protect the CNS: antigen-specific T helper cell responses are inhibited by astrocyte-induced upregulation of CTLA-4 (CD152).

Astrocytes are the first cells that are encountered by T cells invading the central nervous system (CNS) by crossing the blood-brain barrier. We show that primary astrocytes contribute to the immune privilege of the CNS by suppressing Th1 and Th2 cell activation, proliferation and effector function. Moreover, this astrocyte-mediated inhibition of Th effector cells was effective on already activated, proliferating cells. Transforming growth factor (TGF)-β secreted by astrocytes or T cells was not the major factor in the inhibition. The inhibition of T-cell proliferation induced by astrocytes was mainly mediated by upregulation of CTLA-4 on already activated T cells, which occurred both with and without cell-cell contact. Upregulation of the inhibitory molecule CTLA-4 on autoreactive Th cells, as mediated by astrocytes, thus represents a novel mechanism for securing the immune privilege of the CNS.