The Novel Phosphodiesterase 9A Inhibitor BI 409306 Increases Cyclic Guanosine Monophosphate Levels in the Brain, Promotes Synaptic Plasticity, and Enhances Memory Function in Rodents.

N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) is an established cellular model underlying learning and memory, and involves intracellular signaling mediated by the second messenger cyclic guanosine monophosphate (cGMP). As phosphodiesterase (PDE)9A selectively hydrolyses cGMP in areas of the brain related to cognition, PDE9A inhibitors may improve cognitive function by enhancing NMDA receptor-dependent LTP. This study aimed to pharmacologically characterize BI 409306, a novel PDE9A inhibitor, using in vitro assays and in vivo determination of cGMP levels in the brain. Further, the effects of BI 409306 on synaptic plasticity evaluated by LTP in ex vivo hippocampal slices and on cognitive performance in rodents were also investigated. In vitro assays demonstrated that BI 409306 is a potent and selective inhibitor of human and rat PDE9A with mean concentrations at half-maximal inhibition (IC50) of 65 and 168 nM. BI 409306 increased cGMP levels in rat prefrontal cortex and cerebrospinal fluid and attenuated a reduction in mouse striatum cGMP induced by the NMDA-receptor antagonist MK-801. In ex vivo rat brain slices, BI 409306 enhanced LTP induced by both weak and strong tetanic stimulation. Treatment of mice with BI 409306 reversed MK-801-induced working memory deficits in a T-maze spontaneous-alternation task and improved long-term memory in an object recognition task. These findings suggest that BI 409306 is a potent and selective inhibitor of PDE9A. BI 409306 shows target engagement by increasing cGMP levels in brain, facilitates synaptic plasticity as demonstrated by enhancement of hippocampal LTP, and improves episodic and working memory function in rodents. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that BI 409306 is a potent and selective PDE9A inhibitor in rodents. Treatment with BI 409306 increased brain cGMP levels, promoted long-term potentiation, and improved episodic and working memory performance in rodents. These findings support a role for PDE9A in synaptic plasticity and cognition. The potential benefits of BI 409306 are currently being investigated in clinical trials.

K-Lysine acetyltransferase 2a regulates a hippocampal gene expression network linked to memory formation.

Neuronal histone acetylation has been linked to memory consolidation, and targeting histone acetylation has emerged as a promising therapeutic strategy for neuropsychiatric diseases. However, the role of histone-modifying enzymes in the adult brain is still far from being understood. Here we use RNA sequencing to screen the levels of all known histone acetyltransferases (HATs) in the hippocampal CA1 region and find that K-acetyltransferase 2a (Kat2a)--a HAT that has not been studied for its role in memory function so far--shows highest expression. Mice that lack Kat2a show impaired hippocampal synaptic plasticity and long-term memory consolidation. We furthermore show that Kat2a regulates a highly interconnected hippocampal gene expression network linked to neuroactive receptor signaling via a mechanism that involves nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In conclusion, our data establish Kat2a as a novel and essential regulator of hippocampal memory consolidation.

Activated protein C ameliorates diabetic nephropathy by epigenetically inhibiting the redox enzyme p66Shc.

The coagulation protease activated protein C (aPC) confers cytoprotective effects in various in vitro and in vivo disease models, including diabetic nephropathy. The nephroprotective effect may be related to antioxidant effects of aPC. However, the mechanism through which aPC may convey these antioxidant effects and the functional relevance of these properties remain unknown. Here, we show that endogenous and exogenous aPC prevents glomerular accumulation of oxidative stress markers and of the redox-regulating protein p66(Shc) in experimental diabetic nephropathy. These effects were predominately observed in podocytes. In vitro, aPC inhibited glucose-induced expression of p66(Shc) mRNA and protein in podocytes (via PAR-1 and PAR-3) and various endothelial cell lines, but not in glomerular endothelial cells. Treatment with aPC reversed glucose-induced hypomethylation and hyperacetylation of the p66(Shc) promoter in podocytes. The hyperacetylating agent sodium butyrate abolished the suppressive effect of aPC on p66(Shc) expression both in vitro and in vivo. Moreover, sodium butyrate abolished the beneficial effects of aPC in experimental diabetic nephropathy. Inhibition of p66(Shc) expression and mitochondrial translocation by aPC normalized mitochondrial ROS production and the mitochondrial membrane potential in glucose-treated podocytes. Genetic ablation of p66(Shc) compensated for the loss of protein C activation in vivo, normalizing markers of diabetic nephropathy and oxidative stress. These studies identify a unique mechanism underlying the cytoprotective effect of aPC. Activated PC epigenetically controls expression of the redox-regulating protein p66(Shc), thus linking the extracellular protease aPC to mitochondrial function in diabetic nephropathy.

