Cell tracking using iron oxide fails to distinguish dead from living transplanted cells in the infarcted heart.

Recently, debate has arisen about the usefulness of cell tracking using iron oxide-labeled cells. Two important issues in determining the usefulness of cell tracking with MRI are generally overlooked; first, the effect of graft rejection in immunocompetent models, and second, the necessity for careful histological confirmation of the fate of the labeled cells in the presence of iron oxide. Therefore, both iron oxide-labeled living as well as dead epicardium-derived cells (EPDCs) were investigated in ischemic myocardium of immunodeficient non-obese diabetic (NOD)/acid: non-obese diabetic severe combined immunodeficient (NOD/scid) mice with 9.4T MRI until 6 weeks after surgery, at which time immunohistochemical analysis was performed. In both groups, voids on MRI scans were observed that did not change in number, size, or localization over time. Based on MRI, no distinction could be made between living and dead injected cells. Prussian blue staining confirmed that the hypointense spots on MRI corresponded to iron-loaded cells. However, in the dead-EPDC recipients, all iron-positive cells appeared to be macrophages, while the living-EPDC recipients also contained engrafted iron-loaded EPDCs. Iron labeling is inadequate for determining the fate of transplanted cells in the immunodeficient host, since dead cells produce an MRI signal indistinguishable from incorporated living cells.

Left ventricular function in the post-infarct failing mouse heart by magnetic resonance imaging and conductance catheter: a comparative analysis.

AIM: Murine myocardial infarction (MI) models are increasingly used in heart failure studies. Magnetic resonance imaging (MRI) and pressure-volume loops by conductance catheter (CC) enable physiological phenotyping. We performed a comparative analysis of MRI vs. CC to assess left ventricular (LV) function in the failing mouse heart. METHODS: MI was created by LAD ligation. MRI (day 14) and CC (day 15) were used to determine LV end-diastolic volume (EDV), end-systolic volume (ESV) and ejection fraction (EF). RESULTS: Pooled data yielded moderate-to-strong linear correlations: EDV: R = 0.61; ESV: R = 0.72; EF: R = 0.81. We analysed three groups, no MI (sham, n = 10), small MI (<30% of LV, n = 14) and large MI (>30%, n = 20). Volumes and EF were consistently lower by CC than by MRI, but group differences were evident for both techniques. Receiver-operating characteristic analysis indicated good sensitivity and specificity for both techniques, with superior results for MRI. CONCLUSIONS: CC and MRI are highly valuable for evaluation of LV volume and function. MRI is recommended for longitudinal studies, accurate absolute volumes and anatomical information. Unique features of CC are its online signal with high temporal resolution, and advanced analysis of LV function and energetics.

Preservation of left ventricular function and attenuation of remodeling after transplantation of human epicardium-derived cells into the infarcted mouse heart.

BACKGROUND: Proper development of compact myocardium, coronary vessels, and Purkinje fibers depends on the presence of epicardium-derived cells (EPDCs) in embryonic myocardium. We hypothesized that adult human EPDCs might partly reactivate their embryonic program when transplanted into ischemic myocardium and improve cardiac performance after myocardial infarction. METHODS AND RESULTS: EPDCs were isolated from human adult atrial tissue. Myocardial infarction was created in immunodeficient mice, followed by intramyocardial injection of 4x10(5) enhanced green fluorescent protein-labeled EPDCs (2-week survival, n=22; 6-week survival, n=15) or culture medium (n=24 and n=18, respectively). Left ventricular function was assessed with a 9.4T animal MRI unit. Ejection fraction was similar between groups on day 2 but was significantly higher in the EPDC-injected group at 2 weeks (short term), as well as after long-term survival at 6 weeks. End-systolic and end-diastolic volumes were significantly smaller in the EPDC-injected group than in the medium-injected group at all ages evaluated. At 2 weeks, vascularization was significantly increased in the EPDC-treated group, as was wall thickness, a development that might be explained by augmented DNA-damage repair activity in the infarcted area. Immunohistochemical analysis showed massive engraftment of injected EPDCs at 2 weeks, with expression of alpha-smooth muscle actin, von Willebrand factor, sarcoplasmic reticulum Ca2+-ATPase, and voltage-gated sodium channel (alpha-subunit; SCN5a). EPDCs were negative for cardiomyocyte markers. At 6-weeks survival, wall thickness was still increased, but only a few EPDCs could be detected. CONCLUSIONS: After transplantation into ischemic myocardium, adult human EPDCs preserve cardiac function and attenuate ventricular remodeling. Autologous human EPDCs are promising candidates for clinical application in infarcted hearts.

