Brain cell-released Cyclophilin A induces neuroinflammation and exacerbates blood–brain barrier injury in acute ischemic stroke

 What research are you initiating to prevent this from happening? DOING NOTHING, LIKE USUAL?

With NO leadership anywhere in stroke NOTHING IS EVER ACCOMPLISHED!

Our fucking failures of a stroke associations are completely useless in getting stroke solved to 100% recovery. I'll be a bad person and hope every single employee in them get blasted hard by their stroke when they are the 1 in 4 per WHO that has a stroke! 

Brain cell-released Cyclophilin A induces neuroinflammation and exacerbates blood–brain barrier injury in acute ischemic stroke

• 

  Enyue Chen 1

• 

  Qihui Wu 2

• Y

  Yunwen Xue 2

• 

  Shixiong Huang 1*

• 

  Yong Gu 2*

• 1. Department of Neurology, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou, China

• 2. Clinical Research Center, Affiliated Chinese Medicine, Hainan Medical University, Haikou, China

Abstract

Background: 

Excessive neuroinflammation mediates blood–brain barrier (BBB) disruption and poor outcomes after acute ischemic stroke (AIS). Cyclophilin A (CypA), when released into the extracellular space (designated as eCypA), may participate in inflammatory reactions and vascular dysfunction. However, its role in regulating neuroinflammation and BBB injury in AIS, as well as the therapeutic potential of targeting eCypA remain unclear.

Methods: 

ELISA was used to detect eCypA release in serum from 22 AIS patients (17 mild, 5 severe; 13 males, 9 females; mild: age 65.41 ± 10.20 years, NIHSS 2.88 ± 1.45; severe: age 64.00 ± 5.04 years, NIHSS 9.40 ± 3.29; blood sampled within 48 h of onset), in serum/cerebrospinal fluid (CSF) from transient middle cerebral artery occlusion (tMCAO) rats, and in supernatants from BV2 (microglia) or bEnd.3 (brain microvascular endothelial cells) exposed to oxygen–glucose deprivation/reoxygenation (OGD/R) or lipopolysaccharide (LPS). Nine-week-old male Sprague–Dawley rats (n = 5 per group) underwent 1.5 h of tMCAO via the intraluminal suture method followed by 24 h of reperfusion before sampling. These rats received intracerebroventricular injection of cyclophilin A-binding heptameric peptide (C46) before tMCAO establishment. Cerebral infarct volume was measured via TTC staining. BBB permeability was assessed by Evans blue extravasation. Western blot was employed to determine protein levels of tight junction (TJ) proteins, matrix metalloproteinases (MMPs) and proinflammatory mediators. Microglial activation was evaluated by immunofluorescence.

Results: 

eCypA levels were significantly elevated in AIS patient serum (1.74 ± 0.23 ng/mL in mild, 2.39 ± 0.09 ng/mL in severe vs. 1.30 ± 0.19 ng/mL in healthy controls, p < 0.001), in tMCAO rat serum (2.57 ± 0.14 vs. 1.62 ± 0.07 ng/mL, p < 0.001) and CSF (2.14 ± 0.23 vs. 1.47 ± 0.19 ng/mL, p < 0.001), as well as in the supernatants of OGD/R-challenged BV2 (0.92 ± 0.01 vs. 0.55 ± 0.03 ng/mL, p < 0.001) and bEnd.3 cells (1.10 ± 0.05 vs. 0.52 ± 0.03 ng/mL, p < 0.001) and LPS-induced BV2 cells (1.12 ± 0.08 vs. 0.56 ± 0.13 ng/mL, p < 0.001) compared with their respective control groups. The eCypA inhibitory peptide C46 effectively improved neurological function, reduced cerebral infarct volume and edema in tMCAO rats. Moreover, C46 mitigated BBB permeability, preserved the expression levels of TJ proteins, and suppressed the activation of MMPs in tMCAO rats and OGD/R-treated bEnd.3 cells. Meanwhile, C46 administration inhibited microglial activation and downregulated the expression of proinflammatory mediators both in vivo (tMCAO rats) and in vitro (OGD/R- or LPS-induced BV2 microglia).

