Funding was obtained by Medtronic Inc.

Methods/Procedure setup
We included 32 patients with drug-refractory paroxysmal AF and three patients with persistent AF. Informed consent was obtained from all patients before the procedure.
Ablation Procedure
All patients are anticoagulated at least 1 month prior to the procedure. Warfarin is stopped the day prior to the procedure aiming for an international normalized ratio between 1.8 and 2.0 on the day of the procedure. Antiarrhythmic drugs are continued periprocedurally and for three months post procedure. All patients undergo cardiac MRI or cardiac CT imaging before the procedure to assess the left atrial and pulmonary vein anatomy and allow for measurement of PV diameter.
The ablation procedure is performed under conscious sedation. Electrograms are recorded in a specialized EP recording system (EP Med, St Jude Medical, MN, US).
1. Procedure Setup
<ol>
 <li>The patient is draped in the standard way for ablation procedures. Vascular access is obtained in the right and left femoral veins. Systemic arterial pressure is continuously monitored via the left femoral artery through a pressure transducer. The intracardiac (ICE) ultrasound probe (Acuson, Siemens, GER) is advanced to the right atrium.</li>
 <li>Using standard Fluoroscopic views and ICE guidance, trans-septal puncture is obtained using a Preface (Biosense Webster, CA) or SL1 sheath (St Jude Medical, MN). A J tipped 0.035" wire is placed in the left superior PV and the trans-septal sheath is exchanged with the Flexsheath. Before trans-septal access, heparin bolus is infused intravenously and then repeated as needed based on ACT checks performed every 20 min to maintain an activated clotting time over 350 sec for the duration of the procedure. </li>
 <li>Balloon size is selected in accordance with the diameter of the PVs based on cardiac MRI or cardiac CT measurements pre procedure. Typically, a 28 mm cryoballoon is utilized if any of the PV ostia has a diameter larger than 20 mm.</li>
 <li>After appropriate preparation, the cryoballoon is advanced over a wire or the Achieve catheter (Biosense Webster, CA) in the left atrium and then, using ICE and fluoroscopic guidance, into the antrum of each PV.</li>
 <li>The balloon has an inner lumen that is typically used for radioiodine contrast injection through the distal tip of the balloon. A continuous pressure monitoring system is connected to the lumen to allow for pressure waveform analysis. Specifically, a 3-way Manifold (Navylist Medical, CA) (Figure 1) is attached via a Tuohy (Merit Medical, IRE) to the cryoballoon. Removal of air bubbles from the system is critical and care is taken to thoroughly flush the system prior to utilization. The three ports of the manifold are 1. saline flush (under pressure), 2. contrast and 3. pressure monitoring. The pressure monitoring port is then connected to a standard cardiac catheterization laboratory pressure transducer. The pressure waveforms are displayed on the EP recording system live monitor (Figure 2).</li>
 <li>The LA pressure recordings are set at a scale of 25 mmHg and sweep speed at 50 mm/sec. The page has minimal number of channels displayed (one or two surface ECG derivation, CS electrograms and arterial and LA pressure). Electrograms of the PV-LA junctions are recorded prior to ablation (either using the Achieve catheter or a separate multipolar catheter). Continuous recording is displayed on a separate page on the EP system when occlusion is assessed.</li>
</ol>
2. PV Occlusion
<ol>
 <li>The deflated balloon is advanced over the guide-wire or Achieve catheter using standard fluoroscopic views and intracardiac ultrasound and is inflated on the LA side of the PV antrum.</li>
 <li>The pressure page is displayed and the pressure line is opened to the transducer such that pressure is recorded from the tip of the balloon catheter (which dwells in the vein at this stage).</li>
 <li>When the balloon does not occlude the ostium of the PV, a characteristic LA pressure is recorded. During sinus rhythm, A (atrial) and V (ventricular) waves are recorded with the V wave having a typical "isosceles triangle" morphology. During atrial fibrillation however, there is no consistent A wave generated (since there is no atrial contraction) and only the V wave morphology is seen. Using fluoroscopy or ultrasound guidance the balloon is advanced and placed in apposition with the PV ostium.</li>
 <li>When occlusion is achieved, there is an abrupt change in the pressure waveform. During sinus rhythm there is a loss of the A wave and a change of amplitude (increase) and morphology of the V wave. Since now the recording is that of trans-capillary pulmonary arterial pressure, the V wave has the typical characteristic of a more rapid rate of rise and a delayed downstroke. In fact the apex of the V wave triangle moves to the right when compared to the LA pressure V wave recording. This is evident in sinus rhythm (Figure 3) and atrial fibrillation (Figure 4). This characteristic pressure waveform confirms complete occlusion and no further forward pressure or catheter manipulation needs to be used. Contrast use at this time may be utilized for further confirmation of PV occlusion during initial use of this methodology.</li>
 <li>Once occlusion has been achieved, the freezing process can be started. During ablation of right-sided PVs, phrenic nerve pacing is performed from a catheter located in the superior vena cava in order to promptly detect phrenic nerve injury. For the first approximately 15 sec (depending on local blood flow), pressure can still be recorded. Once temperature monitor reaches -10 degrees Celsius, the inner lumen of the balloon freezes and pressure can no longer be recorded. At this stage, we toggle the recording system page to the intracardiac electrogram page to demonstrate "time to effect" of PV isolation. Cryoapplication is continued for 240 sec.</li>
 <li>After the freezing process is complete and thawing has occurred, pressure can again be recorded and the maneuver can be repeated the number of times deemed necessary for permanent PV isolation.</li>
 <li>After cryoablation, every PV is mapped using a 20-pole circular mapping catheter with variable diameter (Biosense Webster, CA). If remnant ostial potentials are still recorded, differential pacing of the atrium near the vein is performed to prove entrance block in the PV. In case of persistent conduction, electrical isolation is completed segmentally using an 8-mm cryoablation catheter (Freezor MaxR, Medtronic, MN) or an irrigated radiofrequency catheter (ThermoCool, Biosense Webster, CA). The endpoint of the procedure is complete electrical PV isolation, documented at least 30 min after the last application.</li>
</ol>
Postablation Care
After the procedure, warfarin is resumed and subcutaneous low molecular weight heparin is administered if necessary until the international normalized ratio is &ge;2.0. Warfarin is continued for at least 3 months. Clinical follow up was performed at six weeks, three months, six months and twelve months and included ECG at each visit and event monitoring at three months. Long term antiarrhythmic and anticoagulation therapy was determined by clinical outcomes.

