Published online before print April 5, 2007, doi: 10.1161/01.RES.0000266606.88463.cb
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© 2007 American Heart Association, Inc.
Integrative Physiology |
Increased Ventricular Preload Is Compensated by Myocyte Proliferation in Normal and Hypoplastic Fetal Chick Left Ventricle
From the Department of Cell Biology and Anatomy (A.dA., T.M., D.S.); Pediatric Cardiology (T.M.), Medical University of South Carolina, Charleston; Institute of Anatomy (D.S.), First Faculty of Medicine, Charles University, Prague, Czech Republic; and Institute of Animal Physiology and Genetics (D.S.), Academy of Sciences of the Czech Republic, Prague.
Correspondence to David Sedmera, Laboratory of Cardiovascular Morphogenesis, Institute of Animal Physiology and Genetics, Videnska 1083, 142 20 Prague 4-Krc, Czech Republic. E-mail sedmera{at}iapg.cas.cz
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Abstract |
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Hemodynamics influence cardiac development, and alterations in blood flow may lead to impaired cardiac growth and malformations. The developing myocardium adapts to augmented workload by increasing cell number (hyperplasia). The aim of this study was to determine the influence of alterations in ventricular preload on fetal myocyte proliferation by manipulation of intracardiac shunting at the atrial level. We hypothesized that partial clipping of the right atrial appendage would increase the blood flow to the left ventricle and, in turn, lead to an increase in chamber volume and myocardial mass based on myocyte proliferation. Using an ex ovo culture setup, we performed partial right atrial clipping on embryonic day 8 chick embryos. Ultrasound imaging was performed before and after the surgery to assess the changes in left ventricular volume. Sampling after 24 hours was preceded by 2 hour of pulse-labeling with 5-bromodeoxyuridine. Ultrasound imaging showed that partial right atrial clipping led to a significant increase in left ventricular end-diastolic volume, demonstrating increased blood flow and preload. Anti–5-bromodeoxyuridine immunolabeling revealed a significant increase in myocyte proliferation in the left ventricle and atrium. No significant changes were found in the right heart structures. Increased left ventricular myocyte proliferation and myocardial mass after right atrial clipping was also observed in embryos with experimental left ventricular hypoplasia. These results demonstrate the ability of fetal myocardium to respond to increased preload by myocyte hyperplasia and support the rationale for prenatal surgical interventions in certain cases of congenital heart disease such as hypoplastic left heart syndrome.
Key Words: chick embryo • hemodynamics • fetal surgery • hypoplastic left heart syndrome
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Introduction |
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Hypoplastic left heart syndrome (HLHS) refers to a group of congenital cardiac anomalies characterized by underdevelopment of the left side of the heart. The anatomic abnormalities include underdevelopment of the left atrium and ventricle and hypoplastic or atretic mitral and aortic valves, and the right ventricle, rather than the left, forms the apex of the heart.1,2 HLHS affects more than 2000 infants a year in the United States alone and accounts for approximately 25% of cardiac deaths within the first year of life.3 The etiology of HLHS is largely unknown. There appears to be a genetic component,4 but the pattern of inheritance is multifactorial, as was demonstrated by a high incidence of heart defects in first-degree relatives of patients with HLHS.5,6 In particular, there is a strong association with other left ventricular outflow tract obstructive lesions, including bicuspid aortic valve.7 Another known association is with terminal deletion of the long arm of chromosome 11.8,9 Nevertheless, no single gene has been implicated to date, and no defined genetic animal models of HLHS exist.
