The Role of Blood-Based Molecular Clock Biomarkers in Predicting the Onset Time of Acute Ischemic Stroke: A Prospective Observational Study

Acute ischemic stroke (AIS) is one of the leading causes of mortality and long-term neurological disability worldwide. The effectiveness of reperfusion therapies, including intravenous thrombolysis and mechanical thrombectomy, is highly dependent on the time elapsed since symptom onset. Determination of the symptom onset time is therefore a critical component of treatment eligibility. However, a substantial proportion of patients present with stroke symptoms of unknown onset, most commonly because the symptoms develop during sleep (wake-up stroke) or because the exact onset time cannot be reliably established. This uncertainty may prevent eligible patients from receiving time-dependent reperfusion therapies.

Current clinical decision-making in patients with uncertain onset time relies largely on imaging-based approaches that attempt to estimate the biological stage of ischemia. The diffusion-weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch concept is the most widely used method and is based on the hypothesis that tissue signal evolution reflects the duration of ischemia. Advanced perfusion imaging techniques have also been incorporated into clinical practice. However, these imaging methods require specialized equipment and expertise, may not be available in all centers, and their interpretation may vary between institutions. Consequently, there is increasing interest in identifying circulating biomarkers that can reflect the biological timing of ischemic injury.

Ischemic brain injury initiates a complex cascade of pathophysiological processes involving astroglial activation, neuronal membrane disruption, axonal injury, inflammatory signaling, cellular stress responses, and vascular dysfunction. These processes evolve dynamically over time and lead to the release of measurable molecular components into the systemic circulation. Biomarkers reflecting different cellular compartments of the neurovascular unit may therefore provide complementary information regarding the temporal evolution of ischemic injury.

Several candidate biomarkers have been identified that represent different biological aspects of this process. Glial fibrillary acidic protein (GFAP) is an astrocyte-specific structural protein that reflects astroglial injury and early glial activation following acute brain damage. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a neuron-specific cytoplasmic enzyme released after neuronal membrane disruption and has been proposed as an indicator of acute neuronal injury. Serum neurofilament light chain (sNfL) is a structural component of the axonal cytoskeleton and is considered a marker of axonal degeneration and structural neuronal damage. Phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitory protein, is involved in the regulation of cellular signaling pathways and stress responses and may reflect cellular adaptation to ischemic stress. Matrix metalloproteinase-9 (MMP-9) is associated with inflammatory activation, extracellular matrix degradation, and blood-brain barrier dysfunction during the evolution of ischemic brain injury.

The combined evaluation of biomarkers representing multiple biological layers of the neurovascular unit may provide a more comprehensive representation of the temporal dynamics of ischemic injury than single-marker approaches. A multi-biomarker strategy has the potential to capture different phases of the ischemic cascade, including glial activation, neuronal damage, axonal injury, cellular stress response, and vascular dysfunction.