Papers by Barbara Hargrave
Europe PMC (PubMed Central), 2015
Electromanipulation of cells as a label free cell manipulation and characterization tool has gain... more Electromanipulation of cells as a label free cell manipulation and characterization tool has gained particular interest recently. However, the applicability of electromanipulation, particularly dielectrophoresis (DEP), to biological cells is limited to cells suspended in buffers containing lower amounts of salts relative to the physiological buffers. One might question the use of low conductivity buffers (LCB) for DEP separation, as cells are stressed in buffers lacking physiological levels of salt. In LCB, cells leak ions and undergo volume regulation. Therefore, cells exhibit time-dependent DEP response in LCB. In this work, cellular changes in LCB are assessed by dielectric spectroscopy, cell viability assay, and gene expression of chondrocytes and Jurkats. Results indicate leakage of ions from cells, increases in cytoplasmic conductivity, membrane capacitance and conductance. Separability factor, which defines optimum conditions for DEP cell separation, for the two cell types is calculated using the cellular dielectric data. Optimum DEP separation conditions change as cellular dielectric properties evolve in LCB. Genetic analyses indicate no changes in expression of ionic channel proteins for chondrocytes suspended in LCB. Retaining cellular viability might be important during dielectrophoretic separation, especially when cells are to be biologically tested at a downstream microfluidic component.
IFMBE proceedings, 2016
Gene electro transfer to the left ventricular myocardium is a promising technique for delivery of... more Gene electro transfer to the left ventricular myocardium is a promising technique for delivery of therapeutic genes for treatment of ischemia, myocardial infarction and heart failure directly to the effected myocardium. There are multiple variables to consider for efficient gene delivery and gene expression modulation, including electro transfer parameters, timing and duration of pulses relative to the electrocardiogram and ischemic state. Here we report establishing a small animal model and a large animal model for gene delivery to non-ischemic and ischemic left ventricular myocardium. Gene expression was evaluated histologically for location of expression within the myocardium as well as quantitatively via ELISA. We developed electro transfer electrodes and protocols that are safe and result robust gene expression. These animal models allow for evaluation of therapeutic potential of particular gene delivery as well as translation to clinical settings.
Life Sciences, 1990
ABSTRACT
PubMed, Dec 1, 2012
In the current study, we used the novel, nonchemical method of nanosecond pulsed electric fields ... more In the current study, we used the novel, nonchemical method of nanosecond pulsed electric fields (nsPEF) to investigate the efficiency of a protocol involving the in vivo treatment of the ischemic and reperfused heart and heart cells in culture with platelet-rich plasma (PRP). Associated with the restoration of blood flow to the ischemic tissue is a phenomenon referred to as "ischemic reperfusion injury." Clinically a type of reperfusion injury occurs during coronary bypass surgery once blood perfusion to the heart is restarted. Although the restoration of oxygen to ischemic myocardial cells is critical for tissue survival, reperfusion causes myocardial oxidative stress, attributable in part to the increased production of reactive oxygen species (ROS). Enhanced ROS production is associated with mitochondrial damage. Adult female New Zealand white rabbits were anesthetized and a left thoracotomy performed to expose the heart. The distal segment of the left anterior descending coronary artery was occluded for 15 minutes and then released so reperfusion of the tissue could occur. PRP (.21 mg/heart) or saline was injected into the ischemic area of the myocardium. Mechanical function of the left ventricle was analyzed using a Millar catheter attached to a Micro-Med Analysis System. H9c2 cells in culture were treated with 1 mL of nsPEF activated PRP (1.05 mg/flask) for 24 hours before analysis for ROS production or mitochondrial depolarization damage). The left ventricle contracted and relaxed faster and infarct size was reduced in hearts treated with PRP compared with saline. ROS production and mitochondrial depolarization were reduced in H9c2 cells treated with PRP and stimulated with hydrogen peroxide. These results provide evidence that nsPEFs can successfully be used to prepare PRP and that the PRP is functional in heart protection possibly by reducing ROS generation and stabilizing the mitochondria of the ischemic/reperfused heart.
Cardiovascular Toxicology, 2005
3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug that has gained immense ... more 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-kB (NF-kB) 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 ´ 10-3 M and 1 ´ 10-2 M, MDMA significantly enhanced NF-kB reporter activity compared with 0 M (medium only) control. This response was mitigated by cotransfection with IkB for 1 ´ 10-3 M but not 1 ´ 10-2 M MDMA. MDMA significantly increased intracellular calcium at concentrations of 1 ´ 10-3 M and 1 ´ 10-2 M and caused mitochondrial depolarization at 1 ´ 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°C to 65°C. Collectively, these results suggest that MDMA may be toxic to the heart through its ability to activate the myocardial NF-kB response, disrupt cytosolic calcium and mitochondrial homeostasis, and alter gene transcription.
