Sustained Baclofen-Induced Activation of GABAB Receptors After Cerebral Ischemia Restores Receptor Expression and Function and Limits Progressing Loss of Neurons

One important function of GABA B receptors is the control of neuronal activity to prevent overexcitation and thereby excitotoxic death, which is a hallmark of cerebral ischemia. Consequently, sustained activation of GABA B receptors with the selective agonist baclofen provides neuroprotection in in vitro and in vivo models of cerebral ischemia. However, excitotoxic conditions severely downregulate the receptors, which would compromise the neuroprotective effectiveness of baclofen. On the other hand, recent work suggests that sustained activation of GABA B receptors stabilizes receptor expression. Therefore, we addressed the question whether sustained activation of GABA B receptors reduces downregulation of the receptor under excitotoxic conditions and thereby preserves GABA B receptor-mediated inhibition. In cultured neurons subjected to oxygen and glucose deprivation (OGD), to mimic cerebral ischemia, GABA B receptors were severely downregulated. Treatment of the cultures with baclofen after OGD restored GABA B receptor expression and reduced loss of neurons. Restoration of GABA B receptors was due to enhanced fast recycling of the receptors, which reduced OGD-induced sorting of the receptors to lysosomal degradation. Utilizing the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia, we verified the severe downregulation of GABA B receptors in the affected cortex and a partial restoration of the receptors after systemic injection of baclofen. Restored receptor expression recovered GABA B receptor-mediated currents, normalized the enhanced neuronal excitability observed after MCAO and limited progressive loss of neurons. These results suggest that baclofen-induced restoration of GABA B receptors provides the basis for the neuroprotective activity of baclofen after an ischemic insult. Since GABA B receptors regulate multiple beneficial pathways, they are promising targets for a neuroprotective strategy in acute cerebral ischemia. Keywords: GABAB receptor; MCAO; OGD; baclofen; cerebral ischemia; neuroprotection

Non-invasive visualization of amyloid-beta deposits in Alzheimer amyloidosis mice using magnetic resonance imaging and fluorescence molecular tomography

Abnormal cerebral accumulation of amyloid-beta peptide (Aβ) is a major hallmark of Alzheimer's disease. Non-invasive monitoring of Aβ deposits enables assessing the disease burden in patients and animal models mimicking aspects of the human disease as well as evaluating the efficacy of Aβ-modulating therapies. Previous in vivo assessments of plaque load have been predominantly based on macroscopic fluorescence reflectance imaging (FRI) and confocal or two-photon microscopy using Aβ-specific imaging agents. However, the former method lacks depth resolution, whereas the latter is restricted by the limited field of view preventing a full coverage of the large brain region. Here, we utilized a fluorescence molecular tomography (FMT)-magnetic resonance imaging (MRI) pipeline with the curcumin derivative fluorescent probe CRANAD-2 to achieve full 3D brain coverage for detecting Aβ accumulation in the arcAβ mouse model of cerebral amyloidosis. A homebuilt FMT system was used for data acquisition, whereas a customized software platform enabled the integration of MRI-derived anatomical information as prior information for FMT image reconstruction. The results obtained from the FMT-MRI study were compared to those from conventional planar FRI recorded under similar physiological conditions, yielding comparable time courses of the fluorescence intensity following intravenous injection of CRANAD-2 in a region-of-interest comprising the brain. In conclusion, we have demonstrated the feasibility of visualizing Aβ deposition in 3D using a multimodal FMT-MRI strategy. This hybrid imaging method provides complementary anatomical, physiological and molecular information, thereby enabling the detailed characterization of the disease status in arcAβ mouse models, which can also facilitate monitoring the efficacy of putative treatments targeting Aβ

Targeting the interaction of GABAB_{B} receptors with CaMKII with an interfering peptide restores receptor expression after cerebral ischemia and inhibits progressive neuronal death in mouse brain cells and slices

