INtra-procedural ultraSound Imaging for DEtermination of atrial wall thickness and acute tissue changes after isolation of the pulmonary veins with radiofrequency, cryoballoon or laser balloon energy: the INSIDE PVs study

Preliminary data in human suggest that both Intracardiac echocardiography (ICE) and Intravascular ultrasound (IVUS) can be used for real-time information on the left atrial (LA) wall thickness and on the acute tissue changes produced by energy delivery. This pilot study was conducted to compare ICE and IVUS for real-time LA wall imaging and assessment of acute tissue changes produced by radiofrequency (RF), cryo and laser catheter ablation. Patients scheduled for RF, cryoballoon or laser balloon Pulmonary Vein Isolation (PVI) catheter ablation were enrolled. Each pulmonary vein (PV) was imaged before and immediately after ablation with either ICE or IVUS. The performance of ICE and IVUS for imaging were compared. Pre- and post-ablation measurements (lumen and vessel diameters, areas and sphericity indexes, wall thickness and muscular sleeve thickness) were taken at the level of each PV ostium. A total of 48 PVs in 12 patients were imaged before and after ablation. Both ICE and IVUS showed acute tissue changes. Compared to IVUS, ICE showed higher imaging quality and inter-observer reproducibility of the PV measurements obtained. Acute wall thickening suggestive of oedema was observed after RF treatment (p = 0.003) and laser treatment (p = 0.003) but not after cryoablation (p = 0.69). Our pilot study suggests that ICE might be preferable to IVUS for LA wall thickness imaging at the LA-PV junctions during ablation. Ablation causes acute wall thickening when using RF or laser energy, but not cryoenergy delivery. Larger studies are needed to confirm these preliminary findings.


Introduction
One of the major limitations of percutaneous catheter ablation is the inability to image the cardiac tissue, thus providing real-time information on wall thickness and on acute tissue changes produced by energy delivery during ablation. Due to the unpredictable variability of the wall thickness in the areas targeted for ablation [1,2], choosing appropriate ablation settings for creation of effective lesions is often challenging. Insufficient ablation will translate into nontransmural and non-durable lesions, while excessive ablation could lead to steam pops, cardiac perforation and damage to surrounding anatomical structures (e.g. the esophagus). Moreover, very little is known about acute tissue changes produced by catheter ablation and the role they might play in lesion failure. Acute development of tissue oedema has been reported when using radiofrequency (RF) energy for ablation [3][4][5] and it has been advocated as a possible mechanism of lesion failure [6,7]. The presence and degree of acute tissue oedema when using cryoenergy or laser energy for ablation is not known.
Ultrasound imaging has previously been used in animal studies for assessment of atrial wall thickness and lesion formation during catheter ablation [8,9]. Preliminary data in human also suggest that ultrasound imaging modalities such as intracardiac echocardiography (ICE) and intravascular ultrasound (IVUS) can be used for left atrial (LA) wall thickness measurements and detection of acute changes produced by ablation [10][11][12].
This pilot study was conducted to compare ICE and IVUS for real-time LA wall imaging and assessment of acute tissue changes produced by different ablation energies during pulmonary vein isolation (PVI).

Study design
The INSIDE PVs study (INtra-procedural ultraSound Imaging for DEtermination of atrial wall thickness and acute tissue changes after isolation of the Pulmonary Veins with radiofrequency, cryoballoon or laser balloon energy) was a single-center prospective pilot study. The trial was approved by the Local Ethics Committee, complied with the Declaration of Helsinki and was registered on www. clini caltr ials. gov (Identifier NCT03372798).
Patients scheduled for RF, cryoballoon or laser balloon PVI catheter ablation for symptomatic, drug-refractory paroxysmal atrial fibrillation (AF) were eligible for the trial. All AF ablations were performed in a standard fashion, as described in detail in the supplementary section.

Protocol for ICE/IVUS imaging of the PVs
As part of the trial, ultrasound imaging of the junctions between LA and pulmonary veins (PVs) was performed with either ICE or IVUS as per operator's discretion.
For ICE imaging, a 9F catheter with a 9 MHz rotational transducer providing a maximal radial depth of 50 mm (Ultra ICE, Boston Scientific) was used. For IVUS imaging, a 20 MHz digital probe with maximum ultrasonic detection depth of 24 mm (Visions PV 0.018, Volcano, San Diego, CA) was chosen and used mounted on a guidewire (0.014-in percutaneous transluminal coronary angioplasty guidewire) as per manufacture instructions.
The ultrasound-imaging probe was introduced into the LA via the trans-septal access and advanced under fluoroscopic guidance distally into each pulmonary vein (PV) with the aid of a long steerable sheath. Images were recorded during slow manual pullback of the probe from each vein into the LA body. Each PV was imaged twice, before and immediately after ablation and acute electrical isolation.

