- To evaluate side-effects, erectile function and capability to preserve adjacent tissues of bilateral focal prostate ablation using low-energy direct current (LEDC) in a canine model.
Irreversible electroporation by application of low-energy direct current (LEDC) is a non-thermal ablative technology that uses electric current pulses of short duration to create pores in cell membranes leading to cell death. LEDC has been shown to effectively ablate prostate cancer cells in vitro , as well as normal prostate tissue in an in vivo canine model .
While previous work showed effective ablation of prostate tissue with distinct margins of transition between necrotic and vital parenchyma after LEDC , side-effects, collateral injuries and functional outcomes have not been assessed. Despite the preliminary findings [2, 3] that LEDC may spare damage to nervous structures and therefore potentially preserve erectile function, there has been no direct study addressing these concerns in vivo.
In the present study, we sought to evaluate erectile function, side-effects and preservation of adjacent tissues after a bilateral focal prostate ablation in vivo using the NanoKnife™ (AngioDynamics Inc., Queensbury, NY, USA) LEDC ablation system in a canine model.
In all, 12 male Beagle dogs (Covance Research Products, Cumberland, VA, USA) aged 3.9–7.4 years and weighting 9.4–12.1 kg were used in this study. The dogs underwent bilateral focal LEDC ablation using the monopolar electrode NanoKnife device. The dogs were assigned to two groups for final pathological evaluation at 7 days (group 1, six dogs) and 27–28 days (group 2, six dogs) after ablation.
The dogs were individually housed in separate cages, and quarantined for 8 days before the beginning of the study. On the day of the procedure, the dogs had a soapy enema before surgery. After premedication (acepromazine 0.02–0.1 mg/kg), i.v. antibiotics (500 mg cefotaxime) and induction (propofol 4–8 mg/kg), anaesthesia was maintained with isofluorane through a facemask. Pancuronium bromide 0.1 mg/kg was used as a muscle relaxant to prevent contractions associated with LEDC treatment. Anaesthetised, the dogs were placed in a dorsal position, prepped and draped for surgery as appropriate. After placement of a urethral catheter, the bladder was partially filled with saline and TRUS was used for prostate measurements and volume calculation (ellipsoid formula: height × width × length × 0.52).
Treatment probes were then positioned percutaneously, through a transperineal 2.5-mm grid under TRUS guidance on one side of the prostate (left or right) using a triangular array (Fig. 1). Probes were placed to maintain a minimum distance of 5 mm from the capsule with 1.5–2 cm active electrode length. Each probe location was sonographically confirmed by sagittal and transverse planes. Distances between the probes, to prostate capsule, urethra and rectum were recorded. The rectum was then separated from the posterior prostate by injection of 5% dextrose solution into Denonvilliers' fascia. A ‘test pulse’ was used to verify effective muscle relaxation. The ablation procedure used 90 pulses of 70 μs duration each. Electric pulses were delivered between each of the electrode pairs (1–2, 2–3 and 1–3). Electric fields generated ranged from 1500 to 1800 V/cm. The procedure was then repeated on the opposite side of the gland. Once both sides were treated, the probes and urethral catheter were removed and the dogs were allowed to recover from anaesthesia. A fentanyl patch (50 μg/h) was used for postoperative pain management as needed; however, fentanyl patches were not required outside of the immediate postoperative period. The dogs were observed by a dedicated veterinarian at least twice daily throughout the study to record potential side-effects, specifically those related to urination and defecation. Haematuria and haematochezia were evaluated upon inspection of the cages for urine and feces. Any red or pink discoloration of the urine was considered as haematuria; frank blood in the stool was considered as haematochezia.
Erectile function was evaluated at baseline (1–2 days before ablation) as well as at 4–5 days (group 1) and 24–25 days (group 2) after ablation. The dogs were observed for erection during physical exercise, physical examination and, if no erection was observed, upon manual stimulation. An erection occurring during exercise, physical examination or manual stimulation was recorded. Female dogs were available if the above mentioned steps failed to produce an erection.
