Dunja Gorup

Toll-like receptor 2 deficiency leads to delayed exacerbation of ischemic injury

BackgroundUsing a live imaging approach, we have previously shown that microglia activation after stroke is characterized by marked and long-term induction of the Toll-like receptor (TLR) 2 biophotonic signals. However, the role of TLR2 (and potentially other TLRs) beyond the acute innate immune response and as early neuroprotection against ischemic injury is not well understood.MethodsTLR2−/− mice were subjected to transient middle cerebral artery occlusion followed by different reperfusion times. Analyses assessing microglial activation profile/innate immune response were performed using in situ hybridization, immunohistochemistry analysis, flow cytometry and inflammatory cytokine array. The effects of the TLR2 deficiency on the evolution of ischemic brain injury were analyzed using a cresyl violet staining of brain sections with appropriate lesion size estimation.ResultsHere we report that TLR2 deficiency markedly affects post-stroke immune response resulting in delayed exacerbation of the ischemic injury. The temporal analysis of the microglia/macrophage activation profiles in TLR2−/− mice and age-matched controls revealed reduced microglia/macrophage activation after stroke, reduced capacity of resident microglia to proliferate as well as decreased levels of monocyte chemotactic protein-1 (MCP-1) and consequently lower levels of CD45high/CD11b+ expressing cells as shown by flow cytometry analysis. Importantly, although acute ischemic lesions (24 to 72 h) were smaller in TLR2−/− mice, the observed alterations in innate immune response were more pronounced at later time points (at day 7) after initial stroke, which finally resulted in delayed exacerbation of ischemic lesion leading to larger chronic infarctions as compared with wild-type mice. Moreover, our results revealed that TLR2 deficiency is associated with significant decrease in the levels of neurotrophic/anti-apoptotic factor Insulin-like growth factor-1 (IGF-1), expressed by microglia in the areas both in and around ischemic lesion.ConclusionOur results clearly suggest that optimal and timely microglial activation/innate immune response is needed to limit neuronal damage after stroke.

Expression analysis of genes involved in TLR2-related signaling pathway: Inflammation and apoptosis after ischemic brain injury

Toll-like receptor 2 (TLR2) is involved in innate immunity in the brain and in the cascade of events after ischemic stroke. The aim of this study was to get an insight into the expression of genes related to TLR2 signaling pathway and associated with inflammation and apoptosis in the later stages of brain response after ischemic injury. Middle cerebral artery occlusion was performed on both wild-type and TLR2(-/-) mice followed by real-time PCR to measure the relative expression of selected genes. In TLR2(-/-) mice expression of genes involved in proinflammatory response was decreased after cerebral ischemia. Tnf was the most prominent cytokine active in the late phase of recovery. Contrary to proinflammatory genes, the expression of Casp8, as a hallmark of apoptosis, was increased in TLR2(-/-) mice, in particular in the late phase of recovery.

Computed microtomography visualization and quantification of mouse ischemic brain lesion by nonionic radio contrast agents

Aim To explore the possibility of brain imaging by microcomputed tomography (microCT) using x-ray contrasting methods to visualize mouse brain ischemic lesions after middle cerebral artery occlusion (MCAO). Methods Isolated brains were immersed in ionic or nonionic radio contrast agent (RCA) for 5 days and subsequently scanned using microCT scanner. To verify whether ex-vivo microCT brain images can be used to characterize ischemic lesions, they were compared to Nissl stained serial histological sections of the same brains. To verify if brains immersed in RCA may be used afterwards for other methods, subsequent immunofluorescent labeling with anti-NeuN was performed. Results Nonionic RCA showed better gray to white matter contrast in the brain, and therefore was selected for further studies. MicroCT measurement of ischemic lesion size and cerebral edema significantly correlated with the values determined by Nissl staining (ischemic lesion size: P=0.0005; cerebral edema: P=0.0002). Brain immersion in nonionic RCA did not affect subsequent immunofluorescent analysis and NeuN immunoreactivity. Conclusion MicroCT method was proven to be suitable for delineation of the ischemic lesion from the non-infarcted tissue, and quantification of lesion volume and cerebral edema.

Increased expression and colocalization of GAP43 and CASP3 after brain ischemic lesion in mouse

GAP43 is a protein involved in neurite outgrowth during development and axon regeneration reflecting its presynaptic localization in developing neurons. Recently, it has been demonstrated that GAP43 is a ligand of CASP3 involved in receptor endocytosis and is also localized post-synaptically. In this study, by using a transgenic mouse strain carrying a bioluminescent reporter for GAP43 combined with an in vivo bioluminescence assay for CASP3, we demonstrated that one day after brain ischemic lesion and, even more pronounced, four days after stroke, expression of both CASP3 and Gap43 in neurons increased more than 40 times. The in vivo approach of CASP3 and GAP43 colocalization imaging was further validated and quantified by immunofluorescence. Importantly, in 82% of GAP43 positive cells, colocalization with CASP3 was present. These findings suggested that one and four days after stroke CASP3 expression, not necessarily associated with neuronal death, increased and suggested that CASP3 and GAP43 might be part of a common molecular pathway involved in early response to ischemic events occurring after onset of stroke.

