An in vitro ischemic penumbral mimic perfusate increases nadph oxidase-mediated superoxide production in cultured hippocampal neurons

An in vitro ischemic penumbral mimic perfusate increasesNADPH oxidase-mediated superoxide production in culturedhippocampal neurons Matthew E. PameSameh S. Qingbo , J. Cameron ,Xiang Q. , Laura L. , Gabriel G. aDepartment of Pediatrics, Division of Respiratory Medicine, University of California San Diego, La Jolla, CA 92093, USAbDepartment of Anesthesiology, University of California San Diego, La Jolla, CA 92093, USAcSchool of Medicine, Department of Geriatric Medicine, University of California San Diego, La Jolla, CA 92093, USAdDepartment of Neuroscience, University of California San Diego, La Jolla, CA 92093, USAeThe Rady Children's Hospital-San Diego, San Diego, CA 92123, USA The currently accepted scheme for reactive oxygen species production during ischemia/ Accepted 1 March 2012 reperfusion injury is characterized by a deleterious mitochondria-derived burst of radical Available online 9 March 2012 generation during reperfusion; however, recent examination of the penumbra suggests acentral role for NADPH-oxidase (Nox)-mediated radical generation during the ischemic pe- riod. Therefore, we utilized a novel in vitro model of the penumbra to examine the free rad- ical profile of ischemic murine hippocampal neurons using electron paramagnetic Membrane permeability resonance spectroscopy, and also the role of Nox in this generation and in cell fate. We re- Two-photon microscopy port that free radical production increased 75% at 2 h of ischemia, and this increase was abolished by: (1) scavenging of extracellular free radicals with superoxide dismutase(SOD), (2) a general anion channel antagonist, or (3) the Nox inhibitor apocynin. Similarly,at 24 h of ischemia, [ATP] decreased >95% and vital dye uptake increased 6-fold relative tocontrols; whereas apocynin, the Cl− channel antagonist 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), or the free radical scavenger N-acetyl cysteine (NAC) each provided mod-erate neuroprotection, ameliorating 13–32% of [ATP]-depletion and 19–56% of vital dye up-take at 24 h. Our results support a cytotoxic role for Nox-mediated free radical productionfrom penumbral neurons during the ischemic period.
Published by Elsevier B.V.
⁎ Corresponding author at: Department of Pediatrics, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093–0735, USA.
Fax: +1 858 534 6972.
E-mail address: (M.E. Pamenter).
Abbreviations: AMPAR, 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptor; Apo, apocynin; APV, (2R)-amino-5-phos- phonovaleric acid; BBB, blood brain barrier; CNQX, 6-cyano-7-nitroquinioxaline-2,3-dione; CrOx, chromium oxalate; DIDS, 4,4-diisothio-cyanatostilbenedisulphonic acid; EPR, electron paramagnetic resonance; IS, ischemic solution; MCAO, middle cerebral artery occlusion;NAC, N-acetyl cysteine; NMDAR, N-methyl-D-aspartic acid receptor; Nox, nicotinamide adenine dinucleotide phosphate H oxidase; NPPB,5-nitro-1-(3-phenylpropylamino)benzoic acid; O•2, superoxide anion; PI, propidium iodide; ROS, reactive oxygen species; SOD, superoxidedismutase; Z-VAD-FMK, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone 1 These authors contributed equally to this work.
0006-8993/$ – see front matter. Published by Elsevier B.V.
