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Eur J Cardiothorac Surg 2002;23:573-577
© 2002 Elsevier Science NL

Clinical histopathology and ultrastructural analysis of myocardium following microwave energy ablation

^a Cardiac Surgery Department, Istituto Clinico Humanitas, University of Milan School of Medicine, Rozzano, Milan, Italy
^b Pathology Department, Istituto Clinico Humanitas, University of Milan School of Medicine, Rozzano, Milan, Italy
^c Pathology Department, Istituto Clinico Humanitas, University of Milan School of Medicine, S. Paolo Hospital, Milan, Italy

Received 2 September 2002; received in revised form 8 December 2002; accepted 11 December 2002.

^* Corresponding author. Via G de Grassi 17, Milan 20123, Italy. Tel.: +39-02-460-442; fax: +39-02-659-6205
e-mail: emanasse{at}inwind.it

	Abstract

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion References

 
Objective: Due to weaknesses of conventional modes for treating atrial fibrillation (AF), surgical energy ablation methods and tools to cure AF have been under rapid development. One of these methods, microwave energy, is beginning to be applied clinically. The purpose of this study was to examine histology and ultrastructure of lesions produced by microwave energy in the myocardium. Methods: Fifteen consecutive patients underwent surgical microwave energy ablation (Microwave Ablation System with FLEX 4 probe, AFx Inc., Fremont, CA) concomitant to a valve procedure. Epicardial ablation was carried out on the beating normothermic heart prior to performing the valve procedure. Two tissue specimens (1 cm²) were obtained from each patient; one from the lesion site (right appendage) and the other from an adjacent, non-ablated site, which was used as control. Tissue samples were fixed and stained as appropriate for histological and ultrastructural analysis. Results: All ablated samples revealed observable microscopic alteration, including loss of nuclei, foci of coagulative necrosis or induced irregular bands of contraction. Ultrastructurally, ablated cells demonstrated architectural disarray, loss of contractile filaments, mitochondrial swelling and focal interruption of plasma membrane. Conclusions: Histologic appearance of lesions created by epicardial microwave energy ablation was consistent over tissue samples, although acute findings demonstrated differences from cryoablation. In most of the cases, lesions were transmural, as was demonstrated by loss of cellular viability throughout the depth of tissue specimens.

Key Words: Cardiac ablation • Microwave • Atrial fibrillation

	1. Introduction

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion References

 
Atrial fibrillation (AF) is the most frequently encountered arrhythmia in clinical practice. Chronic AF is an independent risk factor for early mortality and heightened stroke rate, where risk is increased by two- and fivefold, respectively, in persons who have it [1–4]. Rise in morbidity and mortality associated with AF appears to be most likely due to loss of atrial function, combined with consequent diminished cardiac performance. Traditional treatment for AF, most notably drug therapy, is in most cases a palliative and principally serves to ameliorate symptoms given low rate of stable normal sinus rhythm (NSR) achievable with the drug therapy alone. Use of these types of therapies requires careful risk factor profiling in order to effectively reduce symptoms and modulate, sometimes life-threatening, side effects. Pharmaceutical strategies usually do not eliminate AF, nor is conversion between paroxysmal or persistent AF, and permanent AF prevented [5-7]. With continuing AF and advancing age, risk of stroke and other morbidity, as well as early mortality, remains likely to escalate [7]. Because of these drawbacks, there have been vigorous efforts in the last few years to develop curative techniques designed to eliminate AF altogether.

Curative techniques currently under development include not only novel approaches to surgery, but also a large variety of surgical tools. While the Maze ‘cut and sew’ procedure has provided an excellent model for success in treating AF surgically, it is associated with excess morbidity and mortality and is relatively difficult to perform. Because of this, methods to perform the Maze procedure using energy ablation, including radiofrequency and cryoablation, and more recently, microwave, are becoming widely applied in this clinical arena. The common objective of all these ablation methods is to produce a set of lesions in the atrial wall, which are capable of reversing the aberrant electrical signals associated with AF.