Cardiovascular Toxicology, 2003
This study was designed to determine levels of NF-kB reporter gene activity and free radical gene... more This study was designed to determine levels of NF-kB reporter gene activity and free radical generation in cultured striated myocytes (H9C2 cells) exposed to cocaine or morphine in the presence of free radical scavengers. Cells were transiently transfected with a NF-kB reporter gene and changes in luciferase activity were detected by bioluminescence. Using confocal microscopy and 2',7'-dichlorofluorescin diacetate, cocaine-induced or morphine-induced free radicals were quantified in H9C2 cells. Cocaine and morphine (0-1 ´ 10-2 M) were tested separately. Cocaine but not morphine significantly activated NF-kB reporter gene activity in H9C2 cells. Overexpression of IkB inhibited NF-kB reporter activity at low (1 ´ 10-4 M) but not high (1 ´ 10-2 M) cocaine concentrations. Free radicals were generated in H9C2 cells stimulated with cocaine but not with morphine. The production of free radicals and NF-kB reporter gene activity could be blocked with N-acetylcysteine, glutathione, and, to a lesser extent, lipoic acid. The results suggest that cocaine induces free radical production, which leads to the activation of NF-kB signal transduction and possible inflammatory responses.
Cardiovascular Toxicology, 2005
To determine the cardiovascular molecular events associated with acute exposure to cocaine, the p... more To determine the cardiovascular molecular events associated with acute exposure to cocaine, the present study utilized in vivo analysis of left-ventricular heart function in adult rabbits, fluorescence confocal microscopy of fluo-2, rhod-2, (5-(and-6) carboxy 2',7' dichlorodihydrofluores-cein diacetate (carboxy-H2DCFDA), and JC-1 in H9C2 cells and gene expression microarray technology for analysis of gene activation in both rabbit ventricular tissue and H9C2 cells. In the rabbit, acute cocaine exposure (2 mg/kg) caused left-ventricular dysfunction and 0.1-10 mM cocaine increased cytosolic and mitochondrial calcium activity and mitochondrial membrane depolarization in H9C2 cells. A 3-min pretreatment of H9C2 cells by 10 µM verapamil, nifedipine, or nadolol inhibited calcium increases, but only 1 mM N-acetylcysteine (NAC) or 1 mM glutathione blocked mitochondrial membrane depolarization. Cocaine induced activation of genes in the rabbit heart and H9C2 cells including angiotensinogen, ADRB1, and c-reactive protein (CRP). In H9C2 cells, NAC pretreatment blocked cocaine-mediated increases in CRP, FAS, FAS ligand, and cytokine receptor-like factor1 (CRLF1) expression. Collectively, these data suggest that acute cocaine administration initiates cellular and genetic changes that, if chronically manifested, could cause cardiac deficits similar to those seen in heart failure and ischemia, such as ventricular dysfunction, cardiac arrhythmias, and cardiac remodeling.
Life Sciences, May 1, 1995
Although atrial natriuretic peptide (ANP) has been detected in the plasma of late gestation fetal... more Although atrial natriuretic peptide (ANP) has been detected in the plasma of late gestation fetal sheep, stimuli that control its secretion and its function in regulating fluid volume are unclear. To determine stimuli that increase ANP secretion and to ascertain whether the response is a function of gestation we studied 20 chronically cannulated fetal sheep. The fetuses were divided into 3 groups; Group I (age range 118-129 days), Group II (age range 129-141 days) and Group III (age range 127-137 days) gestation. Arterial pressure was increased to within a physiologic range by infusing phenylephrine at 6 micrograms/min at 0.1 ml/min into the inferior vena cava of the fetus (Groups I and II). Corrected for body weight fetuses in Group I received 2 micrograms/kg/min whereas fetuses in Group II received 1.66 micrograms/kg/min. In the fetuses in Group III preload to the right side of the heart was increased by inflating an occluder cuff was placed around the ductus arteriosus. Systemic mean arterial pressure (MAP) increased significantly in fetuses in Groups I and II. Plasma ANP concentrations increased significantly in all fetuses. Plasma Renin Activity (PRA) decreased significantly in the fetuses in Group II. These results suggest that phenylephrine infusion and closure of the ductus arteriosus in utero increase circulating ANP in fetal sheep.
Anatomy & physiology, 2013
Platelet gel improves left ventricular mechanical function in the ischemic reperfused Langendorff... more Platelet 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.
