Chronic visualization of microcirculation in mice using viral vectors expressing fluorescent protein-fused albumin

Studying blood microcirculation is crucial for understanding vascular diseases, yet current imaging methods involving acute administration of fluorescent dyes are invasive and short-lived. To address this limitation, we present here two novel approaches for longitudinal vasculature study. Given that albumin constitutes ~60% of plasma protein and is​​​​ produced by liver hepatocytes, we developed adeno-associated viral vectors (AAVs) with the hepatocyte-specific P3 optimized transthyretin promoter to express the albumin-mNeonGreen or albumin-mScarlet fusion protein. These AAVs were packaged with the AAV8 capsid because of its known tropism for hepatocytes. Systemic injection of the AAV via the intraperitoneal or intravenous route resulted in the fluorescently labeled blood plasma, which was observable in the cerebral cortex by in vivo two-photon microscopy for more than a few months. While this approach is versatile for imaging in adult mice, the dilution of the episomal AAV genome in the rapidly growing liver in infants precludes chronic neonatal imaging. Thus, we also established a virally induced CRISPR/Cas9-based knock-in of green fluorescent albumin. We constructed an AAV that includes ~1 kb homologous arms around albumin exon 14 to express albumin-mNeonGreen instead of albumin. Systemic injection of this AAV with AAV-CMV-Cas9 in postnatal day 3 mice resulted in two-photon visualization of the cerebral cortex vasculature within ten days and persistent expression of albumin-mNeonGreen for at least the next three months. These techniques represent versatile and powerful tools for micro- and macroscopic imaging of murine vasculature.