Magnetic resonance microscopy at 17.6-Tesla on chicken embryos in vitro.

The non-destructive nature and the rapid acquisition of a three-dimensional image makes magnetic resonance microscopy (MRM) very attractive and suitable for functional imaging investigations. We explored the use of an ultra high magnetic field for MRM to increase image quality per image acquisition time. Improved image quality was characterized by a better signal-to-noise ratio (SNR), better image contrast, and higher resolution compared to images obtained at lower magnetic field strengths. Fixed chicken embryos at several stages of development were imaged at 7.0-T (300 MHz) and at 17.6-T (750 MHz). Maximum intensity projection resulted in three-dimensional vascular images with ample detail of the embryonic vasculature. We showed that at 750 MHz frequency, an image with approximately three times better SNR can be obtained by T1-weighting using a standard gadolinium contrast agent, compared to the same measurement at 300 MHz. The image contrast improved by around 20 percent and the contrast-to-noise ratio improved by almost a factor of 3.5. Smaller blood vessels of the vascular system were identified at the high field, which indicates a better image resolution. Thus, ultra high field is beneficial for MRM and opens new areas for functional imaging research, in particular when SNR, resolution, and contrast are limited by acquisition time.

Magnetic resonance microscopy of mouse embryos in utero.

Magnetic resonance microscopy (MRM) was used to study mouse embryonic development in utero. MRM is a non-invasive imaging technique to study normal and abnormal embryonic development. To overcome image blurring as a result of embryonic movement, fast imaging sequences were used (less than 1 min scanning time). Clear morphologic proton images were obtained by diffusion spin echo and by rapid acquisition with relaxation enhancement (RARE), revealing living mouse embryos with great anatomical detail. In addition, functional information about embryonic blood flow could be obtained, in the absence of a contrast agent. This was achieved by combining two imaging sequences, RARE and very fast gradient echo. We expect that MRM will soon become a feasible method to study longitudinally both normal and abnormal (transgenic) mouse development.

Altered hemodynamics in chick embryos after extraembryonic venous obstruction.

OBJECTIVE: To obtain insight into hemodynamics during abnormal cardiac development, a chick model was developed recently in which a spectrum of conotruncal anomalies, in combination with abnormal semilunar valves and/or pharyngeal arch artery malformations, was induced through extraembryonic venous obstruction (venous clip) at stage 17 (70-h incubation). METHODS: In chick embryos of stage 24 and stage 34 control (n = 8; n = 21) and with venous clip (n = 11; n = 18), we simultaneously measured dorsal flow velocities with a 20-MHz pulsed Doppler velocity meter and dorsal aortic (stage 24) and vitelline artery (stage 34) blood pressures with a servo-null system. After the hemodynamic recordings were collected, all 58 embryos were subjected to morphological examination. The hemodynamic data were correlated with the morphology. Statistical comparison was performed between control and experimental values. RESULTS: At stage 24, venous clip embryos showed impaired looping. Physiologically, only a decrease in peak acceleration was found in these embryos (p < 0.05). At stage 34, a spectrum of conotruncal malformations was seen, that consisted of a ventricular septal defect in combination with abnormal semilunar valves and/or pharyngeal arch malformations. A dextroposed aorta in combination with a ventricular septal defect was diagnosed as double-outlet right ventricle. Hemodynamically, peak systolic and mean velocities, peak systolic and mean blood flows and stroke volume were increased while the heart rate was reduced after placement of the venous clip (p < 0.05). In both stages, pressure readings showed no statistically significant differences between control and experimental embryos. CONCLUSION: Our findings suggest that the hemodynamic changes seen in venous clip embryos reflect the presence of a compensatory mechanism.