doi: that mimic the infarct core, where blood flow is occluded andcells are anoxic ). Conversely, blood flow to Reactive oxygen species (ROS) are important second messen- the penumbral region is hypoperfused, but not blocked; and gers that regulate a myriad of intra- and extracellular signaling thus penumbral cells are hypoxic, and likely exhibit a different pathways In ischemic stroke pathol- free radical profile (Due to technical limita- ogy neuronal ROS production spikes during reperfusion, which tions, ROS generation from penumbral neurons cannot be damages DNA locally, and activates deleterious immune and measured in vivo (therefore, we used an cell death pathways in surrounding tissue electron paramagnetic resonance (EPR) spin-trapping assay ). In addition, ROS to determine the effect(s) of a penumbral mimic (ischemic so- generated during ischemia are a key contributor to the perme- lution: IS )) on ROS production in vitro. In addi- abilization of the blood brain barrier (BBB), which leads to tion, we tested the role of anion channels in ischemic ROS edema and contributes to the formation and expansion of efflux using 4,4-diisothiocyanatostilbenedisulphonic acid the penumbra following stroke (). The penumbra (DIDS); since it is widely used as a general antagonist of is the hypoperfused region surrounding the ischemic core, anion channels ), and because there is ev- and the relatively slow propagation of cell death in the penum- idence that DIDS provides cytoprotection against ischemic in- bra makes this region an attractive target for clinical rescue, sults via regulation of anion channels ( particularly as the majority of stroke-related morbidity and Finally, we examined the cytoprotective effects of mortality is attributable to progressive expansion of the ische- limiting plasmalemmal ROS generation during ischemic insult mic core into the penumbra (). The mechanism(s) of by anion channel or Nox antagonism, or by scavenging ROS cell death here are poorly understood, but are likely initiated with N-acetyl cysteine (NAC).
by deleterious alterations of the local perfusate following therelease of cytoplasmic contents from ruptured core cells (Early BBB breakdown following ische- mic stroke is a key factor in the development of the penumbrasince this event contributes significantly to edema and cell Ischemia increases extracellular ROS accumulation swelling, which in turn underlies cell rupture Therefore, ischemic ROS generation plays a pivotal role in ROS generation in IS-challenged neurons increased 75% rel- the initiation and spread of cell death via a variety of cellular ative to controls (EPR intensity (a.u.): 744.0 ± 82.0 vs. 427.6 and intercellular interactions.
± 27.0, F(2,35) = 7.8, A and B), and computer simulations in- Historically it has been suggested that the primary source dicated that the observed signal was due to spin trapping of of ROS generation is mitochondrial superoxide (O• both hydroxyl and O2. To determine the relative contribution ). Another important free radical source is plasma to the ischemic change in total radical production of intracel- membrane-bound proteins such as the NADPH oxidase lular ROS accumulation compared to extracellular ROS efflux, (Nox), which is a principle enzyme for the production of O•2 we treated samples with chromium oxalate (CrOx, 1.25 μg/μl) critical to intracellular signaling and cell death during ische- or superoxide dismutase (SOD, 100 U/ml), which dissipate mic stress, and which underlies ROS-mediated BBB break- only the resonance signal of, or scavenge, extracellular ROS, down following stroke ( respectively In control experiments, CrOx ). Indeed, neuronal Nox mRNA and protein expres- or SOD decreased ROS generation 30–35%, indicating that at sion are increased following middle-cerebral artery occlusion rest, intracellular versus extracellular ROS accumulation oc- (MCAO), whereas BBB and cellular permeability, infarct curs at a 2:1 ratio (CrOx: 263.3 ± 44.4, F(2,26) = 4.7; SOD: 284.3 spread, and neuronal apoptosis are all reduced or abolished ± 12.6, F(2,23) = 4.18, ). Conversely, either compound entire- by Nox inhibition with apocynin, or by knockout of Nox1, ly abolished the ischemic increase in ROS generation (IS Nox4, or the Nox family subunit gp91phox + CrOx: 273.3 ± 12.6, F(2,24) = 8.7, IS + SOD: 272.3 ± 35.7, F(2,22) = 4.8), and total ROS generation when CrOx or SOD was used to ablate the extracellular ROS signal was not different in IS Regardless of the source of generation, intracellularly de- relative to controls; indicating that the change in ROS genera- rived ROS gain access to the extracellular space primarily via tion during ischemia is attributable to extracellular accumula- plasmalemmal anion channels and tion of ROS.