<ol>
	<li>Pappone, C., Rosanio, S., Oreto, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circumferential+radiofrequency+ablation+of+pulmonary+vein+ostia:+A+new+anatomic+approach+for+curing+atrial+fibrillation.">Circumferential radiofrequency ablation of pulmonary vein ostia: A new anatomic approach for curing atrial fibrillation.</a> Circulation. 102 (21), 2619-2628 (2000).</li><li>Piccini, J. P., Daubert, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoablation+of+atrial+fibrillation.">Cryoablation of atrial fibrillation.</a> J. Interv. Card. Electrophysiol. 32 (3), 233-2342 (2011).</li><li>K&#252;hne, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoballoon+versus+radiofrequency+catheter+ablation+of+paroxysmal+atrial+fibrillation:+biomarkers+of+myocardial+injury,+recurrence+rates,+and+pulmonary+vein+reconnection+patterns.">Cryoballoon versus radiofrequency catheter ablation of paroxysmal atrial fibrillation: biomarkers of myocardial injury, recurrence rates, and pulmonary vein reconnection patterns.</a> Heart Rhythm. 7 (12), 1770-1776 (2010).</li><li>Kojodjojo, P., O'Neill, M. D., Lim, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+venous+isolation+by+antral+ablation+with+a+large+cryoballoon+for+treatment+of+paroxysmal+and+persistent+atrial+fibrillation:+medium-term+outcomes+and+non-randomized+comparison+with+pulmonary+venous+isolation+by+radiofrequency+ablation.">Pulmonary venous isolation by antral ablation with a large cryoballoon for treatment of paroxysmal and persistent atrial fibrillation: medium-term outcomes and non-randomized comparison with pulmonary venous isolation by radiofrequency ablation.</a> Heart. 96 (17), 1379-1384 (2010).</li><li>Chao, T. F., Lin, Y. J., Chang, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+between+renal+function,+atrial+substrate+properties+and+outcome+of+catheter+ablation+in+patients+with+paroxysmal+atrial+fibrillation.">Associations between renal function, atrial substrate properties and outcome of catheter ablation in patients with paroxysmal atrial fibrillation.</a> Circ J. 75 (10), 2326-2332 (2011).</li><li>Van Belle, Y., Janse, P., Rivero-Ayerza, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+Vein+Isolation+Using+an+Occluding+Cryoballoon+for+Circumferential+Ablation:+Feasibility,+Complications,+and+Short-term+Outcome.">Pulmonary Vein Isolation Using an Occluding Cryoballoon for Circumferential Ablation: Feasibility, Complications, and Short-term Outcome.</a> Eur. Heart J. 28 (18), 2231-2237 (2007).</li><li>F&#252;rnkranz, A., K&#246;ster, I., Chun, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoballoon+temperature+predicts+acute+pulmonary+vein+isolation.">Cryoballoon temperature predicts acute pulmonary vein isolation.</a> Heart Rhythm. 8 (6), 821-825 (2011).</li><li>Siklody, C. H., Minners, J., Allgeier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoballoon+pulmonary+vein+isolation+guided+by+transesophageal+echocardiography:+Novel+aspects+on+an+emerging+ablation+technique.">Cryoballoon pulmonary vein isolation guided by transesophageal echocardiography: Novel aspects on an emerging ablation technique.</a> J. Cardiovasc. Electrophysiol. 20 (11), 1197-1202 (2009).</li><li>Sikl&#243;dy, C., Minners, J., Allgeier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-guided+Cryoballoon+Isolation+of+the+Pulmonary+Veins+for+the+Treatment+of+Paroxysmal+Atrial+Fibrillation.">Pressure-guided Cryoballoon Isolation of the Pulmonary Veins for the Treatment of Paroxysmal Atrial Fibrillation.</a> J. Cardiovasc. Electrophysiol. 21 (2), 120-125 (2010).</li><li>Chierchia, G. B., de Asmundis, C., Sorgente, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+extent+of+pulmonary+vein+isolation+after+cryoballoon+ablation+for+atrial+fibrillation:+comparison+between+the+23+and+28+mm+balloons.">Anatomical extent of pulmonary vein isolation after cryoballoon ablation for atrial fibrillation: comparison between the 23 and 28 mm balloons.</a> J. Cardiovasc. Med. 12 (3), 162-166 (2011).</li></ol>