Very-late-antigen-4 (VLA-4)-mediated brain invasion by neutrophils leads to interactions with microglia, increased ischemic injury and impaired behavior in experimental stroke.

Neuronal injury from ischemic stroke is aggravated by invading peripheral immune cells. Early infiltrates of neutrophil granulocytes and T-cells influence the outcome of stroke. So far, however, neither the timing nor the cellular dynamics of neutrophil entry, its consequences for the invaded brain area, or the relative importance of T-cells has been extensively studied in an intravital setting. Here, we have used intravital two-photon microscopy to document neutrophils and brain-resident microglia in mice after induction of experimental stroke. We demonstrated that neutrophils immediately rolled, firmly adhered, and transmigrated at sites of endothelial activation in stroke-affected brain areas. The ensuing neutrophil invasion was associated with local blood-brain barrier breakdown and infarct formation. Brain-resident microglia recognized both endothelial damage and neutrophil invasion. In a cooperative manner, they formed cytoplasmic processes to physically shield activated endothelia and trap infiltrating neutrophils. Interestingly, the systemic blockade of very-late-antigen-4 immediately and very effectively inhibited the endothelial interaction and brain entry of neutrophils. This treatment thereby strongly reduced the ischemic tissue injury and effectively protected the mice from stroke-associated behavioral impairment. Behavioral preservation was also equally well achieved with the antibody-mediated depletion of myeloid cells or specifically neutrophils. In contrast, T-cell depletion more effectively reduced the infarct volume without improving the behavioral performance. Thus, neutrophil invasion of the ischemic brain is rapid, massive, and a key mediator of functional impairment, while peripheral T-cells promote brain damage. Acutely depleting T-cells and inhibiting brain infiltration of neutrophils might, therefore, be a powerful early stroke treatment.

Molecular basis of ß-amyloid oligomer recognition with a conformational antibody fragment.

Oligomers are intermediates of the ß-amyloid (Aß) peptide fibrillogenic pathway and are putative pathogenic culprits in Alzheimer's disease (AD). Here we report the biotechnological generation and biochemical characterization of an oligomer-specific antibody fragment, KW1. KW1 not only discriminates between oligomers and other Aß conformations, such as fibrils or disaggregated peptide; it also differentiates between different types of Aß oligomers, such as those formed by Aß (1-40) and Aß (1-42) peptide. This high selectivity of binding contrasts sharply with many other conformational antibodies that interact with a large number of structurally analogous but sequentially different antigens. X-ray crystallography, NMR spectroscopy, and peptide array measurements imply that KW1 recognizes oligomers through a hydrophobic and significantly aromatic surface motif that includes Aß residues 18-20. KW1-positive oligomers occur in human AD brain samples and induce synaptic dysfunctions in living brain tissues. Bivalent KW1 potently neutralizes this effect and interferes with Aß assembly. By altering a specific step of the fibrillogenic cascade, it prevents the formation of mature Aß fibrils and induces the accumulation of nonfibrillar aggregates. Our data illuminate significant mechanistic differences in oligomeric and fibril recognition and suggest the considerable potential of KW1 in future studies to detect or inhibit specific types of Aß conformers.

SPECT-imaging of activity-dependent changes in regional cerebral blood flow induced by electrical and optogenetic self-stimulation in mice.

Electrical and optogenetic methods for brain stimulation are widely used in rodents for manipulating behavior and analyzing functional connectivities in neuronal circuits. High-resolution in vivo imaging of the global, brain-wide, activation patterns induced by these stimulations has remained challenging, in particular in awake behaving mice. We here mapped brain activation patterns in awake, intracranially self-stimulating mice using a novel protocol for single-photon emission computed tomography (SPECT) imaging of regional cerebral blood flow (rCBF). Mice were implanted with either electrodes for electrical stimulation of the medial forebrain bundle (mfb-microstim) or with optical fibers for blue-light stimulation of channelrhodopsin-2 expressing neurons in the ventral tegmental area (vta-optostim). After training for self-stimulation by current or light application, respectively, mice were implanted with jugular vein catheters and intravenously injected with the flow tracer 99m-technetium hexamethylpropyleneamine oxime (99mTc-HMPAO) during seven to ten minutes of intracranial self-stimulation or ongoing behavior without stimulation. The 99mTc-brain distributions were mapped in anesthetized animals after stimulation using multipinhole SPECT. Upon self-stimulation rCBF strongly increased at the electrode tip in mfb-microstim mice. In vta-optostim mice peak activations were found outside the stimulation site. Partly overlapping brain-wide networks of activations and deactivations were found in both groups. When testing all self-stimulating mice against all controls highly significant activations were found in the rostromedial nucleus accumbens shell. SPECT-imaging of rCBF using intravenous tracer-injection during ongoing behavior is a new tool for imaging regional brain activation patterns in awake behaving rodents providing higher spatial and temporal resolutions than 18F-2-fluoro-2-dexoyglucose positron emission tomography.

