Electroporation Injury Research Articles

Recovery of neurosurgical high-frequency electroporation injury in the canine brain can be accelerated by 7,8-dihydroxyflavone

BackgroundAlthough traumatic brain injury (TBI) occurs in a very short time, the biological consequence of a TBI, such as Alzheimer’s disease, may last a lifetime. To date, effective interventions are not available to improve recovery from a TBI. Herein we aimed to ascertain whether recovery of neurosurgical high-frequency irreversible electroporation (HFIRE) injury in brain tissues can be accelerated by 7,8-dihydroxyflavone (7,8-DHF). MethodsThe HFIRE injury was induced in the right parietal cortex of 8 adult healthy and neurologically intact male dogs. Two weeks before HFIRE injury, each dog was administered orally with or without 7,8-DHF (30 mg/kg) once daily for consecutive 2 weeks (n = 4 for each group). The values of blood-brain barrier (BBB) disruption, brain edema, and cerebral infarction volumes were measured. The concentrations of beta-amyloid, interleukin-1β, interleukin-6 and tumor necrosis factor-α in the cerebrospinal fluid were measured biochemically. ResultsThe BBB disruption, brain edema, infarction volumes, and maximal cross-section area caused by HFIRE injury in canine brain were significantly attenuated by 7,8-DHF therapy (P < 0.0001). Additionally, 7,8-DHF significantly reduced the HFIRE-induced cerebral overproduction of beta-amyloid and proinflammatory cytokines in the cerebrospinal fluid (P < 0.0001) in dogs with HFIRE. ConclusionsRecovery of neurosurgical HFIRE injury in canine brain tissues can be accelerated by 7,8-DHT via ameliorating BBB disruption as well as cerebral overproduction of both beta-amyloid and proinflammatory cytokines.

Excitation and injury of adult ventricular cardiomyocytes by nano- to millisecond electric shocks

Intense electric shocks of nanosecond (ns) duration can become a new modality for more efficient but safer defibrillation. We extended strength-duration curves for excitation of cardiomyocytes down to 200 ns, and compared electroporative damage by proportionally more intense shocks of different duration. Enzymatically isolated murine, rabbit, and swine adult ventricular cardiomyocytes (VCM) were loaded with a Ca2+ indicator Fluo-4 or Fluo-5N and subjected to shocks of increasing amplitude until a Ca2+ transient was optically detected. Then, the voltage was increased 5-fold, and the electric cell injury was quantified by the uptake of a membrane permeability marker dye, propidium iodide. We established that: (1) Stimuli down to 200-ns duration can elicit Ca2+ transients, although repeated ns shocks often evoke abnormal responses, (2) Stimulation thresholds expectedly increase as the shock duration decreases, similarly for VCMs from different species, (3) Stimulation threshold energy is minimal for the shortest shocks, (4) VCM orientation with respect to the electric field does not affect the threshold for ns shocks, and (5) The shortest shocks cause the least electroporation injury. These findings support further exploration of ns defibrillation, although abnormal response patterns to repetitive ns stimuli are of a concern and require mechanistic analysis.

Electrical Injuries in Animals: Causes, Pathogenesis, and Morphological Findings.

Electrical injuries in animals occur most often accidentally. They comprise contact to various forms of currents, including alternating, rotary, or direct currents. Depending on various parameters of the current (including the type of circuit, voltage, current and duration of exposure) and conditions of the animal (such as wet or dry hair coat and pathway of current through the body), lesions may be absent or may include early or localized development of rigor mortis, signs of acute circulatory failure, or severe thermoelectrical burns. Such burns may present as external current marks, singed hair or feathers, metallization of the skin, or occasionally internal electroporation injury resulting in muscle necrosis, hemolysis, vascular damage with thrombosis, injury to brain and spinal cord, or skeletal fractures. Furthermore, lightning strikes occur regularly in grazing animals, which have greater risk of death from step potentials (ground current) in addition to direct strike and contact injury. Such cases may have no lesions, external signs of linear or punctate burns, keraunographic markings, or exit burns on the soles of the hooves or the coronary bands. Besides detailed information about the circumstances at the location where the animal was found, electrical injuries in animals require a thorough morphological workup, including additional investigations in conjunction with certain knowledge about the possible lesion spectrum.