Production and Free Access to this article is sponsored by Medtronic Inc.
Drs. Dan and Wickliffe are consultants for Medtronic Inc. Brian Jones is an employee of Medtronic Inc. Other authors have no conflict of interest to declare.

We present a novel technique of direct pulmonary venous pressure monitoring for assessment of complete pulmonary vein occlusion during cryoballoon ablation for atrial fibrillation. We have demonstrated a steep learning curve during application of the direct pressure monitoring technique and a rapid decrease in radioiodine contrast and fluoroscopy utilization after limited training. The technique can be performed with procedure tools commonly available in most laboratories and the setup requires limited additional preprocedure setup time.
Cryoablation utilizing a cryoballoon has emerged as a novel invasive management strategy for the treatment of paroxysmal atrial fibrillation 2. Procedural success with cryoballoon ablation is highly dependent on correct apposition of the cryoballoon at the pulmonary vein ostium and achievement of complete occlusion of the pulmonary vein 6. Several predictors of PV occlusion and electrical isolation have been studied: 1. the minimum temperature achieved during cryoapplication as well as the slope of rewarming are correlated with electrical isolation 7; however, no precise cut-off values have been assessed and this approach can only be utilized in a retrospective manner and does not allow for confirmation of PV occlusion prior to the initiation of the freezing process 2. Confirmation of complete occlusion is most commonly assessed by intravenous injection of radioiodine contrast after balloon inflation at the pulmonary vein ostium 6. Contrast leak upon balloon inflation is assessed fluoroscopically and confirms incomplete pulmonary vein occlusion with the cryoballoon. Multiple injections are frequently required upon cryoballoon repositioning resulting in repeated patient exposure to radioiodine contrast and radiation. As such, contrast may be contraindicated in patients with chronic kidney disease or known allergic reaction to iodine. Lack of continuous feedback during initial balloon positioning and inflation is an added disadvantage of fluoroscopic assessment for PV occlusion 3. Lastly, TEE-guided balloon positioning has been well described and allows for direct visualization of the antral position of the cryoballoon and continuous reassessment of balloon positioning and PV occlusion with saline infusion 8 as well as evaluation for "leaks" with color flow Doppler; however this technique requires continuous utilization of transesophageal echocardiography under general anesthesia, potentially increasing the risk for periprocedural complications associated with general anesthesia and espophageal intubation.
We observed a steep learning curve allowing for rapid and consistent interpretation of pressure waveforms after limited initial experience. Contrast utilization is directly negatively correlated with acquiring expertise in this technique and utilization of fluoroscopy is also dramatically reduced after initial training on pressure monitoring.
The pressure monitoring described surpasses the pitfalls mentioned above and allows for accurate and continuous assessment of PV occlusion. Currently, one balloon design is available for clinical use (Medtronic Inc., MN). This system includes an over-the-wire balloon catheter, which is cooled using nitrous oxide. Contrast is typically injected via an inner lumen; for the purpose of our described method of pressure monitoring, this inner lumen is utilized for direct assessment of PV pressure with the utilization of a single pressure transducer. As such, other than a single 3-way manifold, no extra catheters are necessary for application of this method. Further, if necessary, contrast may still be utilized if needed for further confirmation of PV occlusion, though we have demonstrated in a single center study that contrast utilization is largely unnecessary once the pressure monitoring technique has been established.
Pressure waveforms are obtained via the pressure transducer connected to the inner lumen and are displayed on the screen. The balloon is further advanced to the pulmonary vein ostium and inflated as done during standard cryoablation; no adjustments in the standard balloon manipulation approach are necessary when the technique of pressure monitoring is applied. Continuous monitoring for acute dislodgement during the initial freezing period is also possible with the pressure monitoring technique, until a measured scavenged gas temperature drop below -10 degrees Celsius. Particularly, balloon dislodgement from the PV ostium is usually observed upon initial refrigerant injection with resultant balloon expansion ("pop-out" phenomenon) and refinement of balloon positioning can be performed safely if the pressure waveform loses the occlusive pressure characteristics. A similar technique has been utilized in patients presenting in sinus rhythm for cryoballoon ablation 9. We have observed accurate determination of occlusive pressure in patients with ongoing atrial fibrillation during the procedure.
