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Objective: Thyroid hormones control a variety of processes in the central nervous system and influence its response to different stimuli, such as ischemic stroke. Post-stroke administration of 3,3',5-triiodo-L-thyronine (T3) has been reported to substantially improve outcomes, but the optimal dosage and time window remain elusive., Methods: Stroke was induced in mice by transient middle cerebral artery occlusion (tMCAO), and T3 was administered at different doses and time points before and after stroke., Results: We demonstrated a dose-dependent protective effect of T3 reducing infarct volumes with an optimal T3 dosage of 25 μg/kg. In addition, we observed a time-dependent effectiveness that was most profound when T3 was administered 1 h after tMCAO (P < 0.001), with a gradual reduction in efficacy at 4.5 h (P = 0.066), and no reduction in infarct volumes when T3 was injected with an 8-h delay (P > 0.999). The protective effect of acute T3 treatment persisted for 72 h post-tMCAO (P < 0.01) and accelerated the recovery of motor function by day 3 (P < 0.05). In-depth investigations further revealed reduced cerebral edema and diminished blood-brain barrier leakage, indicated by reduced extravasation of Evans blue and diminished aquaporin-4 expression., Conclusion: Our findings suggest that T3 may be a promising intervention for ischemic stroke in the acute phase.

Soluble guanylate cyclase (sGC) stands as a pivotal regulatory element in intracellular signalling pathways, mediating the formation of cyclic guanosine monophosphate (cGMP) and impacting diverse physiological processes across tissues. Increased formation of reactive oxygen species (ROS) is widely recognized to modulate cGMP signalling. Indeed, oxidatively damaged, and therefore inactive sGC, contributes to poor vascular reactivity and more severe neurological damage upon stroke. However, the specific involvement of cGMP in redox signalling remains elusive. Here, we demonstrate a significant cGMP-dependent reduction of reactive oxygen and nitrogen species upon sGC activation under hypoxic conditions, independent of any potential scavenger effects. Importantly, this reduction is directly mediated by downregulating NADPH oxidase (NOX) 4 and 5 during reperfusion. Using an in silico simulation approach, we propose a mechanistic link between increased cGMP signalling and reduced ROS formation, pinpointing NF-κB1 and RELA/p65 as key transcription factors regulating NOX4/5 expression. In vitro studies revealed that p65 translocation to the nucleus was reduced in hypoxic human microvascular endothelial cells following sGC activation. Altogether, these findings unveil the intricate regulation and functional implications of sGC, providing valuable insights into its biological significance and ultimately therapeutic potential., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M.H.E., and A.I.C. are inventors of international patent publications WO/2021/167458 titled "Use of a soluble guanylate cyclase (sGC) stimulator or of a combination of a sGC stimulator and an sGC activator for conditions wherein the heme group of sGC is oxidized or wherein sGC is deficient in heme., (Copyright © 2025. Published by Elsevier Masson SAS.)

Background: The discrepancy between experimental research and clinical trial outcomes is a persistent challenge in preclinical studies, particularly in stroke research. A possible factor contributing to this issue is the lack of standardization across experimental stroke models, leading to poor reproducibility in multicenter studies. This study addresses this gap by aiming to enhance reproducibility and the efficacy of multicenter studies through the harmonization of protocols and training of involved personnel., Methods: We established a set of standard operating procedures for various stroke models and the Neuroscore. These standard operating procedures were implemented across multiple research centers, followed by specialized, in-person training for all participants. We measured the variability in infarct volume both before and after the implementation of these standardized protocols and training sessions., Results: The standardization process led to a significant reduction in variability of infarct volume across different stroke models (40%-50% reduction), demonstrating the effectiveness of our harmonized protocols and training. Additionally, the implementation of the Neuroscore system across centers showed low variability and consistent results up to 28 days poststroke, underscoring its utility in chronic phase evaluations., Conclusions: The harmonization of protocols and surgeon training significantly reduced variability in experimental outcomes across different centers. This improvement can increase the comparability of data between research groups and enhance the statistical power of multicenter studies. Our findings also establish the Neuroscore as a reliable tool for long-term assessment in stroke research, paving the way for more consistent and impactful multicenter preclinical studies., Competing Interests: None.

Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) - a key component of the KKS - in the acute phase of ischemic stroke has been reported to reduce thrombosis, inflammation, and damage to the blood-brain barrier. However, the role of PK during the recovery phase after cerebral ischemia is unknown. To this end, we evaluated the effect of subacute PK inhibition starting from day 3 on the recovery process after transient middle artery occlusion (tMCAO). Our study demonstrated a protective effect of PK inhibition by reducing infarct volume and improving functional outcome at day 7 after tMCAO. In addition, we observed reduced thrombus formation in cerebral microvessels, fewer infiltrated immune cells, and an improvement in blood-brain barrier integrity. This protective effect was facilitated by promoting tight junction reintegration, reducing detrimental matrix metalloproteinases, and upregulating regenerative angiogenic markers. Our findings suggest that PK inhibition in the subacute phase might be a promising approach to accelerate the post-stroke recovery process., (© 2024. The Author(s).)