since elevated extracellular ROS can induce cell injury anddeath, it is reasonable to assume that preventing ROS efflux Anion channel-mediated efflux of Nox-derived would limit the spread of cell death following insult. Indeed, radicals underlies ischemic ROS production numerous studies in a variety of pathological models havedemonstrated protective effects of anion channel inhibition In controls, DIDS (400 μM) reduced ROS generation to the same against ROS-mediated inflammation degree as CrOx or SOD (290.0 ± 40.0, F(2,28) = 5.5, A and B), ). Therefore, regulation of ROS transmission and DIDS in the presence of CrOx or SOD did not reduce ROS via blockade of anion channels is an attractive target for clin- generation beyond the effect of any of these compounds ical intervention and may help limit the spread of cell death in alone (DIDS + CrOx: 279.6 ± 14.7, F(2,26) = 4.8; DIDS + SOD: 253.0 the ischemic penumbra.
± 3.5, F(2,22) = 4.2,). Similarly, during ischemia, DIDS-treatment Previous examinations of ROS generation in ischemic brain reduced total radical generation to control levels (396.3 ± 26.7, have predominantly utilized acute models of focal ischemia F(2,31) = 7.9); however, generation remained greater than in

Fig. 1 – IS increases free radical production from neurons. (A)Summary of EPR-revealed changes in superoxide productionfrom neurons treated as indicated for 2 h. (B) Raw EPR traces Fig. 2 – DIDS or Apo prevents IS-mediated increase of ROS from (A). Parentheses ( ) indicate n values. Data are mean generation. (A) Summary of EPR-revealed changes in superoxide ± SEM. Asterisks (*) indicate significant difference from production from neurons treated as indicated for 2 h. (B) Raw control; double daggers indicate significant difference from EPR traces from (A). Parentheses ( ) indicate n values. Data are IS-alone (p < 0.05).
mean±SEM. Dashed and dotted lines indicate normoxic andIS controls from A, respectively. Asterisks (*) indicatesignificant difference from control A); double daggers normoxic DIDS-treated neurons. Therefore during ischemia, indicate significant difference from IS-alone p<0.05).
and with anion channels blocked, ROS accumulate in the cy-tosol. Similarly, the general Nox inhibitor apocynin (Apo:10 μM) reduced the IS-mediated increase in ROS generationto control levels (420.0 ± 27.7, F(2,23) = 3.6, and B), while Nox inhibition or ROS scavenging provide moderate co-treatment of DIDS with Apo did not further reduce ROS neuroprotection against ischemic insult (362.0 ± 72.0).
Next, we assessed the effects of IS ± DIDS ± Apo on neuronal vi- DIDS does not permeate intact neurons ability. Relative to experimental onset (t = 0 h), control cellsmaintained >85% of their [ATP] through 24 h; whereas [ATP] DIDS blocks ROS transmission across multiple lipid mem- dropped progressively to 89.2 ± 0.1, 34.0 ± 3.5, and 3.9 ± 2.0% fol- lowing 2-, 12-, and 24-h IS-treatment, respectively ( therefore, to assess the penetration of DIDS into neurons, n = 8–10 for each treatment). DIDS did not preserve [ATP] in we employed 2-photon confocal microscopy and took advan- IS-treated neurons at 24 h (n = 10 for each), likely due tage of the natural fluorescence of DIDS molecules to secondary intracellular actions of DIDS on cells with dam- In control experiments DIDS did not permeate aged membranes at this time-point (However, another plasma membranes through 2 h (n = 4 for each), general Cl− channel antagonist, 5-nitro-1-(3-phenylpropyla- whereas in IS, DIDS fluorescence was observed in some mino)benzoic acid (NPPB, 100 μM) increased [ATP] 3-fold rela- cells. To determine whether this penetration was due to tive to IS-alone and decreased IS-mediated PI uptake 18.8% membrane degradation, we also measured propidium iodide B and C and D, n = 16 for each). Similarly, Apo or the (PI) uptake. At 2 h, 15% of neurons took up PI, and this up- ROS scavenger NAC had minimal effects on [ATP] in control take coincided with cellular DIDS fluorescence ( experiments, but in ischemia increased [ATP] 7- and 10-fold, n = 10–20 for each). Therefore, the accumulation of DIDS in respectively, and ameliorated 25.7 ± 3.2 and 56.1 ± 4.5% of IS- some cells at 2 h is due to membrane degradation, and mediated PI uptake relative to IS-alone –D, n = 3–10 for DIDS-mediated inhibition of ROS production from intact each [NAC]). When ischemic neurons were treated with both cells at this time-point is likely due to extracellular actions Apo and NAC simultaneously, their neuroprotective effects were not additive.