Currently, two sizes of the cryoballoon catheter are available, 28 mm and 23 mm diameter 10. Usage of the 23 mm balloon is limited, however the pressure monitoring technique is not affected by balloon size. The pressure monitoring technique is independent of rhythm and is applicable to patients presenting in atrial fibrillation. It is imperative that recording noise is eliminated and accurate zeroing is obtained to allow for accurate detection of the point of occlusive pressure. Displaying systemic arterial pressure as well as coronary sinus (CS) or surface ECG tracings are important to determine "live" the presence of A waves and change in V wave morphology. A common ostium is frequently observed, particularly in the left PVs. We observed occlusive pressure waveforms and complete electrical isolation in 16/18 PV with common ostium. Subselective wiring of the inferior and superior branches can be achieved in the presence of common ostium. Given these results, presence of a common PV ostium is not a limiting factor in the utilization of this technique.
Recurrent AF or atrial flutter was noted in fourteen patients (40%) after the initial 3 month blanking period and repeat PVI was performed in twelve patients (35%). Our results are comparable to published literature considering the inclusion of patients with persistent atrial fibrillation as well as six patients who had previously undergone PVI, implying a more extensive AF substrate. Interestingly, recurrences were more common in the first ten cases (6/10) despite higher utilization of contrast on initial testing. Of the last fifteen patients studied with minimal contrast utilization (mean contrast 10.9cc), four had recurrent AF post procedure indicating that the dramatic reduction in contrast utilization did not adversely influence clinical outcomes.
Limitations
Given the inability to record a pressure waveform once the intraluminal temperature decreases to less than -10 degrees, late balloon dislocations cannot be detected. This however is unlikely since at this point cryoadherence has occurred. In addition, perturbations are introduced in the pressure waveform during phrenic nerve pacing and pressure recording characteristics become less accurate immediately upon inflation in the right PVs. Atrial fibrillation is not a limiting factor for pressure monitoring application, however it may make the waveform interpretation more difficult for the inexperienced operator. Potential risks associated with air trapped in the 3-way system are limited but not insignificant. However, similar risk for air embolism is posed by utilization of the inner catheter lumen for contrast injection.
In this pilot study, the positive predictive value was noted to be 99% indicating a high likelihood of complete electrical isolation upon observation of occlusive pressure prior to cryoapplication. In contrast, the negative predictive value was low (40%) and was driven by the frequency of non occlusive pressure waveforms in the RIPV. In all patients, more than one cryoapplications were performed and as such we cannot exclude the possibility of minimal leaks during each cryoapplication with eventual complete electrical isolation due to refined maneuvering in the vein and apposition of the balloon in previously no ablated regions. This is particularly true for the RIPV in which, due to its relatively steep entrance into the antrum, slight changes in balloon orientation are required to achieve effective cryoablation in a segmental approach. As such, pressure monitoring may not be as helpful in the RIPV given this anatomical variation.
In our series, we observed lack of electrical vein isolation despite demonstration of occlusive pressure in two veins. There are several plausible explanations for this discrepancy, including the presence of epicardial muscle fibers between the antrum and the pulmonary vein or balloon-vein mismatch that results in ineffective cryoablation in part of the tissue. This phenomenon occurred during utilization of the first generation cryoballoon and is highly unlikely to occur with the imminent availability of the cryoballoon Advance, which will allow more equal distribution of cryoenergy at the PV ostium. Finally, the technique was primarily applied to patients undergoing cryoballoon ablation under conscious sedation, however assessment of the pressure waveform at end-expiration in the intubated patient should theoretically provide the same accurate results. We have observed consistent results in a small subset of patients undergoing general anesthesia, though this subgroup was not included in the analysis presented here.
Conclusions
The technique of pressure assisted PV isolation is simple, reproducible and safe. It significantly reduces the contrast burden and radiation exposure. Since the pressure can be recorded after the freezing process starts, the risk of loss of occlusion in the initial several seconds of balloon cooling (due to "pop-out" phenomenon) is minimized. Fine adjustments of balloon positioning can be safely made within the first 10 sec of cooling before the freezing occur.

