A new model for mild blast injury utilizing Drosophila melanogaster - biomed 2013.

Current models for blast injury involve the use of mammalian species, which are costly and require extensive monitoring and housing, making it difficult to generate large numbers of injuries. The fruit fly, Drosophila melanogaster, has been utilized for many models of human disease including neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. In this study, a model of blast injury was designed based on Drosophila, to provide a mechanism to investigate blast injury in large numbers and assess biochemical mechanisms of brain injury. Such studies may be used to identify specific pathways involved in blast-associated neurodegeneration, allowing more effective use of mammalian models. A custom-built blast wave simulator (ORA Inc.), comprised of a driver, test section, and wave eliminator, was used to create a blast wave. An acetate membrane was placed between the driver and the rectangular test section before compressed helium caused the membrane to rupture creating the blast wave. Membrane thickness correlates with the blast wave magnitude, which averaged 120 kPa for this experiment. Pressure sensors were inserted into the side of the tube in order to quantify the level of overpressure that the flies were exposed to. Five day old flies were held in a rectangular enclosed mesh fixture (10 flies per enclosure) which was placed in the center of the test section for blast delivery. Sham controls were exposed to same conditions with exception of blast. Lifespan and negative geotaxis, a measurement of motor function, was measured in flies after blast injury. Mild blast resulted in death of 28% of the flies. In surviving flies, motor function was initially reduced, but flies regained normal function by 8 days after injury. Although surviving flies regained normal motor function, flies subjected to mild blast died earlier than uninjured controls, with a 15.4% reduction in maximum lifespan and a 17% reduction in average lifespan, mimicking the scenario observed in humans exposed to mild blast. Although further work is needed, results suggest that utilizing Drosophila as a blast model may provide a rapid, effective means of assessing physiological and biochemical changes induced by mild blast.

Traumatic injury of cultured astrocytes alters inositol (1,4,5)-trisphosphate-mediated signaling.

Our previous studies using an in vitro model of traumatic injury have shown that stretch injury of astrocytes causes a rapid elevation in intracellular free calcium ([Ca2+]i), which returns to near normal by 15 min postinjury. We have also shown that after injury astrocyte intracellular calcium stores are no longer able to release Ca2+ in response to signal transduction events mediated by the second messenger inositol (1,4,5)-trisphosphate (IP3, Rzigalinski et al., 1998). Therefore, we tested the hypothesis that in vitro injury perturbs astrocyte IP3 levels. Astrocytes grown on Silastic membranes were labeled with [3H]-myo-inositol and stretch-injured. Cells and media were acid-extracted and inositol phosphates isolated using anion-exchange columns. After injury, inositol polyphosphate (IPx) levels increased up to 10-fold over uninjured controls. Significant injury-induced increases were seen at 5, 15, and 30 min and at 24 and 48 h postinjury. Injury-induced increases in IPx were equivalent to the maximal glutamate and trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid-stimulated IPx production, however injury-induced increases in IPx were sustained through 24 and 48 h postinjury. Injury-induced increases in IPx were attenuated by pretreatment with the phospholipase C inhibitors neomycin (100 microM) or U73122 (1.0 microM). Since we have previously shown that astrocyte [Ca2+]i returns to near basal levels by 15 min postinjury, the current results suggest that IP3-mediated signaling is uncoupled from its target, the intracellular Ca2+ store. Uncoupling of IP3-mediated signaling may contribute to the pathological alterations seen after traumatic brain injury.

Traumatic injury of cortical neurons causes changes in intracellular calcium stores and capacitative calcium influx.

Using an in vitro traumatic injury model, we examined the effects of mechanical (stretch) injury on intracellular Ca2+ store-mediated signaling in cultured cortical neurons using fura-2. We previously found that elevation of [Ca2+](i) by the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, was abolished 15 min post-injury. In the current studies, pre-injury inhibition of phospholipase C with neomycin sulfate maintained Ca2+-replete stores 15 min post-injury, suggesting that the initial injury-induced store depletion may be due to increased inositol trisphosphate production. Thapsigargin-stimulated elevation of [Ca2+](i) returned with time after injury and was potentiated at 3 h. Stimulation with thapsigargin in Ca2+-free media revealed that the size of the Ca2+ stores was normal at 3 h post-injury. However, Ca2+ influx triggered by depletion of intracellular Ca2+ stores (capacitative Ca2+ influx) was enhanced 3 h after injury. Enhancement was blocked by inhibitors of cytosolic phospholipase A2 and cytochrome P450 epoxygenase. Since intracellular Ca2+ store-mediated signaling plays an important role in neuronal function, the observed changes may contribute to dysfunction produced by traumatic brain injury. Additionally, our results suggest that capacitative Ca2+ influx may be mediated by both conformational coupling and a diffusible messenger synthesized by the combined action of cytosolic PLA2 and P450.