Mechanisms of abiotic horizontal gene transfer: Comment on “Lightning-triggered electroporation and electrofusion as possible contributors to natural horizontal gene transfer” by Tadej Kotnik

Results suggestive of horizontal (lateral) gene transfer, attributed at the time to mutations, have been published since early 20th century [3]. Recent advances in gene sequencing technology have set on solid ground the idea that horizontal (lateral) gene transfer (HTG) is an important factor in evolution [10,11]. There is an increasing interest in this phenomenon [4,8,12], which is now thought to be largely responsible for the growing number of antibiotic resistant bacteria in the world [23]. In this issue, Kotnik, has developed a convincing hypothesis that lightning induced electroporation and electrofusion could be a mechanism for abiotic horizontal gene transfer [13]. Electroporation and electrofusion, described in detail in [13], are mechanisms that involve the application of certain electric fields across a cell and the consequent formation of defects in the cell membrane. These defects can be reversible or irreversible. Reversible electroporation is commonly used for gene transfection and cell fusion while irreversible electroporation is used for cell ablation [13]. Lightning produces a range of electric fields that include those for reversible electroporation, irreversible electroporation and fusion. It is reasonable to assume that microorganisms and viruses exposed to lightning could be exposed to a combination of electric fields that will produce abiotic horizontal gene transfer, as suggested by Kotnik [13]. In fact, the group of Rafael Lee has shown that lightning can cause irreversible electroporation injury to tissue [1,14]. The idea that lighting can cause abiotic lateral gene transfer is somewhat reminiscent of the idea of abiogenesis and the classical experiment of Miller and Urey [17,18]. Could other mechanisms proposed for abiogenesis be also involved in abiotic gene transfer? The clay hypothesis for abiogenesis proposed by Cairns-Smith [2] or the Wachtershauser [7] hypothesis for an iron sulfur world or Mulkidjanian’s [16] hypothesis of the Zn-world theory leads to thinking about micro and nano electroporation [5,6]. Clays and pyrite are structures with local electric charges. We have shown that when cells pass across surfaces with charges separated by nanometer distances the electric fields that are produced are sufficient to cause electroporation and electrolysis induced reversible and irreversible poration of the cell membrane [20,21]. We have also shown that it is sufficient for only part of the cell membrane to be exposed to electroporation fields for electroporation to occur [5,6]. Fig. 1 illustrates the electric field that would develop across a cell membrane when the cell flows across a substrate in which two small charges with a potential difference of 0.1 V are separated by a 100-nanometer gap. Even in the absence of native charges, an electrolytic process

Electroporation induced by internal defibrillation shock with and without recovery in intact rabbit hearts

Defibrillation shocks from implantable cardioverter defibrillators can be lifesaving but can also damage cardiac tissues via electroporation. This study characterizes the spatial distribution and extent of defibrillation shock-induced electroporation with and without a 45-min postshock period for cell membranes to recover. Langendorff-perfused rabbit hearts (n = 31) with and without a chronic left ventricular (LV) myocardial infarction (MI) were studied. Mean defibrillation threshold (DFT) was determined to be 161.4 ± 17.1 V and 1.65 ± 0.44 J in MI hearts for internally delivered 8-ms monophasic truncated exponential (MTE) shocks during sustained ventricular fibrillation (>20 s, SVF). A single 300-V MTE shock (twice determined DFT voltage) was used to terminate SVF. Shock-induced electroporation was assessed by propidium iodide (PI) uptake. Ventricular PI staining was quantified by fluorescent imaging. Histological analysis was performed using Masson's Trichrome staining. Results showed PI staining concentrated near the shock electrode in all hearts. Without recovery, PI staining was similar between normal and MI groups around the shock electrode and over the whole ventricles. However, MI hearts had greater total PI uptake in anterior (P < 0.01) and posterior (P < 0.01) LV epicardial regions. Postrecovery, PI staining was reduced substantially, but residual staining remained significant with similar spacial distributions. PI staining under SVF was similar to previously studied paced hearts. In conclusion, electroporation was spatially correlated with the active region of the shock electrode. Additional electroporation occurred in the LV epicardium of MI hearts, in the infarct border zone. Recovery of membrane integrity postelectroporation is likely a prolonged process. Short periods of SVF did not affect electroporation injury.