Pulmonary vein isolation (PVI) is a widely utilized invasive approach to treatment of atrial fibrillation. Standard PVI techniques include radiofrequency (RF) catheter ablation and, more recently, cryoablation utilizing a cryoballoon (Arctic Front, MDT Inc, MN, USA) 1, 2. Both techniques result in equal long-term efficacy in durable PVI and similar clinical outcomes 3, 4. Given the more rapid vein isolation achieved with the utilization of the cryoballoon, this latter approach has gained widespread acceptance by operators and is currently performed in most centers specializing in atrial fibrillation ablation.
Significant differences between the two techniques include, among others, the requirement of complete pulmonary vein (PV) occlusion with the cryoballoon in order to achieve effective electrical isolation. Apposition of the cryoballoon in the PV ostium is typically assessed fluoroscopically with the utilization of intravenous contrast. As such, the total amount of fluoroscopy utilized during cryoballoon ablation is higher compared to standard catheter ablation, thus increasing the risk of radiation injury. Further, risks associated with contrast infusion, such as renal injury and contrast allergy, are a significant limitation of this technique, given the frequent coexistence of chronic kidney disease in patients with atrial fibrillation 5.
We present a novel method of direct PV pressure monitoring during cryoballoon inflation. This technique was validated in a total of 35 patients and reproducibility and accuracy was assessed.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Standard Angiographic Kit with NAMIC manifold, Three-Valve Classic (Clear) Manifold with Ports on Right and ON Handles</td>
 <td>Navylist Medical, USA</td>
 <td>91301303</td>
 <td>Individual components of the kit can be purchased separately</td>
 </tr>
 <tr>
 <td>Touhey Needle</td>
 <td>Merit Medical, IRL</td>
 <td>MAP 111</td>
 <td></td>
 </tr>
 <tr>
 <td>EP MED system</td>
 <td>St Jude Medical, MN, USA</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pressure Monitoring Kit with TruWave Disposable Pressure Trasnducer</td>
 <td>Edwards Lifesciences, CA, USA</td>
 <td>PX284</td>
 <td></td>
 </tr>
 </tbody>
</table>

direct pressure monitoring accurately predicts pulmonary vein occlusion during cryoballoon ablation

Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.