Astrocytes generate isoprostanes in response to trauma or oxygen radicals.

Previous studies have shown that oxygen radical scavengers prevent the reduced cerebral blood flow that occurs following experimental traumatic brain injury. The exact chemical species responsible for the posttraumatic reduction in flow is unknown. We tested whether isoprostanes, which are formed by non-cyclooxygenase-dependent free radical attack of arachidonic acid and are vasoconstrictors of the cerebral circulation, are increased in astrocytes following stretch-induced trauma or injury with a free radical generating system. Isoprostane (8-epi-prostaglandin F2alpha) was analyzed in cells and in media by immunoassay. Confluent rat cortical astrocytes in culture were injured by a hydroxyl radical generating system consisting of hydrogen peroxide and ferrous sulfate or by rapid stretch of astrocytes grown on a deformable silastic membrane. Some cells were treated with the iron chelator deferoxamine for 1 h before injury. The hydroxyl generating system caused free and cell-bound isoprostanes to increase to more than 400% of control. After trauma, free and membrane bound isoprostanes increased to 321 +/- 34% and 229 +/- 23% of control, respectively, and posttraumatic increases were prevented by deferoxamine. Since astrocytes are in close proximity to cerebral vessels, posttraumatic free radical formation may increase the formation of isoprostanes, which in turn produce vasoconstriction and decrease cerebral blood flow.

Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons.

Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch-induced injury alters mitochondrial membrane potential (delta(psi)m) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, delta(psi)m was measured using rhodamine-123, and ATP was quantified with a luciferin-luciferase assay. In astrocytes, delta(psi)m dropped significantly, and ATP content declined 43-52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, delta(psi)m declined at 15 min only in the severely stretched group. At 48 h postinjury, delta(psi)m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, delta(psi)m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24-48 h. ATP content in mixed cultures declined 22-28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.

Calcium influx factor, further evidence it is 5, 6-epoxyeicosatrienoic acid.

We present evidence in astrocytes that 5,6-epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid, may be a component of calcium influx factor, the elusive link between release of Ca2+ from intracellular stores and capacitative Ca2+ influx. Capacitative influx of extracellular Ca2+ was inhibited by blockade of the two critical steps in epoxyeicosatrienoic acid synthesis: release of arachidonic acid from phospholipid stores by cytosolic phospholipase A2 and cytochrome P450 metabolism of arachidonic acid. AAOCF3, which inhibits cytosolic phospholipase A2, blocked thapsigargin-stimulated release of arachidonic acid as well as thapsigargin-stimulated elevation of intracellular free calcium. Inhibition of P450 arachidonic acid metabolism with SKF525A, econazole, or N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide, a substrate inhibitor of P450 arachidonic acid metabolism, also blocked thapsigargin-stimulated Ca2+ influx. Nano- to picomolar 5, 6-epoxyeicosatrienoic acid induced [Ca2+]i elevation consistent with capacitative Ca2+ influx. We have previously shown that 5, 6-epoxyeicosatrienoic acid is synthesized and released by astrocytes. When 5,6-epoxyeicosatrienoic acid was applied to the rat brain surface, it induced vasodilation, suggesting that calcium influx factor may also serve a paracrine function. In summary, our results suggest that 5,6-epoxyeicosatrienoic acid may be a component of calcium influx factor and may participate in regulation of cerebral vascular tone.

Alterations in calcium-mediated signal transduction after traumatic injury of cortical neurons.