Structural and functional recovery of electropermeabilized skeletal muscle in-vivo after treatment with surfactant poloxamer 188

A critical requirement for cell survival after trauma is sealing of breaks in the cell membrane [M. Bier, S.M. Hammer, D.J. Canaday, R.C Lee, Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells, Bioelectromagnetics 20 (1999) 194–201; R.C. Lee, D.C. Gaylor, D. Bhatt, D.A. Israel, Role of cell membrane rupture in the pathogenesis of electrical trauma, J. Surg. Res. 44 (1988) 709–719; R.C. Lee, J.F. Burke, E.G. Cravalho (Eds.), Electrical Trauma: The Pathophysiology, Manifestations, and Clinical Management, Cambridge University Press, 1992; B.I. Tropea, R.C. Lee, Thermal injury kinetics in electrical trauma, J. Biomech. Engr. 114 (1992) 241–250; F. Despa, D.P. Orgill, J. Newalder, R.C Lee, The relative thermal stability of tissue macromolecules and cellular structure in burn injury, Burns 31 (2005) 568–577; T.A. Block, J.N. Aarsvold, K.L. Matthews II, R.A. Mintzer, L.P. River, M. Capelli-Schellpfeffer, R.L. Wollman, S. Tripathi, C.T. Chen, R.C. Lee, The 1995 Lindberg Award. Nonthermally mediated muscle injury and necrosis in electrical trauma, J. Burn Care and Rehabil. 16 (1995) 581–588; K. Miyake, P.L. McNeil, Mechanical injury and repair of cells, Crit. Care Med. 31 (2003) S496–S501; R.C. Lee, L.P. River, F.S. Pan, R.L. Wollmann, Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo, Proc. Natl. Acad. Sci. 89 (1992) 4524–4528; J.D. Marks, C.Y. Pan, T. Bushell, W. Cromie, R.C. Lee, Amphiphilic, tri-block copolymers provide potent membrane-targeted neuroprotection, FASEB J. 15 (2001) 1107–1109; B. Greenebaum, K. Blossfield, J. Hannig, C.S. Carrillo, M.A. Beckett, R.R. Weichselbaum, R.C. Lee, Poloxamer 188 prevents acute necrosis of adult skeletal muscle cells following high-dose irradiation, Burns 30 (2004) 539–547; G. Serbest, J. Horwitz, K. Barbee, The effect of poloxamer-188 on neuronal cell recovery from mechanical injury, J. Neurotrauma 22 (2005) 119–132]. The triblock copolymer surfactant Poloxamer 188 (P188) is known to increase the cell survival after membrane electroporation [R.C. Lee, L.P. River, F.S. Pan, R.L. Wollmann, Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo, Proc. Natl. Acad. Sci. 89 (1992) 4524–4528; Z. Ababneh, H. Beloeil, C.B. Berde, G. Gambarota, S.E. Maier, R.V. Mulkern, Biexponential parametrization of T2 and diffusion decay curves in a rat muscle edema model: Decay curve components and water compartments, Magn. Reson. Med. 54 (2005) 524–531]. Here, we use a rat hind-limb model of electroporation injury to determine if the intravenous administration of P188 improves the recovery of the muscle function. Rat hind-limbs received a sequence of either 0, 3, 6, 9, or 12 electrical current pulses (2 A, 4 ms duration, 10 s duty cycle). Magnetic resonance imaging (MRI) analysis, muscle water content and compound muscle action potential (CMAP) amplitudes were compared. Electroporation injury manifested edema formation and depression of the CMAP amplitudes. P188 (one bolus of 1 mg/ml of blood) was administrated 30 or 60 min after injury. Animals receiving P188 exhibited reduced tissue edema ( p < 0.05) and increased CMAP amplitudes ( p < 0.03). By comparison, treatment with 10 kDa neutral dextran, which produces similar serum osmotic effects as P188, had no effect on post-electroporation recovery. Noteworthy, the present results suggest that a single intravenous dose of P188 is effective to restore the structural integrity of damaged tissues with intact circulation.