Offline ICE/IVUS quantitative images analysis
Images were stored and analyzed offline by two independent operators, blinded to clinical and procedural data, at the OxACCT CoreLab (Oxford Academic Cardiovascular CT CoreLab) using QIVUS software (Medis Medical, Leiden, The Netherlands).
Images analysis was performed as previously outlined [13,14]. Briefly, the "lumen contour" was identified following the endothelial border, defined as the interface between the hyper-echogenic vessel wall and the an-echogenic lumen area. The "vessel contour", corresponding to the endothelial + media border (e.g. to the external elastic lamina (EEL)), was instead defined following the profile of the an-echogenic structure broadly parallel to the lumen contour. Notably, in segments of completely healthy vessel the distance between the lumen contour and the vessel contour could be absent, resulting in the two lines overlapping. To guarantee consistency and avoiding artificial wall thickness, when the EEL was not detectable for an arch greater than 180°, the vessel contour was drawn as overlapping the lumen contour, making sure that a broadly circular shape of the vessel contour was maintained. In a proportion of cases, it was possible to observe a wide anechogenic structure wrapping around the lumen/vessel profile, according to an angle of variable degree. This structure corresponded to the sleeve of myocardium projecting from the left atrium towards the PV ostium and fusing with the media of the vessel. When present, the myocardial sleeve was used to define the limit of the EEL, and consequently of the vessel contour (Fig. 1).
Lumen and vessel diameters, areas and sphericity indices and wall thickness were measured at the level of the PV ostium. The PV ostium was identified as point of maximal inflection between LA and PV wall, as previously described [15].
Sphericity index was defined as the ratio between minimum and maximum diameter, and it was measured both for lumen and vessel. The wall thickness was defined as the echogenic space between the lumen contour and the vessel contour. Maximum, mean and minimum wall thickness were measured for each vessel. The wall thickness index (WTI) was defined as the area of the surface included between the lumen and vessel contour (Vessel Area − Lumen Area). To facilitate comparisons a percentage wall thickness index (WTI%) was calculated as: WTI = 100 × (Vessel Area − Lumen Area)/Vessel Area. When present, the thickness of the muscular sleeve was also measured.
Comparisons between corresponding PV measurements obtained from ICE and IVUS frames and between pre-and post-ablation measurements were made. Post-ablation morphological changes of the PV wall such as dissection-like changes were also recorded.

Assessment of performance of ICE and IVUS
The time required for imaging and any procedural complications associated with imaging were recorded as indicators of trackability of ICE and IVUS catheters.
The imaging quality of each PV cross-section was defined as good, satisfactory, sub-optimal and poor: good quality if the vessel contour was visible in all 4 quadrants; satisfactory quality if the vessel contour was visible in 3 quadrants; sub-optimal quality if the vessel contour was visible in 2 non-consecutive quadrants; poor quality if not possible to define the vessel contour as visible only in one quadrant or in two consecutive quadrants (Fig. 2). Poor quality images were discarded and not considered for quantitative analyses.
The inter-observer agreement for quantitative data was also assessed for both ICE and IVUS by calculating intraclass correlation coefficient (ICC) values for different PV measurements.

Follow-up data
Although the study was not designed to assess and compare clinical outcomes, long-term data were obtained from standard clinical follow-up appointments. The need of redo catheter ablation due to recurrence of atrial tachyarrhythmias and which PVs were found to be reconnected at the redo procedure were reported.

Statistical analysis
Categorical variables were expressed as absolute number and percentage (%). Continuous variables were expressed as mean and (±) standard deviation (SD) or as median accompanied by interquartile range (IQR), as appropriate after checking for normality using the Shapiro-Wilk's test. ICE and IVUS imaging quality were compared with the use of the Fisher's exact test. Corresponding PV measurements obtained from ICE and IVUS frames were compared with the use of the Student's T-test or Mann Whitney U test, as appropriate according to data distribution. Pre-and postablation measurements were compared with the use of the paired t-test or the Wilcoxon test, as appropriate according to data distribution. The effect of the three ablation technologies on the variation of LA wall thickness was evaluated with an ANCOVA test.
A two-sided P value of less than 0.05 was considered to indicate statistical significance. Data were analysed with the use of SPSS software version 27 (IBM Statistics, Chicago, Illinois).

