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ABSTRACT: Mediated by reactive oxygen species, the damaging effects of high-intensity ionizing irradiation on tissues are dose, frequency, oxygen concentration, and tissue property dependent. Intense ionizing irradiation exposure may cause rapid cellular necrosis by peroxidation of membrane lipids leading to membrane disruption. This leads to a loss of the transmembrane ionic gradients and a subsequent depletion of the cellular ATP store, followed by cellular generation of reactive oxygen species. When membrane disruption is extensive, acute cellular necrosis follows. Triblock copolymer surfactants, such as Poloxamer 188 (P188), are able to seal damaged rhabdomyocyte membranes, increasing post-irradiation viability. Separated rat rhabdomyocytes were exposed to 40 Gy (Co 1.5 Gy min) irradiation and treated at 20 min intervals with combination permutations of P188, N-acetylcysteine (NAC), and Mg-ATP. Cell viability at 18 and 48 h was determined using Calcein-AM and Ethidium Homodimer-1 staining. At 18 h after irradiation, the combined administration of P188, ATP, and NAC restored cell viability rates to near sham-exposed levels of 60%. At 48 h post-irradiation, cell viability dropped substantially to the 7-20% range, regardless of attempted intervention. Nevertheless, the combination of P188, ATP, and NAC more than doubled cell viability at the 48-h time point. Neither 8 kDa polyethylene glycol nor 10 kDa neutral dextran was as effective in enhancing cell viability. These results indicate that antioxidants and cellular energy substrates improve the efficacy of membrane-sealing copolymer surfactants in prolonging cellular viability following massive radiation exposure.
Health physics 12/2011; 101(6):731-8. · 0.92 Impact Factor
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ABSTRACT: Ischemia-reperfusion injury can activate pathways generating reactive oxygen species, which can injure cells by creating holes in the cell membranes. Copolymer surfactants such as poloxamer 188 are capable of sealing defects in cell membranes. The authors postulated that a single-dose administration of poloxamer 188 would decrease skeletal myocyte injury and mortality following ischemia-reperfusion injury.
Mice underwent normothermic hind-limb ischemia for 2 hours. Animals were treated with 150 microl of poloxamer 188 or dextran at three time points: (1) 10 minutes before ischemia; (2) 10 minutes before reperfusion; and (3) 2 or 4 hours after reperfusion. After 24 hours of reperfusion, tissues were analyzed for myocyte injury (histology) and metabolic dysfunction (muscle adenosine 5'-triphosphate). Additional groups of mice were followed for 7 days to assess mortality.
When poloxamer 188 treatment was administered 10 minutes before ischemia, injury was reduced by 84 percent, from 50 percent injury in the dextran group to 8 percent injury in the poloxamer 188 group (p < 0.001). When administered 10 minutes before reperfusion, poloxamer 188 animals demonstrated a 60 percent reduction in injury compared with dextran controls (12 percent versus 29 percent). Treatment at 2 hours, but not at 4 hours, postinjury prevented substantial myocyte injury. Preservation of muscle adenosine 5'-triphosphate paralleled the decrease in myocyte injury in poloxamer 188-treated animals. Poloxamer 188 treatment significantly reduced mortality following injury (10 minutes before, 75 percent versus 25 percent survival, p = 0.0077; 2 hours after, 50 percent versus 8 percent survival, p = 0.032).
Poloxamer 188 administered to animals decreased myocyte injury, preserved tissue adenosine 5'-triphosphate levels, and improved survival following hind-limb ischemia-reperfusion injury.
Plastic and reconstructive surgery 06/2010; 125(6):1651-60. · 2.74 Impact Factor
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Surgery 08/2009; 146(1):40-51. · 3.10 Impact Factor
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ABSTRACT: Electronic stun devices (ESD) often used in law enforcement, military action or self defense can induce total body uncoordinated muscular activity, also known as electromuscular incapacitation (EMI). During EMI the subject is unable to perform purposeful or coordinated movements. The mechanism of EMI induction has not been reported, but has been generally thought to be direct muscle and nerve excitation from the fields generated by ESDs. To determine the neuromuscular mechanisms linking ESD to induction of EMI, we investigated EMI responses using an anesthetized pig model. We found that EMI responses to ESD application can best be simulated by simultaneous stimulation of motor and sensory peripheral nerves. We also found that application of local anesthetics limited the response of ESD to local muscle stimulation and abolished the total body EMI response. Stimulation of the pure sensory peripheral nerves or nerves that are primarily motor nerves induced muscle responses that are consistent with well defined spinal reflexes. These findings suggest that the mechanism of ESD-induced EMI is mediated by excitation of multiple simultaneous spinal reflexes. Although direct motor-neuron stimulation in the region of ESD contact may significantly add to motor reactions from ESD stimulation, multiple spinal reflexes appear to be a major, and probably the dominant mechanism in observed motor response.