The most widely accepted hypothesis is that HLHS develops as a result of embryonic alterations in blood flow, such as premature narrowing of the foramen ovale10 and aortic stenosis.11 However, the primary cause of HLHS remains unclear, as hemodynamic alterations have been shown to lead to secondary mitral stenosis.12 Although the primary cause of HLHS is uncertain, hemodynamic alterations clearly result in the progressive severity of HLHS, as demonstrated by fetal echocardiography in the second and third trimester.13,14 Recent studies15–17 highlighted the possibility of later fetal development of HLHS that could explain the occurrence of neonatal cases who had normal routine 19-week fetal scan. From these reports, we learned that restrictive foramen ovale is not a primary cause in most HLHS cases; many cases (especially those amenable to fetal intervention) are caused so far by poorly characterized events between 19 to 23 weeks that worsen the flow across the aortic valve, and, perhaps most significantly, interventional improvement of this flow by means of balloon dilatation results in significant reversal of left ventricular hypoplasia. From this perspective, it is noteworthy that in postnatal population, 
20% of HLHS patients (excluding variants) present with both mitral and aortic valve patency18,19; this percentage is likely higher in the fetal population, in whom stenosis did not progress yet to complete atresia.20
Without surgery, HLHS is uniformly fatal usually within hours or days of birth.21 Despite ongoing improvements,19 the most common palliative approach (Norwood) consists of 3 major cardiac procedures and leaves the patient with a single-ventricular (Fontan) circulation, with the right ventricle acting as the sole, systemic pump. Biventricular repair is the preferred approach because it restores the 2-ventricular physiology; however, it can only be performed in a very small subset of HLHS patients, in whom the left ventricle is small but potentially functional and both valves are patent.22,23 Therefore, prenatal intervention that would sufficiently rescue the left ventricle for possible restoration of 2-ventricular physiology would greatly improve the management of HLHS and afford patients a near-normal quality of life.
The chick embryo has a long and well-established history as a model system in biology and has contributed major concepts to many other disciplines.24 It is currently the only experimental prenatal model of HLHS, with adequate survival rates that allow for the long-term investigation of interventions. By left atrial ligation (LAL), a phenotype mimicking human HLHS is produced.2 Although not pathogenetically identical with most human cases, the resulting phenotype strikingly recapitulates most features seen in humans, including late-developing premature closure of the foramen ovale and valve atresia.25,26 In this model, the normal pattern of blood flow is redistributed from the developing left ventricle toward the right ventricle. Remodeling of ventricular myocardial architecture can be observed as soon as 2 days after ligation, and left ventricular myocardial volumes are significantly reduced after 4 days.12 These changes in myocardial architecture have been linked to alterations in the proliferative structure of the embryonic ventricle.27
In this study, we used the chick model to test the hypothesis that growth of the hypoplastic left ventricle could be rescued by partial clipping of the right atrial appendage, thereby increasing the blood flow to the left ventricle and, in turn, leading to an increase in chamber volume and myocardial mass based on myocyte proliferation. Using an ex ovo culture setup, ultrasound imaging showed that this procedure indeed led to a significant increase in left ventricular end-diastolic volume, demonstrating increased blood flow and preload. We also demonstrated a significant increase in myocyte proliferation in the left ventricle and atrium, with no significant changes in the right heart structures in both normal and HLHS hearts. In addition, we noted increased left ventricular myocardial volume and improved myocyte differentiation in the hypoplastic left ventricle after clip. These results show that reversal of experimental left ventricular hypoplasia induced by LAL can be achieved with right atrial clipping, thereby redirecting flow by changing atrial compliance. Thus induced myocardial remodeling that is based on increased myocyte proliferation, leading to increased myocardial mass, demonstrates the viability of and rationale for prenatal surgical interventions in certain cases of congenital heart disease such as HLHS by exploiting the unique ability of the fetal myocardium to respond to hemodynamic load by myocyte hyperplasia.
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Materials and Methods |
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For further experimental details on ex ovo culture setup, LAL, immunohistochemistry and image processing, myocardial volume estimation, and statistical analysis, which are all based on published protocols,12,27,28 see the online data supplement at http://circres.ahajournals.org.
Right Atrial Clipping
On embryonic day 8 (E8)/Hamburger–Hamilton stage 34,29 the vascularized chorioallantoic membrane was carefully pushed aside using blunt microsurgical forceps (WPI, Sarasota, Fla), and the amniotic cavity was carefully opened by tearing the membrane. The chest cavity was then opened using fine-curved scissors; we later found that the procedure was shortened and survival improved by opening the chest cavity between E3 and E4 (stages 17 to 24), when the chest wall was still avascular. A fine silver neurosurgical microclip was then placed on the right atrium to reduce its volume and exclude part of it from circulation (Figure 1; for procedure details, see Movie 1 in the online data supplement). After visually verifying the position of the clip, the chorioallantoic membrane was returned to its original position, and the embryos promptly returned to the incubator. Control embryos were treated identically except for placement of the microclip. Surgery on embryos with HLHS was performed in an identical manner but in ovo. This latter technique necessitated coordinated action of 2 operators (D.S. and A.A.); the first placed the clip, while the other held the embryo in a suitable position. The experimental time line and numbers of embryos in each group are summarized in Table.
