Effects of tract embolization on pneumothorax rate after percutaneous pulmonary microwave ablation: a rabbit study

Abstract Purpose To determine the effects of tract embolization with gelatin sponge particles on the prevention of pneumothorax after percutaneous microwave ablation (MWA) in rabbit lungs. Materials and methods Twenty-four New Zealand white rabbits were randomly divided into Group A (MWA followed by tract embolization with gelatin sponge particles, n = 12) and Group B (MWA without tract embolization, n = 12). For each group, CT images were reviewed for the occurrence of pneumothorax within 30 min after MWA. The rate of pneumothorax was compared by Chi-square Test. Lung tissue around the needle tract was harvested after the rabbits were euthanized, and histopathological examinations were performed and studied with hematoxylin and eosin stains. Results Twenty-four animals underwent 47 sessions of MWA (24 sessions in Group A and 23 sessions in Group B). Group A had a statistically lower rate of pneumothorax than Group B (25.0 vs. 56.5%; p = 0.028). The pathological examinations of both groups demonstrated thermal injury of the needle tract characterized by a rim of the coagulated lung parenchyma, which might be responsible for pneumothorax after MWA. Gelatin sponge particles could be arranged in irregular flakes densely to effectively seal the needle tract, thus reducing the occurrence of pneumothorax. The gelatin sponge particles could be almost completely absorbed about 14 days later. Conclusion Results of the present study showed needle tract embolization with gelatin sponge particles after CT-guided pulmonary MWA can significantly reduce the incidence of pneumothorax. Gelatin sponge particles can effectively seal the needle tract after ablation and can be completely absorbed in the body with good safety.


Introduction
Over the past decade, percutaneous image-guided microwave ablation (MWA) has emerged and has been recommended as a safe, effective, and repeatable local therapy for malignant lung tumors [1][2][3][4][5].MWA is used to eradicate focal tumors by applying thermal energy to directly cause irreversible coagulative necrosis.It is more minimally invasive than surgery.Therefore, for patients who cannot tolerate surgery due to comorbidities or other reasons, less invasive treatment methods, such as MWA are increasingly popular among clinicians and patients.
Pneumothorax is the most common complication after percutaneous lung MWA, with an incidence ranging from 37 to 67% and requiring chest tube drainage in 6.0-31.9% of patients [1][2][3][4][5][6][7][8].Although pneumothorax is not associated with increased in-hospital mortality after ablation, the occurrence of pneumothorax leads to increased patient pain, prolonged hospital stays, increased x-ray exposure, and additional treatment cost.Therefore, it is of great interest to reduce the incidence of pneumothorax after ablation to improve economic and social benefits.
Currently, many studies have shown that the application of various materials to seal the puncture tract after lung biopsy reduces the risk of pneumothorax [9][10][11][12].Embolic materials include normal saline, autologous blood clots, hydrogel plug, and gelatin sponge slurry or plug.However, there are few studies on prophylactic measures to reduce the occurrence of pneumothorax by sealing the puncture tract after lung ablation.Two previous studies showed that sealing the pulmonary radiofrequency ablation (RFA) needle tract with the absorbable gelatin sponge significantly reduced the incidence of pneumothorax [13,14].However, its effect and safety remain uncertain.
We hypothesize that the use of gelatin sponge particles will effectively seal the needle tract after MWA to reduce the incidence of pneumothorax.We further hypothesize that the gelatin sponge particles will be completely absorbed with good safety.Accordingly, the purpose of this study is to evaluate the efficacy and safety of this technique in preventing pneumothorax after MWA, to provide a theoretical basis for clinical practice.

Materials and methods
This study was approved by our Institutional Animal Care and Use Committee.The New Zealand white rabbits (3.0-3.5 kg) were used for the in vivo experiments, which were provided by Beijing Keyu Animal Breeding Center (license number: SCXK (Beijing, 2018-0010).All animals were kept in single cages for 5-7 days to adapt to the experimental environment, with a free diet and water intake.