Bioelectromagnetics 05/2009; 30(5):411-21. · 1.84 Impact Factor
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ABSTRACT: Neuropsychological studies in electrical injury patients have reported deficits in attention, learning, and working memory, but the neural substrates of these deficits remain poorly characterized. In this study we sought to examine whether electrical injury subjects demonstrate abnormal patterns of brain activation during working memory and procedural learning tasks. Fourteen electrical injury subjects and fifteen demographically matched healthy control subjects performed a spatial working memory paradigm and a procedural learning paradigm during functional MRI studies. For the spatial working memory task, electrical injury patients exhibited significantly greater activation in the middle frontal gyrus and motor and posterior cingulate cortices. Increased activation in EI subjects also was observed on a visually-guided saccade task in several sensorimotor regions, including the frontal and parietal eye fields and striatum. On the procedural learning task, electrical injury patients exhibited significantly less activation in the middle frontal gyrus, anterior cingulate cortex, and frontal eye fields than controls. This is the first study to document task-dependent, system-level cortical and subcortical dysfunction in individuals who had experienced an electrical shock trauma.
Journal of neurotrauma 04/2009; 26(10):1815-22. · 4.25 Impact Factor
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ABSTRACT: Burn injury leads to a direct damaging effect on cells, disrupting the assembly of the cell and denaturing proteins. Although modern medicine has significantly improved the survival of burn victims, a method to treat injury at the cellular level is presented. In particular, the cell membrane is most vulnerable to heat injury. Copolymer surfactants have been shown to repair the cell membrane, and agents such as poloxamer 188 have demonstrated this effect in numerous studies. Furthermore, copolymer surfactants have been shown to act as molecular chaperones, allowing denatured proteins to regain their native confirmation. Pharmaceutical agents may be developed to repair the cell membrane and refold proteins, mimicking endogenous repair mechanisms and salvaging cells that would otherwise be lost.
The Journal of craniofacial surgery 07/2008; 19(4):903-6. · 0.81 Impact Factor
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ABSTRACT: On the basis of elastic light scattering, we have compared the capacity of the multi-block, surfactant copolymers Poloxamer 108 (P108), Poloxamer 188 (P188), and Tetronic 1107 (T1107), of average molecular weight 4700, 8400, and 15,000, respectively, with that of polyethylene glycol (PEG, molecular weight 8000) to suppress aggregation of heat-denatured hen egg white lysozyme (HEWL) and bovine serum albumin (BSA). We also compared the capacity of P188 to that of PEG to suppress aggregation of carboxypeptidase A denatured in the presence of trifluoroethanol and to facilitate recovery of catalytic activity. In contrast to the multi-block copolymers, PEG had no effect in inhibiting aggregation of HEWL or of carboxypeptidase A with the recovery of catalytic activity. At very high polymer:protein ratios (>or=10:1), PEG increased aggregation of heat-denatured HEWL and BSA, consistent with its known properties to promote macromolecular crowding and crystallization of proteins. At a polymer:protein ratio of 2:1, the tetra-block copolymer T1107 was the most effective of the three surfactant copolymers, completely suppressing aggregation of heat-denatured HEWL. At a T1107:BSA ratio of 10:1, the poloxamer suppressed aggregation of heat-denatured BSA by 50% compared to that observed in the absence of the polymer. We showed that the extent of suppression of aggregation of heat-denatured proteins by multi-block surfactant copolymers is dependent on the size of the protein and the copolymer:protein molar ratio. We also concluded that at least one of the tertiary nitrogens in the ethylene-1,2-diamine structural core of the T1107 copolymer is protonated, and that this electrostatic factor underlies its capacity to suppress aggregation of denatured proteins more effectively than nonionic, multi-block poloxamers. These results indicate that amphiphilic, surfactant, multi-block copolymers are efficient as additives to suppress aggregation and to facilitate refolding of denatured proteins in solution. Because of these properties, multi-block, surfactant copolymers are suitable for application to a variety of biotechnological and biomedical problems in which refolding of denatured or misfolded proteins and suppression of aggregation are important objectives.
Biochimica et Biophysica Acta 01/2008; 1780(1):7-15. · 4.66 Impact Factor
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ABSTRACT: 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.
Biochimica et Biophysica Acta 06/2007; 1768(5):1238-46. · 4.66 Impact Factor
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ABSTRACT: This study investigates whether (99m)Tc 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.
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 (99m)Tc PYP. The radiotracer's temporal behavior among the experimental groups was compared using curve fitting of time-activity curves from the dynamic image data.
