Photoacoustic imaging for in vivo quantification of placental oxygenation in mice

Abstract

Accurate analysis of placental and fetal oxygenation is critical during pregnancy. Photoacoustic imaging (PAI) combines laser technology with ultrasound in real time. We tested the sensitivity and accuracy of PAI for analysis of placental and fetal oxygen saturation (sO₂) in mice. The placental labyrinth (L) had a higher sO₂ than the junctional zone plus decidua region (JZ+D) in C57Bl/6 mice. Changing maternal O₂ from 100 to 20% in C57Bl/6 mice lowered sO₂ in these regions. C57Bl/6 mice were treated with the NO synthase inhibitor L-N^G-nitroarginine methyl ester (L-NAME) from gestational day (GD) 11 to GD18 to induce hypertension. L-NAME decreased sO₂ in L and JZ+D at GD14 and GD18 in association with fetal growth restriction and higher blood pressure. Hypoxia-inducible factor 1α immunostaining was higher in L-NAME vs control mice at GD14. Fetal sO₂ levels were similar between l-NAME and control mice at GD14 and GD18. In contrast to untreated C57Bl/6, L-NAME decreased placental sO₂ at GD14 and GD18 vs GD10 or GD12. Placental sO₂ was lower in fetal growth restriction in an angiotensin-converting enzyme 2 knockout mouse model characterized by placental hypoxia. On phantom studies, patterns of sO₂ measured directly correlated with those measured by PAI. In summary, PAI enables the detection of placental and fetal oxygenation during normal and pathologic pregnancies in mice.-Yamaleyeva, L. M., Sun, Y., Bledsoe, T., Hoke, A., Gurley, S. B., Brosnihan, K. B. Photoacoustic imaging for in vivo quantification of placental oxygenation in mice.

Keywords: HIF-1α; fetal growth restriction; hypertensive pregnancy; oxygen saturation; placental hypoxia.

Figure 1.

In vitro phantom studies. A) PAI probe and a phantom. B) Representative PA signals of blood exposed to nitrogen for 0, 5, 10, and 15 min and corresponding spectra over time. C) sO₂ levels in the blood detected by gas analyzer (direct) after exposure to nitrogen for 0, 5, 10, and 15 min. ^#P < 0.05 vs. 0 and 5 min (n = 4 at each time point). D) sO₂ levels in the blood (phantom) detected by PAI after 0, 5, 10, and 15 min of exposure to nitrogen. *P < 0.05 vs. 0 min (n = 4 at each time point). E) Correlation between sO₂ levels detected by PAI and blood gas analyzer (r = 0.86; P < 0.0001). Data are means ± sem.

Figure 2.

Ultrasound images of placental regions and fetus (A) and placental sO₂ concentrations (B) at GD14 in pregnant C57Bl/6 mice during a change of FiO₂ from 100% (B, C) to 20% (B, D), with each condition lasting for 10 min. L, placental labyrinth. Data are means ± sem. *P < 0.05 vs. 100% FiO₂ in the labyrinth, JZ+D, and whole placenta (total) sO₂; ^#P < 0.05 vs. 100% FiO₂ in the labyrinth; ^P < 0.05 vs. 20% FiO₂ in the labyrinth (n = 6 dams in each group).

Figure 3.

SBPs (A), maternal weights, (B), and fetal (C) weights and litter size at GD14 and -18 in the C57Bl/6 untreated control mice and C57Bl/6 mice treated with L-NAME. Data are means ± sem. *P < 0.05 vs. control GD14 C57Bl/6 mice; ^#P < 0.05 vs. control GD18 C57Bl/6 mice; ^P < 0.05 vs. L-NAME–treated GD14 C57Bl/6 mice (n = 5–7 dams).

Figure 4.

Total placental sO₂ in the C57Bl/6 untreated control mice and C57Bl/6 mice treated with L-NAME. A) Total placental sO₂ throughout gestation (GD10, GD12, GD14, and GD18) in the untreated control C57Bl/6 mice (solid line) and in mice treated with L-NAME C57Bl/6 (dotted line). Data are means ± sem. *P < 0.05 vs. L-NAME–treated C57Bl/6 mice at GD10 and GD12; ^#P < 0.05 vs. GD14 C57Bl/6 untreated control mice (n = 3–8 dams).

Figure 5.

Regional sO₂ differences in the placenta of C57Bl/6 untreated control mice and C57Bl/6 mice treated with L-NAME at GD14 (A) and GD18 (B). L, placental labyrinth. Data are means ± sem. Representative images of regional sO₂ differences in the placenta are shown. *P < 0.05 vs. corresponding untreated control mice; ^#P < 0.05 vs. L in control mice; ^{^}P < 0.05 vs. L in L-NAME–treated mice (n = 5–6 dams for sO₂ at GD14; n = 3–5 dams for sO₂ at GD18).

Figure 6.

Placental HIF-1α immunostaining in C57Bl/6 untreated control mice and C57Bl/6 mice treated with L-NAME at GD14 and data analysis. Nuclear and cytosolic staining for HIF-1α is evident in the placenta. L, placental labyrinth; total, whole placenta. Data are means ± sem. *P < 0.05 vs. L in untreated C57Bl/6 control mice (n = 3–5 dams).

Figure 7.

Representative image (A) and analysis of total fetal sO2 (B) in the C57Bl/6 untreated control mice and C57Bl/6 mice treated with L-NAME at GD14 and GD18 (B). The fetus was scanned in 3-dimensional mode, and the analysis was performed accounting for the total fetal volume.

Figure 8.

Representative images of placental sO₂ in ACE2-KO and C57Bl/6 control mice at GD14. Contours are drawn around placental labyrinth (red), junctional zone and decidua region (green), and total (pink) placenta.