Selective hippocampal neurodegeneration in transgenic mice expressing small amounts of truncated Aß is induced by pyroglutamate-Aß formation.

Posttranslational amyloid-ß (Aß) modification is considered to play an important role in Alzheimer's disease (AD) etiology. An N-terminally modified Aß species, pyroglutamate-amyloid-ß (pE3-Aß), has been described as a major constituent of Aß deposits specific to human AD but absent in normal aging. Formed via cyclization of truncated Aß species by glutaminyl cyclase (QC; QPCT) and/or its isoenzyme (isoQC; QPCTL), pE3-Aß aggregates rapidly and is known to seed additional Aß aggregation. To directly investigate pE3-Aß toxicity in vivo, we generated and characterized transgenic TBA2.1 and TBA2.2 mice, which express truncated mutant human Aß. Along with a rapidly developing behavioral phenotype, these mice showed progressively accumulating Aß and pE3-Aß deposits in brain regions of neuronal loss, impaired long-term potentiation, microglial activation, and astrocytosis. Illustrating a threshold for pE3-Aß neurotoxicity, this phenotype was not found in heterozygous animals but in homozygous TBA2.1 or double-heterozygous TBA2.1/2.2 animals only. A significant amount of pE3-Aß formation was shown to be QC-dependent, because crossbreeding of TBA2.1 with QC knock-out, but not isoQC knock-out, mice significantly reduced pE3-Aß levels. Hence, lowering the rate of QC-dependent posttranslational pE3-Aß formation can, in turn, lower the amount of neurotoxic Aß species in AD.

Insufficient endogenous redox buffer capacity may underlie neuronal vulnerability to cerebral ischemia and reperfusion.

Reactive oxygen species (ROS) are key players in ischemia-induced neurodegeneration. We investigated whether hippocampal neurons may lack sufficient redox-buffering capacity to protect against ROS attacks. Using organotypic hippocampal slice cultures (OHSCs) transiently exposed to oxygen and glucose deprivation (OGD) and gerbils suffering from a two-vessel occlusion (2VO) as complementary ex vivo and in vivo models, we have elucidated whether the intrinsic redox systems interfere with ischemia-induced neurodegeneration. Cell- type-specific immunohistological staining of hippocampal slice cultures revealed that pyramidal neurons, in contrast to astrocytes and microglia, express free thiols only weakly. In addition, free thiol levels were extensively decreased throughout the hippocampal formation immediately after OGD, but recovered within 24 hr after reperfusion. In parallel, progressive glia activation and proliferation were observed. Increased neuronal exposure to ROS was monitored by dihydroethidium oxidation in hippocampal pyramidal cell layers immediately after OGD. Coadministration of reduction equivalents (α-lipoic acid) and thiol-stimulating agents (enalapril, ambroxol) decreased ischemia-induced neuronal damage in OGD-treated OHSCs and in gerbils exposed to 2VO, whereas single drug applications remained ineffective. In summary, limited redox buffering capacities of pyramidal neurons may underlie their exceptional vulnerability to cerebral ischemia. Consistently, multidrug treatments supporting endogenous redox systems may offer a strategy to promote valid neuroprotection.

A population of serum deprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells.

The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures.

Microglia cells protect neurons by direct engulfment of invading neutrophil granulocytes: a new mechanism of CNS immune privilege.

