European Radiology

, Volume 23, Issue 2, pp 375–380

The delayed effects of irreversible electroporation ablation on nerves

Authors

  • Helmut Schoellnast
    • Department of RadiologyMemorial Sloan-Kettering Cancer Center
    • Department of RadiologyMedical University of Graz
  • Sebastien Monette
    • Laboratory of Comparative PathologyMemorial Sloan-Kettering Cancer Center, Weill Cornell Medical College, and the Rockefeller University
  • Paula C. Ezell
    • Research Animal Resource CenterMemorial Sloan-Kettering Cancer Center and Weill Cornell Medical College
  • Majid Maybody
    • Department of RadiologyMemorial Sloan-Kettering Cancer Center
  • Joseph P. Erinjeri
    • Department of RadiologyMemorial Sloan-Kettering Cancer Center
  • Michael D. Stubblefield
    • Department of Neurology, Rehabilitation Medicine ServiceMemorial Sloan-Kettering Cancer Center
  • Gordon Single
    • AngioDynamics Inc
    • Department of RadiologyMemorial Sloan-Kettering Cancer Center
Experimental

DOI: 10.1007/s00330-012-2610-3

Cite this article as:
Schoellnast, H., Monette, S., Ezell, P.C. et al. Eur Radiol (2013) 23: 375. doi:10.1007/s00330-012-2610-3

Abstract

Objective

To evaluate the delayed effects of irreversible electroporation (IRE) ablation on nerves.

Methods

The study was approved by the institutional animal care and use committee. CT-guided IRE-ablation (electric field per distance, 1,500 V/cm; pulse length, 70 μs; number of pulses, 90) of 6 sciatic nerves was performed in 6 pigs that were euthanized 2 months after ablation. The sciatic nerves were harvested immediately after euthanasia for histopathological evaluation. Sections from selected specimens were stained with haematoxylin and eosin (H&E), Masson’s trichrome (MT) method for collagen, and immunohistochemistry was performed for S100 and neurofilaments (markers for Schwann cells and axons, respectively).

Results

All nerves showed a preserved endoneural architecture and presence of numerous small calibre axons associated with Schwann cell hyperplasia, consistent with axonal regeneration. A fibrous scar was observed in the adjacent muscle tissue, confirming ablation at the site examined.

Conclusion

After IRE-ablation of nerves, the preservation of the architecture of the endoneurium and the proliferation of Schwann cells may enable axonal regeneration as demonstrated after 2 months in this study.

Key Points

Irreversible electroporation (IRE) offers promise for non-thermal tumour ablation.

Preservation of endoneural architecture and proliferation of Schwann cells follow IRE-ablation.

Preservation of architecture and proliferation of Schwann cells may enable axonal regeneration.

Despite morphological regeneration, nerve function remains variable after 2 months.

Keywords

Athermal ablationIrreversible electroporationSciatic nerveCT-guidanceAnimal study

Introduction

Irreversible electroporation (IRE) has recently emerged as a promising technique for non-thermal tumour ablation. As opposed to thermal ablation techniques, IRE-ablation does not result in destruction of connective tissue or denaturation of collagen typical of thermal ablation [1, 2]. This selective cell destruction may have important clinical implications such as decreasing the incidence of bile duct, urethral or renal collecting system damage in IRE-ablation of the liver, prostate or kidney, respectively [13]. Similarly, 2 weeks after IRE of the swine sciatic nerve, there is preservation of the endoneural architecture and proliferation of Schwann cells [4]. This suggests that there is potential for axonal regeneration, as preservation of the extracellular matrix and proliferation of Schwann cells organising themselves into columns (bands of Büngner) can provide guidance to regrowing axons [5]. Regeneration of nerves after IRE-ablation may allow this technique to be applied as a treatment for tumours adjacent to nerves, overcoming the permanent nerve damage seen with radiofrequency ablation (RFA) and cryoablation. The purpose of our study was to test the hypothesis obtained from a previous study [4] that the histomorphology of the nerves 2 weeks after ablation with IRE (preservation of the endoneural architecture and proliferation of Schwann cells) actually can result in nerve regeneration, by using the same model and following animals for a longer period of time. For that purpose a follow-up period of 2 months was chosen as one can assume that if axonal regeneration occurs this will be seen after this period.