Conclusion: 

eCypA, released by microglia and brain microvascular endothelial cells under ischemic–hypoxic and inflammatory conditions, serves as a critical pathogenic mediator that drives neuroinflammation and BBB disruption in AIS. Targeting eCypA with C46 peptide effectively abrogates these pathological cascades, thereby supporting eCypA as a novel therapeutic target for AIS.

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Microglial P2RY12 mediates migration to and protection of cerebral microvasculature after ischemia–reperfusion via Caveolin-1

 Have your competent? doctor and hospital either get the EXACT PROTOCOL or get further research done to create such protocol!

Do you prefer your doctor, hospital and board of director's incompetence NOT KNOWING? OR NOT DOING? Your choice; let them be incompetent or demand action!

Microglial P2RY12 mediates migration to and protection of cerebral microvasculature after ischemia–reperfusion via Caveolin-1

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https://doi.org/10.1016/j.expneurol.2026.115662Get rights and content

Highlights

• •
  P2RY12 mediates microglia migration and protects the blood-brain barrier.
• •
  The role of microglial P2RY12 in modulating microvascular protection via Cav-1.
• •
  P2RY12 interacts with the scaffolding domain of Cav-1.

Abstract

Disruption of blood-brain barrier (BBB) integrity after cerebral ischemia-reperfusion (I/R) injury contributes to neuroinflammation and neuronal damage. Microglia plays a significant role in the repair processes of the BBB, and the G protein-coupled receptor P2RY12 is involved in microglial chemotactic migration. However, its precise function and associated downstream mechanisms are unclear. Caveolin-1 (Cav-1), a membrane scaffold protein, plays a key role in signal transduction and cellular motility. This study employed in vivo and in vitro experimental models to explore the functional role of the P2RY12-Cav-1 interaction after ischemic stroke. Blocking P2RY12 with PSB0739 worsened neurological deficits and BBB disruption. In contrast, the P2RY12 agonist 2MeSADP attenuated I/R injury, promoted Bv2 cell migration. Disrupting lipid rafts with methyl-β-cyclodextrin (MβCD) abolished these benefits. Co-immunoprecipitation verified P2RY12 interacts with the scaffolding domain of Cav-1. These findings reveal a possible mechanism by which the P2RY12-Cav-1 signaling axis regulates microglial chemotaxis for microvascular protection, offering a potential therapeutic target for the treatment of ischemic stroke.

Introduction

Stroke, a leading global cause of both mortality and neurological disability, is predominantly characterized by the ischemic subtype, which accounts for approximately 87% of cases (Martin et al., 2025). The associated disease burden continues to escalate (Radak et al., 2017; Rathore et al., 2002). Disruption of the structural integrity and function of the blood-brain barrier (BBB) constitutes a pivotal event in the pathological progression of ischemic brain injury (del Zoppo et al., 2007). While vascular recanalization remains the cornerstone of clinical intervention, reperfusion-induced BBB damage contributes to vascular leakage and secondary brain injury, thus posing a significant limitation to patient prognosis (Krueger et al., 2015). Furthermore, the BBB directly regulates the trans-barrier delivery efficiency of neuroprotective agents (Wang et al., 2024; Yang et al., 2024). Consequently, maintaining BBB integrity carries substantial therapeutic implications, both for preventing further injury and for optimizing the efficacy of neuroprotective treatments.

