Serial left-ventricular biopsy sampling using a minimally invasive trans-thoracic approach in adult dogs
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Myocardial biopsies are an increasingly important tool to unravel the molecular mechanisms of cardiac disease. We evaluate a novel minimally invasive trans-thoracic approach for left-ventricular (LV) intra-mural biopsies, which enables repetitive individual sampling in adult dogs. Forty three generally anaesthesised dogs were studied during sinus rhythm (SR, control) and multiple times after the induction of volume overload hypertrophy (complete atrioventricular block [AVB]). Through a small cutaneous incision, an automatic biopsy needle was advanced into the apicolateral LV, guided by fluoroscopy. Electrocardiography (ECG), LV pressure and echocardiography served to monitor the procedure. One hundred eighty-eight intra-mural LV biopsies were obtained in 82 serial experiments (usually SR, 1, 2 and 6 weeks AVB) with a maximum of 8 repeated biopsies. All biopsies (∼10 mm3) were suitable for simultaneous application of different cell-biological (light and electron microscopy, immunohistochemistry) and molecular techniques (PCR, Western blotting). In chronic experiments, repeated biopsy sampling did not influence haemodynamics, mechanics, electrocardiographic parameters or myocardial remodelling during SR or AVB. The rate of significant complications was as low as 4% of experiments. Minimally invasive sampling of LV needle biopsies enables serial assessment of myocardial remodelling using different molecular techniques in individual animals. The technique is safe and has no long-term effects on cardiac function or structure.
KeywordsInstruments and techniques Myocardial biopsy Myocardial remodelling Ventricles
Myocardial biopsy sampling offers indispensable diagnostic options to unravel the mechanisms of myocardial disease in different conditions [3, 18]. Currently, cardiac biopsies are mostly obtained using a trans-venous catheter-based bioptome enabling to sample sub-endomyocardial tissue from the right ventricle (RV), as introduced in 1962 . This trans-vascular approach is also used trans-arterially to sample from the left ventricle (LV) . In experienced hands, the catheter-based technique is relatively safe with complication rates of 1–4%  and is routinely applied in clinical referral centers . It is important to note that the limited invasiveness of the technique makes it possible to sample cardiac biopsies in a serial manner to monitor specific disease processes in individual patients over time. Yet, sub-endomyocardial tissue samples have significant drawbacks relating to (1) sampling errors  and lack of representativity, which requires to take at least 5 biopsies per session and even more for focal processes ; (2) small sample size (usually <1 mm3)  and (3) poor tissue quality due to squeezing and cutting artefacts in a relatively small tissue sample , which often render the biopsies unsuitable for histology, immunohistochemistry and electron microscopy .
An alternative way to sample ventricular myocardial tissue is by the use of a biopsy needle, which enables to obtain larger, intra-mural samples of superior quality compared to endomyocardial biopsies. This technique was first described using limited thoracotomy , but needle biopsies have generally been confined to the use in patients and animals undergoing standard thoracotomy for direct vision of the puncture site and potential bleeding . Today, needle biopsies can be safely and quickly obtained during open-heart surgery using automatic biopsy devices [14, 20].
In the study reported here, we developed a novel trans-thoracic approach to sample LV needle biopsies from individual anaesthetised dogs in a serial manner and evaluated its application in chronic experiments after the induction of cardiac hypertrophy.
Materials and methods
Experiments were conducted in accordance with the ‘European Directive for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (86/609/EU)’. The Committee for Experiments on Animals of Maastricht University approved the experiments.
Serial trans-thoracic sampling of LV intra-mural biopsies
We studied a total number of 43 adult mongrel dogs of either gender weighing 28 ± 1 kg at baseline during normal sinus rhythm (SR, control). All experiments were performed under general anaesthesia. After overnight fasting, pre-medication (0.15 mg/kg acepromazine, 0.4 mg/kg methadone, 0.06 mg/kg atropine i.m.) was administered. Complete anaesthesia was induced by thiopental (20 mg/kg i.v.) and maintained with halothane (0.5–1%) and O2:N2O (1:2).
