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Our method determines the metal load of metallothioneins but does not differentiate among the different MT isoforms. The cell-specific expression of MTs and their variable metal load and redox states under different experimental conditions continues to be a major challenge in their analysis (Li and Maret 2008). Such a cellular zinc ion deficiency might precede a systemic zinc deficiency that develops in brain trauma patients (McClain et al. Protective effects of zinc supplementation have been observed in both long-term ischemia-reperfusion (Atahan et al. Our findings indicate that a loss of cellular zinc ions is equally, if not more, detrimental as the pathological increase of intracellular zinc ions, especially when sustained for a longer period of time after mild or moderate TBI.

As a matter of fact, our in vitro results support a pro-antioxidant effect of the rise in zinc ion concentrations within the physiological range. The function of such an increase as a physiological response counteracting injury and protecting the cell against further injury may explain the failure of zinc chelation at certain time points as a therapeutic strategy to brain injury. In conclusion, zinc ion fluctuations were detected in sub-lethally injured cells. These fluctuations are within a physiological range of cellular concentrations and may protect the cell against secondary injury. A protective effect of mobilized zinc ions has been observed in ischemic pre-conditioning (Lee et al. However, our results do not rule out that zinc homeostasis and zinc status are compromised over 24 hours after injury when zinc ion concentrations are below baseline and contribute to cell death directly or indirectly. In future studies, the thresholds of cellular zinc buffering, the role of different pathways and compartments in the induction of zinc ion fluctuations at different time points, and the different proteins that become targets at different zinc ions concentrations all need to be considered. Pheochromocytoma (PC12) cells [(Greene and Tischler 1976) ATCC, CRL-1721] were maintained in complete growth medium [RPMI 1640 (GIBCO) supplemented with 10% (v/v) heat-inactivated horse serum (GIBCO), 5% (v/v) fetal bovine serum (FBS) (HyClone) and 1% (w/v) penicillin/streptomycin (GIBCO)] at 37 °C in a humidified, 5% CO 2 incubator. Fluorescence assay for intracellular zinc ion concentrations.

Intracellular zinc ion concentrations were determined with FluoZin-3 (Krezel and Maret 2006). Briefly, cells were detached from culture dishes by gentle scraping. Following two washes with DPBS without Ca 2+ and Mg 2+ at 37 °C, cell suspensions (1 × 10 6 cells/ml) were aliquoted into Eppendorf tubes and incubated at 37 °C for 30 min. For all measurements of intracellular zinc ion concentrations, 0.3 μM FluoZin-3 acetoxymethyl ester (FluoZin-3 AM) (Invitrogen, Molecular Probes), was used. The concentration of FluoZin-3 AM (0.3 μM) was chosen from a quantitative assay that employs a series of different concentrations of FluoZin-3 AM and an extrapolation to a zero probe concentration to determine the absolute intracellular zinc ion concentration (Krezel and Maret 2006). It is the lowest concentration of the probe that allows accurate measurements and exerts a minimal effect on cellular zinc buffering of PC12 cells (Li and Maret 2009). Cells were then washed three times with DPBS without Ca 2+ and Mg 2+ at 37 °C to remove any residual fluorescence probe, and incubated another 30 min at 37 °C. Fluorescence was measured at 25 °C with 492 nm excitation and 517 nm emission in a spectrofluorimeter. Six Eppendorf tubes of cells were prepared for each measurement, and they were randomly assigned into two groups afterwards with three tubes in each group to receive two different treatments for 10 min to measure F min and F max . F min is the background fluorescence of the dye measured in the presence of 50 μM N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) (Sigma-Aldrich), and F max is the maximum of fluorescence in the presence of 100 μM pyrithione (Sigma-Aldrich), a zinc ion ionophore that facilitates zinc entry into cells, and 250 μM ZnSO 4 to saturate the probe. The concentrations of zinc ions were calculated by using the following equation. [Zn 2+ ] = K d (F-F min )/(F max -F) with K d = 8.9 nM. The calibration employing F min and F max makes the determination independent of cell number. Intracellular reactive oxygen species (ROS) was measured with 5′-(and-6′)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester, mixed isomers (CM-H 2 DCFDA) (Invitrogen, Molecular Probes), a probe that detects superoxide (O 2 •− ), hydrogen peroxide (H 2 O 2 ), hydroxyl free radical (HO • ), and peroxynitrite (ONOO − ) (Fekete et al. Cells were detached from culture dishes by gentle scraping. After washing twice with DPBS without Ca 2+ and Mg 2+ at 37 °C, the cell suspensions (1 × 10 6 cells/ml) were aliquoted into Eppendorf tubes and incubated for 30 min with 5 μM CM-H 2 DCFDA with (F max ) or without (F) 300 μM tert-butyl hydroperoxide (Sigma-Aldrich) at 37 °C. Cells were washed three times with DPBS without Ca 2+ and Mg 2+ at 37 °C to remove any residual fluorescence probe, and incubated for another 30 min at 37 °C. Fluorescence was measured at 25 °C with 492 nm excitation and 517 nm emission in a spectrofluorimeter. The level of ROS was calibrated by determination of F min , which is the basal level of ROS in control cells, and F max , which is the maximum fluorescence in the presence of tert-butyl hydroperoxide that is used to oxidize all the probe molecules. The relative normalized fluorescence was calculated by using the equation: The concentration of NO was measured with 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate (Invitrogen, Molecular Probes). Cells were detached from culture dishes by gentle scraping. After washing twice with DPBS without Ca 2+ and Mg 2+ at 37 °C, the cell suspensions (1 × 10 6 cells/ml) were aliquoted into Eppendorf tubes and incubated with 3 μM DAF-FM diacetate at 37 °C for 30 min. Cells were washed three times with DPBS without Ca 2+ and Mg 2+ at 37 °C to remove any residual fluorescence probe, and incubated for another 30 min at 37 °C. Fluorescence was measured at 25 °C with 492 nm excitation and 517 nm emission in a spectrofluorimeter.