At termination, a comprehensive necropsy was performed to evaluate for potential injury to adjacent tissues; prostate, bladder, ureters, urethra as well as rectal wall were harvested en bloc and fixed in 10% neutral buffered formalin for histopathological assessment. Histopathology was performed by a Board Certified veterinary pathologist using standard haematoxylin and eosin staining on 5-mm sections of the specimens. Specific attention was devoted to microscopic examination of the prostatic capsule, urethra, nervous tissues and rectal wall.
The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee and carried out under Good Laboratory Practice conditions.
In all, 12 dogs successfully underwent bilateral focal prostate ablation using LEDC monopolar probes (NanoKnife). The median (range) prostate volume as measured by TRUS was 12.1 (8.9–17.3) mL. The procedural parameters are summarised in Table 1. Monopolar electrodes were placed at a median distance of 0.66, 0.56 and 0.55 cm from the prostate capsule, urethra and rectum, respectively. All procedures were completed successfully with no intraoperative complications recorded. All the dogs recovered well from anaesthesia.
|Prostate volume, mL||12.1 (8.9–17.3)|
|Distance from probes to prostate capsule, cm||0.66 (0.50–0.98)|
|Distance from probes to urethra, cm||0.56 (0.39–0.84)|
|Distance from probes to rectum, cm||0.55 (0.50–0.92)|
|Distance from probe to prostatic apex, cm||0.83 (0.51–1.58)|
|Distance from probe to prostatic base, cm||0.70 (0.51–1.17)|
|Distance between probes, cm||0.72 (0.59–1.20)|
Haematuria was noted in 10 dogs after ablation. Haematuria spontaneously resolved ≤1 day in eight and ≤3 days in the remaining two dogs. No additional treatment or intervention was needed. No episodes of urinary retention were recorded. Blood in the feces was noted in four dogs and resolved spontaneously within 1–2 days. Abnormalities of defecation (consistency, colour, frequency) were self-limiting, resolving ≤7 days after ablation.
In group 1, erections were noted in all six dogs at 4–5 days after ablation (one during exercise, five with manual stimulation). In group 2, erections were also noted in all six dogs (one during physical examination, five with manual stimulation) at 24–25 days after ablation. Overall, all 12 dogs were able to achieve erections at 4–25 days after bilateral focal LEDC ablation of the prostate.
Histopathological examination of specimens revealed necrosis and haemorrhage, inflammation and oedema within the ablation zones 7 days after ablation. At 30 days, acute inflammatory changes had been replaced by fibrous connective tissue. Histopathological assessment showed no clinically significant damage to adjacent anatomical structures. Representative gross and microscopic images are shown in Fig. 2. Notably, pathological examination did not show any significant damage to the urethra or cavernous nerves after LEDC ablation (Fig. 3). There were no injuries to the rectal wall or prostatic capsule upon pathological assessment. Blood vessels remained relatively unaffected at the 7- and 30-day evaluations. Despite instances of partially occluded capsular arterioles adjacent to the area of ablation, larger arteries and veins were not adversely affected by LEDC and maintained patency.
Irreversible electroporation by means of LEDC has been proposed as an effective non-thermal ablative technique. However, its side-effects profile and its effect on erectile function in vivo has not been investigated. In the present study, we performed bilateral focal LEDC ablation of the prostate and evaluated its safety and the impact on erectile function in a canine model. We found that erectile function was preserved even as early as 4–5 days after ablation. The side-effects profile appears to be minimal and transitory and importantly, with careful positioning of the treating probes, damage to adjacent tissues such as the prostatic capsule, urethra and rectal wall can be avoided.
The use of electroporation for tissue ablation has been previously described [1, 2, 4-8]. Irreversible electroporation relies on electrical current to disrupt cell membranes beyond the capacity of the cell to restore the damage . Being a non-thermal ablative technique, LEDC is immune to heat-sink effects, offers short treatment times and appears to spare important structures, e.g. the urethra, nervous tissue and blood vessels . These purported advantages over conventional thermal ablative techniques, e.g. cryoablation and high-intensity focused ultrasound, make LEDC an attractive technique for targeted localised destruction of prostate cancer.