Materials and methods

Animals and clinical course

The study was approved by the institutional animal care and use committee. Percutaneous CT-guided IRE-ablation of six sciatic nerves was performed in six Yorkshire pigs (weight 35–45 kg) from two suppliers (Archer Farms Inc., Darlington, MD. USA; Animal Biotech Industries, Inc., Danboro, PA, USA). Each procedure started with pre-medication using Telazol (4.4 mg/kg) intramuscularly and after intubation general anaesthesia was maintained via continuous inhalation of Isoflurane® (1.5–3 %). All pigs were given buprenorphine 0.01 mg/kg subcutaneously before the procedure and every 12 h for 72 h as needed. Each pig also received meloxicam 0.4 mg/kg orally once a day for 3 days and metronidazole 66 mg/kg once a day for 5 days.

In the post-procedural course the ablation site was assessed for bruising and warmth. The day on which the animal was able to stand without assistance and the day on which the animal was able to bear weight on the treated limb were recorded. All animals had standing wraps (bandages) on both front and hind limbs to provide support and prevent abrasions/bruising during attempts to stand. Once the animals were able to stand unassisted and did not exhibit signs of muscle weakness (i.e. loss of balance when walking) the bandages were removed. Lameness of the treated limb when walking was assessed on a scale of 0–5 (1, minimal; 5, severe) on days 3, 5, 7, 14, 21 and 28.

All animals were kept alive for 2 months. Euthanasia was performed via intravenous injection of Euthasol (pentobarbital sodium 87 mg/kg and phenytoin sodium 11 mg/kg).

IRE-ablation

Pigs were anaesthetised and placed on the CT table (LightSpeed 16, GE Healthcare, Milwaukee, WI, USA) in a lateral position. CT of the pelvis without contrast medium was performed to identify the sciatic nerve and to plan the level and the position of the IRE electrode entry. The skin overlying the entry position of the electrodes was shaved and sterilised in the usual fashion by using alcohol and Betadine. Two single, monopolar electrodes (Nano-Knife IRE System, AngioDynamics, Queensbury, NY, USA) were placed under CT guidance such that the sciatic nerve was bracketed between the active lengths of the electrodes as reported elsewhere [4]. Ablation was performed using an active electrode exposure of 2 cm, an electrode spacing of 1.1–1.4 cm, a voltage of 1,650–2,100 V (voltage was chosen in order to keep a constant voltage per distance in tissue, 1,500 V/cm), and a pulse length of 70 μs. Two ablations were performed per nerve with 90 pulses, each with a change in the direction of the polarity to simulate a clinical ablation protocol, in which a nerve may be ablated twice, a worst case scenario. Pancuronium (0.15 mg/kg) was administered intravenously 10 min before ablation to reduce muscle contractions during the application of the electrical pulses. The adequacy of muscle relaxation was checked with a test pulse before ablation. Correct needle placement was ensured before each ablation and needle displacement due to muscle contraction was ruled out after each ablation with CT.

Nerve conduction studies

Nerve conduction studies were performed on the day of euthanasia using a Cadwell Sierra II EMG machine (Cadwell Laboratories, Inc. Kennewick, WA, USA). Standard machine settings were used to record a compound muscle action potential (CMAP) from the base of the second metatarsal using a disposable 19.05-mm disc electrode (active electrode) referenced to a second electrode placed approximately 2 cm distally (reference electrode). A ground electrode was placed on the dorsum of the hind foot. A prong-type stimulator was used to achieve supramaximal stimulation of the tibial nerve midway between the hock joint and the active electrode and approximately 7 cm above the hock joint. The distances between the proximal and distal stimulation sites were measured so that conduction velocity could be recorded. Measurements of CMAP amplitude, onset latency, and conduction velocity between proximal and distal stimulation sites were recorded.

Gross pathology and histopathological analysis

Gross pathology and histopathological analysis was performed by a board-certified veterinary pathologist.

Immediately after euthanasia, a post-mortem examination limited to the region of the ablation was performed. The portion of the middle gluteal muscle containing the ablation lesion and the adjacent sciatic nerve and associated adipose tissue and blood vessels were fixed by immersion in 10 % neutral buffered formalin. A nerve specimen approximately 10 cm distal to the ablated area was also fixed in formalin. In two animals the contralateral nerve was harvested, fixed in formalin, and used as a control. Muscle, nerve, and associated tissues were routinely processed, embedded in paraffin, cut into 4-μm-thick sections, and stained with haematoxylin and eosin (H&E). Transverse and longitudinal nerve sections were examined from each site. Sections from selected specimens were also stained with Masson trichrome (MT) for collagen. Immunohistochemical staining (IHC) was performed on selected sections for S100 (Schwann cell marker) using polyclonal rabbit anti-S100a antibody (Z0628; Dako, Carpinteria, CA, USA), and for neurofilaments (NF, axonal marker) using monoclonal mouse anti-pan-axonal neurofilaments antibody (SMI-312R; Covance, Princeton, NJ, USA). IHC was performed following the manufacturer’s recommended protocol.

