Cerebrovascular Claudin-5 Isoform Expression Correlates with Worsened Stroke Outcomes Following Thromboembolic Stroke

Background and PurposeClaudin-5 plays a crucial role in the maintenance of the blood-brain barrier (BBB) integrity through its role in endothelial tight junction formation. Alternative splicing of claudin-5 within the microvascular endothelium may modulate BBB structural and functional dynamics, potentially influencing neuronal damage and recovery following ischemic stroke. We hypothesized that ischemic stroke induces temporal changes in claudin-5 protein isoform expression that correlates with worsened neurological outcomes. MethodsMale Wistar rats underwent thromboembolic stroke. Claudin-5 isoform expression was assessed at 3, 6, and 24h post-stroke onset, with additional groups receiving recombinant tissue plasminogen activator (rt-PA) at 4 hours post-stroke. Brain edema, infarct volume, hemorrhage, and cerebral blood flow was 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. In addition, RNA sequencing analysis was performed to identify novel splice variants. ResultsA time-dependent increase in claudin-5 isoform 1 (35kDa) expression levels in the ipsilateral cerebrovasculature at 6 h was observed. Isoform 2 (25kDa) and fragment (10kDa) isoforms of claudin-5 remain unchanged. Treatment with rt-PA maintained the elevated levels of isoform 1 claudin-5 protein expression within the ipsilateral hemisphere. Increased claudin-5 isoform 1 expression within the ipsilateral hemisphere correlated with increased brain edema, hemorrhage, and worsened neurological function at 24h post-stroke onset. RNA sequencing revealed novel CLDN5 splice isoforms in post-stroke rat brain tissue which resemble structural similarity to known human CLDN5 isoforms. ConclusionThese 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 potential indication of novel alternative splice variants suggests that post-transcriptional regulation of claudin-5 represents a previously unrecognized mechanism contributing to endothelial tight junction dysfunction and stroke pathophysiology. These results highlight claudin-5 isoform expression as a potential therapeutic target for preserving BBB integrity following cerebral ischemia.