Treatment with Edoxaban Attenuates Acute Stroke Severity in Mice by Reducing Blood–Brain Barrier Damage and Inflammation
<p>Treatment with edoxaban reduces stroke severity without increasing the risk of intracerebral hemorrhages. (<b>A</b>) (<b>Top</b>): Definition of the best edoxaban treatment strategy in terms of dose and application time (group I–III) for transient middle cerebral artery occlusion (tMCAO) experiments by measuring plasma edoxaban levels. (<b>Bottom</b>): Quantification of edoxaban plasma concentrations of different treatment strategies analyzed by ultra-high-performance liquid chromatography coupled with ultrasensitive tandem mass spectrometer (<span class="html-italic">n</span> = 3/group). (<b>B</b>) (<b>Top</b>): Representative 2,3,5-triphenyltetrazolium chloride (TTC) staining of three corresponding coronal brain sections of vehicle-, edoxaban (Edox)-, or phenprocoumon (Phen)-treated mice euthanized 24 h after tMCAO (scale bar = 10 mm). (<b>Bottom</b>): The infarcts (white) appear smallest in the edoxaban group, and this could be confirmed by infarct volumetry (<span class="html-italic">n</span> = 9). (<b>C</b>) Bederson score (<b>left</b>) and grip test (<b>right</b>) at day 1 after tMCAO in the three mouse groups indicated above (<span class="html-italic">n</span> = 9). (<b>D</b>) (<b>Left</b>): Serial coronal T2-weighted gradient echo magnetic resonance images (MRI) show extensive hyperintense (bright) ischemic lesions in vehicle- and phenprocoumon-treated mice on day 1 and day 7 after tMCAO, whereas the infarcts were smaller with edoxaban treatment. One representative imaging panel per group is depicted. (<b>Right</b>): MRI-based infarct volumetry (<span class="html-italic">n</span> = 3–5). * <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 2
<p>Edoxaban treatment stabilizes the blood–brain barrier and leads to anti-edematous effects in ischemic stroke. (<b>A</b>) <b>Left</b>: Representative corresponding coronal brain sections of vehicle (veh)-, edoxaban (Edox)- or phenprocoumon (Phen)-treated mice after the injection of the vascular tracer Evans Blue. Brains were analyzed at day 1 after transient middle cerebral artery occlusion (tMCAO) (Scale bar = 10 mm). Vascular leakage was decreased in the cortical and subcortical areas after edoxaban treatment. <b>Right</b>: Tissue concentration of Evans Blue in the ischemic (ipsilateral) and contralateral hemispheres of vehicle-, edoxaban-, or phenprocoumon-treated mice 24 h after tMCAO determined by photometry (<span class="html-italic">n</span> = 5–8). (<b>B</b>) Edema formation as measured by brain water content in the ipsilateral and contralateral hemispheres of vehicle-, edoxaban-, or phenprocoumon-treated mice 24 h after tMCAO (<span class="html-italic">n</span> = 4–6). (<b>C</b>) <b>Top</b>: Claudin (Cldn)-3,-5 and Occludin (Occl) protein expression in the ischemic cortices and basal ganglia of vehicle-, edoxaban-, or phenprocoumon-treated mice at day 1 after tMCAO as determined by immunoblot. Actin was used as loading control. <b>Bottom</b>: Densitometric quantification of Cldn-3, -5 and Occl immunoreactivity in the ipsilateral cortex and basal ganglia of vehicle-, edoxaban-, or phenprocoumon-treated mice (<span class="html-italic">n</span> = 4). * <span class="html-italic">p</span> < 0.05.</p> "> Figure 3
