In-utero radiofrequency ablation in fetal piglets: lessons learned

Abstract

Introduction

Radiofrequency ablation (RFA) is increasingly utilized in minimally invasive fetal intervention. However, the response of different fetal tissues to RFA is poorly characterized. We sought to determine the extent of RFA damage in a fetal environment.

Methods

90 day gestation Yorkshire piglets (term 115 days) were subjected to RFA of the chest and abdominal viscera under various temperatures and wattages. The extent of tissue damage was determined by NADPH diaphorase histochemistry.

Results

Tyne temperature was widely variable and displayed varying responses between lung and liver tissue. Tyne exposure to amniotic fluid resulted in an increase in amniotic fluid temperature. Collateral damage, even across the diaphragm, was readily seen, and ultrasonography did not always reflect this injury.

Conclusions

Utilization of extracorporeal tynes heats fluid at a greater rate than solid tissue and reliance on temperature sensitive probes may result in overheating. The extent of injury may extend beyond damage observed by ultrasound examination and varies for different tissues. Additional studies on the use of devices that regulate tyne temperature are needed to define optimal conditions and better define the extent of adjacent tissue injury.

1. Introduction

Fetal surgical intervention is reserved for life-threatening conditions or those associated with severe morbidity. To decrease the morbidity associated with these procedures, there is considerable interest in investigating minimal access techniques for fetal therapy. Fetoscopic urethral valve ablation [1], endoscopic tracheal occlusion for treatment of congenital diaphragmatic hernia [2], endoscopic release of amniotic bands [3], and laser ablation of communicating vessels in twin-twin transfusion [4, 5] are some examples of minimal access fetal interventions. Radiofrequency ablation (RFA) involves the destruction of biological tissues by transferring electricity from a rapidly alternating current at radiofrequencies. Therapeutic frequencies must be high enough to cause molecular friction heating without stimulating the neuromuscular junction and electrolysis, yet low enough to confine energy transmission to a controllable tissue mass. The first electrocautery devise was developed by Bovie and Cushing in the early 1920's [6]. The therapy was modified to target solid organ lesions and is now generally accepted as a first-line treatment for hepatocellular tumors [7, 8]. With the advanced imaging modalities now available for specific and real-time imaging of a lesion, RFA is utilized to treat multiple lesions in organs throughout the body in adults [7, 8].

In the arena of fetal therapy, RFA has been used successfully to obliterate flow in umbilical vessels in cases of twin reversed arterial perfusion (TRAP) sequence with a survival rate as high as 85% or discordant twin pregnancies undergoing selective reduction; both involving fetal demise as a result of the intervention [9-12]. RFA has also been proposed for antenatal intervention for the ablation of fetal tumors such as sacrococcygeal teratoma (SCT) and congenital cystic adenomatoid malformation (CCAM). Following preliminary experiments using fetal animal models, several reports of human fetal intervention were published, some with dire consequences [13]. Of the few cases of intrauterine RFA therapy for SCT, one child was left with an obliterated hip joint and a flaccid limb [14]. In the report by Paek, et al. two of four fetuses with SCT who underwent in-utero ablation of the tumors died and the other two suffered severe perineal injuries [15]. These clinical outcomes highlight the need for the establishment of a clear set of guidelines for the use of RFA in fetal therapy where the fetus is expected to survive.

A review of the published fetal RFA experience demonstrates that instrument settings were often arbitrarily chosen or extrapolated from experience with adult tissues, while the duration of therapy was, in the best of cases, based on temperature change or ultrasound findings. In the case of cord occlusion, therapy was continued until absence of Doppler flow was demonstrated. Otherwise, extent of administration was entirely discretionary and by definition inconsistent. We embarked upon this study in an attempt to better define the use of RFA in fetal tissues, selecting one of the newer RFA probes to test. We aimed to determine the extent of tissue injury following RFA in the fetus, identify any collateral damage that may result from RFA use, determine any difference in the response of different fetal organs to RFA, and determine any evidence of heat dissipation that may affect a co-twin.