Microglial cells maintain the immunological integrity of the healthy brain and can exert protection from traumatic injury. During ischemic tissue damage such as stroke, peripheral immune cells acutely infiltrate the brain and may exacerbate neurodegeneration. Whether and how microglia can protect from this insult is unknown. Polymorphonuclear neutrophils (PMNs) are a prominent immunologic infiltrate of ischemic lesions in vivo. Here, we show in organotypic brain slices that externally applied invading PMNs massively enhance ischemic neurotoxicity. This, however, is counteracted by additional application of microglia. Time-lapse imaging shows that microglia exert protection by rapid engulfment of apoptotic, but, strikingly, also viable, motile PMNs in cell culture and within brain slices. PMN engulfment is mediated by integrin- and lectin-based recognition. Interference with this process using RGDS peptides and N-acetyl-glucosamine blocks engulfment of PMNs and completely abrogates the neuroprotective function of microglia. Thus, engulfment of invading PMNs by microglia may represent an entirely new mechanism of CNS immune privilege.

Time-dependent segmentation of BrdU-signal leads to late detection problems in studies using BrdU as cell label or proliferation marker.

Bromodeoxyuridine incorporates into DNA during mitosis. A long-term stability of the incorporated BrdU is important for the recovery of BrdU-labeled cells. For testing the stability of BrdU incorporation into DNA we pulse-labeled mesenchymal stem cells with BrdU and observed these cells in vitro over 4 weeks. During this time the BrdU-signal was permanently decreasing. Starting with cells containing evenly stained BrdU-nuclei, so-called filled cells, already 3 days after BrdU removal we detected cells containing so-called segmented and punctated BrdU-signals. The number of those labeled cells continuously increased over time. Interestingly, the loss of BrdU in the nucleus was accompanied by an increasing labeling of the cytosol. Further, we injected BrdU intraperitoneally into rats after ischemia and detected BrdU-positive cells in the hippocampus 3 and 23 days after the last BrdU injection. While after 3 days most of the BrdU-positive cells in the hippocampus displayed a filled BrdU-signal, 23 days after BrdU removal an increased number of segmented and punctated BrdU-positive nuclei was detected. The gradual degradation of the BrdU-signal was not caused by cell death. The consequence of this BrdU degradation would be an underestimation of cell proliferation and an overestimation of cell death of newly generated cells.

Inhibition of Calpain Prevents N-Methyl-D-aspartate-Induced Degeneration of the Nucleus Basalis and Associated Behavioral Dysfunction.

N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity is thought to underlie a variety of neurological disorders, and inhibition of either the NMDA receptor itself, or molecules of the intracellular cascade, may attenuate neurodegeneration in these diseases. Calpain, a calcium-dependent cysteine protease, has been identified as part of such an NMDA receptor-induced excitotoxic signaling pathway. The present study addressed the question of whether inhibition of calpain can prevent neuronal cell death and associated behavioral deficits in a disease-relevant animal model, which is based on excitotoxic lesions of the cholinergic nucleus basalis magnocellularis of Meynert. Excitotoxic lesions of the nucleus basalis with NMDA induced a markedly impaired performance in the novel object recognition test. Treatment with the calpain inhibitor, N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1-yl]benzamide (A-705253), dose-dependently prevented the behavioral deficit. Subsequent analysis of choline acetyltransferase in the cortical mantle of the lesioned animals revealed that application of A-705253 dose-dependently and significantly attenuated cholinergic neurodegeneration. Calpain inhibition also significantly diminished the accompanying gliosis, as determined by immunohistochemical analysis of microglia activation. Finally, inhibition of calpain by A-705253 and the peptidic calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO did not impair long-term potentiation in hippocampal slices, indicating that calpain inhibition interrupts NMDA excitotoxicity pathways without interfering with NMDA receptor-mediated signaling involved in cognition. We conclude that inhibition of calpains may represent a valuable strategy for the prevention of excitotoxicity-induced neuronal decline without interfering with the physiological neuronal functions associated with learning and memory processes. Thus, calpain inhibition may be a promising and novel approach for the treatment of various neurodegenerative disorders.

The novel selective PDE9 inhibitor BAY 73-6691 improves learning and memory in rodents.

