In vivo micro particle image velocimetry measurements of blood-plasma in the embryonic avian heart

https://doi.org/10.1016/j.jbiomech.2005.03.015Get rights and content

Abstract

The measurement of blood-plasma velocity distributions with spatial and temporal resolution in vivo is inevitable for the determination of shear stress distributions in complex geometries at unsteady flow conditions like in the beating heart. A non-intrusive, whole-field velocity measurement technique is required that is capable of measuring instantaneous flow fields at sub-millimeter scales in highly unsteady flows. Micro particle image velocimetry (μPIV) meets these demands, but requires special consideration and methodologies in order to be utilized for in vivo studies in medical and biological research.

We adapt μPIV to measure the blood-plasma velocity in the beating heart of a chicken embryo. In the current work, bio-inert, fluorescent liposomes with a nominal diameter of 400 nm are added to the flow as a tracer. Because of their small dimension and neutral buoyancy the liposomes closely follow the movement of the blood-plasma and allow the determination of the velocity gradient close to the wall. The measurements quantitatively resolve the velocity distribution in the developing ventricle and atrium of the embryo at nine different stages within the cardiac cycle. Up to 400 velocity vectors per measurement give detailed insight into the fluid dynamics of the primitive beating heart. A rapid peristaltic contraction accelerates the flow to peak velocities of 26 mm/s, with the velocity distribution showing a distinct asymmetrical profile in the highly curved section of the outflow tract.

In relation to earlier published gene-expression experiments, the results underline the significance of fluid forces for embryonic cardiogenesis. In general, the measurements demonstrate that μPIV has the potential to develop into a general tool for instationary flow conditions in complex flow geometries encountered in cardiovascular research.

Introduction

The need to measure both spatially and temporally resolved fluid velocity fields exists in numerous areas of medical and biological research. For example, two-dimensional blood velocity measurements are suitable for quantitatively monitoring the microcirculation within well-defined regions of the vasculature. Furthermore, the measurement of spatial velocity fields is a minimum requirement for determining hydrodynamic wall shear stresses on the surface of moving boundaries such as those found in the heart. The wall shear stress is an important parameter in fundamental areas of angiogenesis and cardiology and plays a key role in problems such as the pathology of atherosclerosis (Malek et al., 1999, Liepsch, 1990) and the study of cardiogenesis (Hove et al., 2003, Hogers et al., 1999). Experimental values for the wall shear stress, τ, can be extracted from a velocity field by the calculation of the wall-normal velocity gradients, du/dn:τ=ηdudn,where η is the dynamic viscosity of the fluid and is either presumed to be known from literature or can be estimated based on the local flow parameters (i.e., hematocrit value and shear rate).

The development of the method presented in this paper is motivated by the need for a general measurement technique that is applicable to the aforementioned fields of interest. The investigation of the influence of hemodynamically induced wall shear stress on cardiogenesis serves as a particular test case. Placental blood flow is thought to play a significant role in normal and abnormal human heart development (Hogers et al., 1999). For studying this relationship experimentally, an embryonic chicken is used as an animal model. Fig. 1 displays a chicken embryo after approximately 50 h of incubation, which corresponds to development Stage 15 (Hamburger and Hamilton, 1951). On the right and left side of the image one can clearly see extraembryonic vitteline vessels that serve the same function as the placenta in a mammalian embryo. It has been shown that obstructing venous flow by closing one of these vessels with a clip results in re-routing of the venous return to the heart, the alteration of blood flow profiles through the heart, and the development of specific cardiovascular malformations (Hogers et al., 1999).

From in vitro flow studies on the vascular endothelium, it is known that flow induced shear stress modulates gene expression (Topper and Gimbrone Jr., 1999). The present in vivo measurement system has been developed with the long-term goal of combining velocity field measurements with the visualization of gene expression to find a relationship between extraembryonic flow and cardiogenesis. In this paper we describe the measurement technique and the application to the flow in the heart.

Particle image velocimetry (PIV) provides instantaneous velocity fields at a spatial resolution in the order of hundred nanometers under ideal conditions (Tretheway and Meinhart, 2002, Westerweel et al., 2004). The technique is briefly described in the Materials and methods section; detailed descriptions can be found in the works by Westerweel (1993) and Raffel et al. (1998). Advantageous for determining wall shear stress is the fact that our implementation of PIV also allow for a precise determination of the flow boundary. This holds in particular for situations where the boundary is not stationary, like in the case of a beating heart. Such measurements would be impracticable with single point-measurement techniques like laser-Doppler-velocimetry (LDV). MRI is difficult to apply on highly instationary flows, because of its limited temporal resolution (Liepsch, 2002).

In many applications, PIV has developed into the method of choice wherever spatial velocity information is required (Adrian, 1991). More recently Santiago et al. (1998) introduced a modified system which enabled PIV using a microscope, which is commonly referred to as μPIV. Several authors have demonstrated the applicability of μPIV or related techniques to study blood flow. Tangelder et al. (1986) labelled blood platelets with a fluorescent dye to measure steady flow velocities in arterioles of the rabbit mesentery by determining the velocity of individual particles. Individual velocity measurements at different radial positions of the blood vessel were assembled to estimate the velocity profile. Smith et al. (2003) refined this method by injecting fluorescent microspheres (500 nm diameter) into the mouse cremaster muscle venules, although only the velocities of up to four particles at irregular distances above the glycocalyx layer were used to estimate the velocity profile. This method is only useful in the special case of steady flow conditions.