Fig. 3 – DIDS penetrates damaged, but not intact neuronal membranes. (A) Two-photon confocal DIC and Z-stack projection images(2 microns total) of neurons treated without DIDS, or with DIDS during normoxia or IS. Upper panel: DIC images. Lower panel:DIC images (grey) overlaid with PI (red) and DIDS (blue) fluorescent Z-stack projection images. Images are representative of 4separate experiments for each treatment. (B) Summary of PI uptake. Data are mean ± SEM from 10–20 replicates per treatment.
Asterisks (*) indicate significant difference from control (p < 0.05).
Ischemic solution induces mixed modes of cell death in this increase was abolished by co-treatment with Z-VAD-FMK B and C, n = 3).
Finally, since glutamate receptor activation and associated In the infarct core, cell death proceeds primarily via necrosis Ca2+ influx has been linked to ROS and reactive nitrogen spe- due to excitotoxicity, mediated by over-activation of excitatory cies production we examined the effect of AMPAR and NMDAR inhibition on ROS production. In nor- panoic acid and N-methyl-D-aspartic acid receptors (AMPARs, moxia, application of CNQX + APV (50 μM each) increased NMDARs) (Conversely, in the penum- ROS generation 14% from 466.5 ± 34.2 to 545.0 ± 28.7 at 2 h, bra, cell death is thought to occur via a mixture of cell death mo- while during IS-perfusion, CNQX + APV similarly increased dalities, including necrosis, apoptosis, and autophagy ROS generation 23%, from 655.2 ± 18.2 to 854.2 ± 48.1 (a.u., In our 2-photon experi- n = 4 for each).
ments, we observed moderate vital dye uptake at 2 h and B), consistent with the occurrence of rapid necrosis insome cells treated with this mimic solution. Therefore, we next examined the role of necrotic cell death in the IS modelby antagonizing AMPARs and NMDARs with 6-cyano-7-nitro- We demonstrate that deleterious ROS generation is increased quinioxaline-2,3-dione (CNQX) and (2R)-amino-5-phosphono- in hippocampal neurons treated with an in vitro ischemic pen- valeric acid (APV), respectively; and also of apoptosis using the umbral mimic perfusate, and that this increase is primarily due to efflux of Nox-derived ROS via plasma membrane anion channels. This conclusion is supported by our observations In vital dye exclusion assays, none of these treatments that anion channel or Nox antagonism, or ROS scavenging, had a significant effect on cell viability during control ex- each: (1) prevent IS-mediated increases in ROS generation, periments, whereas during IS-treatment, higher concentra- and (2) partially ameliorate IS-mediated neurotoxicity, in non- tions of CNQX + APV (50 μM each) reduced PI uptake 20%, additive manners. In neurons, Nox subunits are found at vari- Z-VAD-FMK (5 or 50 μM) ameliorated approximately one- ous lumen membranes, and following prolonged ischemia, third of IS-mediated PI uptake at 24 h (n = 16–32 for are translocated to plasma, mitochondrial, and endoplasmic each). To confirm the occurrence of apoptosis in this model, reticulum membranes ), and therefore contrib- we next examined the expression of Lamin A, a commonly ute to both cytosolic and extracellular ROS generation. Our re- studied marker of apoptosis whose cleavage is indicative of cas- sults suggest a predominant role for cytosolic Nox-derived pase 6 activation ). In Western blot ROS production in the penumbra since co-antagonism of analysis of Lamin A expression, we observed that IS treatment anion channels and Nox did not reduce EPR spectra beyond ei- (6 h) increased Lamin A 15-fold relative to controls, and that ther treatment alone, as would be expected if ROS were

ROS generation from penumbral neurons cannot presently be measured in vivo because tracers do not reach the ischemictissue (however, our results are consistentwith ex vivo measurements from mouse brain followingMCAO, where Nox-mediated O•2 production was increased inthe penumbral region, but not in the core ).
