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[Preprint]. 2023 Oct 23:2023.10.20.563296.
doi: 10.1101/2023.10.20.563296.

Photoacoustic Imaging for Non-Invasive Assessment of Physiological Biomarkers of Intestinal Injury in Experimental Necrotizing Enterocolitis

Jared A Weis  1   2   3 , Jessica L Rauh  4 , Maryssa A Ellison  5 , Nildris Cruz-Diaz  6   7 , Liliya M Yamaleyeva  6   7 , Cherrie D Welch  8 , Kristen A Zeller  4 , Victoria G Weis  5
Affiliations

Photoacoustic Imaging for Non-Invasive Assessment of Physiological Biomarkers of Intestinal Injury in Experimental Necrotizing Enterocolitis

Jared A Weis et al. bioRxiv. .

Update in

Abstract

Background: Necrotizing enterocolitis (NEC) is an often-lethal disease of the premature infants' intestinal tract that is exacerbated by significant difficulties in early and accurate diagnosis. In NEC disease, the intestine often exhibits hypoperfusion and dysmotility, which contributes to advanced disease pathogenesis. However, these physiological features cannot be accurately and quantitively assessed within the current constraints of imaging modalities frequently used in the clinic (plain film X-ray and ultrasound). We have previously demonstrated the ability of photoacoustic imaging (PAI) to non-invasively and quantitively assess intestinal tissue oxygenation and motility in a healthy neonatal rat model. As a first-in-disease application, we evaluated NEC pathogenesis using PAI to assess intestinal health biomarkers in a preclinical neonatal rat experimental model of NEC.

Methods: NEC was induced in neonatal rat pups from birth to 4 days old via hypertonic formula feeding, full-body hypoxic stress, and lipopolysaccharide administration to mimic bacterial colonization. Healthy breastfed (BF) controls and NEC rat pups were imaged at 2- and 4-days old. Intestinal tissue oxygen saturation was measured with PAI imaging for oxy- and deoxyhemoglobin levels. To measure intestinal motility, ultrasound and co-registered PAI cine recordings were used to capture intestinal peristalsis motion and contrast agent (indocyanine green) transit within the intestinal lumen. Additionally, both midplane two-dimensional and volumetric three-dimensional imaging acquisitions were assessed for oxygenation and motility.

Results: NEC pups showed a significant decrease of intestinal tissue oxygenation as compared to healthy BF controls at both ages (2-days old: 55.90% +/- 3.77% vs 44.12% +/- 7.18%; 4-days old: 56.13% +/- 3.52% vs 38.86% +/- 8.33%). Intestinal motility, assessed using a computational intestinal deformation analysis, demonstrated a significant reduction in the intestinal motility index in both early (2-day) and established (4-day) NEC. Extensive NEC damage was confirmed with histology and dysmotility was confirmed by small intestinal transit assay.

Conclusions: This study presents PAI as a successful emerging diagnostic imaging modality for both intestinal tissue oxygenation and intestinal motility disease hallmarks in a rat NEC model. PAI presents enormous significance and potential for fundamentally changing current clinical paradigms for detecting and monitoring intestinal pathologies in the premature infant.

Keywords: imaging; intestinal motility; intestine; necrotizing enterocolitis; premature infant; tissue oxygenation.

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Conflict of interest statement

Competing interests The authors have no competing interests to declare.

Figures

Figure 1.
Figure 1.
Histological assessment of NEC damage. A. H&E of 4-day old rat pup ileum shows that the NEC model induced severe epithelial damage and loss of crypt morphology as compared to BF. Scale bar = 100 μm. B. Graph of NEC damage grading along the ileal length shows significant amounts of NEC grade damage (≥ Grade 2) as compared to BF. ****p<0.0001.
Figure 2.
Figure 2.
(Left) US and PAI of tissue oxygenation (sO2) in representative BF and NEC 2-day (A) and 4-day (B) old rat pups. Scale bar = 1 mm (Scale bar denotes scale for both US and PAI). (Right) Intestinal tissue oxygenation in both 2-day (A) and 4-day (B) old NEC pups demonstrated a significant reduction compared to BF controls. *p<0.05, **p<0.01.
Figure 3.
Figure 3.
(Left) Volumetric assessment of intestinal tissue oxygenation in representative BF and NEC pups at both 2- and 4-days old. (Right) Quantification of volumetric intestinal tissue oxygenation demonstrated a significant reduction in NEC as compared to BF control at both 2- and 4-days old. **p<0.01. ***p<0.001.
Figure 4.
Figure 4.
(Left) US and ICG-enhanced PAI images (top row) and intestinal motility index maps (bottom row) in representative BF (A) and NEC (B) 2-day old rat pups. Scale bar = 1 mm. (Right) Intestinal motility imaging in 2-day old rat pups showed significant reductions in the motility index in NEC animals as compared to BF control. Note that US and PAI motility index measures are to be separately compared due to differences in imaging data source. ***p<0.001. ****p<0.0001.
Figure 5.
Figure 5.
(Left) US and ICG-enhanced PAI images (top row) and intestinal motility index maps (bottom row) in representative BF (A) and NEC (B) 4-day old rat pups. Scale bar = 1 mm. (Right) Intestinal motility imaging in 4-day old rat pups showed significant reductions in the motility index in NEC animals as compared to BF control. Note that US and PAI motility index measures are to be separately compared due to differences in imaging data source. *p<0.05. **p<0.01.
Figure 6.
Figure 6.
Correlation plots comparing intestinal tissue oxygenation analyzed in 2D vs 3D (A) and correlation plot comparing the intestinal motility index for US (B) and ICG-contrast enhanced PAI (C) analyzed in 2D vs 3D.
See this image and copyright information in PMC

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