Microglia, the resident immune cells of the brain, established connections with the vascular wall via morphological extensions, contributing to vascular homeostasis under physiological conditions (Bisht et al., 2021). Together with neurons and blood vessels, microglia constitute the neurovascular unit and play a critical role in maintaining BBB integrity (Thion et al., 2018; Prinz et al., 2019; Halder and Milner, 2020; Halder and Milner, 2019). P2RY12, a G protein-coupled purinergic receptor first identified in platelets, is a widely studied clinical target in cardiovascular and cerebrovascular diseases (Hollopeter et al., 2001). Its function is cell-type dependent: in platelets, it promotes thrombosis via Gi/o signaling, whereas in microglia, it mediates chemotactic migration (Davalos et al., 2005). By specifically recognizing purinergic signaling molecules (e.g., ATP/ADP) released at injury sites (Davalos et al., 2005). P2RY12-mediated chemotaxis of microglial processes is required for the rapid closure of the BBB, and it protects neurons from damage by forming microglia-neuron connections and plays a significant role in cerebral blood flow regulation (Cserép et al., 2020; Lou et al., 2016). Our preliminary investigations utilizing single-cell scRNA-seq datasets, the R package Cell Chat, and confirmatory experiments suggest that P2RY12 may function as a key trigger molecule through which microglia influence the cerebral microvasculature in ischemic brain injury (Wang et al., 2023). Nevertheless, its precise role and underlying mechanisms remain unclear.

As a key member of the caveolin protein family, Cav-1 functions as both a pivotal architectural protein for the formation of caveolae structures in the cell membrane and as an essential scaffold protein that maintains the structural integrity of specific functional domains within the cell membrane. It plays a crucial role in regulating cell membrane curvature and lipid raft assembly while integrating multiple transmembrane signaling pathways (Liu et al., 2002). In tumor biology, Cav-1 modulates cellular migration and invasion through molecular interactions, signaling pathways, and structural changes in cells (Singh et al., 2024; Du et al., 2009; Martínez et al., 2019; Chanvorachote et al., 2014; Lin et al., 2005; Li et al., 2018; Xiong et al., 2017; Sun et al., 2009). Research has demonstrated that the purinergic receptor P2Y family and Cav-1 regulate the structural and functional coupling between nucleotide receptors on the cell membrane and their downstream effectors, thereby affecting the efficiency of extracellular nucleotide signal transduction (Martinez et al., 2016). Notably, the lipid raft microdomain, serving as the functional platform for caveolae, the ability of Gαi (downstream of the P2Y12 receptor) to potentiate ADP-mediated platelet aggregation is highly dependent upon its localization to lipid rafts (Liu et al., 2022; Quinton et al., 2005). Furthermore, Cav-1 knockdown has been shown to reduce overall microglial migration, suggesting its potential involvement in microglial dynamics relevant to injury responses (Niesman et al., 2013). However, the specific role of Cav-1 in regulating P2RY12-mediated microglial function, particularly its chemotactic migration towards injured vasculature and microvascular protection within the central nervous system (CNS) during ischemic injury, remains unexplored.

This study employs an integrated approach to explore the regulatory mechanisms of the P2RY12/Cav-1 signaling axis in ischemic brain injury. Initially, the functional role of P2RY12 will be investigated through pharmacological inhibition with the antagonist PSB0739. This approach will assess the impact of P2RY12 blockade on cerebral I/R injury outcomes, evaluating its effects on microvascular integrity. Complementary in vitro studies using Bv2 microglial cells will further define the contribution of P2RY12 to microglial migration. Subsequently, both in vivo and in vitro models will be used to elucidate the mechanisms governing microglial chemotactic migration, employing the P2RY12 agonist 2MeSADP and the lipid raft disruptor methyl-β-cyclodextrin (MβCD).Finally, the study will experimentally determine the interaction between Cav-1 and P2RY12.

As a purinergic receptor uniquely expressed in microglia, the signaling network mediated by P2RY12 and its molecular regulatory mechanisms remain incompletely elucidated. Cav-1 may act as a critical molecular hub, potentially involved in P2RY12-induced microglial chemotactic migration. This research elucidates the molecular mechanisms by which microglial P2RY12 regulates the microcirculatory system during stroke pathology. The findings not only expand the theoretical framework of ischemic brain injury but also provide experimental evidence for developing potential therapeutic strategies focused on neurovascular unit protection.