All nerves and associated muscles and tissues were assessed for histopathological findings consistent with nerve injury and repair, such as axonal swelling, fragmentation, loss, and regeneration, Schwann cell loss and proliferation, ellipsoids, inflammatory cell infiltrates, and fibrosis. The percentage of fascicles affected on transverse slices at the site of ablation was recorded.

Results

Clinical course

All animals had minor bruising at the needle puncture site which resolved by days 3–5 post-ablation treatment. Silver sulfadiazine cream was applied on all abrasions to help facilitate healing. Each animal’s recovery progressed differently with regard to the animal’s ability to stand without assistance and bear weight on the treated limb (Table 1). All animals except for one were able to stand on the day of ablation or 1 day after the ablation and all animals except for one were able to put weight on their treated limb within 3 to 4 days after ablation. The lameness scale continuously decreased over time and all animals except for one had no clinical signs of lameness 4 weeks after ablation.
Table 1

Clinical course after the procedure

Animal

Standing

Bearing weight

Lameness (scale 0–5)

Day 3

Day 5

Day 7

Day 14

Day 21

Day 28

1

Day 0

Day 4

4

3

2

1

0

0

2

Day 1

Day 2

2

2

1

1

0

0

3

Day 0

Day 3

3

3

2

1

1

1

4

Day 0

Day 3

3

1

2

1

1

0

5

Day 0

Day 4

4

3

2

1

0

0

6

Day 7

Day 9

5

5

5

3

0

0

Nerve conduction studies

The results of the nerve conduction studies comparing the treated and untreated limbs are presented (Table 2). The drop in CMAP amplitudes ranged from 21 to 97 %. A drop in CMAP of greater than 50 % in the treated limb compared with the untreated limb was considered significant. By this standard, a significant drop occurred in half of the animals with the worst result occurring in the animal that was most severely affected clinically.
Table 2

Compound muscle action potential (CMAP, mV) of the treated and untreated limb

Animal

Treated limb

Untreated limb

% Difference

1

2.74

3.94

30

2

6.39

8.11

21

3

1.31

7.11

82*

4

0.98

9.15

89*

5

5.94

8.87

33

6

0.22

6.86

97*

*Greater than 50 % side to side difference was considered significant

Gross pathology

On gross examination, a well-demarcated focal lesion was observed in the middle gluteal muscle, adjacent to the sciatic nerve in all pigs. The lesions were pale tan, firm, and contained numerous small (1 mm in diameter) hard white foci of calcification. No gross changes were observed in the nerves.

Histopathology

On histopathological examination, marked lesions were observed in all nerves examined. Pathological findings observed 2 months after ablation are summarised in Table 3. Examination of the nerves at the site of ablation revealed changes in 75 to 100 % of fascicles. There was diffuse hypercellularity observed within the affected fascicles, with almost all cells expressing S100, consistent with Schwann cell proliferation. Large numbers of small calibre axons (smaller than normal axons in control nerves) expressing neurofilaments were closely associated with hyperplastic Schwann cells, consistent with axonal regeneration. Ellipsoids, which are hollow spaces in the nerve that result from fragmentation of the myelin sheets and axons, were present in minimal numbers. There was a small increase in the amount of endoneural and perineurial collagen on MT stain.
Table 3

Histopathological findings in the sciatic nerves at the site of IRE-ablation after 2 months

Findings

Incidence

Axonal swelling, fragmentation and loss

0/6

Axonal regeneration

6/6

Schwann cell hyperplasia

6/6

Ellipsoids (collapsed myelin)

6/6

Phagocytes

0/6

Perineurial inflammatory infiltrate

0/6

Perineurial fibrosis

6/6

The specimen obtained distal to the site of ablation showed similar small calibre axons associated with increased numbers of Schwann cells. Ellipsoids were present in moderate numbers and mild endoneural and perineurial fibrosis was observed on MT stain at that site.

Histopathology of the muscle lesion adjacent to the nerve showed a fibrous scar with, in some cases, a small centrally located focus of remaining necrotic and calcified tissue.

