J Vet Sci.  2013 Dec;14(4):433-440. 10.4142/jvs.2013.14.4.433.

Pathology of non-thermal irreversible electroporation (N-TIRE)-induced ablation of the canine brain

• Rossmeisl
• Garcia PA
• Roberston
• Ellis TL
• Davalos RV

• KMID: 1712310
• DOI: http://doi.org/10.4142/jvs.2013.14.4.433

Abstract

This study describes the neuropathologic features of normal canine brain ablated with non-thermal irreversible electroporation (N-TIRE). The parietal cerebral cortices of four dogs were treated with N-TIRE using a dose-escalation protocol with an additional dog receiving sham treatment. Animals were allowed to recover following N-TIRE ablation and the effects of treatment were monitored with clinical and magnetic resonance imaging examinations. Brains were subjected to histopathologic and ultrastructural assessment along with Bcl-2, caspase-3, and caspase-9 immunohistochemical staining following sacrifice 72 h post-treatment. Adverse clinical effects of N-TIRE were only observed in the dog treated at the upper energy tier. MRI and neuropathologic examinations indicated that N-TIRE ablation resulted in focal regions of severe cytoarchitectural and blood-brain-barrier disruption. Lesion size correlated to the intensity of the applied electrical field. N-TIRE-induced lesions were characterized by parenchymal necrosis and hemorrhage; however, large blood vessels were preserved. A transition zone containing parenchymal edema, perivascular inflammatory cuffs, and reactive gliosis was interspersed between the necrotic focus and normal neuropil. Apoptotic labeling indices were not different between the N-TIRE-treated and control brains. This study identified N-TIRE pulse parameters that can be used to safely create circumscribed foci of brain necrosis while selectively preserving major vascular structures.

Keyword

central nervous system; dog; irreversible electroporation; neuropathology

MeSH Terms

Animals
Brain/metabolism/*pathology/surgery/ultrastructure
Caspase 3/metabolism
Caspase 9/metabolism
Dogs
Electroporation/veterinary
Magnetic Resonance Imaging/methods
Microscopy, Electron, Transmission
Necrosis/metabolism/pathology
Neurosurgical Procedures/*adverse effects
Caspase 3
Caspase 9

Figure

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  Fig. 1 Macroscopic morphology of non-thermal irreversible electroporation (N-TIRE) ablation in the right parietal lobe of the canine brain (Dogs 2~5). The caudal aspect of the lesion area in Dogs 2 and 3 is shown along with the rostral lesion area in Dogs 4 and 5. The right side of the brain is on the left side of the images in all panels. (A and B) Normal, pre-treatment T2W MR images. (C and D) Post-treatment T1W MR images showing a T1 iso- to hypointense lesion (white arrow). (E and F) Post-treatment T2W MR images containing focal, ovoid heterogeneously hyperintense lesions. (G and H) Post-treatment, post-contrast T1W MR images demonstrating peripheral contrast enhancement of the ablated regions. (I~L) Subgross (H&E) and gross pathologic features of brain sections corresponding to anatomic levels presented in the MR images in panels E~H. N-TIRE ablative lesions are characterized by malacia and intraparenchymal hemorrhage, and are clearly demarcated from the surrounding normal brain tissues. (M~P) Composite images generated by superimposing H&E-stained brain sections on the corresponding MRI slice. Scale bars = 1 cm (I~L).

• 

  Fig. 2 Adverse effects associated with high-energy N-TIRE in Dog 5. (A) Right unilateral transtentorial herniation resulting in mesencephalic compression (black arrows) in the most caudal aspect of the N-TIRE-affected area (white arrowheads). (B) Cerebrocortical necrosis associated with marked vascular disruption and large artery thrombosis in the N-TIRE treatment region (H&E). Scale bars = 2.0 mm (B), 1cm (A).

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  Fig. 3 Comparative effects of N-TIRE on the superficial parietal cerebral cortex. (A) Untreated, contralateral cortex of Dog 3. (B) Sham-operated cortex demonstrating physical disruption of the neuropil by the electrode in Dog 1. (C) Collateral cortical damage extending into the adjacent neuropil in N-TIRE-affected cortex of Dog 3. H&E, Scale bars = 2.0 mm (A~C).

• 

  Fig. 4 Morphology of N-TIRE-induced lesions in the subcortical white matter of Dog 4. (A) Partial obliteration and vacuolization of the white matter in the corona radiata (luxol fast blue and Periodic Acid Schiff counterstain) with loss of the gray and white matter junctional distinction (inset, control). (B) Disruption of the axonal projections of the corona radiata (inset, control) of Dog 2 (Bielschowsky silver stain). (C) Vacuolar change in the white matter with axonal spheroids (arrows) in Dog 3 (H&E). LV: lateral ventricle. Scale bars = 200 µm (C), 5.0 mm (A, A inset, B and B inset).

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  Fig. 5 Morphology of N-TIRE-induced lesions in the deep cortical gray matter. (A) N-TIRE ablated regions (left side of panel) were sharply demarcated from the adjacent normal brain (right side) identified by their pallor. A focus of intraparenchymal hemorrhage associated with a disrupted capillary bed was visible at the lesion periphery (arrow) in Dog 2. (B) Necrotic neurons (arrowheads) and degenerate free glial nuclei in the N-TIRE-associated lesion core in Dog 4. (C) Gitter cells and intact vessels (arrows) were present in the transition zone between the affected and normal brain tissue in Dog 3. H&E. Scale bars = 200 µm (B and C inset), 500 µm (C), 2.0 mm (A).

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  Fig. 6 Vascular sparing effects of N-TIRE. (A) A medium sized arteriole (*) at the periphery of the N-TIRE ablation region remained intact despite diffuse damage to the surrounding neuropil in Dog 3 (H&E). (B) Electron photomicrograph of a large vessel within the core N-TIRE ablation zone of Dog 4 in which the endothelial cytoarchitecture and intravascular erythrocytes were preserved. (C) Intact vascular collagenous scaffolds in N-TIRE-treated and control (inset) gray matter of Dog 4 (trichrome). (D) CD31-positive vascular endothelial immunoreactivity and perivascular inflammation in the core N-TIRE treatment region (inset, CD31 control). (E) Electron photomicrograph of an untreated control cerebral capillary. (F) Electron photomicrographs of capillaries within the core N-TIRE ablation zone with cytoplasmic and capillary luminal disruption (*), extravasted erythrocytes, and endothelial nuclear pyknosis (inset, arrow) in Dog 2. CL: capillary lumen. Scale bars = 2 µm (E, F and F inset), 10 µm (B), 200 µm (C, C inset, D and D inset), 500 µm (A).

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  Fig. 7 N-TIRE-induced lesion transition zone (TZ). The broken gray line defines the boundary between the N-TIRE ablated region and TZ. (A) The neuropil in the TZ of Dog 2 was diffusely vacuolated and contained perviascular inflammatory cuffs (arrows; H&E). (B) An astroglial reaction in the TZ of Dog 1 (glial fibrillary acidic protein; GFAP). (C) More severe parenchymal vacuolization and astrogliosis in Dog 4 were associated with higher treatment energies (GFAP). Scale bars = 200 µm (A), 500 µm (B and C).

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  Fig. 8 Immunohistochemical evaluation of apoptosis. No differences in Bcl-2, caspase-3, and casapse-9 immunoreactivity were observed between N-TIRE-treated and control brain tissues.

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