Molecular Therapy, May 1, 2015
American Journal of Physiology-endocrinology and Metabolism, Apr 1, 1986
Plasma adrenocorticotropin (ACTH) and cortisol (F) responses to 15-min intravenous infusions of s... more Plasma adrenocorticotropin (ACTH) and cortisol (F) responses to 15-min intravenous infusions of synthetic ovine corticotropin-releasing factor (CRF) were measured by radioimmunoassay in three groups of chronically cannulated ovine fetuses. Fetuses (n = 7) in group I were 107 +/- 2 days gestation (0.72 G) and fetuses (n = 7) in group II were 126 +/- 2 days gestation (0.86 G). Group III fetuses (n = 5) were studied at 5-day intervals during the last 2 wk of gestation. Resting fetal plasma ACTH levels were not significantly different among the three experimental groups. Infusions of CRF (50 ng X kg-1 X min-1) provided similar, significant increases in fetal plasma ACTH concentrations in the three groups. In the young fetuses (group I) the ACTH responses to 500 ng X kg-1 X min-1 infusions of CRF were greater than responses to 50 ng X kg-1 X min-1 infusions P less than 0.001. In group II both doses of CRF produced equivalent increases in ACTH that were comparable with the responses to 50 ng X kg-1 X min-1 infusions in Group I. This suggests that fetal ACTH responsiveness to large doses of CRF decreases between 0.72 and 0.86 G. Resting fetal plasma F levels increased during gestation (P less than 0.01). There was an F response (P less than 0.001) to CRF in all groups with greater responses observed in the older fetuses. This suggests that fetal adrenal responsiveness to ACTH or other peptides released by CRF increases during gestation.
Physiological Reports, Jul 1, 2015
Platelet-rich plasma is a therapeutic strategy used for accelerating wound healing of a wide rang... more Platelet-rich plasma is a therapeutic strategy used for accelerating wound healing of a wide range of tissues through the release of platelet growth factors. Here, we describe a nonchemical, safe method for preparing platelet-rich plasma using nanosecond-pulsed electric fields (nsPEFs) and investigated the effect of this platelet-rich plasma on reperfusion of blood in large skin flap or ischemic hind limb wounds in New Zealand White rabbits. Laser Doppler images of blood flow to the dorsal surface of skin flap wounds or to ischemic hind limb wounds were obtained from wounds treated with 0.9% saline or nanosecond-pulsed electric field prepared platelet-rich plasma (nsPRP). Reperfusion in the skin flap wounds was greater in the nsPRP-treated wounds than in the wounds treated with saline on postoperative days 3 (P < 0.001) and 21 (P < 0.03). Reperfusion in the ischemic hind-limb treated with nsPRP was greater than in the saline-treated limb on post-operative Day 3 (P < 0.001), post-operative week 1 (P < 0.025) and post-operative week 4 (P < 0.015). In the hind limb ischemic tissue, the number of endothelial cells, collagen, and cells containing vascular endothelial growth factor (VEGF) was greater in the nsPRP-treated tissue. These results demonstrate that nsPRP improves blood flow in large surgical skin wounds and in ischemic wounds.
Archives of Biochemistry and Biophysics, Mar 1, 2008
Nanosecond pulse stimulation of a variety of cells produces a wide range of physiological respons... more Nanosecond pulse stimulation of a variety of cells produces a wide range of physiological responses (e.g., apoptosis, stimulation of calcium (Ca 2+) fluxes, changes in membrane potential). In this study, we investigated the effect of nanosecond pulses, which generate intense electric fields (nsPEFs), on human platelet aggregation, intracellular free Ca 2+ ion concentration ([Ca 2+ ] i) and platelet-derived growth factor release. When platelet rich plasma was pulsed with one 300 ns pulse with an electric field of 30 kV/cm, platelets aggregated and a platelet gel was produced. Platelet aggregation was observed with pulses as low as 7 kV/cm with maximum effects seen with approximately 30 kV/cm. The increases in intracellular Ca 2+ release and Ca 2+ influx were dose dependent on the electrical energy density and were maximally stimulated with approximately 30 kV/cm. The increases in [Ca 2+ ] i induced by nsPEF were similar to those seen with thapsigargin but not thrombin. We postulate that nsPEF caused Ca 2+ to leak out of intracellular Ca 2+ stores by a process involving the formation of nanopores in organelle membranes and also caused Ca 2+ influx through plasma membrane nanopores. We conclude that nsPEFs dose-dependently cause platelets to rapidly aggregate, like other platelet agonists, and this is most likely initiated by the nsPEFs increasing [Ca 2+ ] i , however by a different mechanism.
IEEE Transactions on Dielectrics and Electrical Insulation, Oct 1, 2007
Electrical models for biological cells predict that reducing the duration of applied electrical p... more 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.