The present study investigated the putative pro-cognitive effects of the novel selective PDE9 inhibitor BAY 73-6691. The effects on basal synaptic transmission and long-term potentiation (LTP) were investigated in rat hippocampal slices. Pro-cognitive effects were assessed in a series of learning and memory tasks using rodents as subjects. BAY 73-6691 had no effect on basal synaptic transmission in hippocampal slices prepared from young adult (7- to 8-week-old) Wistar rats. A dose of 10 microM, but not 30 microM, BAY 73-6691 enhanced early LTP after weak tetanic stimulation. The dose effective in young adult Wistar rats did not affect LTP in hippocampal slices prepared from young (7- to 8-week-old) Fischer 344 X Brown Norway (FBNF1) rats, probably reflecting strain differences. However, it increased basal synaptic transmission and enhanced early LTP after weak tetanic stimulation in hippocampal slices prepared from very old (31- to 35-month-old) FBNF1 rats. BAY 73-6691 enhanced acquisition, consolidation, and retention of long-term memory (LTM) in a social recognition task and tended to enhance LTM in an object recognition task. Bay 73-6691 attenuated the scoplamine-induced retention deficit in a passive avoidance task, and the MK-801-induced short-term memory deficits in a T-maze alternation task. The mechanism of action, possibly through modulation of the NO/cGMP-PKG/CREB pathway, is discussed. Our findings support the notion that PDE9 inhibition may be a novel target for treating memory deficits that are associated with aging and neurodegenerative disorders such as Alzheimer's disease.

Anti-inflammatory treatment with the p38 mitogen-activated protein kinase inhibitor SB239063 is neuroprotective, decreases the number of activated microglia and facilitates neurogenesis in oxygen-glucose-deprived hippocampal slice cultures.

We investigated the effect of the p38 mitogen-activated protein kinase inhibitor SB239063 on inflammation and neurogenesis after ischemia in organotypic hippocampal slice cultures. Our study shows that after oxygen-glucose deprivation, the p38 mitogen-activated protein kinase (MAPK) and the extracellular-signal-regulated kinase 1/2 (ERK1/2) are strongly activated. The p38 MAPK phosphorylation returned to basal levels within 1 h after oxygen-glucose deprivation, whereas the ERK1/2 phosphorylation reached the basal level only after 24 h. Treatment with 20 microM and 100 microM SB239063 strikingly reduced cell death after oxygen-glucose deprivation and significantly diminished microglia activation in the cornu ammonis (CA-region), but not in the area dentata. Levels of the pro-inflammatory cytokine IL-1beta were reduced by 84% after treatment with SB239063 whereas the cytokines IL-6 and TNF-alpha were not affected. After 6 days, neurogenesis was significantly increased in the posterior periventricle. Based on these findings, our study shows that anti-inflammatory treatment with SB239063 reduces cell death, inflammation and microglia activation and, at high concentrations, enhances the oxygen-glucose deprivation-induced neurogenesis in the posterior periventricle.

Pattern of time-dependent reduction of histologically determined infarct volume after focal ischaemia in mice.

The mouse model of transcranial permanent occlusion of the middle cerebral artery (tpMCAO) is widely used in stroke research. Here we quantified infarct size using a conventional histological method at several post-ischaemic times, going beyond the commonly analysed period of up to 2 days, following artery occlusion. Two different mouse strains, which are widely used for pharmacological studies of neuroprotection and for genetic engineering, were used. A drill whole was made into the skull of anaesthetised mice and ischaemia was induced by electrocoagulation of the middle cerebral artery. In both mouse strains tested (C57Black/6 and NMRI), the measured infarct volumes decreased significantly during the first days after tpMCAO. Notably, 13 days after surgery, ischaemic and sham-operated animals had indistinguishably small lesions, which where in the range of only 5% of the infarct size on day 2 post-ischaemia. The standard method of calculating oedema and shrinkage correction provided no sufficient explanation for this significant decrease in infarct volume. There was, however, evidence that structural changes in the residual ipsilateral hemisphere may compromise the significance of results arising from the method of calculating oedema and shrinkage correction. In conclusion, our study indicates that the pronounced and fast, time-dependent decrease in histologically defined infarct volume can compromise results when studying the lasting neuroprotective effects of potential drugs.

Beware the intruder: Real time observation of infiltrated neutrophils and neutrophil-Microglia interaction during stroke in vivo.

Inflammation plays an important role in the pathogenesis of ischemic stroke including an acute and prolonged inflammatory process. The role of neutrophil granulocytes as first driver of the immune reaction from the blood site is under debate due to controversial findings. In bone marrow chimeric mice we were able to study the dynamics of tdTomato-expressing neutrophils and GFP-expressing microglia after photothrombosis using intravital two-photon microscopy. We demonstrate the infiltration of neutrophils into the brain parenchyma and confirm a long-lasting contact between neutrophils and microglia as well as an uptake of neutrophils by microglia clearing the brain from peripheral immune cells.

The late maintenance of hippocampal LTP: requirements, phases, 'synaptic tagging', 'late-associativity' and implications.

