Electrical models for biological cells predict that reducing the duration of applied electrical pulses to values below the charging time of the outer cell membrane (which is on the order of 100 ns for mammalian cells) causes a strong increase in the probability of electric field interactions with intracellular structures due to displacement currents. For electric field amplitudes exceeding MV/m, such pulses are also expected to allow access to the cell interior through conduction currents flowing through the permeabilized plasma membrane. In both cases, limiting the duration of the electrical pulses to nanoseconds ensures only nonthermal interactions of the electric field with subcellular structures. This intracellular access allows the manipulation of cell functions. Experimental studies, in which human cells were exposed to pulsed electric fields of up to 30 MV/m amplitude with durations as short as 3 ns, have confirmed this hypothesis and have shown that it is possible to selectively alter the behavior and/or survival of cells. Observed nanosecond pulsed effects at moderate electric fields include intracellular release of calcium and enhanced gene expression, which could have long term implications on cell behavior and function. At increased electric fields, the application of nanosecond pulses induces a type of programmed cell death, apoptosis, in biological cells. Cell survival studies with 10 ns pulses have shown that the viability of the cells scales inversely with the electrical energy density, which is similar to the "dose" effect caused by ionizing radiation. On the other hand, there is experimental evidence that, for pulses of varying durations, the onset of a range of observed biological effects is determined by the electrical charge that is transferred to the cell membrane during pulsing. This leads to a similarity law for nanosecond pulse effects, with the product of electric field intensity, pulse duration, and the square root of the number of pulses as the similarity parameter. The similarity law allows one not only to predict cell viability based on pulse parameters, but has also been shown to be applicable for inducing platelet aggregation, an effect which is triggered by internal calcium release. Applications for nanosecond pulse effects cover a wide range: from a rather simple use as preventing biofouling in cooling water systems, to advanced medical applications, such as gene therapy and tumor treatment. Results of this continuing research are leading to the development of wound healing and skin cancer treatments, which are discussed in some detail.

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Background Therapeutic delivery of angiogenic growth factors is a promising approach for treating ischemia observed in skin flaps and chronic wounds.Several studies have demonstrated that vascular endothelial growth factor(VEGF) helps mitigate skin flap necrosis by facilitating angiogenesis. The present study aimed to demonstrate an electrically-mediated nonviral gene delivery approach using a non-invasive multi-electrode array (MEA) for effective treatment of ischemic skin flaps.Methods We used a standard random dorsal skin flap model in rats. The study aimed to determine the optimal treatment sites on the skin flap, optimal plasmid dose and timing of the treatment for preventing distal flap necrosis.Results We determined that two treatment sites on the ischemic flap with a plasmid dose of 50-100 μg per treatment site proved adequate to prevent 95% flap necrosis, and that this was significantly better than the no treatment or injection only group. A 2-day window was critical to deliver the VEGF to achieve flap survival and prevent necrosis. Histological examination demonstrated minimal electro transfer associated tissue damage.Conclusions Our results demonstrate that MEA can be used as a non-invasive physical gene delivery method for plasmid VEGF, resulting in a significant reduction of necrosis in ischemic wounds. We propose that this method could be translated into a potential therapeutic approach to deliver growth factors to prevent ischemia in cases of chronic wounds, burns and skin flap necrosis.

In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using 3 different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the ECG were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on the anterior wall of the left ventricle were treated. Animals were euthanized 48 hours after injection and electroporation and gene expression was determined. Results were compared to sites in the heart that received plasmid injection but no electric pulses or were not treated. Gene expression was higher in all electroporated sites when compared to injection only sites demonstrating the robustness of this approach. Our results provide evidence that in vivo electroporation can be a safe and effective non-viral method for delivering genes to the heart, in vivo.

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug that has gained immense popularity among teenagers and young adults. The cardiovascular toxicological consequences of abusing this compound have not been fully characterized. The present study utilized a transient transfection/dual luciferase genetic reporter assay, fluorescence confocal microscopy, and gene expression macroarray technology to determine nuclear factor-kappaB (NF-kappaB) activity, intracellular calcium balance, mitochondrial depolarization, and gene transcription profiles, respectively, in cultured rat striated cardiac myocytes (H9c2) exposed to MDMA. At concentrations of 1 x 10(-3) M and 1 x 10(-2) M, MDMA significantly enhanced NF-kappaB reporter activity compared with 0 M (medium only) control. This response was mitigated by cotransfection with IkappaB for 1 x 10(-3) M but not 1 x 10(-2) M MDMA. MDMA significantly increased intracellular calcium at concentrations of 1 x 10(-3) M and 1 x 10(-2) M and caused mitochondrial depolarization at 1 x 10(-2) M. MDMA increased the transcription of genes that are considered to be biomarkers in cardiovascular disease and genes that respond to toxic insults. Selected gene activation was verified via temperature-gradient RT-PCR conducted with annealing temperatures ranging from 50 degrees C to 65 degrees C. Collectively, these results suggest that MDMA may be toxic to the heart through its ability to activate the myocardial NF-kappaB response, disrupt cytosolic calcium and mitochondrial homeostasis, and alter gene transcription.