Extraembryonic venous obstructions lead to cardiovascular malformations and can be embryolethal.

OBJECTIVE: To expand our knowledge concerning the effect of placental blood flow on human heart development, we used an embryonic chicken model in which extraembryonic blood flow was manipulated. METHODS: First, one of the three major vitelline veins was ligated, while blood flow was visualized with Indian ink. In this way, we could study the effect of different ligation positions on intracardiac flow patterns. Secondly, these vitelline veins were ligated permanently with a microclip until cardiac septation was completed, thereafter, the hearts were morphologically evaluated. In this way, we could study the impact of the ligation position on the severity and frequency of heart malformations. On combining the results, we were able to study the effect of different intracardiac flow patterns on heart development. RESULTS: Although ligation of each vein resulted in different intracardiac flow patterns, long-term ligation resulted in similar cardiovascular malformations in survivors. These consisted mainly of ventricular septum defects (VSDs), semilunar valve anomalies, and pharyngeal arch artery malformations. There was no significant difference (p > 0.05) between the ligation position and the incidence of cardiovascular malformations. However, the percentage mortality after clipping the left lateral vitelline vein was significantly higher (p < 0.05) than after ligation of either the right lateral or posterior vitelline vein. CONCLUSIONS: Early extraembryonic venous obstruction leads to altered flow patterns, which probably result in shear stress changes. In postseptation stages, these result in a spectrum of cardiovascular malformations irrespective of the ligation position. A diminished incidence of VSDs in the oldest stage was attributed to delayed closure of the interventricular foramen.

Unilateral vitelline vein ligation alters intracardiac blood flow patterns and morphogenesis in the chick embryo.

To study the role of blood flow in normal and abnormal heart development, an embryonic chicken model was developed. The effect of altered venous inflow on normal intracardiac blood flow patterns was studied by visualization of blood flow with India ink. At stage 17, India ink was injected into a capillary or small venule within a specific yolk sac region. After determination of the normal intracardiac flow pattern, the right lateral vitelline vein was ligated, and the new intracardiac flow pattern was studied. Ligation resulted in disturbance of normal intracardiac flow patterns, which was most obvious in the conotruncus. The long-term effect of these abnormal intracardiac flow patterns on the development of the heart and pharyngeal arch arteries was investigated by permanent ligation in ovo with a microclip at stage 17 and subsequent evaluation at stages 34, 37, and 45. These experiments revealed anomalies of the vascular system in 58 of the 91 ligated embryos studied. We observed intracardiac malformations consisting of subaortic ventricular septal defects (n = 52), semilunar valve anomalies (n = 19), atrioventricular anomalies (n = 7), and pharyngeal arch artery malformations (n = 32). It is concluded that abnormal intracardiac blood flow, resulting from hampered venous inflow, may result in serious intracardiac and pharyngeal arch artery malformations comparable to defects observed in embryonic chicken models subjected to neural crest ablation, cervical flexure experiments, and excessive retinoic acid treatment.

Intracardiac blood flow patterns related to the yolk sac circulation of the chick embryo.