Our review focuses on the mechanisms which enable the late maintenance of hippocampal long-term potentiation (LTP; >3h), a phenomenon which is thought to underlie prolonged memory. About 20 years ago we showed for the first time that the maintenance of LTP - like memory storage--depends on intact protein synthesis and thus, consists of at least two temporal phases. Here we concentrate on mechanisms required for the induction of the transient early-LTP and of the protein synthesis-dependent late-LTP. Our group has shown that the induction of late-LTP requires the associative activation of heterosynaptic inputs, i.e. the synergistic activation of glutamatergic and modulatory, reinforcing inputs within specific, effective time windows. The induction of late-LTP is characterized by novel, late-associative properties such as 'synaptic tagging' and 'late-associative reinforcement'. Both phenomena require the associative setting of synaptic tags as well as the availability of plasticity-related proteins (PRPs) and they are restricted to functional dendritic compartments, in general. 'Synaptic tagging' guarantees input specificity and thus the specific processing of afferent signals for the establishment of late-LTP. 'Late-associative reinforcement' describes a process where early-LTP by the co-activation of modulatory inputs can be transformed into late-LTP in activated synapses where a tag is set. Recent evidence from behavioral experiments, which studied processes of emotional and cognitive reinforcement of LTP, point to the physiological relevance of the above mechanisms during cellular and system's memory formation.

Microglia provide neuroprotection after ischemia.

Many neurological insults are accompanied by a marked acute inflammatory reaction, involving the activation of microglia. Using a model of exogenous application of fluorescence-labeled BV2 microglia in pathophysiologically relevant concentrations onto organotypic hippocampal slice cultures, we investigated the specific effects of microglia on neuronal damage after ischemic injury. Neuronal cell death after oxygen-glucose deprivation (OGD) was determined by propidium iodide incorporation and Nissl staining. Migration and interaction with neurons were analyzed by time resolved 3-D two-photon microscopy. We show that 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 or HL-60 granulocytes. The induction of migration and neuron-microglia interaction deep inside the slice was markedly increased under OGD conditions. Lipopolysaccharide-prestimulated microglia failed to provide neuroprotection after OGD. Pharmacological interference with microglia function resulted in a reduced neuroprotection. Microglia proved to be neuroprotective even when applied up to 4 h after OGD, thus defining a "protective time window." In acute injury such as trauma or stroke, appropriately activated microglia may primarily have a neuroprotective role. Anti-inflammatory treatment within the protective time window of microglia would therefore be counterintuitive.

Detrimental effects of halothane narcosis on damage after endothelin-1-induced MCAO.

The influence of anaesthesia in experimental stroke research is controversial. We addressed this problem using the model of endothelin-1-induced occlusion of the middle cerebral artery (eMCAO). This model provided the opportunity to compare the infarct volumes of rats which were under halothane anaesthesia during eMCAO induction with the lesions of rats which were without anaesthesia during eMCAO. All animals were implanted with guide cannulae which allowed the induction of ischaemia in freely moving animals. For comparison, one group of animals was exposed to halothane during the induction of ischaemia. Seven days after eMCAO, the average infarct volume of halothane-anaesthetised rats was significantly larger than the lesion in freely moving animals. This difference was mainly due to increased cortical damage, whereas the striatum was much less influenced. The cortical infarct volume 21 days after induction of eMCAO under anaesthesia was significantly reduced compared to the infarct volume 7 days after eMCAO under anaesthesia. Our results indicate that halothane anaesthesia during eMCAO can cause a transient cortical increase in ischaemic infarct volume. The influence of volatile anaesthetics on ischaemic pathophysiology should be taken into consideration when preclinically testing potential neuroprotective drugs for clinical applications.

Protease-activated receptor-1 induces generation of new microglia in the dentate gyrus of traumatised hippocampal slice cultures.

The protease thrombin is not only known as a major component in the blood coagulation cascade but is also involved in proinflammatory processes in the central nervous system (CNS). Here we used an in vitro model, to investigate the effect of thrombin and protease-activated receptor-1 (PAR-1) on proliferation and microgliosis after traumatic injury. A 5-day exposure to thrombin after cutting the Schaffer collaterals induced a proliferation and microgliosis in the dentate gyrus of organotypic slice cultures. This effect is at least partially mediated by PAR-1 since the selective peptide agonist TRag shows similar effects. Thus, thrombin effects after injury might involve microglial proliferation.