The level of NO was normalized to total cell numbers, which were determined using a Coulter Counter, Model Z F (Coulter Electronics, Inc., Hialeah, FL). Rapid stretch injury (RSI) Intracellular zinc ions and generation of reactive species were monitored fluorimetrically over a time period of up to 24 hours after injuring PC12 cells with the rapid stretch injury model described by Ellis et al., 1995. To perform RSI, cells were plated in 6-well Flex I ® culture plates with a silastic membrane bottom coated with collagen I (FlexCell International Corporation) at a density of 1 × 10 5 /ml in 1 ml complete culture medium. After 24 hours of incubation, the culture plates were connected to a 94A Cell Injury Controller (Biomedical Engineering Facility, Medical College of Virginia), which employs a nitrogen gas pulse to deform the silastic membrane and achieve a predetermined degree of stretch for a predetermined duration (Ellis et al. Viability of PC12 cells 24 hours after different levels of stretch injury (0, 20, 30, 40, 50 and 60 psi for a duration of 50 msec) were determined by propidium iodide (PI, Calbiochem, San Diego, CA) staining. Cells were stained with 5 μM PI solution in medium for 30 min and counted under fluorescent microscope. For all of the RSI experimental procedures, the pulse pressure/duration was 50 psi/50 msec, which generated sub-lethal mechanical stretch to PC12 cells.

After RSI, cells were kept in complete culture medium at 37 °C in a humidified, 5% CO 2 incubator for 0.5, 1, 3, 6, 12 and 24 hours before measuring intracellular zinc ion concentrations or reactive species. For the NOS inhibition experiment, 500 μM N ω -nitro-L-arginine methyl ester hydrochloride (L-NAME) (Sigma-Aldrich) was added immediately after RSI and the cells were kept at 37 °C in a humidified, 5% CO 2 incubator before measuring intracellular zinc ion concentrations. PC12 cell cultures were divided randomly into four groups: Group 1 served as a control without either RSI or L-NAME treatment; Group 2 was treated only with L-NAME; Group 3 received only RSI; and Group 4 received both RSI and treatment with L-NAME. Since the highest intracellular zinc ion concentrations were observed one hour after RSI, L-NAME was added to cell cultures with or without RSI one hour before zinc ions were measured.

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