<p>Edoxaban exerts anti-inflammatory effects in ischemic stroke. (<b>A</b>) Relative gene expression of tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the ischemic cortices (<b>top</b>) and basal ganglia (<b>bottom</b>) of vehicle-, edoxaban (Edox)-, or phenprocoumon (Phen)-treated mice 24 h after transient middle cerebral artery occlusion (tMCAO) (<span class="html-italic">n</span> = 7–10). (<b>B</b>) <b>Left</b>: Representative immunohistochemical staining of CD3<sup>+</sup> T cells (green) and nuclei (blue) in the ischemic hemisphere of vehicle-, edoxaban-, or phenprocoumon-treated mice 24 h after tMCAO (scale bar = 50 µm). <b>Right</b>: Quantification of CD3<sup>+</sup> T cells per slice in the infarcted hemispheres at day 1 after tMCAO (<span class="html-italic">n</span> = 5). (<b>C</b>,<b>D</b>) <b>Left</b>: Representative immunocytochemical staining of Ly-6B.2<sup>+</sup> neutrophils (<b>C</b>) and CD11b<sup>+</sup> macrophages/microglia (<b>D</b>) in the ischemic hemisphere of vehicle-, edoxaban-, or phenprocoumon-treated mice 24 h after tMCAO (scale bar = 100 µm). <b>Right</b>: Quantification of Ly6B.2<sup>+</sup> neutrophils and CD11b<sup>+</sup> macrophages/microglia per slice in the infarcted hemispheres at day 1 after tMCAO (<span class="html-italic">n</span> = 5). * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 4
<p>Edoxaban reduces intracerebral thrombosis. (<b>A</b>) <b>Top</b>: Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein expression in the ischemic cortices and basal ganglia of vehicle (veh)-, edoxaban (Edox)-, or phenprocoumon (Phen)-treated mice at day 1 after tMCAO as determined by immunoblot. Actin was used as loading control. <b>Bottom</b>: Densitometric quantification of ICAM-1 and VCAM-1 immunoreactivity in the ipsilateral cortex and basal ganglia of vehicle-, edoxaban-, or phenprocoumon-treated mice (<span class="html-italic">n</span> = 4). (<b>B</b>) <b>Top</b>: Representative H&E stains in the ischemic hemisphere of vehicle-, edoxaban-, or phenprocoumon-treated mice 24 h after tMCAO (Scale bar = 50 µm). Thrombotic vessels were shown in vehicle- and phenprocoumon-treatment groups, whereas an open vessel is shown for the edoxaban group. <b>Bottom</b>: Percentage of occluded vessels in the ischemic hemispheres of vehicle-, edoxaban-, or phenprocoumon-treated mice at day 1 after tMCAO (<span class="html-italic">n</span> = 5). * <span class="html-italic">p</span> < 0.05.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Treatment with Edoxaban Improves Outcome after Stroke in Mice
2.2. Edoxaban Stabilizes the BBB after Stroke
2.3. Treatment with Edoxaban Reduces Cerebral Inflammation after tMCAO
2.4. Treatment with Edoxaban Does Not Alter Cellular Adhesion but Reduces Intracerebral Thrombosis
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Animal Treatment
4.3. Measurement of Edoxaban Plasma Concentrations
4.4. Ischemia Model
4.5. Exclusion Criteria
4.6. Assessment of Functional Outcome
4.7. Triphenyltetrazolium Chloride (TTC) Staining
4.8. Magnetic Resonance Imaging
4.9. Determination of BBB Leakage and Brain Edema Formation
4.10. Protein Extraction and Western Blot Analysis
4.11. Quantitative Real-Time PCR
4.12. Histology and Immunohistochemistry
4.13. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Bieber, M.; Foerster, K.I.; Haefeli, W.E.; Pham, M.; Schuhmann, M.K.; Kraft, P. Treatment with Edoxaban Attenuates Acute Stroke Severity in Mice by Reducing Blood–Brain Barrier Damage and Inflammation. Int. J. Mol. Sci. 2021, 22, 9893. https://doi.org/10.3390/ijms22189893
Bieber M, Foerster KI, Haefeli WE, Pham M, Schuhmann MK, Kraft P. Treatment with Edoxaban Attenuates Acute Stroke Severity in Mice by Reducing Blood–Brain Barrier Damage and Inflammation. International Journal of Molecular Sciences. 2021; 22(18):9893. https://doi.org/10.3390/ijms22189893
Chicago/Turabian StyleBieber, Michael, Kathrin I. Foerster, Walter E. Haefeli, Mirko Pham, Michael K. Schuhmann, and Peter Kraft. 2021. "Treatment with Edoxaban Attenuates Acute Stroke Severity in Mice by Reducing Blood–Brain Barrier Damage and Inflammation" International Journal of Molecular Sciences 22, no. 18: 9893. https://doi.org/10.3390/ijms22189893