Animal study design
The study was designed to evaluate the statistically significant differences in pneumothorax rate.Twenty-four New Zealand White rabbits were randomly assigned into two groups in a 1:1 ratio: (1) group A (n ¼ 12), MWA followed by needle tract embolization with gelatin sponge particles; (2) group B (n ¼ 12), MWA without needle tract embolization.
MWA was performed with healthy lung tissue in subpleural locations, with a distance from the pleura of about 10 mm in the lower lobes in both groups.MWA was performed once in each lower lobe.The right lower lobe was chosen for the first ablation and used as the specimen for pathological examination.The second MWA was performed in the left lower lobe only for pneumothorax evaluation before sample collection.The details were illustrated in Figure 1.

MWA procedure
General anesthesia was administered with 10% chloral hydrate solution (2.5 ml/kg) for all procedures.Animals were placed in the prone position with a special fixed box.MWA was performed under CT guidance according to the protocols.Briefly, a 15-gauge coaxial needle (Argon Medical Device, Inc.) was introduced into the healthy lung tissue at a distance from the pleura of about 10 mm with a single pleural puncture (Figure 2(A)).A 17-gauge water-cooled microwave ablation antenna (ECO Medical Device, China), with a 3 mm active tip, was then advanced into the lung parenchyma about 20 mm below the pleura through the coaxial needle (Figure 2(B)).The ablation was initiated at a power of 20 W for a duration of 2 min.One ablation per lower lobe was attempted in different sessions for each animal.Tract cautery was not performed after MWA.

Needle tract sealing procedure
Gelatin sponge particle embolic agent (Alicon, Hangzhou, China) was supplied in size ranges of 1000-1400 lm containing !100 mg.Each vial of gelatin sponge particles was mixed with 3 ml of sterile saline to obtain a thick suspension.
A helical CT was immediately performed in group A to confirm whether the coaxial needle was located in the lung parenchyma after ablation.If the needle was confirmed in the lung parenchyma, the needle tract embolization was performed using prepared gelatin sponge particle suspension.One milliliter of the suspension was firstly gently injected into the needle tract through the coaxial needle.The stylet was then replaced into the trocar during the needle removal to the intercostal space to fill the track (Figure 2(C,D)).If the coaxial needle was out of the pleura, the needle was directly removed without sealing the tract.For animals of group B, the coaxial needle and antenna were completely removed after MWA without tract embolization.

Pneumothorax
A helical CT of the chest was performed immediately and 30 min after all procedures to evaluate the occurrence of pneumothorax.The incidence of pneumothorax within 30 min post-ablation in each group was recorded.Pneumothorax that caused dyspnea or increased in size rapidly in 30 min was treated with aspiration.Aspiration was performed using a coaxial needle.A chest CT was reexamined 30 min after aspiration to assess whether there was any new pneumothorax.If the pneumothorax was mild, no aspiration was performed.

Histopathologic evaluation
For both groups, every two rabbits were euthanized immediately, 1, 3, 7, 10, and 14 days after MWA.Animals were euthanized with 10% chloral hydrate.The right lower lobe of the lung was surgically removed.The specimens around the needle tracts were identified and fixed in 10% formalin and embedded in paraffin for pathological examination.Each slide of the specimen was stained with Hematoxylin Eosin (H&E).The specimen was examined microscopically for assessment of the post-ablation modifications of the needle tract, and the presence and appearance of gelatin sponge particles.

Statistical analysis
Technical success was defined as the completion of tract embolization with 1 ml of gelatin sponge particles after MWA.SPSS 25.0 (SPSS, Chicago, IL, USA) statistical software was used for data analysis.The Chi-square test or Fisher's exact test was used for pneumothorax analysis.A p-value of <0.05 was considered statistically significant.