Cerebral ischemia is the leading cause for long-term disability and mortality in adults due to massive neuronal death. Currently, there is no pharmacological treatment available to limit progressive neuronal death after stroke. A major mechanism causing ischemia-induced neuronal death is the excessive release of glutamate and the associated overexcitation of neurons (excitotoxicity). Normally, GABA$_{B}$ receptors control neuronal excitability in the brain via prolonged inhibition. However, excitotoxic conditions rapidly downregulate GABA$_{B}$ receptors via a CaMKII-mediated mechanism and thereby diminish adequate inhibition that could counteract neuronal overexcitation and neuronal death. To prevent the deleterious downregulation of GABA$_{B}$ receptors, we developed a cell-penetrating synthetic peptide (R1-Pep) that inhibits the interaction of GABA$_{B}$ receptors with CaMKII. Administration of this peptide to cultured cortical neurons exposed to excitotoxic conditions restored cell surface expression and function of GABA$_{B}$ receptors. R1-Pep did not affect CaMKII expression or activity but prevented its T286 autophosphorylation that renders it autonomously and persistently active. Moreover, R1-Pep counteracted the aberrant downregulation of G protein-coupled inwardly rectifying K$^{+}$ channels and the upregulation of N-type voltage-gated Ca$^{2+}$ channels, the main effectors of GABA$_{B}$ receptors. The restoration of GABA$_{B}$ receptors activated the Akt survival pathway and inhibited excitotoxic neuronal death with a wide time window in cultured neurons. Restoration of GABA$_{B}$ receptors and neuroprotective activity of R1-Pep was verified by using brain slices prepared from mice after middle cerebral artery occlusion (MCAO). Treatment with R1-Pep restored normal GABA$_{B}$ receptor expression and GABA receptor-mediated K$^{+}$ channel currents. This reduced MCAO-induced neuronal excitability and inhibited neuronal death. These results support the hypothesis that restoration of GABA$_{B}$ receptor expression under excitatory conditions provides neuroprotection and might be the basis for the development of a selective intervention to inhibit progressive neuronal death after ischemic stroke

In vivo alkaline comet assay: Statistical considerations on historical negative and positive control data

The alkaline comet assay is frequently used as in vivo follow-up test within different regulatory environments to characterize the DNA-damaging potential of different test items. The corresponding OECD Test guideline 489 highlights the importance of statistical analyses and historical control data (HCD) but does not provide detailed procedures. Therefore, the working group “Statistics” of the German-speaking Society for Environmental Mutation Research (GUM) collected HCD from five laboratories and >200 comet assay studies and performed several statistical analyses. Key results included that (I) observed large inter-laboratory effects argue against the use of absolute quality thresholds, (II) > 50% zero values on a slide are considered problematic, due to their influence on slide or animal summary statistics, (III) the type of summarizing measure for single-cell data (e.g., median, arithmetic and geometric mean) may lead to extreme differences in resulting animal tail intensities and study outcome in the HCD. These summarizing values increase the reliability of analysis results by better meeting statistical model assumptions, but at the cost of information loss. Furthermore, the relation between negative and positive control groups in the data set was always satisfactorily (or sufficiently) based on ratio, difference and quantile analyses

Cross-talk between GABAergic postsynapse and microglia regulate synapse loss after brain ischemia

Microglia interact with neurons to facilitate synapse plasticity; however, signal(s) contributing to microglia activation for synapse elimination in pathology are not fully understood. Here, using in vitro organotypic hippocampal slice cultures and transient middle cerebral artery occlusion (MCAO) in genetically engineered mice in vivo, we report that at 24 hours after ischemia, microglia release brain-derived neurotrophic factor (BDNF) to downregulate glutamatergic and GABAergic synapses within the peri-infarct area. Analysis of the cornu ammonis 1 (CA1) in vitro shows that proBDNF and mBDNF downregulate glutamatergic dendritic spines and gephyrin scaffold stability through p75 neurotrophin receptor (p75NTR) and tropomyosin receptor kinase B (TrkB) receptors, respectively. After MCAO, we report that in the peri-infarct area and in the corresponding contralateral hemisphere, similar neuroplasticity occurs through microglia activation and gephyrin phosphorylation at serine-268 and serine-270 in vivo. Targeted deletion of the Bdnf gene in microglia or GphnS268A/S270A (phospho-null) point mutations protects against ischemic brain damage, neuroinflammation, and synapse downregulation after MCAO

