Brain cell-released Cyclophilin A (eCypA) exacerbates neuroinflammation and blood-brain barrier injury in acute ischemic stroke
Background
Acute ischemic stroke (AIS) is a leading cause of death and disability, with limited therapies effectively addressing its complex pathophysiology. A critical component of post-stroke injury is excessive neuroinflammation, which mediates blood-brain barrier (BBB) disruption and contributes to poor patient outcomes. Cyclophilin A (CypA), particularly when released into the extracellular space (eCypA), is implicated in inflammatory reactions and vascular dysfunction. However, its precise role in regulating neuroinflammation and BBB injury in AIS, and the therapeutic potential of targeting eCypA, remained largely unexplored.
Study Design
Researchers utilized ELISA to measure eCypA in serum from 22 AIS patients (mild/severe) and healthy controls, as well as in serum/CSF from transient middle cerebral artery occlusion (tMCAO) rats. In vitro, ELISA detected eCypA in supernatants from BV2 microglia and bEnd.3 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 tMCAO followed by 24 h reperfusion. These rats received intracerebroventricular injection of cyclophilin A-binding heptameric peptide (C46) before tMCAO. Primary endpoints included cerebral infarct volume (TTC staining), BBB permeability (Evans blue extravasation), tight junction proteins, matrix metalloproteinases (MMPs), and proinflammatory mediators (Western blot), alongside microglial activation (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). Similar elevations were observed in tMCAO rat serum (2.57 ± 0.14 ng/mL vs. 1.62 ± 0.07 ng/mL, p < 0.001) and CSF (2.14 ± 0.23 ng/mL vs. 1.47 ± 0.19 ng/mL, p < 0.001), and in OGD/R-challenged BV2 cell supernatants (0.92 ± 0.01 ng/mL vs. 0.55 ± 0.03 ng/mL, p < 0.001). > Intracerebroventricular administration of C46 significantly reduced cerebral infarct volume and BBB permeability in tMCAO rats. This intervention also attenuated the decrease in tight junction proteins (ZO-1, occludin), suppressed the upregulation of MMP-2 and MMP-9, and decreased proinflammatory mediators like TNF-α, IL-1β, and iNOS. Furthermore, C46 inhibited microglial activation, suggesting a broad anti-inflammatory and neuroprotective effect.
Key Findings
- eCypA levels were significantly elevated in AIS patient serum (p < 0.001) and tMCAO rat serum/CSF (p < 0.001).
- eCypA release was observed in
OGD/R-challenged BV2 microglia and bEnd.3 endothelial cells. - Intracerebroventricular C46 reduced cerebral infarct volume and BBB permeability in tMCAO rats.
- C46 attenuated tight junction protein (
ZO-1,occludin) degradation and suppressedMMP-2/MMP-9upregulation. - C46 decreased proinflammatory mediators (
TNF-α,IL-1β,iNOS) and inhibited microglial activation.
Why It Matters
This research identifies extracellular Cyclophilin A (eCypA) as a critical mediator of neuroinflammation and blood-brain barrier disruption following acute ischemic stroke, offering a novel therapeutic target. Targeting eCypA with peptides like C46 could represent a new strategy to mitigate post-stroke brain injury and improve outcomes. While C46 was administered intracerebroventricularly in this preclinical model, future research could explore systemic delivery or modified peptide forms for clinical translation. This finding opens avenues for developing new interventions beyond current standard-of-care, potentially reducing infarct size and preserving BBB integrity in stroke patients. It highlights the importance of modulating inflammatory pathways for neuroprotection.