Gene Therapy, May 12, 2016
This study aimed to assess safety and therapeutic potential of gene electrotransfer as a method f... more This study aimed to assess safety and therapeutic potential of gene electrotransfer as a method for delivery of plasmid encoding vascular endothelial growth factor A to ischemic myocardium in a porcine model. Myocardial ischemia was induced by surgically occluding the left anterior descending coronary artery in swine. Gene electrotransfer following plasmid encoding vascular endothelial growth factor A injection was performed at four sites in the ischemic region. Control groups either received injections of the plasmid without electrotransfer or injections of saline vehicle. Animals were monitored for seven weeks and hearts were evaluated for angiogenesis, myocardial infarct size, and left ventricular contractility. Arteriograms suggest growth of new arteries as early as two weeks post treatment in electrotransfer animals. There is a significant reduction of infarct area and left ventricular contractility is improved in gene electrotransfer treated group compared to controls. There was no significant difference in mortality of animals treated with gene electrotransfer of plasmid encoding vascular endothelial growth factor A from control groups. Gene delivery of plasmid encoding vascular endothelial growth factor A to ischemic myocardium in a porcine model can be accomplished safely with potential for myocardial repair and regeneration.
Physiological Reports, Feb 1, 2016
Ischemia and reperfusion (I/R) of the heart is associated with biochemical and ionic changes that... more Ischemia and reperfusion (I/R) of the heart is associated with biochemical and ionic changes that result in cardiac contractile and electrical dysfunction. In rabbits, platelet-rich plasma activated using nanosecond pulsed electric fields (nsPRP) has been shown to improve left ventricular pumping. Here, we demonstrate that nsPRP causes a similar improvement in mouse left ventricular function. We also show that nsPRP injection recovers electrical activity even before reperfusion begins. To uncover the mechanism of nsPRP action, we studied whether the enhanced left ventricular function in nsPRP rabbit and mouse hearts was associated with increased expression of heat-shock proteins and altered mitochondrial function under conditions of oxidative stress. Mouse hearts underwent 30 min of global ischemia and 1 h of reperfusion in situ. Rabbit hearts underwent 30 min of ischemia in vivo and were reperfused for 14 days. Hearts treated with nsPRP expressed significantly higher levels of Hsp27 and Hsp70 compared to hearts treated with vehicle. Also, pretreatment of cultured H9c2 cells with nsPRP significantly enhanced the "spare respiratory capacity (SRC)" also referred to as "respiratory reserve capacity" and ATP production in response to the uncoupler FCCP. These results suggest a cardioprotective effect of nsPRP on the ischemic heart during reperfusion.
Pathogens and Disease, Oct 23, 2015
Acinetobacter baumannii is an environmentally resilient healthcare-associated opportunistic patho... more Acinetobacter baumannii is an environmentally resilient healthcare-associated opportunistic pathogen responsible for infections at many body sites. In the last 10 years, clinical strains resistant to many or all commonly used antibiotics have emerged globally. With few antimicrobial agents in the pharmaceutical pipeline, new and alternative agents are essential. Platelets secrete a large number of proteins, including proteins with antimicrobial activity. In a previous study, we demonstrated that donor platelet supernatants and plasma significantly inhibited the growth of a reference strain of A. baumannii in broth and on skin. This inhibition appeared to be unrelated to the platelet activation state. In this study, we demonstrate that this growth inhibition extends to clinical multidrug resistant isolates. We also demonstrate that there is no relationship between this activity and selected platelet-derived antimicrobial proteins. Instead, the donor plasma components complement and alpha-2 macroglobulin are implicated.
Life Sciences, Apr 1, 1995
To assess the effects of cocaine, administered to the ewe, on the secretion of atria1 natriuretic... more To assess the effects of cocaine, administered to the ewe, on the secretion of atria1 natriuretic peptide (ANP), Plasma Renin Activity (PRA) and hypoxanthine in the fetus we studied 6 chronically cannulated sheep fetuses late in gestation. The ewe was given an intravenous injection of cocaine (2 mg/kg). Maternal and fetal arterial blood samples were withdrawn prior to the injection and at 2, 5, 10, 15, 45 and 60 min after the injection for the measurement of ANP, PRA and hypoxanthine. Fetal arterial blood pressure (MAP), plasma ANP and protein levels increased and pH and p0, decreased after cocaine was administered to the ewe. Fetal plasma hypoxanthine and PRA did not change. These results suggest that cocaine administration to the ewe is associated with fetal hypertension, hypoxemia and acidemia all of which may serve as stimuli for the secretion of ANP.
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Papers by Barbara Hargrave