2. Materials and Methods

2.1 Radiofrequency ablation procedure

Following approval by the Institutional Animal Care and Use Committee at Baylor College of Medicine, one time-dated pregnant Yorkshire sow at 90 days gestation (term = 115 days) was sedated with Ketamine 15 mg/kg and Xylazine 1mg/kg and anesthetized with isoflurane 0.5-4% in oxygen. A midline abdominal incision was performed to gain access to the uterine horns containing fetuses. The pig fetuses at this gestation are about 550 – 650 grams and correlate, by size, to a 23- 24 week gestation human fetus. Following laparotomy, the uterine horns were sequentially exposed and interrogated with an Acuson Sequoia^® ultrasound (Siemens Inc., New York, NY) with a 3.5 or 6 MHz probe to assess the orientation and number of fetuses in-utero. A Starburst XL 17 gauge probe (RITA Medical Systems, Fremont, CA) with 4 expandable tynes was connected to a RITA continuous 460 kHz generator (RITA Medical Systems, Fremont, CA) and was inserted into the intact uterus and guided to the fetal organs under sonographic visualization for each fetus. The generator was capable of monitoring the temperature at each tyne of the probe and dispersed energy varied from 18 to 105 watts. Tyne deployment was to a diameter of 2 cm. Target temperature was set to an assigned temperature of either 75°C or 90°C and ablation was stopped when this target temperature was reached for 2 to 3 minutes, regardless of the degree of damage to surrounding tissue as visualized on the ultrasound image. A thermometer was inserted into the uterus to measure the temperature of the amniotic fluid subsequent to RFA. A total of thirteen fetal piglets from the sow were evaluated. Baseline amniotic fluid temperature was measured in untreated litter-mates while each sequential fetus was being treated. Upon completion of therapy, the fetuses were immediately removed from the uterus and euthanized. They then underwent necropsy. The RFA lesion in each piglet was located and photographed, and serial tissue samples were excised at 1, 2, 3, and 4 cm from the center of the lesion.

2.2 Tissue preparation and staining

The extent of gross tissue injury from the probe insertion site was measured and photographed. Tissue samples were either flash frozen in liquid nitrogen or fixed using Histochoice^® synthetic fixative (Amresco Inc., Parkway Solon, OH). Frozen specimens were cut into 20μm sections and processed for NADPH diaphorase enzyme histochemistry, a reliable method for assessing tissue viability post-RFA [16, 17]. The procedure was performed as described previously [18]. Fixed samples were dehydrated in alcohol, paraffin embedded, sectioned (5μm), and stained with hematoxylin and eosin. All sections were observed under a light microscope.

3. Results

A total of thirteen fetal pigs at 90 days of gestation were detected by ultrasonography. Eleven were treated with RFA and two served as controls to monitor amniotic fluid temperature (Table 1). On occasion, upon targeting of the umbilical cord, one of the tynes would be located within the amniotic fluid. Continued monitoring of the tyne temperature allowed for prompt recognition of this issue, as noted in Fig. 1. In an attempt to compensate for this heat sink when located within the amniotic fluid and to attain the set goal temperature, some of the tynes reached a temperature greater than the set limit. Some newer generation instruments allow individual discordant tynes to be shut down and eliminated from treatment.

Table 1.

Tyne temperature and duration of deployment to target tissue for each piglet are detailed below. The resulting areas of injury are also noted.

Piglet Identification	Tyne Temperature (Celsius)	Assigned Duration of Tyne Deployment (minutes)	Target	Injured Areas
1	75	2	lung	lung
2	75	2	lung	lung
3	75	3	lung	lung
4	90	2	lung	axilla
5	90	3	lung	lung
6	75	2^	liver	liver
7	75	2^	liver	lung, diaphragm, liver, stomach
8	90	2^	liver	lung, diaphragm, liver
9	75	3	umbilical cord	cord occlusion
10	90	3	umbilical cord	cord occlusion
11	90	3	umbilical cord	cord, lung, diaphragm, liver
12 (control)	-	-	-	-
13 (control)	-	-	-	-

^{^}Tyne deployment aborted <1 minute due to temperatures exceeding set limit

Figure 1.

Generator display of temperature at each tyne was possible and varied significantly. Tyne position 2 registers 59° C while tyne position 4 registers 100° C.

3.1 Amniotic Fluid Temperature

Baseline amniotic fluid temperature in fetal pigs was between 38°C and 39°C prior to manipulation. RFA treatment did not significantly increase the amniotic fluid temperature (38.1-39.2°C) unless the tynes were not completely intracorporeal. With a tyne in the amniotic fluid, the fluid temperature increased to as high as 43°C. The impact this may have on a co-twin is unclear.