Hitt et al. (1996) applied a correlation technique to video images of the venous flow in the hamster cremaster muscle. Tsukada et al. (2000) and Sugii et al., 2002a, Sugii et al., 2002b used μPIV to measure red blood cell velocity profiles in mesentery vessels of rats. Hove et al. (2003) followed the course of small groups of erythrocytes through the heart of a zebrafish embryo. In all of these studies, erythrocytes were used as tracer particles.

As will be explained in the Discussion section of this paper, tracer particles that are significantly smaller than the large erythrocytes (which have a diameter of eight to ten micrometers) enhance resolution and reliability of the measurement. Accordingly the present work focuses on the enhancement of the accuracy of in vivo μPIV measurements by using fluorescent, long-circulating liposomes that have a nominal diameter of 400 nm as tracer particles. The liposome tracer particles are illuminated by a pulsed laser that is used in conjunction with an acoustic pulsed Doppler velocimeter probe (Ursem et al., 2001) to provide time-resolved ensemble average measurements throughout the cardiac cycle.

Section snippets

Measurement principle

The basic principle behind PIV uses two sequential digital images that are taken from a flow which is visualized by adding small tracer particles. The displacement of the particles in the second image, relative to the position of the particles in the first image, is a measure of the motion of the fluid. The displacement of the particles is obtained by means of a two-dimensional cross-correlation using a computer. This means that a small interrogation window of the first image is correlated with

Results

Measurements are made at nine discrete points in the cardiac cycle, and are separated from each other by 50 ms. Each measurement represents the ensemble averaged evaluation of up to 50 image pairs with an interframing time of 0.5–4 ms, depending on the average magnitude of the velocities at a given point in the cycle. Given that a chicken embryo typically has a heart rate of 2 Hz, it follows that a single ensemble measurement requires 25 s to complete. The spatial resolution of the velocity data is

Discussion

In the post-genomics era, there exists not only an increasing interest in the functional aspects of (altered) gene expression, but also in the physical determinants of gene expression regulation. For example, lessons from knockout studies in mice have taught us the pivotal role of the growth hormone endothelin-1 in cardiovascular development (Yanagisawa et al., 1998), and in vitro studies with cultured endothelial cells showed a clear role for blood flow and shear stress on the regulation of

Acknowledgements

The authors would like to thank Patrick A.M. van Wieringen from Leica who generously provided the microscope, Ann L.B. Seynhaeve for preparing the lipsomal formulations, and Dr. Hans Vink for useful comments and suggestions during the preparation of the manuscript. This project is funded by the Dutch Technology Foundation STW (DSF.5695), the Netherlands Heart Foundation (NHF 2000.016; BPH, SS, BCWG), and the Dutch Cancer Foundation (DDHK2000-2224).

References (33)

  • V. Hamburger et al.

    A series of normal stages in the development of the chick embryo

    Journal of Morphology

    (1951)
  • D. Hitt et al.

    A new method for blood velocimetry in the microcirculation

    Microcirculation

    (1996)
  • B. Hogers et al.

    Extraembryonic venous obstructions lead to cardiovascular malformations and can be embryolethal

    Cardiovascular Research

    (1999)
  • J. Hove et al.

    Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis

    Nature

    (2003)
  • R. Keane et al.

    Theory of cross-correlation analysis of PIV images

    Applied Scientific Research

    (1992)
  • Liepsch, D. (Ed.), 1990. Blood Flow in Large Arteries: Application to Atherogenesis and Clinical Medicine. Monographs...
  • Cited by (175)

    • The zebrafish cardiovascular system

      2019, The Zebrafish in Biomedical Research: Biology, Husbandry, Diseases, and Research Applications
    • Cost effective measuring technique to simultaneously quantify 2D velocity fields and depth-averaged solute concentrations in shallow water flows

      2018, Flow Measurement and Instrumentation
      Citation Excerpt :

      Applications of PIV range from slowly creeping flows such as those examined by [33], who measure both instantaneous and mean velocity flow in micro-scale fluid devices using a micro-scale PIV; to detonations lasting only a few tens of microseconds such as those examined in [25], who applied the PIV technique to study moving millimeter shock waves, from nanoscale flow phenomena [43], who used a novel non-intrusive technique to obtain the shape of walls studying flow around them with a precision of nanometers, to motion in the atmosphere of Jupiter [44]. Moreover, PIV application range goes from the motion in the beating heart of vertebrate embryos [16,46], where velocity distribution of blood were studied to obtain shear stress distributions to the accidental release of oil at the bottom of the Gulf of Mexico [23,22] where flow rate of the oil escaping from the well to the sea was studied. What all of these studies show is that PIV is an incredibly versatile and data-rich technique, but they all use equipment that is relatively expensive (such as lasers, microscopes, cameras) for optimal results, prohibiting the widespread implementation of PIV, particularly in challenging environments.

    • Influence of blood flow on cardiac development

      2018, Progress in Biophysics and Molecular Biology
    View all citing articles on Scopus
    View full text