Ischemic stroke is associated with exacerbated tissue damage caused by the activation of immune cells and the initiation of other inflammatory processes. Dimethyl fumarate (DMF) is known to modulate the immune response, activate antioxidative pathways, and improve the blood-brain barrier (BBB) after stroke. However, the specific impact of DMF on immune cells after cerebral ischemia remains unclear. In our study, male mice underwent transient middle cerebral artery occlusion (tMCAO) for 30 min and received oral DMF (15 mg/kg) or a vehicle immediately after tMCAO, followed by twice-daily administrations for 7 days. Infarct volume was assessed on T2-weighted magnetic resonance images on days 1 and 7 after tMCAO. Brain-infiltrating immune cells (lymphocytes, monocytes) and microglia were quantified using fluorescence-activated cell sorting. DMF treatment significantly reduced infarct volumes and brain edema. On day 1 after tMCAO, DMF-treated mice showed reduced lymphocyte infiltration compared to controls, which was not observed on day 7. Monocyte and microglial cell counts did not differ between groups on either day. In the acute phase of stroke, DMF administration attenuated lymphocyte infiltration, probably due to its stabilizing effect on the BBB. This highlights the potential of DMF as a therapeutic candidate for mitigating immune cell-driven damage in stroke.

Background Typically defined as a thromboinflammatory disease, ischemic stroke features early and delayed inflammatory responses, which determine the extent of ischemia-related brain damage. T and natural killer cells have been implicated in neuronal cytotoxicity and inflammation, but the precise mechanisms of immune cell-mediated stroke progression remain poorly understood. The activating immunoreceptor NKG2D is expressed on both natural killer and T cells and may be critically involved. Methods and Results An anti-NKG2D blocking antibody alleviated stroke outcome in terms of infarct volume and functional deficits, coinciding with reduced immune cell infiltration into the brain and improved survival in the animal model of cerebral ischemia. Using transgenic knockout models devoid of certain immune cell types and immunodeficient mice supplemented with different immune cell subsets, we dissected the functional contribution of NKG2D signaling by different NKG2D-expressing cells in stroke pathophysiology. The observed effect of NKG2D signaling in stroke progression was shown to be predominantly mediated by natural killer and CD8 + T cells. Transfer of T cells with monovariant T-cell receptors into immunodeficient mice with and without pharmacological blockade of NKG2D revealed activation of CD8 + T cells irrespective of antigen specificity. Detection of the NKG2D receptor and its ligands in brain samples of patients with stroke strengthens the relevance of preclinical observations in human disease. Conclusions Our findings provide a mechanistic insight into NKG2D-dependent natural killer- and T-cell-mediated effects in stroke pathophysiology.

Platelets are key mediators of thrombus formation and inflammation during the acute phase of ischaemic stroke. Particularly, the platelet glycoprotein (GP) receptors GPIbα and GPVI have been shown to mediate platelet adhesion and activation in the ischaemic brain. GPIbα and GPVI blockade could reduce infarct volumes and improve functional outcome in mouse models of acute ischaemic stroke, without concomitantly increasing intracerebral haemorrhage. However, the functional role of platelets during long-term stroke recovery has not been elucidated so far. Thus, we here examined the impact of platelet depletion on post-stroke recovery after transient middle cerebral artery occlusion (tMCAO) in adult male mice. Platelet depleting antibodies or isotype control were applied from day 3-28 after tMCAO in mice matched for infarct size. Long-term functional recovery was assessed over the course of 28 days by behavioural testing encompassing motor and sensorimotorical functions, as well as anxiety-like or spontaneous behaviour. Whole brain flow cytometry and light sheet fluorescent microscopy were used to identify resident and infiltrated immune cell types, and to determine the effects of platelet depletion on the cerebral vascular architecture, respectively. We found that delayed platelet depletion does not improve long-term functional outcome in the tMCAO stroke model. Immune cell abundance, the extent of thrombosis and the organisation of the cerebral vasculature were also comparable between platelet-depleted and control mice. Our study demonstrates that, despite their critical role in the acute stroke setting, platelets appear to contribute only marginally to tissue reorganisation and functional recovery at later stroke stages., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Despite decades of promising preclinical validation and clinical translation, ischemic stroke still remains as one of the leading causes of death and disability worldwide. Within its complex pathophysiological signatures, thrombosis and inflammation, that is, thromboinflammation, are highly interconnected processes leading to cerebral vessel occlusion, inflammatory responses, and severe neuronal damage following the ischemic event. Hence, we here review the most recent updates on thromboinflammatory-dependent mediators relevant after stroke focusing on recent discoveries on platelet modulation, a potential regulation of the innate and adaptive immune system in thromboinflammation, utterly providing a thorough up-to-date overview of all therapeutic approaches currently undergoing clinical trial.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion., (© 2022. The Author(s).)