In Vivo Measurement of Mouse Endothelial Surface Layer in Surface Cortical Microvasculature

Abstract

Sepsis, a systemic inflammatory response to infection, is the most common cause of in‐hospital mortality in the United States. Sepsis survivors often demonstrate chronic neurocognitive dysfunction. However, little is known about the mechanisms underlying the development of neurocognitive dysfunction during sepsis. We have previously demonstrated that septic lung and kidney injury is mediated by degradation of the endothelial surface layer (ESL), a thick glycosaminoglycan‐rich layer lining the pulmonary and glomerular microcirculation, respectively. We now postulate that degradation of the neurovascular ESL may contribute to septic neurocognitive dysfunction. To pursue this hypothesis, we have adapted a surgical approach to in vivo brain confocal microscopy that allows for direct visualization of the ESL in surface cortical microvasculature. We placed a cranial window in anesthetized mice. After allowing for 4 days of recovery, we re‐anesthetized mice and administered an intravenous bolus of 200 μl Fluorescein isothiocyanate (FITC)‐labeled 150 kDa dextran (excluded by the ESL), followed by a bolus of 200 μl Tetramethylrhodamine isothiocyanate (TRITC)‐labeled 40 kDa dextran (which can penetrate the ESL). Using an in‐focus frame, we identified surface cortical microvessels (< 20 μm diameter); at least 3 microvessels are typically found on a single frame. We performed in vivo confocal microscopy through the cranial window, simultaneously measuring TRITC (inclusive of the ESL) and FITC (exclusive of the ESL) microvessel widths. Assuming equal ESL thickness at both edges of the vessel, the ESL size is defined by one‐half the difference between TRICT‐ and FITC‐dextran vascular widths. After measurement of baseline ESL thickness, mice were injected with lipopolysaccharide (LPS, 20 μg/g body weight in 100 μl saline) to model sepsis. ESL thickness was followed every 30 min thereafter. The baseline ESL thickness of surface cortical microvessels was 0.51 μm, a size less than that which we have previously observed in lungs (1.67 μm) but similar to that observed in systemic vessels (0.6 – 0.7 μm). Endotoxemia led to a rapid loss of ESL thickness (0.08 μm vs. 0.71 μm 30 min after LPS or saline, respectively). In conclusion, by use of surgical placement of a cranial window, we have developed an experimental model that allows for the detailed observation of the neurovascular ESL. Using this model, we have demonstrated that the neurovascular ESL thickness is approximately 0.51 μm in thickness and may be degraded during endotoxemia, similar to other vascular beds of clinical relevance in sepsis.Support or Funding InformationR01 HL125371 K08 HL105538This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.