Neural stem cells from mouse strain Thy1 YFP-16 are a valuable tool to monitor and evaluate neuronal differentiation and morphology.

To analyse events following transplantation of stem cells in the brain robust tools for tracing stem cells are required. Here we took advantage of the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J (Thy1 YFP-16), where yellow fluorescent protein (YFP) is under control of the promoter of Thy1 gene. This allows visualising whole neurons, i.e. their cell body, axons and dendrites. In this work fluorescent cells were followed during embryonic development, in vitro differentiation, and after transplantation in the healthy and stroke-affected mouse brain. During embryonic development Thy1-YFP positive cells were first observed on E12.5 and subsequently located in the prosencephalon, rhombencephalon, spinal cord and peripheral nerves. Quantitative analysis by RT-PCR and immunocytochemistry revealed that Thy1-YFP positive cells during embryo development and in vitro differentiation were expressing nestin and SOX2 then MAP2, β3-tubulin and NeuN. Thy1-YFP positive cells isolated from E14.5 represented 21.88±053% (SD) of the cultivated neurons and this remained constant along in vitro differentiation. On the other hand, proportion of Thy1-YFP positive cells reached 50% of neurons in perinatal and one month old mouse brain. Neural stem cells isolated from Thy1 YFP-16 mouse strain transplanted near hippocampus of the healthy and stroke-affected brain were distinguishable by YFP fluorescence. They differentiated into mature neurons and were detectable even 14 weeks after transplantation, the end point of our experiment. In conclusion, stem cells originating from Thy1 YFP-16 mice represent an outstanding tool to monitor neurogenesis enabling morphological analyses of new neurons and their projections, in particular after transplantation in the brain.

Urodilatin reverses the detrimental influence of bradykinin in acute ischemic stroke

Occlusion of cerebral arteries leads to ischemic stroke accompanied by subsequent brain edema. Bradykinin (BK) is involved in the formation of cerebral edema, and natriuretic peptides (NPs) potentially have beneficial effects on brain edema formation via a still unknown mechanism. The aim of this study was clarifying the mechanisms of action of NPs on BK signaling, and their interactive effects after ischemic brain injury. We used a mouse model for stroke, the middle cerebral artery (MCA) occlusion. Brain lesion and edema were measured by microcomputerized tomography volumetric measurements. To determine the effects of NPs on the BK signaling pathway in the MCAs we measured changes in vessel diameter and membrane potentials in endothelial cells. To determine the effects of NPs on BK signaling pathway in isolated astrocytes and neurons, membrane potentials and intercellular Ca2+ concentrations were measured. Urodilatin inhibited and when applied together with BK, reduced the formation of the ischemic lesion via activation of G-Protein-Signaling Protein Type 4 at the cellular (atrocities, neurons) and blood vessel (endothelial cells and isolated MCA) level as well as in in vivo experiments. The results of this study show the existence of a natural antagonist of BK in the brain, and the possible use of NPs in the treatment of stroke.

Stem cells and stroke—how glowing neurons illuminate new paths

ABSTRACT A reliable method of cell tracing is essential in evaluating potential therapeutic procedures based on stem cell transplantation. Here we present data collected using neural stem cells isolated from a transgenic mouse line Thy1-YFP. When transplanted into a stroke affected brain these cells give rise to neurons that express a fluorescent signal which can be used for their detection and tracing. Observed processes were compared with those taking place during normal embryonic neurogenesis as well as during in vitro differentiation. Since the same neurogenic patterns were observed, we confirm that neural stem cell transplantation fits well into the paradigm of neuronal birth and differentiation.

The effect of natriuretic peptides and bradykinin on development of brain oedema after ischemic stroke

Background Ischemic stroke is characterized by a rapid loss of brain function due to disturbance in blood supply to a part of the brain. Due to fixed intracranial space, any increase in intracranial fluid volume, or progressive brain oedema formation, contributes to further deterioration of the already impaired brain function. Bradykinin (BK), which levels increase during ischemic stroke, promotes blood– brain barrier permeability and raises intracranial capillary blood pressure, leading to brain oedema formation. Furthermore, BK induces glutamate release from neurons and astrocytes via activation of BK receptor type 2. suggesting involvement of BK in glutamate neurotoxicity. It has been recently shown that humans without functional natriuretic peptides (NPs) suffer from massive stokes [1,2]. NPs can reduce brain oedema and have a neuroprotective role in acute ischemic stroke as well as during recovery after stroke. Although mechanisms are still not clear, it appears that NPs enhance angiogenesis, neurogenesis and oligodenrogenesis [3,4]. One of the possible beneficiary effects of NPs during the stroke could be an inhibition of BK pathological function. Materials and methods Aim of our study is to determine beneficial effects of the NPs in stroke development in murine model (middle cerebral artery occlusion – MCAO). The symptoms of the stroke are determined by behavioural studies. The sizes of the lesion and brain oedema are established by μCT. Furthermore, we determined the effects of NPs on the BK signalling pathway in primary culture of neurons and astrocytes using whole cell patch clamp experiments to measure membrane potential and measurements of intracellular Ca 2+ concentration.