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Microglia provide neuroprotection after ischemia

Jens Neumann, Matthias Gunzer^||, Herwig O. Gutzeit, Oliver Ullrich, Klaus G. Reymann^*, and Klaus Dinkel^,1

^* Leibniz Institute for Neurobiology, Project Group Neuropharmacology, Magdeburg, Germany;
Institute for Applied Neuroscience (FAN gGmbH), Magdeburg, Germany;
Institute of Immunology, University Hospital Magdeburg, Magdeburg, Germany;
Institute of Zoology, Technical University Dresden, Dresden, Germany; and
^|| German Research Centre for Biotechnology, Research Group Immunodynamics, Braunschweig, Germany

¹Correspondence: Jerini AG, Invalidenstr. 130, Berlin 10115, Germany. E-mail: dinkel{at}jerini.com

SPECIFIC AIMS

Ischemic injury (stroke) represents the third leading cause of death in the developed countries. Yet the associated mechanisms and especially the role of post-stroke neuroinflammation are still poorly understood. Our aim was to elucidate the highly debated (harmful or beneficial) role of microglia in the context of an ischemic insult. Our unique experimental design includes the exogenous application of fluorescently labeled microglial cell line BV2 to transgenic (fluorescently labeled neurons) organotypic hippocampal cultures. We could thus modify microglia prior to application, reach pathophysiologically relevant microglia numbers, and ensure that the observed effects were mediated exclusively by the microglia.

PRINCIPAL FINDINGS

1. Microglia protect against ischemia-induced neuronal cell death within a protective time window before and after the ischemic insult
The focus of this study was to elucidate the role of microglial cells in a model of ischemic injury in vitro (see Fig. 1 A for experimental setup). Direct application of the microglial cell line BV2 (up to 8x10⁴ cells/slice) onto otherwise untreated organotypic hippocampal slice cultures had no effect on neuronal viability in the CA area after 24 h (control in Fig. 1B, C ). For all the following experiments involving direct application a concentration of 8x10⁴ microglia was used. Since only 20% of the applied microglia adhered to or invaded into the slice, this exogenous application resulted in in vitro microglia (OX–42+) concentrations (1.9x10³) that are also found in vivo in the hippocampus after global ischemia (2.3x10³). When microglia were transferred directly 24 h before oxygen-glucose deprivation (OGD), we observed a significant reduction (***P<0.001 vs. OGD) of the OGD-induced neuronal damage (Fig. 1B ). This microglial neuroprotection was also effective when the microglia were added 1 h or 4 h after OGD (Fig. 1B ). At 6 h after OGD, however, the application of microglia was no longer protective (Fig. 1B ). Representative fluorescent images for the densitometric quantification (Fig. 1B ) are shown in Fig. 1C . Microglia viability was monitored with CMFDA vital staining before application. On the slice border viable (CMFDA+) microglia showed some PI uptake under control conditions, which was dramatically increased after OGD (Fig. 1C , arrowhead).

View larger version (38K): [in this window] [in a new window]   Figure 1. Protection against OGD-induced neuronal damage by microglia directly applied onto hippocampal slice cultures. BV2 microglia were applied onto the OHCs 24 h before or 1, 4, or 6 h after oxygen-glucose deprivation (OGD) (A). Quantification of neuronal death in cornu amonis (CA) by propidium iodide (PI) incorpoartion was determined after 24 h (***P<0.001 vs. OGD; n=6–8/bar) (B). Representative PI fluorescent images showing neuronal death in CA (C). Arrowhead indicates PI-positive microglia on the slice border.

2. Microglial migration and interaction with neurons is massively induced by an ischemic insult
These results indicated that the microglial neuroprotection might be based on a specific interaction between microglia and neurons in the slice cultures. Previous studies showed that exogenously applied microglia migrated into slice cultures under normal and NMDA-excitotoxic conditions. We used a novel 2-photon microscopy approach to study the interaction of fluorescence labeled microglia with fluorescence-labeled neurons in living brain tissue of transgenic animals.