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Vascular recanalization exacerbates BBB permeability after ischemic stroke

You've known of blood brain barrier problems post stroke, WHY THE FUCK AREN'T YOU SOLVING THEM?

 Inflammatory action leaking through the blood brain barrier.  May 2013

A decade of complete incompetence and you're continuing that incompetence.  You'll want a solution when you are the 1 in 4 per WHO that has a stroke

 and by then it will be too late for them to change their recovery trajectory! 

 Vascular recanalization exacerbates BBB permeability after ischemic stroke

Rong-Fei Wang^1,2,3Jing Liu³Chang-Hui Chen^2*

• ¹Guangzhou University of Chinese Medicine, Guangzhou, China
• ²Department of Neurology, The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China
• ³Department of Neurology, The Affiliated Brain Hospital of Guangzhou Medical University, Guangzhou, China

Introduction: Ischemic stroke is a common and serious neurological disease. After cerebral ischemia occurs, the integrity of the BBB is disrupted, leading to increased permeability, causing pathophysiological changes such as brain edema and hemorrhagic transformation, which aggravates neuronal damage. Such changes become more obvious after the recovery of blood flow. However, the effect of vascular recanalization on blood–brain barrier leakage is poorly known.

Methods: Mice were divided into the recanalization group and the non-recanalization group. Mice in the recanalization group suffered from the middle cerebral artery occlusion and were reperfused 60 min later. Mice in the non-recanalization group suffered from permanent occlusion of the middle cerebral artery. The permeability of the blood–brain barrier was tested using fluorescence staining, and the expression of tight junction proteins and transcytosis-related proteins were analyzed by western blot.

Results: The IgG results revealed a significantly larger area of leakage in the recanalization group compared to the non-recanalization group. A consistent trend was observed in the FITC-dextran leakage experiment. Moreover, after blood flow recanalization, there was a significant reduction in tight junctions-related proteins, occludin and ZO-1. Meanwhile, both ischemia and reperfusion caused changes in the ratio of transcytosis related protein Caveolin-1 /MFSD2a, and this is more obvious in the blood flow recanalization group.

Conclusion: Vascular recanalization can exacerbate blood–brain barrier disruption, concurrently impairing both the paracellular and transcytosis pathways. This finding provides a rationale for exploring new approaches for protecting the integrity of the blood–brain barrier, reducing its permeability, and lowering the risk of hemorrhagic transformation.

Phased blood-brain barrier disruption in ischaemic stroke: implications for therapy?

 Why listen to you when you don't know that stroke has been called neurological disease by the WHO since 2006 not cerebrovascular, once again proving the stroke medical world doesn't keep up to date in their field!

Phased blood-brain barrier disruption in ischaemic stroke: implications for therapy?

• Alissia Blase, 
• Costanza Giovene di Girasole, 
• Laura Benjamin & 
• Patric Turowski 

Fluids and Barriers of the CNS volume 22, Article number: 90 (2025) Cite this article

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Abstract

Cerebrovascular disease, which primarily affects the brain’s blood vessels, remains a major global cause of death and disability. Among its clinical manifestations, ischaemic stroke is by far the most common. Prolonged oedema due to blood vessel leakage is detrimental to the delicate neuronal environment throughout the ischaemic and reperfusion phase and contributes to the mortality, morbidity, and disabilities associated with this devastating condition. Under physiological conditions, an intact blood-brain barrier (BBB) protects and regulates solute and cell transit in and out of the central nervous system. Indeed, dysfunction of this formidable cerebrovascular regulator has been functionally linked to adverse outcomes in stroke. While our knowledge of the underlying mechanism is incomplete, increasing evidence, particularly from studies using models of rodents exposed to middle cerebral artery occlusion (MCAO), supports a biphasic breakdown of the BBB in ischemic stroke. However, debate persists regarding the precise mechanisms of BBB dysfunction. Understanding this pathobiology is essential for developing targeted interventions to improve clinical outcomes in stroke patients. In this review, we provide a summary of the structure and function of the BBB as well as the cellular and molecular determinants of leakage pathways present in pathological conditions, and evaluate medical strategies aimed at reducing BBB disruption in stroke. We also discuss the potential for selectively targeting specific phases of BBB leakage.