The washout kinetics of (99m)Tc PYP changed in proportion to the electric current magnitude that produced electroporation. Also, (99m)Tc PYP washout from electroporated muscle differed between poloxamer-188 treatment and saline treatment. Finally, 10-kDa dextran treatment of electroporated muscle altered (99m)Tc PYP washout less than poloxamer-188 treatment.
Behavior of (99m)Tc 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 (99m)Tc PYP uptake. Coupled to results showing poloxamer-188 seals ruptured cellular membranes, lessens the extent of electroporation injury and improves cell viability, (99m)Tc PYP imaging appears to be a useful in vivo monitoring tool for the extent of electroporation injury.
Burns 10/2006; 32(6):755-64. · 1.96 Impact Factor
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ABSTRACT: We investigated the ability of certain triblock copolymer surfactant poloxamers of the form polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO), to prevent formation of stable aggregates of heat denatured hen egg lysozyme. Differential scanning calorimetry (DSC) and synchrotron small angle x-ray scattering (SAXS) experiments were performed to study the thermodynamics and solution structures of lysozyme at temperatures between 20 and 90 degrees C in the presence and absence of poloxamers with various molecular weights (8.4-14.3 kDa), but similar hydrophile/hydrophobe (PEO:PPO) ratio of 80%. Poloxmer 188 was found to be very effective in preventing aggregation of heat denatured lysozyme and those functioned as a synthetic surfactant, thus enabling them to refold when the conditions become optimal. For comparison, we measured the ability of 8 kDa polyethylene glycol (PEG) to prevent lysozyme aggregation under same conditions. The results of these studies suggest that poloxamers are more efficient than PEG in preventing aggregation of heat denaturated lysozyme. To achieve equivalence, more than an order of magnitude higher concentration of PEG concentration was needed. Apparently, the presence of a hydrophobic segment in the poloxamers increases their ability to target the hydrophobic region of the unfolded proteins and protect them from self association. Given their biocompatibility and the low concentrations at which they effectively facilitate refolding of denatured proteins, they may be useful in the treatment of burns and other conditions resulting in the denaturation of proteins.
Annals of Biomedical Engineering 08/2006; 34(7):1190-200. · 2.37 Impact Factor
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ABSTRACT: Effects of imposed large supraphysiological transmembrane potential (TP) pulses on channel proteins, particularly on the voltage-gated Na channels, were investigated. Voltage clamp techniques were used to deliver both shock and stimulation pulses, and to monitor changes in the channel functions. Our experimental results indicated that more than one 4 ms duration TP shock of -450 mV resulted in electroconformational denature of voltage-gated Na channels. This resulted in functional reductions in muscle cells' excitability. We quantified the TP shock-induced decrease in the Na channel currents, compared the pre- and post-shocked Na channel currents' voltage dependency, and studied the reversibility of the electroconformationally denatured ion channel proteins. These observations are particularly relevant to the problem of explaining the neuromuscular damage following high voltage electrical shock injuries despite no evidence of a thermal injury component.
Burns 03/2006; 32(1):52-9. · 1.96 Impact Factor
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ABSTRACT: The clinical presentation of electrical injury commonly involves physical, cognitive, and emotional complaints. Neuropsychological studies, including case reports, have indicated that electrical injury (EI) survivors may experience a broad range of impaired neuropsychological functions, although this has not been clarified through controlled investigation. In this study, we describe the neuropsychological test findings in a series of 29 EI patients carefully screened and matched to a group of 29 demographically similar healthy electricians. Participants were matched by their estimated premorbid intellectual ability. Multivariate analysis of variance was used to assess group differences in the following neuropsychological domains: attention and mental speed, working memory, verbal memory, visual memory, and motor skills. EI patients performed significantly worse on composite measures of attention/mental speed and motor skills, which could not be explained by demographic differences, injury parameters, litigation status, or mood disturbance. Results suggest that cognitive changes do occur in patients suffering from electrical injury.
Journal of the International Neuropsychological Society 02/2006; 12(1):17-23. · 2.76 Impact Factor
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ABSTRACT: The triblock copolymer poloxamer 188 is a non-cytotoxic, nonionic surfactant with both hydrophobic and hydrophilic domains. We show that P188 is able to facilitate the recovery of catalytic activity of heat-denatured lysozyme in dilute solution at low molar ratios of P188:enzyme. Heat-denatured enzyme retained 55% of native activity. After treatment with P188, the enzyme's activity was 85% of native. Because of the low molar ratios used and the non-cytotoxic nature of the compound, P188 may be of potential use in burn therapy.