Tc-99m pyrophosphate imaging of poloxamer-treated electroporated skeletal muscle in an in vivo rat model

Objective This study investigates whether 99mTc pyrophosphate (PYP) imaging provides a quantitative non-invasive assessment of the extent of electroporation injury, and of the effect of poloxamer in vivo on electroporated skeletal muscle. Methods High-voltage electrical shock was used to produce electroporation injury in an anesthetized rat's hind limb. In each experiment, the injured limb was treated intravenously by either poloxamer-188, dextran, or saline, and subsequently imaged with 99mTc PYP. The radiotracer's temporal behavior among the experimental groups was compared using curve fitting of time–activity curves from the dynamic image data. Results The washout kinetics of 99mTc PYP changed in proportion to the electric current magnitude that produced electroporation. Also, 99mTc PYP washout from electroporated muscle differed between poloxamer-188 treatment and saline treatment. Finally, 10-kDa dextran treatment of electroporated muscle altered 99mTc PYP washout less than poloxamer-188 treatment. Conclusions Behavior of 99mTc PYP in electroporated muscle appears to be an indicator of the amount of electroporation injury. Compared to saline, intravenous polaxamer-188 treatment reduced the amount of 99mTc PYP uptake. Coupled to results showing poloxamer-188 seals ruptured cellular membranes, lessens the extent of electroporation injury and improves cell viability, 99mTc PYP imaging appears to be a useful in vivo monitoring tool for the extent of electroporation injury.

Magnetic Resonance Imaging of Changes in Muscle Tissues after Membrane Trauma

A pure electroporation injury leads to cell membrane disruption and subsequent osmotic swelling of the tissue. The state of water in the injured area of a tissue is changed and differs from a healthy tissue. Magnetic resonance imaging (MRI), which is very sensitive to the quality of the interaction between mobile (water) protons and a restricted (protein) proton pool, is therefore a useful tool to characterize this injury. Here, we present a protocol designed to measure the difference between the values of the transverse magnetic relaxation time (T2) in MRIs of healthy and electrically injured tissue. In addition, we present a method to evaluate the two main contributions to the MRI contrast, the degree of structural alteration of the cellular components (including a major contribution from membrane pores), and edema. The approach is useful in assessing the level of damage that electric shocks produce in muscle tissues, in that edema will resolve in time whereas structural changes require active repair mechanisms.

Development and application of a small gamma camera

This work investigates the design, construction, and application of a portable gamma camera based on a single position‐sensitive photomultiplier tube (PSPMT) rather than an array of conventional phototubes. The PSPMT is an innovation in photomultiplier design that allows two‐dimensional position information to be obtained from a single phototube. PSPMT‐based portable gamma cameras can have several distinct advantages over portable systems using conventional technology: lower weight, reduced electronics, and smaller size. This design can be incorporated as modules in conjugate imaging, orthogonal view, or ring detector systems, or even in large‐area planar imagers. The PSPMT design is applicable for diagnostic clinical procedures and for basic biomedical research. Two small gamma camera (SGC) systems have been constructed. Computer simulations and physical measurements have been applied to the performance characterization of the SGC. The SGC incorporates a maximum‐likelihood position estimation scheme. The intrinsic resolution of the SGC is 1.7 mm FWHM (3.2 mm FWTM) at the center of the 48 mm×48 mm field of view. The SGC has been evaluated for several nuclear medicine imaging applications as well as laboratory research imaging. The clinical applications include planar and tomographic imaging. Radiotracer imaging with the SGC has been applied to the in vivo investigation of electroporation injury and tissue repair.