Figure 1 illustrates the histological findings in the nerves after IRE-ablation at 2 weeks and 2 months.
https://static-content.springer.com/image/art%3A10.1007%2Fs00330-012-2610-3/MediaObjects/330_2012_2610_Fig1_HTML.gif
Fig. 1

Transverse histological sections of sciatic nerves. H&E staining, neurofilaments staining for axons, S100 staining for Schwann cells and Masson trichrome (MT) staining for collagen of an untreated control nerve, at 2 weeks after IRE-ablation, and at 8 weeks after IRE-ablation. At 2 weeks, a significant loss of axons is observed (f) combined with hypercellularity (arrow) and ellipsoids (arrowhead) on H&E stain (e), with most cells identified as Schwann cells on the basis of S100 expression (b, brown colour). At 8 weeks, there is still hypercellularity due to S100+ Schwann cells (i, k), but ellipsoids have resolved (i). Numerous small calibre axons are present in close association with Schwann cells. (j, k). The collagenous matrix shows preservation of endoneural architecture at 2 weeks (h) and 8 weeks (l), with an increased amount of collagen. Images a, e, g, and h are reused from Schoellnast H, Monette S, Ezell PC et al. (2011) Radiology 260:421–427 with permission. Scale bars 20 μm. Original magnification ×600

Discussion

The findings of this study on the delayed effects of IRE-ablation support the hypothesis that nerves can regenerate after IRE, in accordance with our initial results obtained at 2 weeks after IRE-ablation [4]. Similar to the subacute (2-week) study, affected fascicles varied from 75 to 90 %. However, previous findings of subacute nerve damage such as axonal swelling, fragmentation, and loss as well as perineurial inflammatory infiltrate were not seen in our chronic study. There was still diffuse hypercellularity observed within the affected fascicles, with almost all cells expressing S100, representing Schwann cell proliferation with formation of bands of Büngner which were also demonstrated after 2 weeks [4]. Schwann cells play an important role in nerve regeneration at the site of injury. Schwann cells and phagocytes are recruited to the injury site, and phagocytise myelin which is collapsed into ellipsoids. Proliferating Schwann cells organise themselves into bands of Büngner and the regenerating axons associate with them by growing distally between their basement membranes. The most important finding in our study was a large number of small calibre axons expressing neurofilaments which were closely associated with the hyperplastic Schwann cells, consistent with axonal regeneration [6].

The findings of this study show some concordance with a recent study on the effects of IRE on nerves in rats [6]. In that study, 10 pulses of 3,800 V/cm, each 100 μs long were applied directly to rat sciatic nerves. Electrophysiological and functional studies as well as histological analysis were performed immediately and up to 10 weeks following surgery. The authors reported that 3 days after IRE-ablation, some of the myelin sheath structures disintegrated and 1 week after IRE-ablation, myelin sheath structures barely existed. Three and 5 weeks after IRE-ablation, some myelin sheath structures had been restored, and 10 weeks after IRE-ablation, many nerve fibres had regenerated, and most of the myelin sheath structures had been restored. In our study, axonal regeneration was observed after 2 months. However, the regenerated axons were smaller than normal axons of an untreated control nerve.

In the study by Li et al. [6], CMAP were markedly restored 7 and 10 weeks after IRE-ablation, and there were no differences between the IRE group and a control group. Our nerve conduction studies 2 months after IRE-ablation showed a significant difference in CAMP amplitude between the treated and the untreated limb in 50 % suggesting limited functional recovery. The different animal model with different sizes of the sciatic nerve may explain the different results of the nerve conduction studies. On the basis of the significant proliferation of Schwann cells detected at 2 months after IRE-ablation in our study, one can postulate that the axons will show further regeneration after 2 months leading to improvement of nerve conduction. However, most animals showed rapid recovery after IRE-ablation and were able to bear weight on their treated limb after a few days. The lameness scale continuously decreased and most animals did not show clinical evidence of lameness 1 month after IRE-ablation. Therefore, although nerve conduction studies showed limited function in half of the animals, the animals were able to compensate for the limited function. This may be explained by the fact that the gluteal muscles, and the extensors and adductors of the thigh are not affected by palsy of the sciatic nerve.

This study has several limitations. The small sample size and site-specific nature of the ablation make it difficult to extrapolate these results to nerve injury during IRE-ablation of other parts of the body, especially when IRE is performed on small non-myelinated, non-sheath nerves instead of the sciatic nerve. The specific IRE protocol may also have influenced the results. Some new developments in IRE may provide a method of IRE without muscle contractions and our results may not apply to these new methods [7]. The observation period of 2 months after IRE-ablation may be considered as a further limitation of this study. Nerve conduction studies showed a significant difference between the treated and the untreated limb in 50 % and observations longer than 2 months may be necessary to prove further functional improvement.

In conclusion, after IRE-ablation of nerves, the preservation of the architecture of the endoneurium and the proliferation of Schwann cells enabled axonal regeneration as demonstrated after 2 months in this study.

Acknowledgments

SB Solomon received grants from Angio Dynamics, GE Healthcare, Johnson & Johnson and G Single is an employee of Angio Dynamics.

Copyright information

© European Society of Radiology 2012