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Echocardiography of Chick Embryos
Access to the embryo from a variety of imaging planes was made possible by the use of the ex ovo culture system. For imaging, we used a 55-MHz scanhead on the Vevo 660 ultrasound biomicroscopy system (VisualSonics LTD; Figure 1). The temperature of the embryos was maintained at 37.5±0.5°C during examination by using a combination of a Radnoti circulating water-jacketed incubator and a heat lamp. After preoperative imaging, the embryos were allowed to recover for 1 hour before clipping; after clipping, an additional 1 hour was allowed for recovery before postoperative imaging. For examples of echocardiographic images before and after surgery, see supplemental Movies 2 and 3. We found that optimal survival was achieved when both surgery and imaging time was limited to 5 minutes. For the quantitative estimation of left ventricular cavity volume, end-systolic and end-diastolic area and minor diameter (width; cavity cross-section was approximated to an ellipse; Figure 1) from 3 cardiac cycles acquired at 30 frames per second were measured by planimetry using bundled software by a single, nonblinded observer (D.S.). Blinding of the observer was not practical, because the silver microclip was visible in most of the postclip recordings that included the ventricles (Figure 1). All individual measured and calculated values can be found in supplemental Table I. The volume (V) was calculated using the formula V=2/3x areaxwidth, assuming geometry of a prolate ellipsoid with equal minor axes.30 Cycle length, determined visually as number of cycles during 10-second loops (300 frames) of B-mode recordings and confirmed by peak-to-peak contraction measurements in M-mode, was similar between the recordings using this protocol. It was impossible to obtain simultaneous ECG tracings with this setup, optimized for adult mice, because of its insufficient sensitivity for cardiac mass of a chick embryo that is several orders of magnitude smaller.
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Results |
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Right Atrial Clipping Increases Blood Flow to the Left Heart Structures
The results of echocardiographic examination before and after clipping showed an increase more than preclip values to a varied degree in all 8 hearts examined (62% on average; Figure 1 and supplemental Table I). This increase was not attributable to a change in cycle length, as the basic functional parameters of the left ventricle established at baseline (heart rate, 181±10 bpm; ejection fraction, 63±3%) were not significantly changed; however, the stroke volume was increased, in accordance with the Frank–Starling relationship. Thus, right atrioplasty resulted in increased preload of the left ventricle. Despite the low survival of ex ovo LAL embryos to E8, 3 survivors were ultimately obtained, of which 2 had a distinct HLHS phenotype and were subjected to right atrial clipping and echocardiographic measurements (supplemental Figures 1 and 2 and supplemental Movies 4 and 5). In both cases, the procedure led to an increase in left ventricular end-diastolic volume of a magnitude similar to normal embryos.
Increased Preload Is Compensated by Myocyte Hyperplasia
We were interested to see whether the increased volume loading would translate into myocardial remodeling. Cumulative 24-hour survival of embryos clipped ex ovo at E8/stage 34 was 28% (improving with learning curve; mortality most often caused by clip-related bleeding). Histological analysis of 5 E9/stage 35 survivors showed increased distension of the left ventricular cavity, and the position of the clip on the right atrium was ascertained (Figure 2). However, despite the left ventricular cavity being larger, the signs of decompensated dilation (globular shape of the heart, increased transverse to longitudinal diameter) were absent, indicating good compensation of increased volume loading. Similarly, no venous congestion or edema characteristic of embryonic heart failure was noted.
