Photoacoustic Imaging as a Novel Non-invasive Biomarker to Assess Intestinal Tissue Oxygenation and Motility in Neonatal Rats

Abstract

Background: Within the premature infant intestine, oxygenation and motility play key physiological roles in healthy development and disease such as necrotizing enterocolitis. To date, there are limited techniques to reliably assess these physiological functions that are also clinically feasible for critically ill infants. To address this clinical need, we hypothesized that photoacoustic imaging (PAI) can provide non-invasive assessment of intestinal tissue oxygenation and motility to characterize intestinal physiology and health.

Methods: Ultrasound and photoacoustic images were acquired in 2-day and 4-day old neonatal rats. For PAI assessment of intestinal tissue oxygenation, an inspired gas challenge was performed using hypoxic, normoxic, and hyperoxic inspired oxygen (FiO2). For intestinal motility, oral administration of ICG contrast agent was used to compare control animals to an experimental model of loperamide-induced intestinal motility inhibition.

Results: PAI demonstrated progressive increases in oxygen saturation (sO2) as FiO2 increased, while the pattern of oxygen localization remained relatively consistent in both 2-day and 4-day old neonatal rats. Analysis of intraluminal ICG contrast enhanced PAI images yielded a map of the motility index in control and loperamide treated rats. From PAI analysis, loperamide significantly inhibited intestinal motility, with a 32.6% decrease in intestinal motility index scores in 4-day old rats.

Conclusion: These data establish the feasibility and application of PAI to non-invasively and quantitatively measure intestinal tissue oxygenation and motility. This proof-of-concept study is an important first step in developing and optimizing photoacoustic imaging to provide valuable insight into intestinal health and disease to improve the care of premature infants.

Keywords: Imaging; Intestinal motility; Intestine; Necrotizing enterocolitis; Premature infant; Tissue oxygenation.

Figure 1.

Schematic of the intestinal motility index calculation. Acquired cine time series images (6 Hz acquisition frame rate) from US and PAI imaging of the neonatal rat intestine are registered to the initial image using a nonrigid image registration algorithm to calculate displacements in the x- and y-directions for each image in the time series. Spatial gradients of the deformation fields are then used to create a deformation Jacobian matrix [21] at each pixel location and the determinant of this matrix is used to quantify volumetric expansion/contraction for each pixel location for each image in the time series. The motility index is then calculated based on the standard deviation across the time series. Breathing motion is not directly corrected in the analysis of this metric. Scale bar = 1 mm (Scale bar denotes scale for both US and PAI).

Figure 2.

Intestinal oxygen saturation in 2-day old pups at 5%, 21%, and 100% FiO2. Progressive increases in intestinal tissue oxygenation (blue=low oxygen saturation, red=high oxygen saturation) are seen when increasing the fraction of inspired oxygen. Graphs depict quantification of intestinal tissue oxygenation in 2-day old rat pups at 5%, 21%, and 100% FiO2 with bars indicating statistical significance at the p < 0.05 (*) and p < 0.001 (***) levels. Scale bar = 1 mm (Scale bar denotes scale for both US and PAI).

Figure 3.

Intestinal oxygen saturation in 4-day old pups at 5%, 21%, and 100% FiO2. Progressive increases in intestinal tissue oxygenation (blue=low oxygen saturation, red=high oxygen saturation) are seen when increasing the fraction of inspired oxygen. Graphs depict quantification of intestinal tissue oxygenation in 4-day old rat pups at 5%, 21%, and 100% FiO2 with bars indicating statistical significance at the p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) levels. Scale bar = 1 mm (Scale bar denotes scale for both US and PAI).

Figure 4.

Intestinal motility in 4-day old control and loperamide-treated animals. Left panels are representative images from 4-day old animals. A significant global reduction in the intestinal motility index is seen in loperamide-treated animals as compared to controls. Graphs depict quantification of intestinal motility index in 4-day old rat pups in the control and loperamide-treated groups using both US and PAI images. (**denotes p < 0.01, *** denotes p < 0.001). Scale bar = 1 mm (Scale bar denotes scale for US, PAI, and Motility Index images).

Figure 5.

Small intestinal transit assay by methylene blue oral gavage in 4-day old control and loperamide-treated animals. The transit assay confirms a significant reduction in methylene blue transit with the graph demonstrating quantification of small intestinal transit velocity. (*** denotes p < 0.001. Scale bar = 1 cm).