3.2 Extent of Tissue Injury

An assigned temperature of either 75°C or 90°C and duration of 2 or 3 minutes was preset per piglet. The effects of tissue ablation were observed via ultrasound, and in some cases “bubbling” of the tissues noted at the site of treatment. In many instances, no ultrasound changes were noted despite the fact that the preset temperature had been attained. Gross examination of the tissues revealed injury beyond the 2 cm diameter of the probe. With lung ablation, collateral damage to the heart, stomach, liver, and chest wall were noted (Fig. 2A). These appeared to be effects of heat rather than direct necrosis caused at the probe site, as suggested by the fact that these injuries were all intracorporeal in nature. Damage seen at the 75°C setting was as great as that observed at 90°C.

Figure 2.

A) Ablation of lung tissue for 3 minutes produced significant damage in adjacent organs (arrows). Lesion diameter is approximately 1.7 cm. B) 1.7 cm lung lesion (arrow) after RFA ablation at target temperature of 90° C. C) 2.1 cm lung lesion (arrow) after RFA ablation at target temperature of 75° C. D) Probe insertion site was minimal in size and redness. E) Irreversible liver damage was evident after only 5 seconds of liver-targeted ablation. F) Abdominal wall necrosis subsequent to RFA at insertion point of umbilical cord.

Targeted lung ablations were performed in only 5 fetuses, and the time required to reach target temperature from room temperature of the probe (28°C) was typically 1 minute. It became evident that variations in temperature occurred between the various tynes even after target temperature was reached Lesions measured at a maximum of 8 cm in diameter on gross examination and varied up to 4 cm when histologically analyzed, regardless of whether the probe temperature was set to 75°C or 90°C (Fig. 2B, 2C). Target temperature was maintained for a preset 3 minutes and reproducibly achieved irreversible damage, as indicated by H+E staining (Fig. 3) and NADPH diaphorase histochemistry (Fig. 4). After ablation, the chest wall insertion site was inspected and found to be quite small. Minimal erythema was noted surrounding the insertion site (Fig. 2D). During ablation within the chest cavity, multiple instances of damage to surrounding organs were observed.

Figure 3.

Tissue samples were excised, prepared, and stained with hematoxylin and eosin. Lung samples are shown here at 10× (left) and 25× (right) magnification. Healthy tissues are seen above (A and B) while damaged tissue is displayed below (C and D). While a healthy airway and alveolar tissue are depicted in A and B, inflammation and edema are noted with arrows with infiltration into the alveolar space in C and D.

Figure 4.

Tissue samples were excised and analyzed with NADPH diaphorase enzyme histochemistry. Liver tissue samples are shown at 10×. Slices of tissue are at (A) 1 cm, (B) 2 cm, (C) 3 cm, and (D) 4 cm from the probe. The resultant brown color of the staining indicates injured tissue in which there is no formazan product, seen in the samples taken near the probe, whereas the black hue from formazan product is visualized in the samples of healthy tissue obtained further from the probes.

In 3 fetuses, the RFA probe was inserted into the fetal liver. Notably, in all of these cases, the temperature of the tynes quickly shot up to the target temperature or above, causing significant liver damage within a short period of time (Fig. 2E). In all the liver treatments we had to abruptly terminate the treatment (less than 1 minute) because the temperature rose higher than the set limit and destruction of the liver tissue was noted on ultrasound. This suggests differences in tissue response to RFA between the liver and lung. Necrotic fetal liver became adherent to the tyne and appeared to render ineffective the feedback temperature regulation from the generator.

3.3 Cord Occlusion

Three minutes of ablation at the set temperature had varying effects on the cord occlusion. Successful cord occlusion was confirmed by Doppler blood flow. Location of a tyne within the amniotic fluid delayed attainment of target temperature and increased amniotic fluid temperatures. The cord insertion to the abdomen proved to be the most effective location for placement of the probe. This site is, however, associated with abdominal wall necrosis (Fig. 2F) though it is not a clinical concern in the treatment of a non-viable fetus.