Microglia were labeled with cell tracker orange (CTO), then directly applied onto organotypic hippocampal cultures prepared from transgenic B6.Cg-TgN (Thy1-YFP)16Jrs mice. In these transgenic mice a subset of neurons (particularly the CA1 area) is labeled by expression of EYFP. 2-photon microscopy was performed at 1 h, 7 h, and 20 h after OGD and 16 h after microglia application under basal conditions. Representative images are shown as 3-D reconstruction (Fig. 2 A, A*) and a 2-D collapsed side view (Fig. 2B, C ) of the individual confocal planes. Under basal conditions the majority of microglia stayed on the surface of the slice and rarely migrated into the slice. At 1 h after OGD the majority of microglia were still on the slice surface however most of these microglia showed a "capping" interaction with the first neuronal layer (Fig. 2A ). This interaction is best described as a capping of the neuron by the microglia (Fig. 2A *).

View larger version (62K): [in this window] [in a new window]   Figure 2. Migration of microglia into organotypic hippocampal cultures and capping interaction with neurons under normal and ischemic conditions. BV-2 microglia were labeled with CTO (red), then directly applied onto organotypic hippocampal cultures (OHCs) prepared from transgenic B6.Cg-TgN (Thy1-YFP)16Jrs mice (neurons: green). At indicated time points (Basal+16 h, OGD+1 h, 20 h) Z-stacks were performed from living OHCs using 2-photon microscopy. Images show 3-D reconstruction (A, A*) and 2-D collapsed side view of the individual confocal planes (B, C). Images show a 180° view of a capping interaction between a neuron (green) and a BV-2 microglia (red) (A*). Capping is marked by arrows showing double labeling (yellow) in panel C. White brackets indicate first neuronal layer; white dashed lines indicate depth in the slice (B, C). C)Asterisks indicate neuronal fragments.

At 16 h under basal conditions the microglia did not infiltrate the slice and the neuronal layer was undamaged (Fig. 2B ). At 20 h after OGD the majority of microglia had migrated into the slice (80 µm depth) and the top neuronal layer was destroyed except for a few EYFP+ fragments (Fig. 2C ). We could observe a capping interaction of microglia with these fragments on the surface, but the majority of capping was detected inside of the slice (80 µm depth; Fig. 2C ).

3. Microglial integrin CD11a expression is crucial for migration and neuroprotection
Based on the 2-photon data, we suggested that microglial migration into the slice and subsequent interaction with neurons was necessary to provide neuroprotection. To elucidate the molecular basis for the microglia-mediated neuroprotective effect after OGD we generated BV2 microglia that are unable to express the integrin CD11a, antisense CD11a transfected BV2 (asCD11a). We found that asCD11a microglia failed to provide neuroprotection after OGD but were still engaging in a capping interaction with the neurons on the slice surface 1 h after OGD. This suggested that the capping interaction occurred independently of CD11a expression and was not crucial for the neuroprotective effect. However, the migration of asCD11a microglia deep into the slice culture after OGD was severely inhibited. The asCD11a microglia were unable to migrate deep (max. 50 µm) into the injured slice and were not colocalized at that depth with neurons while wild-type BV2 microglia did colocalize with neurons of the deeper layers (80 µm) These data indicate that CD11a-mediated microglial migration into the injured slice is necessary for neuroprotection.

4. Microglia-mediated neuroprotection is cell-specific and stimulus-specific
To investigate whether the observed neuroprotective effect was specific for microglia, we performed experiments using the granulocyte cell line HL60. HL60 cells caused significant neuronal cell death under basal conditions and failed to ameliorate neuronal cell loss after OGD, thus confirming the specificity of the neuroprotective microglia effect.