Study population
Twelve patients undergoing PVI catheter ablation for paroxysmal AF were enrolled. The patients' baseline characteristics are presented in Table A of the Supplementary  Section. Pulmonary vein isolation was performed with RF in 5 patients (20 PVs), with cryoballoon in 4 patients (16 PVs) and with laser balloon in 3 patients (12 PVs). All 48 PVs were isolated. A total of 28 PVs in 7 patients were imaged before and after PVI with ICE, while 20 veins in 5 patients were imaged with IVUS.

Performance of ICE/IVUS for PV imaging
No significant differences in terms of trackability were observed between ICE and IVUS catheters (additional procedural time required for imaging: ICE = 24.8 ± 3.4 min, IVUS = 25 ± 3.5 min, p = 0.95; no procedural complications).
Compared to IVUS, ICE also performed better in terms of inter-observer reproducibility of PV measurements, as showed in Table B

ICE/IVUS images analysis
Out of the 48 PV cross-sections, 3 were discarded because of poor quality and 45 (28 ICE and 17 IVUS frames) were considered adequate for quantitative analysis.
Compared to the IVUS cross-sections, the ICE ones showed larger lumen and vessel diameters and areas, larger wall thicknesses and significantly lower lumen and vessel sphericity indexes, indicative of more oval/elliptical shape of both lumen and vessel contours ( Table 2). Of note, median pre-and post-ablation wall thickness index percentage (WTI%) and muscular sleeve thicknesses did not differ between ICE and IVUS-imaged PVs.
As detailed in Table 3, statistically significant increases of mean wall thickness, wall thickness index (WTI) and WTI%, suggestive of acute wall thickening, were observed after ablation in both ICE and IVUS cross-sections. A significant reduction of the thickness of the muscular sleeve was also observed after ablation [ICE cross-sections: median (IQR) percentage reduction = 8.2 (0-45.9) %; IVUS cross-sections: median (IQR) percentage reduction = 31.68 (14.54-81.64) %], with complete disappearance of the muscular sleeve after ablation in 6 PVs out of 45.
When grouping by ablation technology used for PVI (RF, cryo or laser), the increase in WTI% was observed only after RF treatment (p = 0.003) and laser treatment (p = 0.003), whilst no significant changes in wall thickness were observed after cryo ablation (p = 0.69) (Figs. 3, 4; Table 4). Statistical analysis with ANCOVA testing confirmed the effect of ablation modality on LA wall thickness changes [F (2,41) = 4.468; p = 0.018). A single case of vessel dissection was documented after ablation (right inferior pulmonary vein after cryoballoon ablation) (Fig. 5).

Follow-up data
Patients were followed up for 26.5 ± 4.1 months. Six patients (50%) required a redo procedure due to recurrence of atrial tachyarrhythmias 3 months or later after the AF ablation. Of them, 67% (4 patients) had reconnected PVs, for a total of 7 reconnected PVs out of the 24 checked during the redo procedures.
Reconnected and isolated PVs at the second ablation procedure showed similar degrees of wall thickness increase

Discussion
This pilot study was conducted to compare ICE and IVUS for real-time LA wall imaging and for detection of acute tissue changes produced by different ablation energies during PVI catheter ablation. Although both ICE and IVUS probes image in the axial plan, providing cross-sectional images of the vessel (or chamber of interest) and surrounding tissues, their transducers use different ranges of ultrasound frequencies. The catheter designs are also different: the IVUS catheter has a smaller size (6F) and uses a mono-rail system, with the distal portion of the catheter advanced over a guidewire for better support and stability, while the ICE catheter has a bigger size (9F) and no central lumen. We chose to compare a 9 MHz ICE catheter with a 20 MHz IVUS probe to investigate which ultrasound frequency would give the best compromise between contrast resolution and image penetration for PVs imaging.

Performance of ICE and IVUS
In our study, ICE performed better than IVUS with regards to quality of imaging provided and to inter-observer reproducibility of measurements obtained. These findings are likely due to the lower ultrasound frequency used by ICE, which was advantageous in terms of acoustic penetration without a significant loss in spatial resolution. When using ICE, the outer vessel circumference was well-defined in most or all image quadrants, as well as inner structures such as lumen circumference and wall. We observed no significant differences between the two technologies in terms of trackability: similar additional procedural times were needed for ICE and for IVUS imaging and no procedural complications occurred as result of imaging.
Both lumen and vessel diameters and areas were consistently larger in the ICE-imaged PVs compared to the IVUS-imaged PVs. Moreover, although both imaging techniques showed an elliptical shape of the PV cross-sections, in keeping with previous CT and MRI studies [16,17], lumen and vessel sphericity indexes were lower in the ICE-imaged PVs, which is indicative of a more elliptical shape of the PV cross-sections. Our PV measurements determined by IVUS are in line with previously reported PV measurements, obtained from both IVUS images and histological sections [14]. Taken together, these data might suggest that ICE overestimated the PVs sizes due to non-coaxial cross-sectioning, as indicated by the lower sphericity index when compared to the IVUS images. ICE catheters are not advanced or pulled back over a wire and