Intracardiac flow patterns during heart development were studied by injection of india ink into the yolk sac circulation of chick embryos at Hamburger-Hamilton stages 10 to 17. We injected india ink into a small venule or capillary, carefully preventing application of overpressure to the vascular system, and recorded the intracardiac route by video. From stage 12 onward, blood flow was laminar, and separate intracardiac currents were visualized. The yolk sac was divided into a left and a right half. Blood coursed through each half in concentric loops, ranging from the marginal sinus to the sinus venosus. This parallel array persisted within the heart. Bilateral to the embryo, two lateral regions arose that extended wedge-like within each half, resulting in six equally sized yolk sac regions at stage 16. The process of heart looping was not accompanied by a change in flow pattern. However, developmental changes of the yolk sac circulation were reflected in alteration of the intracardiac flow pattern. From stage 16 onward, the intracardiac flow pattern was no longer determined by the left- or right-hand side of the yolk sac but by bilateral anterior, lateral, and posterior regions of the yolk sac. Blood from the lateral regions of the yolk sac was preferentially distributed to the head. The results show that in preseptation stages a relatively stable flow pattern is present. We suggest that alterations in blood flow could influence the process of normal heart development.

Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras.

The endothelium of the coronary vascular system has been described in the literature as originating from different sources, varying from aortic endothelium for the main coronary stems, endocardium for the intramyocardial network, and sinus venosus lining for the venous part of the coronary system. Using an antibody against quail endothelial cells (alpha-MB1), we investigated the development of the coronary vascular system in the quail (Hamburger and Hamilton stages 15 to 35) and in a series of 36 quail-chicken chimeras. In the chimeras, pieces of quail epicardial primordium and/or liver tissue were transplanted into the pericardial cavity of a chicken host. The results showed that the coronary vascular endothelial distribution closely followed the formation of the epicardial covering of the heart. However, pure epicardial primordium transplants did not lead to endothelial cell formation, whereas a liver graft with or without an epicardial contribution did have this capacity. The first endothelial cells were seen to reach the heart at the sinus venosus region, subsequently spreading through the inner curvature to the atrioventricular sulcus and the outflow tract and, last of all, over the ventricular surfaces. At these sites, the precursor cells and small vessels were seen to invade the sinus venosus wall, the ventricular and atrial myocardium, and the mesenchymal border of the aortic orifice. Connections with the endocardium of the heart tube were only observed in the right ventricular outflow region. Initially, the connections with the aortic endothelium were multiple, but later in development only two of these connections persisted to form the proximal part of the two main coronary arteries. Connections to the pulmonary orifice were never observed. Our transplantation data showed that the entire coronary endothelial vasculature originated from an extracardiac source. Moreover, using the developing subepicardial layer as a matrix, we showed that the endothelial cells reached the heart from the liver region. Ingrowth into the various cardiac segments was also observed. Implications for the relation to specific congenital cardiac malformations are discussed.

The development of the vascular system in quail embryos: a combination of microvascular corrosion casts and immunohistochemical identification.

Although vascular casts, obtained by injection with methacrylates, are frequently used to investigate the adult vascular system, little data are available for embryonic stages. In this paper we use Mercox in quail embryos in the period of 2 to 7 days after incubation. The microvascular corrosion casts were evaluated in the scanning electron microscope (SEM) with special attention to the development and remodelling of the large arteries and veins. Our results show that the remodelling of the large arteries and veins together with their developing tributary vessels can be visualized from very early embryonic stages onwards. However, complete replication of a developing vascular system depends on diameter and regularity of the lumen. In the stages investigated, the vascular lumen, even of the largest vessels, is still very irregular. Detailed cellular characteristics like nuclear impressions of endothelial cells, as often seen in adult material, were seldom found in the embryos. To examine whether blind-ending sprouts are completely or incompletely replicated in a developing vascular system, additional series of quail embryos were stained immunohistochemically with a monoclonal antibody (MB1) specific for endothelial and hemopoietic cells. It seems that a plexus consisting of endothelial precursors (endothelial cells lacking a lumen) is present in the developing organ before the formation of a lumen and assembly into vessels, which are connected to an adjacent artery or vein. Expansion of the vascular system may in part be due to incorporation of these endothelial precursors in the wall of existing vessels.