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We next analyzed the proliferative structure of the myocardium to examine the cellular basis of the adaptive response of fetal myocardium to such hemodynamic alteration. First, we observed a dramatic decrease (drop to 48% of the nonclipped part, P=0.005 by paired t test) in proliferation in the excluded portion of the right atrium (Figure 2). Determination of the percentage of S-phase cells in matched areas of different heart compartments showed a significant increase in the labeling index in the left atrium and ventricle, with no significant changes in the right heart structures (Figure 2). In control hearts, the labeling was higher in the right atrium than in the left, whereas, after clipping, this relationship was inverted. Myocyte proliferation in both left atrium (+42%, P<0.001) and left ventricle (+38%, P=0.009) was significantly higher than nonclipped, E9/stage 35 ex ovo controls.
Decreased Myocyte Proliferation in the Hypoplastic Left Ventricle Is Rescued by Increased Hemodynamic Loading
Because of the severe difficulties of survival until E8/stage 34 of the embryos subjected to LAL ex ovo, we modified the clipping technique to perform the surgery in ovo. After modifying the technique to gently lifting the embryo from the egg by its neck using a fine glass hook, we produced a group of 9 embryos with established left ventricular hypoplasia subjected to clip that survived for an additional 24 hours until E9 (stage 35). In a control group of 16 in ovo embryos with HLHS, a small, but not statistically significant decrease in left ventricular myocyte labeling was noted when compared with normal in ovo embryos. This finding was in agreement with our previous study.27 In HLHS embryos with right atrioplasty, we noted the inversion of the right atrium>left atrium proliferation gradient similar to that of the ex ovo group (Figure 2). Specifically, whereas, in "plain" HLHS embryos, the right atrial labeling index was 30% higher than in the left atrium, after clip, it was 5% lower; however, these changes were not statistically significant. The myocyte labeling index was significantly increased in the hypoplastic left ventricle, actually reaching normal values (Figure 3). In contrast to untreated HLHS hearts, the values were also greater than in the right ventricle (also described previously27). There were no significant changes in the right ventricle (Figure 3). One of the embryos was excluded from the analysis, because it was moribund at the time of sampling (dilated vasculature, slow heart rate), and proliferation in all compartments was less than half of the group average. Semiquantitative evaluation of myocyte differentiation based on sarcomeric actin immunofluorescence showed recovery of staining to normal levels (98.5% of normal left ventricle). Inspection of the whole hearts showed (with the exception of 1 heart with a very severe phenotype) homogenous staining throughout the myocardium in contrast to a telltale decrease (–41.5%) in the hypoplastic left ventricle previously described.27 In contrast, no significant changes were noted between normal and normal+clip left ventricles. Last, the analysis of myocyte proliferation and differentiation in the ligated portion of the left atrium (excluded from circulation for 5 days) showed a decrease in proliferation (drop to 54% of values of the neighboring, nonexcluded part; P=0.03), as well as a decrease in differentiation indicated by reduced immunostaining (drop to 57.5%) for sarcomeric actin (Figure 2).
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Increased Myocyte Proliferation in Rescued Hypoplastic Left Ventricle Translates Into Increase in Myocardial Volume
For a success of any fetal cardiac procedure, it is necessary to see the measured experimental parameter (myocyte proliferation) translated into an actual increase of myocardial mass. Figure 4 shows clearly that increased proliferation results in significantly increased ventricular myocardial volume and cell number in the hypoplastic but, interestingly, not in the normal left ventricle. If expressed as an absolute number of myocytes, the observed increase would be 2 400 000 myocytes.