Results
A total of 47 sessions of MWA were performed on 24 animals (24 sessions in group A and 23 in group B).One rabbit in group B received only one session of MWA, which died of a severe pneumothorax after the first MWA in the right lower lobe.Tract embolization was completed in all 12 animals of Group A. The technical success rate was 100%.The lung tissue samples around the needle path were obtained in all animals.

Pneumothorax
There were 19 cases of pneumothorax after 47 sessions of MWA.The incidence of pneumothorax in group A was 25.0% (6/24).Two cases of pneumothorax were treated with aspiration.The incidence of pneumothorax in group B was 56.5% (13/23).Six cases of pneumothorax were treated with aspiration.One rabbit was still found to have a large amount of pneumothorax after aspiration and died of dyspnea.
Group A had a significantly lower incidence of pneumothorax than Group B (25.0 vs. 56.5%;p ¼ 0.028; Table 1).Aspiration rates were 8.3% (2/24) vs. 26.1% (6/23) (p ¼ 0.137) comparing Group A with Group B. There was no significant difference in aspiration rate between the two groups.No complications relative to the gelatin sponge particles were encountered during our study.

Histopathologic assessment
Tissue around the needle tract showed thermal injury in all animals and in some instances, with fistulous tracts between the pleura and ablation zone.The needle path after MWA showed a circular bilayer structure.The inner layer was thermal coagulation with compressed and deformed alveolar walls (Figure 3(A)).In several cases of pneumothorax requiring aspiration, an open needle tract was observed between the ablation zone and the pleura, forming a fistula.Congestion with hemorrhage was seen in the outer layer, and exudation was visible in the alveolar lumen.These findings were true for both groups reviewed histologically.
Gelatin sponge particles were found in all specimens immediately after MWA.They were slightly basophilic.The gelatin sponge particles were arranged in small irregular stripes, occupying the needle tract densely (Figure 3(B)).On days 1 and 3, gelatin sponge particles were visible in all four specimens, with morphology similar to samples immediately after MWA.
On day 7, gelatin sponge particles appeared in all specimens.Lysis of the spongy material had occurred.Gelatin sponge particles were visible in only 1 specimen on days 10 and 14.Most of the spongy materials were degraded and absorbed.Fibrotic changes around the needle tract and pleural thickening around the puncture point were evident.Specifically, there was no evidence of migration to a pulmonary artery or vein in any specimen.