In the present study, erectile function was preserved in all the dogs and erections were noted as soon as 4 days after bilateral focal LEDC ablation of the prostate. The preservation of erectile function seen in the present study may be, at least in part, attributed to the ability of LEDC to preserve nervous structures. In fact, while previous work has suggested preservation of nervous structures , Schoellnast et al.  directly assessed the damage induced by LEDC (1500 V/cm, 90 pulses of 70 ms) to a porcine sciatic nerve at 3, 6 and 14 days. They found that while signs of injury were present at all time-points (axonal swelling and inflammatory infiltrates), the endoneurium and perineurium remained intact. Moreover, the pigs regained function and were able to stand without assistance after 1–3 days, and were able to bear weight after 2–7 days. Li et al.  have recently reported on the effects of irreversible electroporation on rat sciatic nerves; after applying 10 100-ms pulses (3800 V/cm) to the sciatic nerve they reported a full recovery of function ≤7 weeks from the injury.
There was transient haematuria in most of the dogs (10/12) but this resolved spontaneously after 1–3 days without additional treatment. Haematuria could also be attributed to trauma during catheterisation of the dogs before ablation. Upon histopathological analysis there were no clinically significant findings regarding the urethra and only minor submucosal haematomas were seen. Similarly, transient haematochezia was recorded in four dogs and was attributed to minor rectal wall trauma during enema and/or TRUS-probe placement. No dogs required additional treatment for these occurrences. These events are common in percutaneous procedures under TRUS guidance and have been described after prostate cryoablation as well , and specifically in a canine model , as the TRUS probe is designed for human use. Moreover, the definition of haematuria used in the present study by visually inspecting the cage, as well as removal of the catheter immediately after ablation may have led to an overestimation of the incidence. Nevertheless, in no case were these side-effects clinically significant or required further attention.
Histopathology results largely confirmed previous reports suggesting LEDC can spare the urethra, nerves and blood vessels . Furthermore, despite the probes being positioned as close as 3.9, 5 and 5 mm from the urethra, rectum and capsule, respectively, there was no evidence of injury to these crucial structures, indicating that LEDC can be applied with great accuracy and precisely target the zones of interest while effectively sparing adjacent structures.
The results of this canine study of bilateral focal LEDC ablation of the prostate can be used as a foundation for future research and translation of the potential benefits of this technology to human application. Along with lack of histological and functional injury to the adjacent tissues and the excellent preservation of erectile function after bilateral focal ablation, these results indicate that LEDC is a promising technology for targeted ablation of the prostate.
The present study was performed in a canine model and the results are not necessarily applicable to their full extent to humans. However, the present study provides the necessary background in a preclinical model and delineates a potential morbidity profile associated with targeted focal LEDC ablation. The study was neither designed as a whole-gland ablation model nor was it used with nerve-sparing intention. Further, this canine prostate model did not harbour prostate cancer. Moreover, the extremely small prostate size in this canine model rendered electrode positioning difficult, with little margin for probe placement while maintaining proper distance from the prostate capsule to allow for assessment of potential collateral damage to adjacent structures. In addition, the present study is limited by the lack of long-term follow-up. Erectile function outcomes in the present study need to be interpreted considering healthy young dogs that do not necessarily resemble the potential human patient population for prostate ablation.
In conclusion, in the present study bilateral focal prostate LEDC ablation was safe with only minor self-limiting and expected side-effects. Application of LEDC in the present protocol did not result in injuries to adjacent tissues (prostatic capsule, urethra, rectal wall). Erectile function outcomes were remarkable with all the dogs achieving erections as early as 4 days after ablation. Herein, LEDC prostate ablation is shown to be a safe non-thermal ablative technique capable of preserving the adjacent structures and physiological functions. Further studies are needed to fully explore the advantages of LEDC compared with other ablative techniques.
TJP had a consultancy agreement with Angiodynamics at the time this work was completed.
low-energy direct current