After 3 years experience with cryoablation, we have recently started to use microwave energy ablation to surgically treat AF using an epicardial approach. This approach, through the epicardial approach might allow reduction or even elimination of the aortic cross-clamp time and permits a surgeon to avoid an atriotomy when not required for treating the principal cardiac pathology. Clinical histologic study of myocardial lesions produced using cryoablation and radiofrequency have been previously conducted in different subsets but are helpful in understanding the tissue interactions and reaction to this kind of procedure [8–11]. The aim of this investigation was to examine histopathology and ultrastructure of epicardial lesions especially produced clinically by microwave energy. Different studies have focused on this topic but very little is known so far, about the performance of this energy source in a clinical setting. Furthermore the efficacy and the histological effect of the microwave applied on the beating heart have never been considered.

	2. Patients and methods

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion References

 
Subjects (n=15) were consecutive patients presenting to our clinic for routine valve surgery who also had documented permanent AF for at least 6 months. All data collection took place during concomitant procedures that were performed between January and October 2001.

The ablation procedure and tissue sample collection for the study were conducted on the right atrial appendage prior to the valve procedure. The right atrium was chosen because of its anatomical exposure, and because its excision would not burden any other risk to the patients. After double atrial cannulation was performed to institute cardiopulmonary bypass but before instituting the extracorporeal circulation, epicardial ablation was carried out using a Microwave Ablation System and FLEX 4 probe (AFx Inc., Fremont, CA). Ablations were carried out using a power setting of 65 W, with ablation time of 90 s. Because of energy losses in the connecting coaxial cable between the generator and the microwave antenna, 40% of the power was lost between the generator and the antenna. Therefore, 39 W were emitted into the tissue, which corresponds to a power output of 9.75 W/cm along the antenna. The ablation procedure was performed on the beating normothermic heart.

Ablation and sample collection procedures were carried out in the same manner for all patients. Immediately following the ablation, two 1x1 cm² transmural tissue specimens were obtained from each patient (one from the ablation site and one outside the ablated area selected as a control over the right appendage). Each specimen was placed on tissue paper, carefully sectioned through its center and immediately fixed in 4% formalin. Tissue specimens were dehydrated and embedded in paraffin for histological and histochemical analysis. From each specimen, 3 µm thick sections were obtained, stained using Haematoxylin and Eosin and evaluated with Masson's Trichrome and Periodic Acid-Schiff (PAS) techniques.

Furthermore, samples obtained from five randomly selected patients were divided into two fragments. The first fragment was fixed in formalin and embedded in paraffin as initial samples. The second fragment, intended for ultrastructural studies, was reduced to small cubes of 1x2x2 mm², fixed in 2.5% glutaraldehyde in 0.13 M phosphate buffer, which had pH of 7.2–7.4, and embedded in epoxy resin. All resin-embedded samples were cut into semithin 0.5 µm sections and stained with tholuidin blue for microscopic study. Four out of seven selected samples were cut into ultrathin sections, counterstained with uranyl acetate and lead citrate, and examined using a JEM transmission electron microscope (Jeol, Tokyo, Japan). One of these four samples consisted of normal tissue and served as a control.

	3. Results

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion References

 
3.1. Microscopic findings
Lesions ranged from 0.4 to 1 cm in length. Specimens were characterized by damage involving the full thickness of the atrial wall (Fig. 1 ). Histologically, ablations had ill-defined borders and myocellular damage was geographically distributed continuously throughout the myocardial wall. All samples contained clear foci of coagulative necrosis, and often demonstrated irregular or complete loss of membranous borders with shrunken, hyper-eosinophilic cytoplasms of myocardial cells (Fig. 2 ). Diffuse, nuclear pycnotic changes were evident and, in small areas, a complete loss of nuclear structures was observed (Fig. 3 ). The geographical distribution of the damage was probably due to the presence of normal viable myocytes, sometimes detected, in the thickness of the atrial wall (Fig. 4 ).

View larger version (170K): [in this window] [in a new window]   Fig. 1. Histologic sample of a transmural cross-section collected from the atrial wall shows acute response following epicardial (open arrow) ablation with microwave energy. Damage to cardiomyocytes is evident over full thickness of the transmural sample (40x, Hematoxylin and Eosin).