Furthermore, our data are also consistent with measurementsfrom penumbral epithelial cells and arteries in brain in whichROS production increases markedly during ischemia via aNox-dependant mechanism Conversely, ROS production does not increase in ische-mic core arteries. These results from three penumbral tissuesfurther demonstrate that the profile of ROS generation in thepenumbral region is markedly different from the infarctcore, and in particular support a dominant contribution fromNox. Based on our present findings and these recent examina-tions, we conclude that Nox-mediated O•2 production is a keyregulator of deleterious ROS signaling in the penumbral re-gion, and that neuronal Nox contribute to this mechanism.
In the penumbra, oxidative phosphorylation persists due to a steady, albeit restricted supply of oxygen. This persistentreduced level of oxygen availability likely partially preservescells' ability to generate ATP, and thus opposes excitotoxic de-polarization and early rapid necrotic cell death (). However, this temporary preservation of ATP pro-duction allows for the induction of apoptosis and autophagyduring prolonged insult ), as occurs in thepenumbra. As a result, a variety of cell death mechanisms ap-pear to be concomitantly activated in this model present report), and this ex-plains why cell viability was only partially preserved in our ex-periments by ROS scavenging, or by glutamate receptor orcaspase antagonism.
In conclusion, we have used the present gold standard for free radical measurements (EPR), to record ROS productionfrom live neurons treated with a novel ischemic penumbralmimic perfusate for the first time. We conclude that: (1) neu- Fig. 4 – Nox inhibition or ROS scavenging is moderately protective ronal ROS production is increased during IS and this increase against prolonged ischemic insult. (A) Summary of [ATP] is manifested extracellularly, (2) this production is due to in- changes. (B) Dose–response curve of [ATP] vs. [NAC] at 24 h.
creased Nox-derived superoxide production, (3) the efflux of (C) Summary of [ATP] changes from neurons treated as Nox-derived superoxide occurs via anion channels, (4) Nox in- indicated for 24 h. Red and blue dashed lines represent hibition provides mild protection against IS insult in neurons, baseline and ischemic controls, respectively, from Fig. 4A. (D) and (5) neuronal cell death is mediated by a variety of path- Summary of PI uptake by neurons treated as indicated for 24 h.
ways during IS insult.
Red and blue dashed lines represent baseline and ischemiccontrols, respectively, from B. Data are mean ± SEM from8 to 10 replicates per treatment. Asterisks (*) indicate Experimental Procedures significant difference from control (Fig. 4A); double daggersindicate significant difference from IS (Fig. 4A; p < 0.05).
HT22 mouse hippocampal neurons (a generous gift from Dr.
Pam Maher, The Salk Institute, La Jolla, CA) were cultured produced by plasma membrane-bound Nox. Therefore, unlike in Dulbecco's Modified Eagle Medium (DMEM, ATCC) sup- in anoxic infarct core cells where ROS production is low during plemented with 10% bovine calf serum (Hyclone, Santa ischemia and cytotoxicity is predominately mediated by a del- Clara, CA) and 100 U/ml penicillin/streptomycin (Invitrogen, eterious burst of ROS during reperfusion Carlsbad, CA) and grown at 37 °C in a 5% CO2 incubator.
hypoperfused penumbral neurons maintain elevation of the Cells were grown for 5–8 passages and split when they generation, efflux, and extracellular accumulation of ROS, reached 60–80% confluence. For experiments, cells were which are known to contribute to the spread of cell injury and seeded into 96-well microplates (Corning, Lowell, MA), death in the penumbra ).