Throughout the procedure, a standard 6-lead ECG was registered and the LV intra-cavitary blood pressure was monitored in a sub-set of experiments (Fig. 2a and Fig. 2b). After sampling, all animals were followed-up echocardiographically (Fig. 2c) for at least 60 min to monitor LV contractility and potential occurrence of pericardial effusion. Echocardiograms were usually performed every 5 min during the first 20 min after sampling. If pericardial effusion was absent or had not increased to a maximum of more than ∼5 mm, anaesthesia was terminated. After detubation, additional echocardiograms were performed before and after transportation to the cage, usually at 30, 45 and 60 min after sampling. When pericardial effusion increased to more than ∼10 mm in the first hour after sampling, we decided to intervene surgically by left-lateral thoracotomy, pericardiotomy, evacuation of blood from the pericardial space and local haemostasis of the bleeding site. LV haemodynamics, mechanics and echocardiographic estimates of LV mass were assessed as described before [4, 5]. The complete procedure is described as a practical step-by-step approach in Flowchart 1.
All dogs were studied serially during SR and after the induction of complete atrioventricular block (AVB). AVB was induced by radiofrequency (RF) catheter ablation of the His bundle. Sampling was performed under baseline conditions at sinus rhythm (SR; control) and, in the same animals, at different time points after AVB ranging from 2 days up to 6 weeks. The dogs were sacrificed several weeks later and the hearts were excised and inspected macroscopically.
Data are presented as the mean ± standard error of the mean (SEM). Data were compared using Student’s t-test for unpaired data. Differences were considered statistically significant if P < 0.05.
In 82 experiments, a total number of 188 intra-mural LV biopsies was obtained (2.6 ± 0.6 biopsies per experiment). In individual animals, up to 4 consecutive experiments were conducted in which maximally 8 repeated biopsies were taken, typically at SR, 1, 2 and 6 weeks of AVB.
Serial LV function and structure in bioptised vs non-bioptised dogs
Procedural experiences and complications
Generally, the animals recovered from the procedure within a few hours and post-operative wound infection was not observed. In the first 34 experiments, we observed cardiac tamponade in 5 (15%) experiments. Four dogs died (12%) and 1 underwent successful left-lateral thoracotomy, pericardiotomy and operative evacuation of the haemopericardium. It is important to note that all these dogs were heparinised (1,000 IU i.v.) around biopsy sampling, as routinely applied in our operation room to avoid catheter-induced thromboembolism and only 3 dogs had been haemodynamically monitored during the experiment.
Identification of sampling site
Sample in apicolateral LV, avoid thin-walled apex and coronaries in the lateral LV
Cardiac tamponade, coronary perforation
Feel mild resistance and cardiac pulsations on needle shaft before intra-myocardial insertion
Extra-myocardial biopsy, no biopsy
Monitor the occurrence of ventricular extra-systoles originating from the biopsy sitea
Extra-myocardial biopsy, no biopsy
Use standard histological staining to ensure tissue quality and intra-myocardial extent, e.g. haematoxylin/eosin, toluidin blue, Masson’s trichrome
Light microscopy allows to visualise endocardium/epicardium and myocardium in the same section
Monitor standard leads throughout the experiment
Sinus tachycardia and signs of ischaemia can aid to diagnose a cardiac tamponade in time
Assess cardiac contractility and pericardial effusion directly after sampling and repeat frequently in the first hour
Cardiac tamponade can be diagnosed in time
LV intra-cavitary pressure catheter (optional)
Monitor LV systolic blood pressure throughout the experiment (optional)
Progressive decrease of systolic LV blood pressure can aid to diagnose a cardiac tamponade in time
In the present study, we demonstrate how LV needle biopsies can be repeatedly obtained in dogs undergoing chronic experiments. A trans-thoracic approach together with optimal safety monitoring ensures a low complication rate, quick post-operative recovery and optimal tissue quality.