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Echocardiography Provides Proof of Loading After Right Atrial Clip
In the clinical setting, a subgroup of HLHS patients with a premature closure of the foramen ovale has a particularly poor prognosis postnatally. To alleviate this problem of blood flow restriction to the fetal left ventricle (that could contribute to phenotype progression and valve atresia), Marshall et al31 attempted fetal balloon septoplasty. Despite a technically successful procedure, the transseptal flow to the left atrium was minimal, pointing out the need of an additional procedure to force the blood to the left heart structures. Although a stent that maintains the opening in the interatrial septum might serve this purpose in human patients (A. C. Marshall, personal communication), we have tested in our studies a right atrial volume reducing procedure that would redirect the blood toward the smaller, most likely less compliant left atrium. This approach was substantiated by the fact that in the chick model of HLHS, premature closure of the interatrial communication occurs rarely and never before E10 (stage 36). By using embryonic echocardiography, we were able to verify that right atrial clipping indeed succeeded in increasing the blood flow to the left ventricle. The same effect was noted also in 2 embryos with hypoplastic left ventricles that survived ex ovo until E8 (Table), suggesting that the smaller left ventricle has sufficient compliance to respond by an increase in end-diastolic volume. Histological examination at a later time point showed that this enhanced volume loading resulted in adaptive myocardial remodeling. For technical reasons (the shape not easily approximated to geometric solid, difficult delineation of actual lumen because of more extensive trabeculation), analysis of right ventricular volume could not be performed, but it is likely that the amount of blood flowing through the right atrioventricular valve was reduced by right atrial clipping. However, volume load and pressure load are linked. We are basing our terminology on original experimental embryonic cardiac manipulations of Clark and colleagues,35 who distinguish modified afterload (eg, in conotruncal banding model) and preload (in venous end manipulations, such as LAL or venous clip model32). Indeed, because conotruncal banding increases ventricular end-diastolic volume, there is undoubtedly also volume load component, and, vice versa, left/right atrial clipping is accompanied by pressure changes. Unfortunately, direct pressure measurements in embryos are a nonsurvival procedure and probably impossible to achieve in highly mobile embryos at later fetal stages (the published data go only as far as E6/stage 29). Longitudinal examination of left ventricular systolic function (supplemental Table II) showed an increase in ejection fraction from 45% to 68% and average stroke volume from 0.52 to 0.79 mm3 between E6 and E8, correlating with left ventricular wall compaction,33,34 completion of ventricular septation, and beginning of coronary perfusion.
Increased Volume Loading Is Compensated by Fetal Myocyte Proliferation
Previous studies have shown that increased pressure loading in the fetal heart is compensated for by myocyte hyperplasia.35,36 However, our own study of volume-overloaded preseptation right ventricle in the chick experimental model of HLHS27 cast some doubt on whether the same is true for volume overload stimulus, because minimal changes in the myocardial proliferative structure were noted in the dilated right ventricle.26 Here we show that at least for postseptation early fetal chick left ventricle, increased preload is a stimulus for increased myocardial growth. This apparent discrepancy could be explained by the different compliance and geometry between the left and right ventricle, making the right ventricle more prone to dilation rather than adaptive chamber wall thickening in response to increased hemodynamic loading.37 Along this line, the changes of myocyte proliferation in the right ventricle after clip were minimal and statistically not significant, and the sampling interval was probably too short to see any "shrinking." Even in severe left ventricular hypoplasia,12 the right ventricular myocardial volume is increased only slightly, suggesting its high functional plasticity. Because apoptosis in developing myocardium is a rare event, and remodeling of myocardial architecture during this period is based on modulation of myocyte proliferation,12,27,38 we would expect the gradual involution of the dilated right ventricle to be based on a small decrease of proliferation over a longer time period (similar to mechanism of experimental left ventricular hypoplasia), which is consistent with our observations. Of note, it is likely that increased preload is actually a combination of volume and pressure increase; however, direct pressure measurements were impossible in this setup. Despite its statistical significance, the magnitude of changes in myocardial proliferation appears rather small; this is because of the necessity of the myocardium to maintain adequate pumping function even while continuing myocyte division. In addition, even minor changes in proliferation measured at a single time point are, over time, translated into appreciable differences in phenotype, and the role of proliferation can be confirmed using the method of radiolabel dilution.27,38
Findings of decreased proliferation in both acutely and chronically excluded parts of the atria correlates with previously published data.27 The differentiation status of this in vivo unloaded myocardium varied, with no change in the relative amount of contractile protein apart from some myofibrillar disorganization in acutely (24 hours) and significantly less of both actin and myosin in chronically (4 to 5 days) excluded portions.