(So, you described a problem; PROVIDED NO SOLUTION; You're fired!)

Protection of Stroke-induced Blood-Brain Barrier Disruption by Guanxinning Injection and Its Active-component Combination via TLR4/NF-κB/MMP9-mediated Neuroinflammation

 Is this solving this problem? 

Inflammatory action leaking through the blood brain barrier.

Have your competent? doctor answer that and when human testing will start. Doesn't know and won't figure out human testing; THEN YOU HAVE AN INCOMPETENT DOCTOR! Fire them!

Protection of Stroke-induced Blood-Brain Barrier Disruption by Guanxinning Injection and Its Active-component Combination via TLR4/NF-κB/MMP9-mediated Neuroinflammation

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https://doi.org/10.1016/j.phymed.2025.157162Get rights and content

Highlights

• •
  A cell-based 3D blood-brain barrier (BBB) organoids successfully resemble ischemic damage's structural and functional characteristics.
• •
  The TLR4/NF-κB/MMP9 axis critically safeguards BBB integrity by modulating neuroinflammatory signaling.
• •
  SAB and SI combination in GXNI synergizes BBB protection by targeting MMP9 protein.

Abstract

Background

: The blood-brain barrier (BBB) is essential for central nervous system (CNS) homeostasis, yet neuroinflammatory mechanisms driving BBB disruption remain poorly understood.

Purpose

: To explore the oxygen-glucose deprivation/reoxygenation (OGD/R)-induced BBB dysfunction and evaluate the therapeutic effects of Guanxinning injection (GXNI), a Danshen-Chuanxiong herbal compound, targeting neuroinflammatory pathways.

Methods

: A 3D-BBB organoid composed of human brain microvascular endothelial cells, human astrocytes, and primary human brain microvascular pericytes was constructed, and conditions for OGD/R that simulate ischemic stroke were established. Structure and function of the in vitro BBB were evaluated by morphology, paracellular permeability, and tight junction proteins ZO-1, claudin-5, and occludin expression. In vivo, infarct volume and BBB leakage were measured in a mid-cerebral artery occlusion-induced cerebral ischemia-reperfusion injury model. RNA-seq and network pharmacology analysis were used to identify key genes and pathways for ischemic BBB disruption. HPLC-MS was performed to identify and quantify active components. Molecular docking, SPR, and molecular dynamics were performed to predict and confirm the interaction of active compounds and target proteins.

Results

: A Danshen-Chuanxiong double herbal medicine, GXNI, mitigated these effects, restoring transport capacity, reducing oxidative stress (ROS), and enhancing basement membrane components (laminin, collagen IV). In vivo, GXNI alleviated cerebral ischemia-reperfusion injury (CIRI), decreasing BBB leakage, infarct volume, and neurological deficits. The pivotal role of TLR4/NF-κB/MMP9 neuroinflammatory axis for GXNI BBB protection was identified through transcriptomic analysis and validated via immunofluorescence in BBB spheroids. Molecular docking revealed Danshen-derived salvianolic acid B (SAB) as a high-affinity MMP9 binder, confirmed by quantitative binding assays. The SAB and Chuanxiong-derived senkyunolide I (SI) combination achieved more prominent upregulation of tight junction proteins and suppression of MMP9.

Conclusion

: Our findings further confirm neuroinflammation as a central driver of ischemic BBB damage and demonstrate that GXNI preserves BBB integrity by targeting TLR4/NF-κB/MMP9 signaling in 3D models and CIRI mice, with SAB-SI synergistically contributing to enhanced therapeutic efficacy.

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