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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session D

Poster F7 - Optical Quantification of Intestinal Oxygenation and its Effects on Gut Microbiota Composition in Mice

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The intestinal lumen is nearly devoid of oxygen, with the partial pressure of oxygen increasing radially toward the serosa. This oxygen gradient creates microenvironments that are essential in maintaining the diverse group of organisms residing within the gut. These organisms, known as the gut microbiota, play a key role in digestion, immune responses, and chemical communication within the body. Changes in the composition of the gut microbiota can hinder these functions and lead to disease.
Disruption of the intestinal oxygen gradient has not only been linked to decreased microbial diversity in the gut but also to the degradation of the intestinal barrier. Increased permeability of the intestinal epithelial barrier allows for the unregulated circulation of the gut microbiota and its metabolites, resulting in widespread inflammation.
Chronic inflammatory diseases such as inflammatory bowel disease and diabetes have been independently linked to a decrease in gut microbiota diversity and disruption of the intestinal oxygen gradient. However, a quantitative relationship between intestinal oxygenation and gut microbiota diversity has yet to be defined. Using two-photon phosphorescence lifetime microscopy, we are able to quantitatively measure oxygenation and red blood cell flux throughout the intestinal wall in mice. These optical microscopy measurements, in combination with metagenomic analysis of fecal samples, will be used to establish a quantitative relationship between the gut microbiota composition and oxygen metabolism in the intestinal capillary bed.

Materials and

Methods:  Throughout the 35 days of the experiment, three experimental groups are exposed to varying concentrations of inhaled air for 8 hours per day: 21% oxygen (control), 28% oxygen (hyperoxia), 13% oxygen (hypoxia). For all other hours the three groups are housed in normal air conditions.

The effect of the altered air conditions on oxygen metabolism in the cecum is measured every three days through a silicone window implanted in the abdomen. The silicone window uses a glueless and sutureless design to seal the skin surrounding the window, and the cecum is secured against the window in a basket made of sutures.

The partial pressure of oxygen and red blood cell flux in the intestinal capillary plexus are measured through the window using two-photon phosphorescence lifetime microscopy. The technique involves the simultaneous absorption of two photons by the injectable phosphorescent probe Oxyphor2P. Oxygen quenches the probe, resulting in a faster decay in the number of photons emitted over time. The probe is calibrated so that the length of the decay can be quantitatively related to the partial pressure of oxygen present in the sample.

Prior to these microscopy measurements, the three groups are fasted for six hours to minimize differences in blood perfusion in the gut due to digestion. Fecal samples are also collected during each imaging session. The fecal samples are analyzed using 16S rRNA sequencing to quantitatively determine gut microbiota diversity.

Results, Conclusions, and Discussions: Preliminary data was collected through a terminal version of this experiment that involved removing the cecum through an incision in the abdomen to collect optical measurements. When mice were exposed to normal oxygen conditions, the data did not show the expected increase in the partial pressure of oxygen (pO2) from the lumen to the serosa. However, this increase in pO2 was observed in the mice exposed to pure inhaled oxygen. The mean pO2 across the cecum wall increased from 60 mmHg in normal air conditions to 120 mmHg when mice were exposed to pure inhaled oxygen as expected. Additionally, red blood cell (RBC) flux increased from an average of 23 to 31 red blood cells per second when mice were exposed to normal and pure air conditions respectively.

This data demonstrates that two-photon microscopy is capable of quantitatively measuring pO2 and RBC flux across the cecum wall in mice. Much of the data collected from terminal experiments had to be excluded, however, due to tissue injury that may be associated with removing the cecum from the body. Transitioning from a terminal to a longitudinal model will allow us to improve the outcomes of our experiments in addition to exploring other experimental parameters like microbiota diversity and intermittently altered air conditions.

Combining the optical microscopy measurements with fecal analysis will enable us to define a quantitative relationship between gut microbiota composition, intestinal pO2, and RBC flux.
Based on the literature, we expect to observe a decrease in microbiota diversity for the groups undergoing altered air conditions in the longitudinal experiments. We expect to specifically observe an increase in the proportion of anaerobes following chronic exposure to hypoxia and an increase in aerobic microorganisms following hyperoxia exposure.

Microbiota composition, oxygenation, and blood flow are important indicators of gut health.The results of this study will contribute to the emerging literature on the prevention and treatment of intestinal diseases such as inflammatory bowel disease. It will further inform future research that relates gut microbiota composition to the health and function of gut, brain, and beyond.

Acknowledgements (Optional):  Sencan I, Zong W, Alfadhel MA, et al. Two-photon phosphorescence lifetime microscopy of intestinal capillary pO2 and RBC flux. In: Periasamy A, So PTC, König K, eds. Multiphoton Microscopy in the Biomedical Sciences XXI. Vol 11648. SPIE; 2021:116480H. doi:10.1117/12.2583123

Jacquemin G, Benavente-Diaz M, Djaber S, et al. Longitudinal high-resolution imaging through a flexible intravital imaging window. Sci Adv. 2021;7(25):eabg7663. doi:10.1126/sciadv.abg7663