We found that both normal and hypoplastic left ventricular myocardium responded to increased volume loading in a similar fashion, ie, by increased myocyte proliferation. This is a significant finding, and gives hope to the idea that if a way is ascertained to hemodynamically reload the hypoplastic left ventricle prenatally, this chamber could reasonably be expected to respond by augmented growth and contractile differentiation. In addition, this increased cell cycling translated into increased myocyte numbers in the hypoplastic but not the normal left ventricle. Given the average cell cycle duration of less than 16 hours at that stage, this increase in myocardial mass is plausible, with an observed increase in cell cycling. Similarly, there was an improvement (to near-normal values) of differentiation status of myocytes in the reloaded hypoplastic left ventricle.
Significance of Prenatal Cardiac Repair
Our experience showed that to achieve optimal survival, careful "dosing" is necessary. Together with the learning curve, our survival rates improved over time, from 20% to 80% of operated embryos. From clinical settings, it is known that the immature heart does not easily tolerate sudden hemodynamic changes and, hence, is among the reasons why surgical palliation for HLHS is performed in 3 steps. To gauge the effects of such interventions in practice, careful monitoring of the hemodynamic response to "dosed" ligation of the right atrium, either echocardiographically or by means of pressure measurements during cardiac catheterization would be necessary, with possible adjustments based on measured values. Such monitoring is now standard practice in postoperative follow up in pediatric cardiology.2
The limitations of this experimental study are several. The chick model, despite its 4-chambered heart and almost identical patterns of prenatal and postnatal circulation, is not an etiological model of the human condition (most often likely to be genetic in origin) but a hemodynamically created phenocopy. Its different size means that our findings should be ideally verified in a larger fetal mammalian model, such as sheep11 or pig.39 Nevertheless, it is a useful model of aberrant myocardial loading, highly relevant to understanding the biology of ventricular myocardium in a variety of congenital heart disease, of which HLHS is among the most significant. Furthermore, the majority of patients18,19 present with aortic and/or mitral atresia, which would necessitate modification of the interventional approach to account for this complication. Current clinical experience with prenatal balloon dilatation of critical aortic stenosis suggests that careful patient selection, together with technical precision can result in favorable outcomes,15–17 making fetal interventions a viable alternative to postnatal palliation and our model timely.
Because the clipping of LAL embryos had to be performed in ovo, we had to rely on external visual inspection rather than more objective echocardiographic evaluation of the phenotype. We reached a complete agreement on the phenotype of further operated embryos between 2 observers with a cumulative experience with LAL exceeding 10 years, and correlation with histological examination (supplemental Figures 1 and 2) was excellent. Selective survival of "milder" phenotypes, which could provide an alternative explanation of observed phenotypic "rescue," is also unlikely, because routine autopsy of all nonsurvivors did not show any overt difference in phenotypic severity between these groups.
We did not aim for survival past the set 24-hour interval, because we anticipated that the rather large silver microclip would interfere with chest wall closure and long-term survival. We are currently working on technical aspects of the procedure that would allow us to follow up the survival until hatching to compare the rates and address this issue.
In conclusion, we have shown that volume-displacing surgical procedures at the atrial level in the fetal heart can influence ventricular loading and that such an increase in ventricular preload in the early postseptation left ventricle is compensated by myocardial hyperplasia in both normal and hypoplastic left ventricle. Reversal of experimental left ventricular hypoplasia induced by LAL can be achieved with right atrial clipping, thereby redirecting flow by changing atrial compliance. The underlying mechanism of this myocardial remodeling is increased myocyte proliferation, leading to increased myocardial mass. These results provide hope that carefully designed human fetal procedures that seek to restore normal cardiac structure and function in the settings of severe congenital heart disease such as HLHS will be successful.
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Acknowledgments |
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Sources of Funding
This project was supported in part by NIH grant RR16434, March of Dimes grant 5-FY02–269, Ministry of Education of the Czech Republic grant VZ 206100–3, Institutional Research Plan IAPG AVOZ50450515 from the Academy of Sciences of the Czech Republic, and a Purkinje Fellowship from the Academy of Sciences of the Czech Republic. This work was conducted (in part) in a facility constructed with support from the NIH (grant C06 RR018823) and the Extramural Research Facilities Program of the National Center for Research Resources.
Disclosures
None.
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Footnotes |
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Original received September 29, 2006; resubmission received January 9, 2007; revised resubmission received February 28, 2007; accepted March 27, 2007.
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