Myocardial ischemia can damage heart muscle and reduce the heart's pumping efficiency. This study used an ischemic swine heart model to investigate the potential for gene electro transfer of a plasmid encoding vascular endothelial growth factor for improving perfusion and, thus, for reducing cardiomyopathy following acute coronary syndrome. Plasmid expression was significantly greater in gene electro transfer treated tissue compared to injection of plasmid encoding vascular endothelial growth factor alone. Higher gene expression was also seen in ischemic versus non-ischemic groups with parameters 20 Volts (p<0.03), 40 Volts (p<0.05), and 90 Volts (p<0.05), but not with 60 Volts (p<0.09) while maintaining a pulse width of 20 milliseconds. The group with gene electro transfer of plasmid encoding vascular endothelial growth factor had increased perfusion in the area at risk compared to control groups. Troponin and creatine kinase increased across all groups, suggesting equivalent ischemia in all groups prior to treatment. Echocardiography was used to assess ejection fraction, cardiac output, stroke volume, left ventricular end diastolic volume, and left ventricular end systolic volume. No statistically significant differences in these parameters were detected during a 2-week time period. However, directional trends of these variables were interesting and offer valuable information about the feasibility of gene electro transfer of vascular endothelial growth factor in the ischemic heart. The results demonstrate that gene electro transfer can be applied safely and can increase perfusion in an ischemic area. Additional study is needed to evaluate potential efficacy.

SummaryPlatelet gel improves left ventricular mechanical function in the ischemic reperfused Langendorff rabbit heart, decreases ROS production and mitochondrial depolarization in HUV-EC cells (Homo sapiens, human Umbilical Vein Endothelium) in vitro, protects catalase and superoxide dismutase when prepared using nanosecond pulsed electric fields rather than bovine thrombin and decreases the metalloproteinase MMP-2 and increases the metalloproteinase inhibitor TIMP-1 in H9c2 cells (Rattus norvegicus H9c2 cardiac myoblast cells) in vitro. IntroductionIn patients admitted to a hospital with myocardial infarction, some of the myocardial tissue is irreversibly damaged by necrosis, but a larger part is under ischemic stress and may still be saved by appropriate intervention. It is essential to restore coronary flow to the ischemic region (reperfusion), but if no additional measures are taken, reperfusion causes the production of reactive oxygen species (ROS), which can lead to substantial tissue death (reperfusion injury) [1].Autologous Platelet-rich plasma (PRP) or platelet gel is emerging as a biological tool to reduce cardiovascular reperfusion injury in animal experiments [2]. PRP is made from an animal's own whole blood (autologous). The platelets are concentrated and the release of growth factors from the platelets is induced, typically by combining the platelet concentrate with bovine thrombin and calcium. The resulting "activated" PRP contains a super-physiological concentration of growth factors, cytokines, and other proteins [3]. Injecting it into the myocardium after infarction and prior to reperfusion significantly improves left ventricular mechanical function during reperfusion in vivo [4] in rabbit hearts and promotes angiogenesis and mitogenesis in the sheep heart when injected 3 weeks after coronary ligation and evaluated 9 weeks later [5]. In addition to the growth and healing promoting proteins, PRP provides a scaffold that traps cells such as stem cells in the region of injury giving the proteins time to help the cells differentiate into cardiac myocytes. Several problems have so far precluded the use of activated PRP in the cardiovascular system of patients: 1) injecting a PRP that contains red blood cells into the heart may lead to thrombi that can cause stroke or myocardial infarction; 2) thrombin used to prepare PRP can cause serious bleeding abnormalities in some patients who develop antibodies against certain clotting factors as well as other adverse events 3 (thrombin, itself, causes the generation of ROS and 4) lack of a mechanism explaining the mode of action of activated PRP. AbstractBackground: The prompt restoration of blood flow (reperfusion) to the ischemic myocardium after an acute myocardial infarction is critical to the survival of non damaged heart tissue. However, reperfusion is responsible for additional myocardial damage. Our objective was to investigate the role of autologous platelet rich plasma or platelet gel prepared using nanosecond pulsed electric fields (nsPEFs) in imp...

Activation of human platelets produces a gel-like substance referred to as platelet rich plasma or platelet gel. Platelet gel is used clinically to promote wound healing; it also exhibits antimicrobial properties that may aid in the healing of infected wounds. The purpose of this study was to quantify the efficacy of human platelet gel against the opportunistic bacterial wound pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus on skin. These opportunistic pathogens may exhibit extensive antibiotic resistance, necessitating the development of alternative treatment options. The antimicrobial efficacy of platelet gel supernatants was quantified using an in vitro broth dilution assay, an ex vivo inoculated skin assay, and in an in vivo skin decontamination assay. Human platelet gel supernatants were highly bactericidal against A. baumannii and moderately but significantly bactericidal against S. aureus in vitro and in the ex vivo skin model. P. aeruginosa was not inactivated in vitro; a low but significant inactivation level was observed ex vivo. These supernatants were quite effective at inactivating a model organism on skin in vivo. These results suggest application of platelet gel has potential clinical applicability, not only in the acceleration of wound healing, but also against relevant bacteria causing wound infections.