Serial myocardial sampling using needle biopsies in a chronic experimental setting is generally limited by the invasiveness of a thoracotomy, which results in only few sampling points, e.g., at the control stage and before sacrifice of the dogs . We circumvented this by choosing a trans-thoracic, percutaneous approach, thus avoiding the thoracotomy. Historically, different closed-chest approaches have been reported in canine and human hearts to sample ventricular needle biopsies from the LV anterior wall, apical area  and the inter-ventricular septum  under local or general anaesthesia. After the 1970s, percutaneous needle biopsy techniques have not been widely applied in patients or experimental animals, and no further technical developments have been reported. This might be largely due to the advent of catheter-based techniques, which are less invasive, relatively easy to use and safe, and, moreover, suitable for repeated biopsies.
The novel trans-thoracic approach we described in this study shares many advantages with the trans-vascular technique: (1) it is minimally invasive; (2) it has a low rate of potentially life-threatening complications (∼4%) despite the limited number of experiments we performed, which is comparable to the clinically accepted trans-vascular techniques ; (3) it is suitable for repeated biopsies and thereby allows individual follow-up studies; (4) it can be performed within a few minutes; (5) it can be easily performed in a standard experimental animal laboratory (see Flowchart 1) with experienced bio-technicians reaching a sufficient level of competence after having performed ∼25 biopsies. Moreover, the approach can likely be applied in other large animal species. This assumption is based on comparative anatomical considerations and reports of experiments with minimally invasive thoracoscopic cardiac access in sheep , pigs  and goats.
Multiple serial intra-mural biopsies from the same LV area did not influence functional and structural remodelling processes that occur after AVB. All analysed mechanical parameters were compatible with our earlier work [4, 5]. Also, under control conditions, repeated biopsy sampling from the apicolateral LV wall did not significantly influence the expression of cardiomyocyte-specific proteins that have been shown to be altered in remodelling processes early after the induction of AVB . These findings support the minimally invasive character of the technique and are in line with the macroscopic finding of only slight local fibrosis at barely visible puncture sites. Thus, it can be concluded that myocardial damage is confined to the puncture site. However, the total number of biopsies taken in individual animals might be relevant in this regard. We obtained a maximum of eight serial needle biopsies in up to four consecutive closed-chest experiments per dog. Higher numbers of serial ventricular needle biopsies in chronic experiments have only been reported in studies using cardiac allograft biopsies in dogs undergoing orthotopic  or heterotopic  heart transplantation. In these studies, post-operative fibrosis and adhesions after open-heart surgery likely play a role in reducing the risk of haemopericardium and tamponade, but may also increase the probability of biopsy-induced functional and structural myocardial changes.
Needle biopsies offer ample opportunities for a detailed myocardial analysis (Fig. 3, Fig. 4 and Fig. 7). The tissue specimens are relatively large, cutting artefacts are limited to the border zones and they represent a cross-section through the ventricular wall potentially extending into the (sub)endocardium (Fig. 3). Our serial approach allows to discern even mildly fluctuating expression patterns in long-term studies because individual dogs serve as their own controls [4, 16]. These subtle changes are likely missed in group comparisons due to inherent inter-individual differences. These aspects meet contemporary issues in cardiac research, aimed at a comprehensive multi-level molecular analysis in individual subjects.
A novel minimally invasive trans-thoracic approach for serial LV intra-mural needle biopsies has been successfully developed in adult dogs. The procedure is safe and allows repeated sampling in individual animals without influencing cardiac function or structure in long-term experiments. Different cell-biological and molecular techniques can be simultaneously applied in these biopsy specimens. The approach is potentially applicable to other animal models of cardiac disease and offers the opportunity to study molecular mechanisms relevant for myocardial remodelling in chronic experiments relating to various pathological conditions.
This study was financially supported by the ‘Stichting Hartsvrienden Rescar’, Maastricht, The Netherlands. Dirk W. Donker was supported by Medtronic, The Netherlands. The authors wish to thank Jet Beekman (Dept. of Med. Physiol., Utrecht), Helma Kuijpers and Fons Verheyen (Dept. of Mol. Cell Biol., Maastricht) for technical assistance.
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