5. Pharmacological interference with microglia function causes reduced neuroprotection
We investigated whether the observed microglial neuroprotective effect could be influenced by impairment of microglial function. When microglia were pretreated with the protein synthesis inhibitor anisomycine or the monocyte inhibitor minocycline, we noted a significant reduction in neuroprotection compared with untreated microglia. This indicates that de novo protein synthesis seems necessary to achieve the highest neuroprotective microglia effect. Simultaneous application of microglia with minocycline after OGD also resulted in a significantly lower neuroprotection.

CONCLUSIONS AND SIGNIFICANCE

We demonstrate for the first time that microglia in pathophysiologically relevant numbers protect against ischemia-induced neuronal damage and engage in close contact with neurons in living brain tissue after the insult. Based on our 3-D reconstruction images we propose the following mode of action for the microglia-mediated protection: immediately after OGD microglia are activated by injured neurons and engage in close cell-cell contact with the first neuronal layer (capping). Since these neurons die later on, this capping could ensure the early recognition and fast phagocytic removal of dying/dead neurons. This mechanism would provide protection for the neurons in the deeper layers of the slice by minimizing the exposure time and dose of these cells to cytotoxic cell contents/debris released from dead/dying neurons. At later time points, we detected microglia that had migrated 80 µm into the slice. These microglia could provide trophic support by the release of growth factors to improve the survival of the neurons in the deeper layers of the slice. The close proximity of injured neurons to microglia may also facilitate the targeted delivery of neurotrophic growth factors.

The demonstrated neuroprotective effect of microglia seems to be in sharp contradiction to previous studies suggesting that activation of microglia contributes to ischemic and excitotoxic cell death. However, many of the information on the neurotoxic properties of activated microglia is derived from in vitro studies using monolayer dissociated cell cultures or are drawn from in vivo studies without differentiation between microglia, macrophages, neutrophils and their individual effects and reactions to various treatments. As a consequence of this generalization microglia suffer from a bad reputation as contributors of cell death in ischemic and neurodegenerative disorders. There is also considerable evidence that microglial activation after acute CNS injury is triggered by injured/dying neurons and results in reduction of neuronal damage and tissue repair. There is no doubt that microglia can not only secrete potentially neurotoxic substances, but also can secrete factors to promote neuronal survival and tissue repair. It seems likely that the type and degree of injury (e.g., stroke: acute ischemia; Alzheimer’s disease: chronic neurodegeneration) activates microglia differently toward performing neuroprotective or neurodestructive effects.

View larger version (63K): [in this window] [in a new window]   Figure 3. Schematic diagram. We investigated the specific effects of microglia on neuronal damage after ischemic injury. A) Fluorescence-labeled BV2 microglia (red) were applied directly onto transgenic organotypic hippocampal slice cultures (neurons: green) after oxygen glucose deprivation (OGD). B) Migration and interaction with neurons was analyzed by time-resolved 3-D, 2-photon microscopy. Induction of migration and neuron-microglia interaction deep inside the slice was markedly increased under OGD conditions. C) Microglia protect against OGD-induced neuronal damage and engage in close physical cell-cell contact with neurons in the damaged brain area. Neuroprotection and migration of microglia were not seen with integrin regulator CD11a-deficient microglia. Prestimulation of BV2 microglia with LPS as well as pretreatment with minocycline or anisomycine resulted in significant reduction or loss of neuroprotection. Respective pretreatments are in brackets. We propose that activated microglia engage in cell-cell contact with dying neurons to ensure early recognition and fast phagocytic removal of cell debris. Microglia also migrate into the deeper neuronal layers, where they seem to provide neuroprotection. D) Microglia proved to be neuroprotective when applied up to 4 h after OGD, thus defining a protective time window. These data indicate that in acute injury appropriately activated microglia primarily have a neuroprotective role.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4882fje;

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This Article Full Text (PDF) All Versions of this Article: 20/6/714 05-4882fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Neumann, J. Articles by Dinkel, K. Search for Related Content PubMed PubMed Citation Articles by Neumann, J. Articles by Dinkel, K.