We assessed the accuracy of pressure monitoring in predicting complete PV occlusion in 35 patients (28 males) with paroxysmal (n=32) or persistent (n=3) AF. Six patients had a prior AF ablation (17%). A total of 128 PVs were assessed with pressure monitoring during balloon inflation. Occlusive pressure was demonstrated with balloon inflation in 113 PVs, of which 111 were electrically isolated with CBA (98.2%). Representative results are shown in Figure 5.
Lack of occlusive pressure waveform was more frequently noted in the RIPV (11/15 PVs). Two veins with demonstrated occlusive pressure waveform were still electrically connected despite concurrent demonstration of PV occlusion by contrast venography. The positive predictive value was 99%. In the RIPV, we observed a requirement for up to three cryoapplications per vein when occlusive pressure was observed in 90% of RIPVs, whereas 10% of veins with occlusive pressure required 4 cryoapplications. In contrast, when occlusive pressure was not observed in the RIPV, 4 or 5 cryoapplications were performed in 70% of RIPVs.
A common PV ostium was observed in nine patients (27.1%). Left common PV ostium was noted in eight patients and a right common PV in one patient. Occlusive pressure was achieved in 16/18 pulmonary veins with common ostium.
We observed a significant effect of the pressure monitoring technique in reducing total fluoroscopy time and radioiodine contrast utilization. Mean fluoroscopy time was 25.2&plusmn;11.2 min, mean contrast utilization 38&plusmn;33cc and mean LA time 109&plusmn;30.8 min during cryoballoon ablation. Contrast and fluoroscopy time decreased significantly after the initial ten procedures utilizing the technique (34.4&plusmn;10.1 vs. 21.6&plusmn;9.4 min of fluoroscopy, p=0.001 and 74&plusmn;28 vs 23.6&plusmn;23cc of contrast, p=0.00), indicating a very steep learning curve. No complications related to the utilization of the pressure monitoring technique were observed in this series of patients.
Post procedure arrhythmia free survival in one year was 60%. Recurrence of AF or atrial flutter was noted in 40% of patients leading to repeat PVI in 35% of patients. 3/12 patients with recurrent AF had previously documented persistent AF and 6/12 patients had undergone PVI prior to the index procedure.
<img src="/files/ftp_upload/50247/50247fig1.jpg" alt="Figure 1"/> 
 Figure 1. A 3-way manifold is connected via tubing to the inner lumen of the cryoballoon and a single pressure transducer for continuous pressure recording.
<img src="/files/ftp_upload/50247/50247fig2.jpg" alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50247/50247fig2highres.jpg"/> 
 Figure 2. Standard monitoring screen during live recording. Surface ECG, intracardiac CS recording and venous and arterial pressure recording are displayed. <a href="https://www.jove.com/files/ftp_upload/50247/50247fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/50247/50247fig3.jpg" alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50247/50247fig3highres.jpg"/> 
 Figure 3. PV pressure waveform recording during sinus rhythm. Upon initial apposition of the deflated balloon (left panel), characteristic LA waveform is recorded with A and V waves noted. With initial inflation (middle panel), a change in the waveform is noted with more prominent V waves and persistent lower amplitude A waves, suggesting incomplete venous occlusion. Further inflation and with successful circumferential apposition results in disappearance of the A wave and a larger V wave displaced towards mid diastole, confirming complete PV occlusion (right panel). <a href="https://www.jove.com/files/ftp_upload/50247/50247fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/50247/50247fig4.jpg" alt="Figure 4" fo:content-width="4.8in" fo:src="/files/ftp_upload/50247/50247fig4highres.jpg"/> 
 Figure 4. During atrial fibrillation, continuous atrial activity (A waves) with small amplitude V waves is observed when the PV is not occluded. With further manipulation of the cryoballoon in the PV ostium, abrupt increase in the V wave amplitude with loss of the small continuous atrial A waves is noted in the pressure waveform, confirming PV occlusion during atrial fibrillation. <a href="https://www.jove.com/files/ftp_upload/50247/50247fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<table border="1">
<tbody>
 <tr>
 <td>Total PVs, n</td>
 <td>128</td>
 </tr>
 <tr>
 <td>PVs with occlusive pressure, n</td>
 <td>113/128</td>
 </tr>
 <tr>
 <td>PVs isolated with CB when occlusive pressure is demonstrated (n) and PPV (%)</td>
 <td>111/113, 99</td>
 </tr>
 <tr>
 <td>Occlusive pressure in PVs with common ostia , n</td>
 <td>16/18</td>
 </tr>
 <tr>
 <td>Fluroscopy utilization change (25 procedures vs initial ten procedures), min</td>
 <td>21.6&plusmn;9.4 vs 34.4&plusmn;10.1 </td>
 </tr>
 <tr>
 <td>Contrast utilization change (25 procedures vs initial ten procedures), cc</td>
 <td>23.6.6&plusmn;23 vs 74&plusmn;28</td>
 </tr>
 <tr>
 <td>12-month arrhythmia free survival after ablation, %</td>
 <td>60</td>
 </tr>
 </tbody>
</table>
 Figure 5. Representative results.
Abbreviations
PV=pulmonary vein, CBA=cryoballoon ablation, AF=atrial fibrillation, ICE=intracardiac echocardiography, TEE=transesophageal echocardiography, LA=left atrium

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Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

Cryoballoon  ablation (CBA) is an established therapy for atrial fibrillation (AF).  Pulmonary vein (PV) occlusion is essential for achieving antral ...

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19.9K Views.  Piedmont Heart Institute. Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

Video: Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Reduction of Iatrogenic Atrial Septal Defects with an Anterior and Inferior Transseptal Puncture Site when Operating the Cryoballoon Ablation Catheter

