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 (sO) in mice. The placental labyrinth (L) had a higher sO than the junctional zone plus decidua region (JZ+D) in C57Bl/6 mice. Changing maternal O from 100 to 20% in C57Bl/6 mice lowered sO in these regions. C57Bl/6 mice were treated with the NO synthase inhibitor L-N-nitroarginine methyl ester (L-NAME) from gestational day (GD) 11 to GD18 to induce hypertension. L-NAME decreased sO 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 control mice at GD14. Fetal sO levels were similar between l-NAME and control mice at GD14 and GD18. In contrast to untreated C57Bl/6, L-NAME decreased placental sO at GD14 and GD18 GD10 or GD12. Placental sO was lower in fetal growth restriction in an angiotensin-converting enzyme 2 knockout mouse model characterized by placental hypoxia. On phantom studies, patterns of sO 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 quantification of placental oxygenation in mice.

X-linked myotubular myopathy (XLMTM) is an isogenic muscle disease characterized by progressive wasting of skeletal muscle, weakness, and premature death of affected male offspring. Recently, the XLMTM gene knock-in mouse, Mtm1 p.R69C, was found to have a similar phenotype as the MTM1 gene mutation in humans (e.g., central nucleation of small myofibers, attenuated muscle strength, and motor unit potentials). Using this rodent model, we investigated whether syngeneic cell therapy could mitigate muscle weakness. Donor skeletal muscle-derived myoblasts were isolated from C57BL6 wild-type (WT) and Mtm1 p.R69C (KI) mice for transplantation into the gastrocnemius muscle of recipient KI mice. Initial experiments demonstrated that donor skeletal muscle-derived myoblasts from WT and KI mice remained in the gastrocnemius muscle of the recipient KI mouse for up to 4 weeks posttransplantation. KI mice receiving syngeneic skeletal muscle-derived myoblasts displayed an increase in skeletal muscle mass, augmented force generation, and increased nerveevoked skeletal muscle action potential amplitude. Taken together, these results support our hypothesis that syngeneic cell therapy may potentially be used to ameliorate muscle weakness and delay the progression of XLMTM, as application expands to other muscles.

Pregnancy complications can seriously impact fetal well-being. However the tools used for the evaluation of fetal oxygenation in clinic are limited to the monitoring of fetal heart rate or invasive techniques (fetal scalp pH and PO 2 ). Therefore, the development of methods enabling accurate, non-invasive and in real-time assessment of fetal status throughout pregnancy is warranted. In this study we tested the combination of photoacoustic imaging (PAI) and 3D power Doppler for the assessment of fetal distress. PAI combines optical contrast of photoacoustic laser technology with high spatial resolution of ultrasound. Power Doppler is a sensitive technique for the detection of blood flow. We hypothesize that a combination of PAI with 3D power Doppler can non-invasively and in real time establish fetal oxygenation, volume, and tissue vascularity. Pregnant C57Bl/6 mice were infused with nitric oxide synthase inhibitor, L-NAME via osmotic minipumps (50 mg/kg/day; days 11 to 14 of gestation) to induce the hypertensive phenotype. At day 14 of gestation, systolic blood pressures were higher in L-NAME-treated vs. untreated C57Bl/6 mice although these values did not reach hypertensive levels (90.8±3.1 vs. 104.4±3.7 mmHg, p<0.05, n=4-5). Fetal weights were lower in the L-NAME-infused mice versus controls (0.12±0.01 vs. 0.17±0.01 g fetal weight per cm tibia length, p<0.05, n=5-6). Fetal liver sO2 was lower in L-NAME-infused mice (47.5±2.1 vs. 56.1±1.6 %, n=4, p<0.05), while no differences were found in fetal brain sO2. Total fetal volume was lower in L-NAME-treated mice (213.8±26.5 vs. 399±45.1 %, n=5, p<0.05) and positively correlated with fetal body weights obtained postmortem in study groups. Fetal tissue vascularity was also lower in L-NAME-infused mice possibly due to limited vascular branching or blood flow (40.8±1.5 vs. 47.7±1.8%, p<0.05). These changes were associated with a compensatory increase in fetal heart rate in L-NAME-treated mice (170.7±9.1 vs. 135.8±5.7 %, p<0.05). We conclude that a combination of PAI with 3D power Doppler provides valuable information about fetal oxygenation and growth in association with tissue vascularity thus permitting non-invasive, in real-time analysis of fetal well-being.

Placental hypoxia/ischemia induces abnormal maternal and neonatal outcomes including preeclampsia and intrauterine growth restriction (IUGR). The ability to accurately determine placental oxygenation in a non-invasive way and in real-time is highly important during pregnancy as it may allow for the early diagnosis of IUGR and preeclampsia. Photoacoustic imaging (PA) is a novel preclinical and emerging clinical tool that combines optical contrast of photoacoustic laser technology with high spatial resolution of ultrasound. PA measures tissue oxygen saturation (sO2) that reflects differences in absorption spectra for oxygenated and deoxygenated hemoglobin. By using photoacoustic features of VEVO LAZR high resolution ultrasound system (VisualSonics) in a three-dimensional mode we investigated the sensitivity and accuracy of PA for placental oxygenation in C57Bl/6 mice at day 14 of gestation. Furthermore, since nitric oxide deficiency is associated with upregulation of circulatory hypoxia markers, C57Bl/6 mice were chronically treated with the nitric oxide inhibitor, L-NAME via osmotic minipumps (50 mg/kg/day; days 13 to 18 of gestation). The comparisons between scanned vs. not scanned uteroplacental units showed that PA had no effect on fetal (scanned: 0.028±0.001 vs. not scanned: 0.03±0.001 g/maternal body weight; p>0.05) or placental (scanned: 0.002±0.003 vs. not scanned: 0.002±0.002 g/maternal body weight; p>0.05) weights in C57Bl/6 mice. Changing inhaled O2 from 100- to 20% resulted on average in 12.5% reduction in total placental sO2. Systolic blood pressures were higher in L-NAME-treated vs. C57Bl/6 mice (215.8±0.8 vs. sham 99.3±4.4 mmHg; p<0.05). L-NAME infusion decreased sO2 in all areas of the placenta: labyrinth (73.6±0.97 vs. 58.6±3.4%, p<0.05), mesometrial triangle (63.0±2.0 vs. 48.9±1.0%, p<0.05), and total placenta (68.2±1.7 vs. 54.4±2.2%, p<0.05). Placental labyrinth had higher sO2 vs. mesometrial triangle area in both L-NAME infused (58.6±3.4 vs. 48.9±1.0%, p<0.05) and in C57Bl/6 (73.6±0.97 vs. 63.0±2.0%, p<0.05) mice reflecting elaborate branching morphology of the labyrinth. Our data suggest that PA imaging can detect regional differences in placental sO2 non-invasively and at different physiological states.