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.
Purpose
Stroke is one of the most prevalent vascular diseases. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and follow alterations ...of receptor expression and metabolism in ischemic stroke. The aim of this study was to assess the cannabinoid type 2 receptor (CB
2
R) levels in transient middle cerebral artery occlusion (tMCAO) mouse models at subacute stage using positron emission tomography (PET) with our novel tracer
18
FRoSMA-18-d6 and structural imaging by magnetic resonance imaging (MRI).
Procedures
Our recently developed CB
2
R PET tracer
18
FRoSMA-18-d6 was used for imaging neuroinflammation at 24 h after reperfusion in tMCAO mice. The RNA expression levels of CB
2
R and other inflammatory markers were analyzed by quantitative real-time polymerase chain reaction using brain tissues from tMCAO (1 h occlusion) and sham-operated mice.
18
Ffluorodeoxyglucose (FDG) was included for evaluation of the cerebral metabolic rate of glucose (CMRglc). In addition, diffusion-weighted imaging and T
2
-weighted imaging were performed for anatomical reference and delineating the lesion in tMCAO mice.
Results
mRNA expressions of inflammatory markers
TNF-α
,
Iba1
,
MMP9
and
GFAP
,
CNR2
were increased to 1.3–2.5 fold at 24 h after reperfusion in the ipsilateral compared to contralateral hemisphere of tMCAO mice, while mRNA expression of the neuronal marker
MAP-2
was markedly reduced to ca. 50 %. Reduced
18
FFDG uptake was observed in the ischemic striatum of tMCAO mouse brain at 24 h after reperfusion. Although higher activity of
18
FRoSMA-18-d6 in
ex vivo
biodistribution studies and higher standard uptake value ratio (SUVR) were detected in the ischemic ipsilateral compared to contralateral striatum in tMCAO mice, the
in vivo
specificity of
18
FRoSMA-18-d6 was confirmed only in the CB
2
R-rich spleen.
Conclusions
This study revealed an increased
18
FRoSMA-18-d6 measure of CB
2
R and a reduced
18
FFDG measure of CMRglc in the ischemic striatum of tMCAO mice at subacute stage.
18
FRoSMA-18-d6 might be a promising PET tracer for detecting CB
2
R alterations in animal models of neuroinflammation without neuronal loss.
The abnormal deposition of fibrillar beta-amyloid (Aβ) deposits in the brain is one of the major histopathological hallmarks of Alzheimer’s disease (AD). Here, we characterized curcumin-derivative ...CRANAD-2 for multi-spectral optoacoustic tomography and fluorescence imaging of brain Aβ deposits in the arcAβ mouse model of AD cerebral amyloidosis. CRANAD-2 showed a specific and quantitative detection of Aβ fibrils in vitro, even in complex mixtures, and it is capable of distinguishing between monomeric and fibrillar forms of Aβ. In vivo epi-fluorescence microscopy and optoacoustic tomography after intravenous CRANAD-2 administration demonstrated higher cortical retention in arcAβ compared to non-transgenic littermate mice. Immunohistochemistry showed co-localization of CRANAD-2 and Aβ deposits in arcAβ mouse brain sections, thus verifying the specificity of the probe. In conclusion, we demonstrate suitability of CRANAD-2 for optical detection of Aβ deposits in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting Aβ deposits.
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 (p75
) 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
gene in microglia or
S268A/S270A (phospho-null) point mutations protects against ischemic brain damage, neuroinflammation, and synapse downregulation after MCAO.
Jet-mixing and residence time in a rectangular water storage tank with a constant water level are investigated using the tools of Computational Fluid Dynamics (CFD). A set of Unsteady ...Reynolds-Averaged Navier-Stokes (URANS) equations using a realisable k-ε model for different inlet configurations has been used. Numerical simulations were validated by means of experimental measurements. A saline inflow was simulated and the computed salinity in the outflow was compared with the measured values, with the aim of improving the tank performance based only on simple modifications of the inlet position and inflow rate. The results show that the URANS technique is able to adequately capture the experimental dilution curve measured at the outlet of the tank. The residence time is mainly influenced by advective transport. Modifications of the horizontal angle and Reynolds number of the inflow jet produce changes in the mixing characteristics when different performance indexes are compared.
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.
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, GABAB receptors control neuronal excitability in the brain via prolonged inhibition. However, excitotoxic conditions rapidly downregulate GABAB 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 GABAB receptors, we developed a cell‐penetrating synthetic peptide (R1‐Pep) that inhibits the interaction of GABAB receptors with CaMKII. Administration of this peptide to cultured cortical neurons exposed to excitotoxic conditions restored cell surface expression and function of GABAB 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 Ca2+ channels, the main effectors of GABAB receptors. The restoration of GABAB receptors activated the Akt survival pathway and inhibited excitotoxic neuronal death with a wide time window in cultured neurons. Restoration of GABAB 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 GABAB 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 GABAB 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.
There is no pharmacological treatment available to limit progressing neuronal death after cerebral ischemia. Here we show that targeting the interaction of GABAB receptors with CaMKII using a synthetic interfering peptide after an excitotoxic insult in cultured neurons or in brain slices from MCAO‐treated mice restored downregulated GABAB receptor expression and function, normalized aberrant expression of their major effectors, reduced neuronal excitability, activated the Akt survival pathway, and inhibited progressing neuronal death.
Near-infrared fluorescence (NIRF) imaging enables non-invasive monitoring of molecular and cellular processes in live animals. Here we demonstrate the suitability of NIRF imaging to investigate the ...neutrophil response in the brain after transient middle cerebral artery occlusion (tMCAO). We established procedures for ex vivo fluorescent labelling of neutrophils without affecting their activation status. Adoptive transfer of labelled neutrophils in C57BL/6 mice before surgery resulted in higher fluorescence intensities over the ischaemic hemisphere in tMCAO mice with NIRF imaging when compared with controls, corroborated by ex vivo detection of labelled neutrophils using fluorescence microscopy. NIRF imaging showed that neutrophils started to accumulate immediately after tMCAO, peaking at 18 h, and were still visible until 48 h after reperfusion. Our data revealed accumulation of neutrophils also in extracranial tissue, indicating damage in the external carotid artery territory in the tMCAO model. Antibody-mediated inhibition of α4-integrins did reduce fluorescence signals at 18 and 24, but not at 48 h after reperfusion, compared with control treatment animals. Antibody treatment reduced cerebral lesion volumes by 19%. In conclusion, the non-invasive nature of NIRF imaging allows studying the dynamics of neutrophil recruitment and its modulation by targeted interventions in the mouse brain after transient experimental cerebral ischaemia.
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
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.
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
receptors control neuronal excitability in the brain via prolonged inhibition. However, excitotoxic conditions rapidly downregulate GABA
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
receptors, we developed a cell-penetrating synthetic peptide (R1-Pep) that inhibits the interaction of GABA
receptors with CaMKII. Administration of this peptide to cultured cortical neurons exposed to excitotoxic conditions restored cell surface expression and function of GABA
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
channels, the main effectors of GABA
receptors. The restoration of GABA
receptors activated the Akt survival pathway and inhibited excitotoxic neuronal death with a wide time window in cultured neurons. Restoration of GABA
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
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
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.
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