Calcium influx and elevation of intracellular free calcium ([Ca2+]i), with subsequent activation of degradative enzymes, is hypothesized to cause cell injury and death after traumatic brain injury. We examined the effects of mild-to-severe stretch-induced traumatic injury on [Ca2+]i dynamics in cortical neurons cultured on silastic membranes. [Ca2+]i was rapidly elevated after injury, however, the increase was transient with neuronal [Ca2+]i returning to basal levels by 3 h after injury, except in the most severely injured cells. Despite a return of [Ca2+]i to basal levels, there were persistent alterations in calcium-mediated signal transduction through 24 h after injury. [Ca2+]i elevation in response to glutamate or NMDA was enhanced after injury. We also found novel alterations in intracellular calcium store-mediated signaling. Neuronal calcium stores failed to respond to a stimulus 15 min after injury and exhibited potentiated responses to stimuli at 3 and 24 h post-injury. Thus, changes in calcium-mediated cellular signaling may contribute to the pathology that is observed after traumatic brain injury.

Intracellular free calcium dynamics in stretch-injured astrocytes.

We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca2+]i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura-2 microphotometry. Our results indicate that astrocyte [Ca2+]i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury-induced [Ca2+]i elevation is not sustained and returns to near-basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca2+]i returns to normal after injury, we have identified persistent injury-induced alterations in calcium-mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate-sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor-mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate-linked metabotropic glutamate receptor agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid with an elevation in [Ca2+]i. We hypothesize that changes in calcium-mediated signaling pathways, rather than an absolute elevation in [Ca2+]i, is responsible for some of the pathological consequences of traumatic brain injury.

Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock.

Anandamide, an endogenous cannabinoid ligand, binds to CB1 cannabinoid receptors in the brain and mimics the neurobehavioural actions of marijuana. Cannabinoids and anandamide also elicit hypotension mediated by peripheral CB1 receptors. Here we report that a selective CB1 receptor antagonist, SR141716A, elicits an increase in blood pressure in rats subjected to haemorrhagic shock, whereas similar treatment of normotensive rats or intracerebroventricular administration of the antagonist during shock do not affect blood pressure. Blood from haemorrhaged rats causes hypotension in normal rats, which can be prevented by SR141716A but not by inhibition of nitric oxide synthase in the recipient. Macrophages and platelets from haemorrhaged rats elicit CB1 receptor-mediated hypotension in normotensive recipients, and incorporate arachidonic acid or ethanolamine into a product that co-elutes with anandamide on reverse-phase high-performance liquid chromatography. Also, macrophages from control rats stimulated with ionomycin or bacterial phospholipase D produce anandamide, as identified by gas chromatography and mass spectrometry. These findings indicate that activation of peripheral CB1 cannabinoid receptors contributes to haemorrhagic hypotension, and anandamide produced by macrophages may be a mediator of this effect.

Alterations in phosphatidylcholine metabolism of stretch-injured cultured rat astrocytes.

The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic-bottomed tissue culture wells in medium that was usually supplemented with 10 microM unlabeled arachidonate. Cell injury was produced by stretching (5-10 mm) the Silastic membrane with a 50-ms pulse of compressed air. Stretch-induced cell injury increased the incorporation of [3H]choline into PC in an incubation time- and stretch magnitude-dependent manner. PC biosynthesis was increased three- to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch-induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A2 (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch-induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretreating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n-acetylcysteine). Arachidonate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non-arachidonate-enriched cells. In summary, the data suggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-catalyzed Haber-Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch-induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch-induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.

Effect of Ca2+ on in vitro astrocyte injury.

Current literature suggests that a massive influx of Ca2+ into the cells of the CNS induces cell damage associated with traumatic brain injury (TBI). Using an in vitro model for stretch-induced cell injury developed by our laboratory, we have investigated the role of extracellular Ca2+ in astrocyte injury. The degree of injury was assessed by measurement of propidium iodide uptake and release of lactate dehydrogenase. Based on results of in vivo models of TBI developed by others, our initial hypothesis was that decreasing extracellular Ca2+ would result in a reduction in astrocyte injury. Quite unexpectedly, our results indicate that decreasing extracellular Ca2+ to levels observed after in vivo TBI increased astrocyte injury. Elevating the extracellular Ca2+ content to twofold above physiological levels (2 mM) produced a reduction in cell injury. The reduction in injury afforded by Ca2+ could not be mimicked with Ba2+, Mn2+, Zn2+, or Mg2+, suggesting that a Ca(2+)-specific mechanism is involved. Using 45Ca2+, we demonstrate that injury induces a rapid influx of extracellular Ca2+ into the astrocyte, achieving an elevation in total cell-associated Ca2+ content two- to threefold above basal levels. Pharmacological elevation of intracellular Ca2+ levels with the Ca2+ ionophore A23187 or thapsigargin before injury dramatically reduced astrocyte injury. Our data suggest that, contrary to popular assumptions, an elevation of total cell-associated Ca2+ reduces astrocyte injury produced by a traumatic insult.