The cryoballoon catheter ablates atrial fibrillation (AF) triggers in the left atrium (LA) and pulmonary veins (PVs) via transseptal access. The typical transseptal puncture site is the fossa ovalis (FO) &#8211; the atrial septum&#8217;s thinnest section. A potentially beneficial transseptal site, for the cryoballoon, is near the inferior limbus (IL). This study examines an alternative transseptal site near the IL, which may decrease the frequency of acute iatrogenic atrial septal defect (IASD). Also, the study evaluates the acute pulmonary vein isolation (PVI) success rate utilizing the IL location. 200 patients were evaluated by retrospective chart review for acute PVI success rate with an IL transseptal site. An additional 128 IL transseptal patients were compared to 45 FO transseptal patients by performing Doppler intracardiac echocardiography (ICE) post-ablation to assess transseptal flow after removal of the transseptal sheath. After sheath removal and by Doppler ICE imaging, 42 of 128 (33%) IL transseptal patients demonstrated acute transseptal flow, while 45 of 45 (100%) FO transseptal puncture patients had acute transseptal flow. The difference in acute transseptal flow detection between FO and IL sites was statistically significant (P &#60;0.0001). Furthermore, 186 of 200 patients (with an IL transseptal puncture) did not need additional ablation(s) and had achieved an acute PVI by a &#8220;cryoballoon only&#8221; technique. An IL transseptal puncture site for cryoballoon AF ablations is an effective location to mediate PVI at all four PVs. Additionally, an IL transseptal location can lower the incidence of acute transseptal flow by Doppler ICE when compared to the FO. Potentially, the IL transseptal site may reduce later IASD complications post-cryoballoon procedures.

The goal of this study is to demonstrate the preferential location of transseptal puncture during a cryoballoon catheter ablation procedure for the treatment of atrial fibrillation.

The cryoballoon catheter ablates atrial fibrillation (AF) triggers in the left atrium (LA) and pulmonary veins (PVs) via transseptal access. The typical ...

Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise

Exercise is a stress to the pulmonary vasculature. With incremental exercise, the pulmonary diffusing capacity (DLCO) must increase to meet the increased oxygen demand; otherwise, a diffusion limitation may occur. The increase in DLCO with exercise is due to increased capillary blood volume (Vc) and membrane diffusing capacity (Dm). Vc and Dm increase secondary to the recruitment and distension of pulmonary capillaries, increasing the surface area for gas exchange and decreasing pulmonary vascular resistance, thereby attenuating the increase in pulmonary arterial pressure. At the same time, the recruitment of intrapulmonary arteriovenous anastomoses (IPAVA) during exercise may contribute to gas exchange impairment and/or prevent large increases in pulmonary artery pressure.
We describe two techniques to evaluate pulmonary diffusion and circulation at rest and during exercise. The first technique uses multiple-fraction of inspired oxygen (FIO2) DLCO breath holds to determine Vc and Dm at rest and during exercise. Additionally, echocardiography with intravenous agitated saline contrast is used to assess IPAVAs recruitment.
Representative data showed that the DLCO, Vc, and Dm increased with exercise intensity. Echocardiographic data showed no IPAVA recruitment at rest, while contrast bubbles were seen in the left ventricle with exercise, suggesting exercise-induced IPAVA recruitment.
The evaluation of pulmonary capillary blood volume, membrane diffusing capacity, and IPAVA recruitment using echocardiographic methods is useful to characterize the ability of the lung vasculature to adapt to the stress of exercise in health as well as in diseased groups, such as those with pulmonary arterial hypertension and chronic obstructive pulmonary disease.

To assess the pulmonary diffusion and vasculature responses to exercise, we describe the multiple-inspired oxygen diffusion capacity technique to determine capillary blood volume and membrane diffusing capacity, as well as agitated saline contrast echocardiography to assess the recruitment of intrapulmonary arteriovenous anastomoses.

Exercise is a stress to the pulmonary vasculature. With incremental exercise, the pulmonary diffusing capacity (DLCO) must increase to meet the increased ...

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to pulmonary hypertension. For a better understanding and treatment of this disease, precise hemodynamic monitoring of left and right ventricular parameters is important. For this reason, it is essential to establish experimental pig models of cardiac hemodynamics and measurements for research purpose. 
This article shows the induction of ARDS by using oleic acid (OA) and consequent right ventricular dysfunction, as well as the instrumentation of the pigs and the data acquisition process that is needed to assess hemodynamic parameters. To achieve right ventricular dysfunction, we used oleic acid (OA) to cause ARDS and accompanied this with pulmonary artery hypertension (PAH). With this model of PAH and consecutive right ventricular dysfunction, many hemodynamic parameters can be measured, and right ventricular volume load can be detected. 
All vital parameters, including respiratory rate (RR), heart rate (HR) and body temperature were recorded throughout the whole experiment. Hemodynamic parameters including femoral artery pressure (FAP), aortic pressure (AP), right ventricular pressure (peak systolic, end systolic and end diastolic right ventricular pressure), central venous pressure (CVP), pulmonary artery pressure (PAP) and left arterial pressure (LAP) were measured as well as perfusion parameters including ascending aortic flow (AAF) and pulmonary artery flow (PAF). Hemodynamic measurements were performed using transcardiopulmonary thermodilution to provide cardiac output (CO). Furthermore, the PiCCO2 system (Pulse Contour Cardiac Output System 2) was used to receive parameters such as stroke volume variance (SVV), pulse pressure variance (PPV), as well as extravascular lung water (EVLW) and global end-diastolic volume (GEDV). Our monitoring procedure is suitable for detecting right ventricular dysfunction and monitoring hemodynamic findings before and after volume administration.

