Cerebrovascular claudin-5 isoform expression correlates with worsened stroke outcomes following thromboembolic stroke

Abstract Background and purpose Claudin-5 plays a crucial role in maintaining blood-brain barrier (BBB) integrity through endothelial tight junction formation. Alternative splicing of claudin-5 within the microvascular endothelium may modulate BBB structural and functional dynamics, thereby affecting neuronal damage and recovery after ischemic stroke. The aim of this study was to investigate temporal and hemisphere-specific changes in claudin-5 isoform expression after ischemic stroke and to evaluate their correlation with BBB dysfunction and neurological outcomes. Methods Male Wistar rats underwent thromboembolic stroke. Claudin-5 isoform expression was assessed at 3, 6, and 24 h post-stroke onset, with additional groups receiving recombinant tissue plasminogen activator (rt-PA) at 4 h post-stroke. Brain edema, infarct volume, hemorrhage, and cerebral blood flow were evaluated using 9.4T MRI. Ipsilateral and contralateral cerebrovascular claudin-5 expression was quantified via western blotting while neurological function was assessed by 28-point neuroscore. RNA sequencing analysis was performed to identify novel splice variants. In addition, molecular dynamics simulations of AlphaFold-predicted claudin-5 isoform 1 and 2 tetramers embedded in lipid bilayers were performed to quantify steric–electrostatic barrier properties and ion permeability. Results A time-dependent increase in claudin-5 isoform 1 (35 kDa) expression levels in the ipsilateral cerebrovasculature at 6 h was observed. Isoform 2 (25 kDa) and fragment (10 kDa) isoforms of claudin-5 remained unchanged. Treatment with rt-PA maintained the elevated levels of isoform 1 claudin-5 protein expression within the ipsilateral hemisphere. Increased isoform 1 expression correlated with edema, hemorrhage, and worsened neurological function at 24 h post-stroke onset. RNA sequencing revealed novel CLDN5 splice isoforms in post-stroke rat brain tissue resembling known human CLDN5 isoforms. Computational modeling suggested that isoform 1 forms a wide but electrostatically exclusionary pore with strong anion selectivity, whereas isoform 2 permits markedly greater cation permeability. These isoform-specific barrier properties are consistent with the in vivo association of isoform 1 upregulation with BBB dysfunction after stroke. Conclusion These findings demonstrate that ischemic stroke induces temporal, hemisphere-specific alterations in claudin-5 isoform expression that correlate with BBB dysfunction and poor neurological outcomes. The combination of RNA sequencing and molecular dynamics simulations indicates that alternative splice variants of claudin-5 confer distinct structural and permeability profiles, representing a previously unrecognized mechanism of endothelial tight junction dysfunction in stroke. These results highlight claudin-5 isoform expression as both a novel biologically relevant and a potential therapeutic target for preserving BBB integrity following cerebral ischemia.