Visualization and Curve-Parameter Estimation Strategies for Efficient Exploration of Phenotype Microarray Kinetics

The Phenotype MicroArray (OmniLog® PM) system is able to simultaneously capture a large number of phenotypes by recording an organism's respiration over time on distinct substrates. This technique targets the object of natural selection itself, the phenotype, whereas previously addressed '-omics' techniques merely study components that finally contribute to it. The recording of respiration over time, however, adds a longitudinal dimension to the data. To optimally exploit this information, it must be extracted from the shapes of the recorded curves and displayed in analogy to conventional growth curves.The free software environment R was explored for both visualizing and fitting of PM respiration curves. Approaches using either a model fit (and commonly applied growth models) or a smoothing spline were evaluated. Their reliability in inferring curve parameters and confidence intervals was compared to the native OmniLog® PM analysis software. We consider the post-processing of the estimated parameters, the optimal classification of curve shapes and the detection of significant differences between them, as well as practically relevant questions such as detecting the impact of cultivation times and the minimum required number of experimental repeats.We provide a comprehensive framework for data visualization and parameter estimation according to user choices. A flexible graphical representation strategy for displaying the results is proposed, including 95% confidence intervals for the estimated parameters. The spline approach is less prone to irregular curve shapes than fitting any of the considered models or using the native PM software for calculating both point estimates and confidence intervals. These can serve as a starting point for the automated post-processing of PM data, providing much more information than the strict dichotomization into positive and negative reactions. Our results form the basis for a freely available R package for the analysis of PM data

Extracerebral Tissue Damage in the Intraluminal Filament Mouse Model of Middle Cerebral Artery Occlusion

Middle cerebral artery occlusion is the most common model of focal cerebral ischemia in the mouse. In the surgical procedure, the external carotid artery (ECA) is ligated; however, its effect on the tissue supplied by the vessel has not been described so far. C57BL/6 mice underwent 1 h of transient MCAO (tMCAO) or sham surgery. Multi-spectral optoacoustic tomography was employed at 30 min after surgery to assess oxygenation in the temporal muscles. Microstructural changes were assessed with magnetic resonance imaging and histological examination at 24 h and 48 h after surgery. Ligation of the ECA resulted in decreased oxygenation of the left temporal muscle in most sham-operated and tMCAO animals. Susceptible mice of both groups exhibited increased T2 relaxation times in the affected muscle with histological evidence of myofibre degeneration, interstitial edema, and neutrophil influx. Ligatures had induced an extensive neutrophil-dominated inflammatory response. ECA ligation leads to distinct hypoxic degenerative changes in the tissue of the ECA territory and to ligature-induced inflammatory processes. An impact on outcome needs to be considered in this stroke model

Noninvasive detection of acute cerebral hypoxia and subsequent matrix-metalloproteinase activity in a mouse model of cerebral ischemia using multispectral-optoacoustic-tomography

Oxygen metabolism and matrix metalloproteinases (MMPs) play important roles in the pathophysiology of cerebral ischemia. Using multispectral optoacoustic tomography (MSOT) imaging, we visualized in vivo changes in cerebral tissue oxygenation during 1 h of transient middle cerebral artery occlusion (tMCAO) and at 48 h after reperfusion together with MMP activity using an MMP-activatable probe. The deoxyhemoglobin, oxyhemoglobin, and MMP signals were coregistered with structural magnetic resonance imaging data. The ipsi-/contralateral ratio of tissue oxygen saturation (SO2) was significantly reduced during 1 h of tMCAO and recovered after 48 h of reperfusion in tMCAO compared with sham-operated mice (n  =  8 to 10 per group). A higher ipsi-/contralateral MMP signal ratio was detected at 48 h after reperfusion in the lesioned brain regions of tMCAO compared with the sham-operated animal (n  =  4 to 6 per group). Ex vivo near-infrared fluorescence imaging of MMP signal in brain slices was used to validate in vivo MSOT measurements. In conclusion, noninvasive MSOT imaging can provide visualization of hemodynamic alterations and MMP activity in a mouse model of cerebral ischemia