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In contrast to ischemic brain disease (Sensi et al. 2009), only a few investigations have addressed the role of zinc in TBI. After TBI, zinc staining with intracellular zinc indicators, such as N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) decreases in the presynaptic neurons, but increases in the postsynaptic hippocampal neurons (Suh et al.

Intracerebroventricular injection of CaEDTA, an extracellular zinc chelator, after transient global cerebral ischemia prevents neuronal death (Koh et al. In TBI of rats, zinc up-regulates neuroprotective genes (Hellmich et al. 2004), but it does not improve learning and memory deficits in behavioral tests following TBI (Hellmich et al. It has been suggested that release of zinc ions from presynaptic vesicles does not contribute to neuronal damage after TBI (Doering et al. Thus, intracellular zinc ions mobilized as a consequence of oxidative stress rather than extracellular zinc ions could be the source of potentially cytotoxic zinc ions (Maret 1994;Maret 1995;Frederickson et al. From a large body of data, the sequence of events that links increased cellular zinc ion concentrations and neuronal cell death is thought to begin with glutamate excitotoxicity and to involve calcium influx through calcium channels, calcium-activated nitric monoxide (NO) production by NO synthase (NOS), an increase in zinc ion concentrations, mitochondrial production of reactive oxygen species (superoxide), release of more zinc ions from proteins such as metallothionein (MT), and activation of the mitochondrial pathway of apoptosis (Bossy-Wetzel et al. Increased concentrations of intracellular zinc ions may promote neuronal death by inhibiting cellular energy production, increasing cellular reactive oxygen species (ROS), changing the mitochondrial membrane potential, and reducing cellular ATP levels (Dineley et al. A major issue in defining the functions of zinc ions in these pathways inducing cellular injury is their cellular concentrations and the capacity of the cell to control them (Maret and Li 2009). Total cellular zinc concentrations are a few hundred micromolar.

Most zinc is bound to proteins with high affinity; therefore, the concentrations of cellular free zinc ions are very low. Estimates put them in the picomolar to nanomolar range (Krezel and Maret 2006;Bozym et al. 2006), but higher concentrations can occur when oxidative stress releases zinc from proteins that utilize sulfur ligands for zinc binding (Maret and Vallee 1998;Maret 2006). Thus, exposing cells to oxidizing agents or generating a redox signal, e.g. NO, within the cell, increases cellular free zinc ion concentrations (Turan et al. Such increased free zinc ion concentrations are very potent effectors of proteins (Maret et al. The threshold for cellular zinc buffering demarcates cytoprotective (pro-antioxidant) from cytotoxic (pro-oxidant) effects of zinc ions (Hao and Maret 2005). Although these dual activities of zinc ions may appear paradoxical (Cuajungco and Faget 2003), they merely reflect the actions of zinc ions at different concentrations: physiological concentrations confer neuroprotection while pathophysiological concentrations are neurotoxic (Hao and Maret 2005;Maret 2008). For example, sub-lethal ischemia triggers a neuroprotective increase in free zinc ion concentrations in postsynaptic neurons, and this ischemic preconditioning can be blocked by chelating zinc ions with CaEDTA (Lee et al. Dysregulation of neuronal zinc ion homeostasis, how much cellular free zinc ions increase, and to which targets the released zinc ions bind, are important questions in elucidating the mechanisms of cell death following TBI. In this investigation, changes of zinc ion concentrations during an early phase of sub-lethal injury (within hours) were quantified. Both in vitro (rapid stretch injury, RSI) and in vivo (fluid percussion TBI) models of brain injury were employed. The results demonstrate induced fluctuations of free zinc ions within a physiological range of concentrations and suggest a cytoprotective effect of the released zinc ions in this time period after injury. The free zinc ion concentrations in normal (uninjured) PC12 cells are 0.97 nM in serum-containing media and transiently reach 1.4 nM during proliferation (Li and Maret 2009). Remarkably, during serum withdrawal, PC12 cells mobilize zinc ions from an intracellular source (Li and Maret 2009). With time, free zinc ion concentrations decrease below the normal level and growth arrest ensues (Li and Maret 2009). Thus, measurements of zinc ion concentrations provide critical information on whether or not cells are viable and healthy (Li and Maret 2009). RSI caused biphasic zinc ion fluctuations in PC12 cells. After subjecting PC12 cells to RSI (up to 60 psi, 50 ms), less than 10% cell death was detected by PI staining ( Figure 1 ). Accordingly, 50 psi (50 ms) was employed as sublethal stretch injury in the following experiments. Within 24 hours, there was a biphasic fluctuation of intracellular zinc ion concentrations ( Figure 2 ). The highest concentration was observed at one hour post-injury ( 1.4 nM), followed by a sharp decline below the baseline ( 0.9 nM). The concentrations are then maintained at this low level ( PC12 cell viability measured with propidium iodide (PI) staining after rapid stretch injury.

(A) Phase contrast and fluorescence microscopic images of PC12 cell cultures at 24 hours after different levels of RSI (0, 20, 30, 40, 50 and 60 psi for a duration of 50 msec). (B) Percentage of dead cells relative to the total number of cells. Rapid stretch injury-induced fluctuations of intracellular zinc ion concentrations in PC12 cells. Intracellular zinc ion concentrations were measured fluorimetrically with 0.3 μM FluoZin-3 AM in 1 ml DPBS without Ca 2+ and Mg 2+ at 37 °C for 30 min at 0, 0.5, 1, 3, 6, 12 and 24 hours after RSI. Data are represented as mean ± SD (n = 3, p Figure 3A ). Following the peak at one hour, ROS decreased slightly but increased again and remained high even when intracellular zinc ion concentrations were low.

Levels of NO in PC12 cells, measured by DAF-FM over time, displayed a slightly different profile peaking at three hours rather than at one hour after RSI ( Figure 3B ). Nonetheless, NO levels began to increase immediately after injury. However, they decreased after the peak rather than increased again as observed for ROS. Effect of zinc chelation on ROS generation in PC12 cells. CONTROL: no RSI, no TPEN treatment; TPEN: treated with 50 nM TPEN in complete culture medium for 1 hour; RSI: 1 hour after RSI; and RSI+TPEN: treated with 50 nM TPEN immediately after RSI for 1 hour before measurement.


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