Discussion
Pneumothorax is the most common complication of percutaneous MWA for pulmonary malignancies.The incidence of pneumothorax after CT-guided thermal ablation varies, from 37 to 67% [3,7].The present study showed a rate of 56.5% pneumothorax without tract embolization.Therefore, it is of great significance to use effective techniques to reduce the incidence of pneumothorax.
The most studied technique is using various sealant materials to embolize the needle path to reduce the risk of pneumothorax after CT-guided lung biopsy.The rationale is that the mechanism of pneumothorax after lung biopsy is air leakage from pleural rupture caused by mechanical injury [9].Therefore, sealing the pleural rupture during needle withdrawal may reduce the occurrence of pneumothorax.The idea of tract embolization was first reported in 1974 [15].Many subsequent studies have shown that the technique of tract embolization can significantly reduce the risk of pneumothorax after lung biopsy with various materials, such as gelatin sponge, autologous blood patch, normal saline, or hydrogel plugs [9][10][11][12]16].
However, the mechanism of pneumothorax after lung thermal ablation is more complex than lung biopsy.There are both mechanical and thermal injury factors.The study confirms thermal injury around the needle tract after MWA.Histopathology demonstrates coagulative changes in the lung tissue around the needle path.A porcine model after radiofrequency also identifies pathological damage around the needle track [17].This study shows that the damage causes the formation of a coagulated pulmonary parenchyma rim along the needle pathway.Both studies imply thermal injury could induce the formation of a fistula between the ablation zone and pleura, which is significantly associated with the occurrence of pneumothorax.These findings obtained from animal experiments were verified by clinical observation.Lignieres et al. [18] reported the visualization of an air path between the pleura and ablation zone immediately after the RF needle withdrawal was significantly associated with a high risk of pneumothorax and tube drainage.Therefore, further study is necessary to determine the effectiveness of this sealing technique.
Only two studies have evaluated the efficacy of tract embolization after lung RFA both with gelatin sponge [13,14].One study utilized gelfoam torpedoes to embolize the RFA pathway, while the other sealed the tract with gelatin sponge slurry.Both studies demonstrated a significantly decreased incidence of pneumothorax and chest tube placement after tract embolization.
The present study shows that tract embolization with gelatin sponge particle suspension significantly reduced pneumothorax rate, which is consistent with previous studies.The study also demonstrated for the first time that gelatin sponge particles could effectively fill and seal the tract after thermal ablation from the perspective of histopathology.There are several differentiations from the previous two studies.The gelatin sponge particles are relatively uniform in size, shape, and quantity.They are really easy to use and reproducible among different operators, without additional preparation.Gelfoam used in the previous studies is made into a sheet and must be cut into fragments before use, varying in size, shape, and quantity, which may induce unpredictable results.In addition, Izaaryene et al. [13] reported gelfoam torpedo was limited in the application of subpleural lesions, which made it difficult to release accurately.The study of tract embolization with gelatin sponge slurry excluded lesions in subpleural locations [14].Our study showed that for lesions close to 1.0 cm of pleura, gelatin sponge particle suspension can be effectively used to seal the tract and reduce the rate of pneumothorax, with a technical success rate of 100%.
Gelatin sponge particles have good absorbability and safety.The study shows gelatin sponge particles can be degraded and absorbed in vivo.One safety concern for clinicians is the risk of migration of gelatin sponge particles to pulmonary vessels.The risk of migration to pulmonary veins or arteries potentially exists.However, there seems to be no evidence of any sealant material migrating into the pulmonary vessels of any specimen in our study.Such a complication has never been reported in clinical practice [11,13,14,16].The present study implies that the use of large-size gelatin sponge particles to embolize the needle tract under the pleura may help to decrease the risk of migration.Renier et al. [11] also used the technique of withdrawing the needle tip at a distance of 1 cm from the pleura before embolization to reduce such a complication.The rationale is that vessels become anatomically fewer and smaller in the sub-pleural lung.In addition, confirming that there is no blood return before injecting gelatin sponge particles can reduce the risk of migration [14].
This study has several limitations.First, ablation in normal lung tissue rather than tumor does not fully reflect the real situation.No subpleural lesions have been evaluated in the study.Second, the small sample size may induce bias.Finally, specimens obtained at each time point were too few to be analyzed quantitatively.

Conclusions
Results of the present study showed the incidence of pneumothorax can be significantly reduced by tract embolization with gelatin sponge particles after CT-guided lung MWA.The results of this study will provide important information for clinical application.Of course, it is still necessary to confirm the safety and efficacy of such an embolic agent in the prospective studies of larger patient samples.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.

Figure 1 .
Figure 1.Flowchart of the experimental protocol.

Figure 2 .
Figure 2. CT-guided lung microwave ablation and tract embolization in the right lower lobe.(A) A 15-G coaxial needle was introduced into the lung tissue $10 mm of the pleural surface under CT guidance.(B) Placement of the MW antenna.(C) Removal of the coaxial needle and sealing of the needle tract with gelatin sponge particle suspension (arrow).(D) CT performed 2 weeks after the procedure shows complete absorption of the gelatin sponge particles.The ablation zone is visible (arrowhead).

Figure 3 .
Figure 3. Rabbits were euthanized immediately after microwave ablation.(A) The histopathologic view (H and E, Â20) shows bilayer modifications around the needle tract: compressed and deformed alveolar walls in the inner layer (arrow), and congestion in the outer layer (arrowhead).Visualization of an open needle tract (star).(B) The slightly basophilic gelatin sponge particles fill the needle tract densely (H and E, Â40).

Table 1 .
Pneumothorax and aspiration rates in the two groups.