Quantifying Intestinal Capillary Oxygenation Using Two‐photon Phosphorescence Lifetime Microscopy

Abstract

Impaired oxygen delivery reduces the diversity of the gut microbiota1,2. Quantifying the microvascular oxygenation in the small intestines may help us better understand progression of the intestinal diseases such as inflammatory bowel disease (IBD), their relation with the changes in the gut microbiota, as well as implications to the microbiota–gut–brain axis. To start addressing these questions, we applied two-photon phosphorescence lifetime microscopy (2PLM) to quantify partial pressure of oxygen (pO2) and Red Blood Cell (RBC) flux in the capillary plexus through entire cecum wall in healthy, 2-month-old, female C57BL6 mice. Mice were anesthetized with isoflurane mixed with air (3% during surgery and 2% during imaging). The animals were breathing spontaneously through a loose mask. A small incision was made into the abdomen and the cecum was gently pulled out and positioned into a custom-made chamber with a cover glass on top. The exposed intestine was kept moist with a warmed 1x phosphate buffered saline (PBS) solution. Both core body temperature and temperature over the cecum were maintained at 37 °C. Experiments were terminated when the peristaltic movement prevented microscopic measurements. We measured pO2 and RBC flux during normoxia (n=4 mice, mean pO2 = 60 ± 6 mmHg, mean capillary RBC flux = 23 ± 8 RBCs/s) and hyperoxia (100% O2 inhalation, n=3, mean pO2= 120 ± 15 mmHg, mean capillary RBC flux = 31 ± 17 RBCs/s), as a function of depth in the cecum wall from 0 to 120 µm. Our results show expected significant increase in pO2 in response to hyperoxia, small capillary mean RBC flux values, and approximately uniform capillary pO2 across the thickness of the cecum wall during normoxia. Future steps include investigation of the capillary oxygenation and RBC flux in pathological conditions and in relation with the diversity of the gut microbiome.