Optical coherence tomography captures rapid hemodynamic responses to acute hypoxia in the cardiovascular system of early embryos

Abstract

Background: The trajectory to heart defects may start in tubular and looping heart stages when detailed analysis of form and function is difficult by currently available methods. We used a novel method, Doppler optical coherence tomography (OCT), to follow changes in cardiovascular function in quail embryos during acute hypoxic stress. Chronic fetal hypoxia is a known risk factor for congenital heart diseases (CHDs). Decreased fetal heart rates during maternal obstructive sleep apnea suggest that studying fetal heart responses under acute hypoxia is warranted.

Results: We captured responses to hypoxia at the critical looping heart stages. Doppler OCT revealed detailed vitelline arterial pulsed Doppler waveforms. Embryos tolerated 1 hr of hypoxia (5%, 10%, or 15% O(2) ), but exhibited changes including decreased systolic and increased diastolic duration in 5 min. After 5 min, slower heart rates, arrhythmic events and an increase in retrograde blood flow were observed. These changes suggested slower filling of the heart, which was confirmed by four-dimensional Doppler imaging of the heart itself.

Conclusions: Doppler OCT is well suited for rapid noninvasive screening for functional changes in avian embryos under near physiological conditions. Analysis of the accessible vitelline artery sensitively reflected changes in heart function and can be used for rapid screening. Acute hypoxia caused rapid hemodynamic changes in looping hearts and may be a concern for increased CHD risk.

Figure 1

Survival of quail embryos post hypoxic exposure. The quail embryos (stage 17) were exposed to different degrees of hypoxia for one hour. Normal oxygen (21%) was restored at the end of the hypoxic exposure period, represented in the figure as 0 hours. Embryos were cultured for another 48-hour period, and the numbers of surviving embryos were plotted as a percentage at different time points.

Figure 2

Doppler OCT can provide high resolution pulsed Doppler waveforms at the position of the vitelline artery for hemodynamic measurements. (1A), Microscopic view of a stage 17 quail embryo connected to the extraembryonic vasculature spreading over the yolk in an ex ovo shell-less culture. The black bar indicates the location of the vitelline vessels that was scanned for OCT imaging. (1B). B-scan OCT image of the vitelline vessel. LOMA - left omphalomesenteric artery; LOMV - left omphalomesenteric vein. (1C). Colored Doppler image at the midline of LOMA (dashed line in Figure 1B). The red color indicates forward blood flow, and the blue color indicates retrograde blood flow. (1D). Pulsed Doppler images at the center of LOMA (blue dashed line in Figure 1C). The magnitude of the signal indicates relative flow velocity with positive values representing forward blood flows. Max – maximal flow, Min – minimal flow (or maximal retrograde flow), and Avg – timed-average flow.

Figure 3

Changes in hemodynamic parameters, bradycardia and missing contractions were observed under hypoxic exposure. (A and B). Pulsed Doppler images of a stage 17 quail embryo before and after exposure to 10% oxygen for 5 minutes. Arrow in (B) indicates increased retrograde blood flow. (C). Embryo heart rate under different degrees of hypoxia for one hour. Numbers were averaged from 6 individual embryos in each group, and plotted as averages and standard errors (indicated by the error bars). (D). Pulsed Doppler image from a stage 17 quail embryo exposed to 10% oxygen for 1 hour. Arrow indicates a missing contraction with increased retrograde blood flow during the missing beat. Dotted lines indicate time intervals between consecutive heart beats.

Figure 4

Hypoxic exposure alters the systolic time, the diastolic time, and the relative shoulder height. Quail embryos were exposed to various degrees of hypoxia for 1 hour, and systolic and diastolic time was measured based on the pulsed Doppler images taken from LOMA. Relative shoulder height is defined as the ratio of the secondary peak velocity to the primary peak velocity. All numbers are averaged from 6 independent observations, and plotted as averages and standard errors. Asterisks indicate that values were statistically different from controls (prior to the hypoxic exposure) with a p-value less than 0.05.