Reduction of voltage-dependent Mg2+ blockade of NMDA current in mechanically injured neurons.

Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury. Here, the effects of mechanical injury on the voltage-dependent magnesium (Mg2+) block of NMDA currents in cultured rat cortical neurons were examined. Stretch-induced injury was found to reduce the Mg2+ blockade, resulting in significantly larger ionic currents and increases in intracellular free calcium (Ca2+) concentration after NMDA stimulation of injured neurons. The Mg2+ blockade was partially restored by increased extracellular Mg2+ concentration or by pretreatment with the protein kinase C inhibitor calphostin C. These findings could account for the secondary pathological changes associated with traumatic brain injury.

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells.

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A2 (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219-225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced as significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA2 and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA2 activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMPL was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMPL-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA2 rather than secretory PLA2. Inhibition of PLA2 with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA2-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.

Cellular regulation of arachidonate mobilization and metabolism.

Synthesis of eicosanoids is initiated by signal transduction cascades which result in the hydrolysis of free arachidonic acid from membrane phospholipids. Both a cytosolic 85 kDa and a nonpancreatic 14 kDa PLA2 may contribute to cellular arachidonate mobilization. In many cells, agonist-stimulated fatty acid release is dependent upon increases in intracellular free calcium and is enhanced by pretreatment with agents such as phorbol esters and soluble diglycerides. The response is specific for arachidonate and structurally similar polyunsaturated fatty acids containing a cis 5, 6 double bond. DMSO-differentiation of U937 cells markedly enhances the A23187-stimulated release of [3H]arachidonate, which appears to be coupled to differentiation-induced enhancement of capacitance calcium entry. Although both phorbol esters and soluble diglycerides enhance subsequent fMLP or A23187-stimulated arachidonate release in human neutrophils, several lines of evidence indicate that the effects of oleoylacetylglycerol and 1,2-dioctanoylglycerol are protein kinase C-independent. Soluble diglycerides, but not phorbol esters, enhance the coupling of arachidonate mobilization to subsequent leukotriene B4 synthesis. Further studies will be required to elucidate the mechanisms which regulate activation of cellular phospholipases and subsequent synthesis.

Effects of DMSO-induced differentiation on arachidonate mobilization in the human histiocytic lymphoma cell line U937: responsiveness to sub-micromolar calcium ionophore A23187 and phorbol esters.

The human histiocytic lymphoma cell line, U937, is a rich source for isolation and purification of the 85 kDa cytosolic phospholipase A2 (cPLA2). Recent studies suggest that this enzyme catalyzes the agonist-stimulated release of arachidonate from membrane phospholipids, thereby initiating eicosanoid synthesis. We therefore investigated in situ regulation of phospholipase A2 activity in intact U937 cells. The results indicate that calcium ionophore A23187 stimulatable release in intact undifferentiated U937 is low and only weakly dose dependent. Dimethyl sulfoxide (DMSO) differentiation of U937 cells results in a dramatic increase of A23187-stimulated arachidonate mobilization. Consistent with the characteristics of cPLA2 in vitro, A23187-stimulated arachidonate release in differentiated U937 cells is highly specific for arachidonate and is activated by submicromolar A23187 concentrations. Phorbol myristate acetate (PMA) further potentiates arachidonate release in differentiated U937 cells by 4--6-fold over A23187 alone. However, treatment of differentiated U937 cells with PMA alone is an ineffective stimulus for arachidonate release, suggesting that a calcium transient is necessary for in situ arachidonate mobilization. A23187-stimulated arachidonate release increases during distinct temporal phases of differentiation (0-36 h, 84-96 h). By contrast PMA enhancement of the response to A23187 develops early in differentiation, and is complete by 36 h. These results suggest that differentiation-induced alterations in cPLA2 regulatory elements, such as intracellular free calcium and/or phosphorylation, may regulate mobilization of arachidonate in U937 cells.