Acute changes produced by the different ablation energies
In our study, the LA wall thickness was found to increase similarly at the level the PV ostia following ablation when using RF or laser balloon energy, while no increase in wall thickness was observed when using the cryoballoon. While acute development of tissue oedema is well known after RF [9,18,19], limited data are available regarding acute tissue changes after laser energy delivery [12,20,21]. Apart from the lack of direct contact of the energy source with the tissue (the optical fiber delivering arc of laser energy is in a balloon), laser energy as with RF produces tissue damage through heating and is delivered in a point-by-point fashion. Thus, it is not surprising that the two energy modalities might share similar mechanisms of tissue injury, including acute wall thickness increase  from oedema. In the study by Mangrum et al. [12], a significantly more pronounced wall thickening was observed after RF ablation than after laser ablation, however a lower RF power was used.
In another IVUS study [10], tissue oedema was reported in 90% of the PVs after cryoablation (and similar number of freezes per vein). Of note, in this study dissection-like changes were also observed, together with oedema, in most of the PVs, while in our study dissection-like changes were observed only in one vein after cryoballoon ablation and, interestingly, this occurred in the context of acute wall tissue thickening. It could be hypothesized that in these veins the oedema was due to the mechanical injury associated with dissection, rather than being a direct consequence of cryoenergy delivery. In the sequential process of tissue injury produced by cryoenergy [22,23], tissue oedema is thought to occur only at a late stage, once the tissue has thawed, following freezing, and has become hyperaemic, and to gradually progress over subsequent hours [24]. Concordantly, early PV imaging in our study showed no acute wall thickening suggestive of development of oedema.
The different morphological changes produced by the different ablation energies could suggest different mechanisms of lesion failure. Recent data suggest that the adjustment of the ablation settings based on baseline LA wall thickness can improve the procedure outcome and reduce the risk of collateral injury [11]. A further adjustment based on the acute wall thickening produced by energy delivery for ablation could also be beneficial when using RF or laser energy and could potentially highlight gaps between lesions.
Apart from acute wall thickening, we observed a reduction of the thickness of the PV muscular sleeve after PVI catheter ablation. Myocardial sleeves are known to extend from the left atrium into the PVs walls and to be a source of focal activity triggering AF [25]. The thickness reduction after ablation could indicate damage, translating to elimination of the PV potentials and acute electrical isolation of the vein and may also explain why it is often impossible to get local capture during pacing to demonstrate exit-block. Whether durable PV isolation correlates with a certain degree of wall thickness reduction or complete disappearance of the muscular sleeve after catheter ablation is unclear. We did not observe a correlation between degree of wall thickness increase or muscular sleeve thickness reduction after the first catheter ablation procedure and evidence of PV reconnection at the second catheter ablation procedure, however only a small number of PVs were checked with a second ablation procedure in our study.

Limitations
There are some limitations in our work that must be acknowledged.
First, results need to be interpreted as hypothesis-generating. Statistical significances need to be taken with caution due to the higher chance of type II hyperinflation error related to the small and heterogeneous sample size.
Another major limitation of the study is the lack of an alternative imaging modality like CT, or histopatology, to use as reference for the PVs sizes measured with ICE and IVUS. Moreover, no direct comparisons between ICE and IVUS were made by imaging the same PVs with both modalities. As result, it was not possible to ascertain which of the two imaging modalities gave more accurate measurements. However, this did not preclude confirming the feasibility of both ICE and IVUS for LA wall imaging, since comparable wall thickness measurements were obtained and similar acute changes in wall thickness were detected with both imaging modalities.
Pullback was manual rather than automatic. This precluded the precise comparison of distal cross-sections before and after ablation. The white arrow indicates acute tissue thickening suggestive of oedema, while the white asterisks indicate a dissection flap Imaging during energy delivery was not attempted as the same trans-septal access was used for either ablation catheter or imaging catheter. However, simultaneous imaging might not have been possible due to spatial interference, especially when using cryo or laser balloon catheters, and/or due to artifacts created by irrigation of the RF catheter.

Conclusions
In our pilot study, ICE and IVUS showed similar ability to detect acute tissue changes related to catheter ablation. Acute wall thickening was observed at the PV ostia after RF and laser energy delivery and not after cryoenergy delivery. Larger studies are needed to confirm these preliminary findings.