4. Discussion

Radiofrequency ablation offers the promise of a minimally invasive, image-guided fetal therapy. Single access with an instrument of approximately 14-18 gauge size reduces the risk of membrane-related complications. Radiofrequency ablation is a proven effective, minimally invasive technique used to ablate solid organ tumors in adults. It took a worldwide effort by many investigators to achieve consensus on the safest and most efficacious ablation techniques [19]. A similar effort is required in the fetal surgery/intervention community as guidelines for RFA use in an adult organ cannot readily be applied for use in fetal tissues in-utero, a fact that is demonstrated by the results of the experiment. Differences in tissue characteristics and the inutero fluid environment must be taken into account.

Using a porcine fetal model, we have studied the effects of RFA in the fetal environment. We have attempted to determine optimal settings using one of the RFA generators commercially available (RITA Medical Systems, Fremont, CA). As opposed to other generators, the RITA generator is aimed at achieving target probe temperature [8]. Our study confirms that RFA is indeed capable of ablating fetal tissue in the in-utero fluid environment. However, these injuries were created within a short period of time, especially in comparison to adult tissue [20, 21]. No difference in the degree of damage obtained at 75°C and 90°C was observed, suggesting that 75°C is sufficient to cause fetal tissue necrosis. Further studies will be needed to determine if still lower temperatures can achieve this result. Although varying amounts of energy were required to attain the target temperature, the elapsed time remained constant at 3 minutes. Setting the generator at a defined wattage may therefore produce a variety of temperatures in diverse tissues, resulting in poor control of the therapy.

In addition, extensive injury beyond the diameter of the tynes became apparent. Our NADPH diaphorase staining confirmed a zone of cell necrosis up to 4 cm in diameter though the diameter of the probe tynes was set to 2 cm. A review of the adult literature revealed that internally-cooled electrodes are often employed to target pulmonary lesions close to the pleura or pulmonary hilum [20]. This is a technique that may also be useful in fetal pulmonary lesions to minimize injury to adjacent structures, especially given the limited size of the chest cavity, and should be explored further. It was further found that ultrasonography was a poor assessor of impact and extent of lung injury. In the fetal liver, however, injury occurred more quickly to the degree where the tynes were found adherent to necrotic tissue, readily observed by ultrasound. Tynes with continuous temperature measurements may more accurately determine the impact of injury. In the case of cord occlusion, Doppler assessment remains the most reliable means of assessing successful occlusion.

Increase in amniotic fluid temperature was observed when one of the tynes was extra-corporeal. Despite the one report of sciatic nerve injury and structural loss of the pelvis and femur during an attempt at fetal tumor ablation, damage secondary to increase in amniotic fluid temperature has not been reported in the clinical experience, and has not been assessed in the literature [15]. The significant change in amniotic fluid temperature caused by an exposed tyne may be amplified in our fetal pig model, in which the amniotic fluid volume is less than that in humans. Nevertheless, practitioners should be cognizant of discordant temperature readings on the tynes as this may result in heating of the amniotic fluid. The effect this may have on multiple gestations, if any, is unknown. Some newer generation instruments allow individual discordant tynes to be shut down and eliminated from treatment. It would be of considerable benefit to investigate a refined model of the tynes that ideally become inactive when not attached to tissue. Potential elimination of continuous heat release from nonfunctional tynes may decrease harm to surrounding structures and multiple gestations. A better understanding of the unique characteristics of various fetal tissue, the higher fluid content of fetal tissues and the energy needed to create injury in various organs may aid in the development of devices that can more efficiently distribute energy to a limited areas with feedback sensors from each tyne that can regulate the energy dissipated to each region. Even such technology may still have limited used in heterogeneous tissues as found in sacrococcygeal teratomas.

This study is limited by being an acute, non-survival preparation; the effect of RFA on fetal survival was not assessed. In addition, actual extent of injury may be beyond the 4 cm margin of necrosis observed in the acute preparation. Further studies are needed to elucidate this and would require tissue sampling of areas up to 6 cm. Tissue would be evaluated in a similar fashion by analyzing tissue by both gross and microscopic assessment.

5. Conclusion

In summary, the extent of tissue injury caused by RFA in the fetus is unpredictable. It is effective for cord occlusion, but this study demonstrated that it is accompanied by the risk of increasing amniotic fluid temperature. The extent of tissue injury varies between fetal tissues, cannot be accurately assessed by ultrasonography and collateral damage is common. As a result, until better measures of the extent of injury are developed, clinical use of RFA in fetal medicine should be limited to cord occlusion (such as in cases of TRAP).