Annals of the New York Academy of Sciences 01/2006; 1066:321-7. · 3.15 Impact Factor
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Raphael C Lee
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ABSTRACT: The molecular architecture of biological systems is heavily influenced by the highly polar interactions of water. Thus, macromolecules such as proteins that are highly water soluble must be electrically polar. Energy generation methods needed to support cell metabolic processes depend on compartmentalizing mobile ions and thus require electrical ion transport barriers such as membranes. One consequence of these biological design constraints is vulnerability to injury by electrical forces. Supraphysiological electric forces cause damage to cells and tissues by disrupting cell membranes and altering the conformation of biomolecules. In addition, prolonged passage of electrical current leads to damage by thermal mechanisms. This review will focus on the non-thermal effects.
Annals of the New York Academy of Sciences 01/2006; 1066:85-91. · 3.15 Impact Factor
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ABSTRACT: The volume fraction occupied by the dry matter of the cell can be as large as 40%, of which more than half (approximately 60%) are proteins. Thus, cellular proteins and protein assemblies occupy a large volume that can have a profound effect on their own native-state stabilities and on their unfolding/refolding rates. In addition, macromolecular crowding can change the properties of a significant fraction of the water in the cell. We review features of the molecular crowding effect which are relevant for describing the microscopic mechanism of thermal injuries.
Annals of the New York Academy of Sciences 01/2006; 1066:54-66. · 3.15 Impact Factor
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ABSTRACT: Our cells and tissues are challenged constantly by exposure to extreme conditions that cause acute and chronic stress. Wounding at the cellular level is a common event, and results from cell exposure to supra-physiologic forces, or is the consequence of action by reactive chemical agents. An individual cellular wound results from either the alteration of protein or DNA structure, or the disruption of molecular assemblies, the most important of which is the cell's membranes. Tissue healing at the macroscopic level is a complex and coordinated process involving many different cell types while, in contrast, the wounds of individual cells heal primarily via biomolecular interactions. Like tissue wound healing, cellular wound healing involves the upregulation or acceleration of processes that are constitutively expressed in routine physiologic repair of cellular structures In addition, recent advances have been made in the identification of pharmaceutical strategies to aid the cellular repair response. Many of these strategies offer promise for augmenting the already present cellular repair mechanisms.
Annals of the New York Academy of Sciences 01/2006; 1066:295-309. · 3.15 Impact Factor
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ABSTRACT: Crowding can substantially affect the transition of a protein between its native (N) and unfolded (U) states via volume exclusion effects. Also, it influences considerably the aggregation (A) of unfolded proteins. To examine the details, we developed an approach for computing the kinetic rates of the process N <--> U --> A in which the concentration of the protein is explicitly taken into account. We then compute the relative change with temperature of the protein denaturation for various fractional volume occupancies and partition of proteins in solution. The analysis indicates that, in protein solutions in which the average distance between proteins is comparable with the radius of gyration of an unfolded protein, steric effects increase the stability of the proteins which are in compact, native states. In heterogeneous protein solutions containing various types of proteins with different thermal stabilities, the unfolding of the most thermolabile proteins will increase the stability of the other proteins. The results shed light on the way proteins change the thermal stability of a cell as they unfold and aggregate. This study may be valuable in questions related to the dynamics of thermal injuries.
Annals of Biomedical Engineering 08/2005; 33(8):1125-31. · 2.37 Impact Factor
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ABSTRACT: The pathogenesis and progression of wound-healing involve intricate pathways and numerous chemical mediators. This remains an area of intense study as undesirable results of this process, such as hypertrophic scars and keloids, can result in significant morbidity. These lesions are distinct in their characteristics, although they are similar in their distribution in patients with darker skin colors. There is a robust inflammatory mechanism behind the formation of hypertrophic scars and keloids. Furthermore, their development may be intimately related to vitamin D-3, which has been shown to be a powerful anti-inflammatory agent. This chemical is made in the skin, whose production is influenced by various factors of which the amount of melanin is a crucial one. More specifically, an increase in pigmentation has been shown to decrease the amount of vitamin D-3 synthesis in the skin. Thus, this paper proposes the hypothesis linking the propensity of inflammation and subsequent scarring in darker-skinned individuals to the reduced levels of vitamin D-3 production in their skin.
Journal of the National Medical Association 08/2005; 97(7):1004-9. · 1.16 Impact Factor
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Neurorehabilitation 02/2005; 20(1):53-62. · 1.63 Impact Factor
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ABSTRACT: When a cell's transmembrane potential is increased from a physiological one to more than 370 mV, the transmembrane current increases more than hundredfold within a millisecond. This is due to the formation of conductive pores in the membrane. We construct a model in which we conceive of pore formation as a voltage sensitive chemical reaction. The model predicts the logarithm of the pore formation rate to increase proportionally to the square of the voltage. We measure currents through frog muscle cell membranes under 8 ms pulses of up to 440 mV. The experimental data appear consistent with the model.
Bioelectromagnetics 01/2005; 25(8):634-7. · 1.84 Impact Factor