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to ...

A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are therefore warranted in order to investigate disease mechanisms and effects of potential therapeutic strategies. Banding of the pulmonary trunk is a common method to induce isolated RV hypertrophy but in general, previously described models have not succeeded in creating a stable model of RV hypertrophy and failure.
We present a rat model of pressure overload induced RV hypertrophy caused by pulmonary trunk banding (PTB) that enables different phenotypes of RV hypertrophy with and without RV failure. We use a modified ligating clip applier to compress a titanium clip around the pulmonary trunk to a pre-set inner diameter. We use different clip diameters to induce different stages of disease progression from mild RV hypertrophy to decompensated RV failure.
RV hypertrophy develops consistently in rats subjected to the PTB procedure and depending on the diameter of the applied banding clip, we can accurately reproduce different disease severities ranging from compensated hypertrophy to severe decompensated RV failure with extra-cardiac manifestations.
The presented PTB model is a valid and robust model of pressure overload induced RV hypertrophy and failure that has several advantages to other banding models including high reproducibility and the possibility of inducing severe and decompensated RV failure.

We present a surgical method to induce right ventricular hypertrophy and failure in rats.

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary ...

Cooling or Warming the Esophagus to Reduce Esophageal Injury During Left Atrial Ablation in the Treatment of Atrial Fibrillation

Ablation of the left atrium using either radiofrequency (RF) or cryothermal energy is an effective treatment for atrial fibrillation (AF) and is the most frequent type of cardiac ablation procedure performed. Although generally safe, collateral injury to surrounding structures, particularly the esophagus, remains a concern. Cooling or warming the esophagus to counteract the heat from RF ablation, or the cold from cryoablation, is a method that is used to reduce thermal esophageal injury, and there are increasing data to support this approach. This protocol describes the use of a commercially available esophageal temperature management device to cool or warm the esophagus to reduce esophageal injury during left atrial ablation. The temperature management device is powered by standard water-blanket heat exchangers, and is shaped like a standard orogastric tube placed for gastric suctioning and decompression. Water circulates through the device in a closed-loop circuit, transferring heat across the silicone walls of the device, through the esophageal wall. Placement of the device is analogous to the placement of a typical orogastric tube, and temperature is adjusted via the external heat-exchanger console.

The goal of this protocol is to describe the use of esophageal temperature modulation to counteract esophageal thermal injury from left atrial ablation for the treatment of atrial fibrillation.

Ablation of the left atrium using either radiofrequency (RF) or cryothermal energy is an effective treatment for atrial fibrillation (AF) and is the most ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

Real-Time Electrocardiogram Monitoring During Treadmill Training in Mice

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. However, in certain cardiac diseases such as inherited arrhythmia syndromes, e.g., arrhythmogenic cardiomyopathy (ACM) or myocarditis, physical exercise may have negative effects on the heart leading to a proarrhythmogenic substrate production. Currently, the underlying molecular mechanisms of exercise-related proarrhythmogenic remodeling are largely unknown, thus it remains unclear which frequency, duration, and intensity of exercise can be considered safe in the context of disease(s).
The proposed method allows to study proarrhythmic/antiarrhythmic effects of physical exercise by combining treadmill training with real-time monitoring of the ECG. Implantable telemetry devices are used to continuously record the ECG of freely moving mice over a period of up to 3 months both at rest and during treadmill training. Data acquisition software with its analysis modules is used to analyze basic ECG parameters such as heart rate, P wave duration, PR interval, QRS interval, or QT duration at rest, during and after training. Furthermore, heart rate variability (HRV) parameters and occurrence of arrhythmias are evaluated. In brief, this manuscript describes a step-by-step approach to experimentally explore exercise induced effects on cardiac electrophysiology, including potential proarrhythmogenic remodeling in mouse models.

Electrocardiogram (ECG) is the key variable to understanding cardiac electrophysiology. Physical exercise has beneficial effects but may also be harmful in the context of cardiovascular diseases. This manuscript provides a method of recording real-time ECG during exercise, which can serve to investigate its effects on cardiac electrophysiology in mice.

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.