View larger version (129K): [in this window] [in a new window]   Fig. 2. A characteristic histology sample collected from ablated tissue demonstrates clear foci of coagulative necrosis (N), irregular or complete loss of membranous borders and shrunken, hyper-eosinophilic cytoplasms of myocardial cells (200x, Hematoxylin and Eosin).

View larger version (149K): [in this window] [in a new window]   Fig. 3. Ablated tissue sample: complete or partial loss of membrane borders and hyper-eosiniphilic cytoplasms of cardiomyocytes with pycnotic changes was noted (200x Hematoxylin and Eosin).

View larger version (174K): [in this window] [in a new window]   Fig. 4. Following ablation with microwave energy, cellular structures were destroyed (head arrow) but viable myocells throughout the lesions were also observed in some cases (open arrow) (100x, Hematoxylin and Eosin).

3.2. Ultrastructural findings
The control sample showed only focal mild ultrastructural alteration (Fig. 5A ). Focal hypercontraction patterning was noted, most likely due to peri-operative handling, while plasma membrane and organelles were well preserved. All ablated cardiomyocyte samples demonstrated variable degrees of ultrastructural degeneration. Characteristic features included architectural disarray and loss of contractile filaments (Fig. 5B) and mitochondria swelling and focal interruption of plasma membrane (Fig. 5C). These tissue degeneration features were identifiable only in the regions that appeared to be damaged at the H&E staining, and not in normal looking areas.

View larger version (98K): [in this window] [in a new window]   Fig. 5. (A) Two myocytes without relevant ultrastructural alterations (7200x, electron microscopy). (B) Microwave energy ablated sample: a myocyte shows architectural disarray and severe loss of contractile filaments (5000x, electron microscopy). (C) Microwave energy ablated sample: swollen mitochondria with cristae rupture (M) and interrupted plasma membrane (arrows) in a hyper contracted myocyte (17,000x, electron microscopy).

	4. Discussion

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion References

Acute morphologic features demonstrated after both microwave energy and cryoablation included clear foci of coagulative necrosis, irregular or complete loss of membranous borders, loss of nuclei, and eosinophilic bands of hypercontraction [14], morphologic features which are the sign of an irreversible injury and cannot be reconciled with cell survival and life. In contrast, edema and increased distance between myocardial cells, was not observed with microwave as it was following cryoablation. This difference may be due to the effects of water crystallization occurring with cryoablation, resulting in increased intercellular spaces following ablation.

Although this might be only one of the possible manner that cryoablation might affect tissues [16], an important osmotic effect is generated by ‘fast forming’ ice, and this might be reproduced by vaporization of water with a powerful enough heat source. Another big role might be played by the denaturation of proteins: it is well known that overheating proteins above 56°C breaks hydrogen bonds, that play a key rule in determining 3D protein structure, especially for the membrane lipid embedded ones. Cryoablation might play a stronger role in blocking the lipid floating surface and altering the membrane permeability.

Furthermore, physics of microwave allows ‘focusing’ of energy on the most water containing structures [15], and thus to overcome fat tissue barriers, that are well known good isolators for cryobased ablations. Although this might look superfluous for the endocardial approach (the widest used, and the mostly so far accepted), it could be vital in the attempt of developing an epicardial closed heart approach. It could be hypothesized that with using microwave to focus on the energy at different depths according to tissue thickness, the ablation devices could be optimized.

In sum, histologic appearance of the epicardial lesions created by microwave energy was consistent and appeared to conform to findings observed using other energy ablation sources. In all tissue samples, regions containing necrotic cells were extensive and transmural. Nonetheless, there were several tissue samples where viable-looking cells were detected within heavily severed tissue. It is unclear whether these cells would remain viable over time to be capable of delivering electrical re-entrant activity associated with AF.

For these reasons, longer term follow-up is necessary to examine the histological and conductive properties of chronic lesions following microwave energy ablation to evaluate if the full thickness lesion is a constant effect of microwaves. To date, it is not clear whether transmurality is a prerequisite for a lesion set to be effective. The interruption of an electrical pathway may be effective although not all the fibers are destroyed. On the other hand, a thinner tissue might conduce slower enough to interrupt reentrance phenomena and be sufficient to restore regular atrial actrivity.

	References

Top Abstract 1. Introduction 2. Patients and methods 3. Results 4. Discussion References

 

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