glass-bottom 35-mm culture dishes (MatTek, Ashland, MA),

Fig. 5 – Multiple cell death pathways are activated in IS-treated neurons. (A) Summary of PI uptake by neurons treated as indicatedfor 24 h. Red and blue dashed lines represent baseline and ischemic controls, respectively, from B. Data are mean ± SEMfrom 16–32 replicates per treatment. (B) Summary of Lamin A expression in neurons treated as indicated for 6 h. Data arenormalized to expression of α-actin on the same blot. (C) Sample paired Western blot images of Lamin A α-actin expressionfrom (B). Images are representative of 3 separate experiments. (D) Summary of EPR-revealed changes in superoxide productionfrom neurons treated as indicated for 2 h. Parentheses ( ) indicate n values. Asterisks (*) indicate significant difference fromcontrol; double daggers indicate significant difference from IS (p < 0.05).
or poly-L-lysine coated custom designed plastic coverslips pre- was designed to incorporate and mimic key changes previous- cisely cut to the dimensions of the EPR tissue culture chamber ly described in the literature from in vivo examinations of the (Fisher, Pittsburgh, PA). Cells were allowed to grow to 80% con- penumbral rim (the region of tissue immediately adjacent to fluence before experimentation and samples were treated as the infarct core, and thus most effected by local cell rupture), specified in the experimental design section (below). To reduce and the individual effects of each of these alterations on cell shear stress, cells seeded into 96-well microplates were gently viability have been carefully examined elsewhere washed with a TECAN PW96/384 Washer (TECAN, San Jose, CA) and then examined visually to ensure cells had not beenwashed away.
Determination of superoxide production in live neuronal cultures by electron paramagnetic resonance (EPR) spectroscopy Experimental design Measurement of O•2 production in live neuronal cultures was Samples were treated for 2, 6, 12 or 24 h (as indicated) in two carried out by EPR spin trapping method using 70 mM of the treatment groups: cell death-negative control (DMEM/F12 spin trap agent DEPMPO (5-(diethylphosphoryl)-5-methyl-1- media (Invitrogen), pH 7.4, gassed with 21% O2, 5% CO2, bal- pyrroline-N-oxide). DEPMPO readily permeates mouse heart, ance N2), or an ischemic penumbral perfusate mimic (IS, in liver, and brain tissues in vivo and is evenly distributed within mM: K+ 64, Na+ 51, Cl− 77.5, Ca2+ 0.13, Mg2+ 1.5, glucose 3, glu- 15 min (); and DEPMPO-spin adducts diffuse tamate 0.1, [315 mOsM, pH 6.5, 1.5% O2, 15% CO2, balance N2]) through lipid bilayers effectively (Samples (). APV (5 or 50 μM), CNQX (5 or 50 μM), were treated for 2 h before transferring the coverslip to the CrOx (1.25 μg/μl), NAC (0.1–1000 μM), and SOD (100 U/ml) were EPR tissue culture chamber containing the spin trap agent in dissolved in water. SOD does not penetrate the cell membrane 40 μL of normoxic or IS gas-equilibrated media. The EPR cell and therefore specifically scavenges extracellular superoxide was then tightly covered to avoid media leakage and inserted (Apo (10 mM), DIDS (400 μM), in the EPR cavity of a MiniScope MS200 Benchtop spectrome- NPPB (100 μM), and Z-VAD-FMK (5 or 50 μM) were dissolved ter maintained at 37 °C. EPR spectra were recorded using the in DMSO to a final bath [DMSO] < 0.01%, and all solutions following spectral settings: microwave frequency, 9.45 GHz; were made fresh daily. Chemicals were purchased from microwave power, 5 mW; modulation amplitude, 0.2 mT; Sigma unless otherwise indicated (Sigma-Aldrich, St. Louis, modulation frequency, 100 kHz; sweep width, 15 mT centered MO). It is important to note that atmospheric [O2] (21%) is at 335 mT; scan rate, 0.75 mT s−1 and each spectrum was the hyperoxic relative to in situ tissue pO2. Nonetheless, 21% [O2] average of 10 scans.
is commonly used as "normoxic" control in in vitro experimen- EPR traces were quantified by measuring the relative signal tation due to convenience and cost-related complications, amplitudes detected automatically using the Magnettech and therefore we utilized 21% as our baseline control [O2] in Analysis Software (Version 2.02; Berlin, Germany). The average order to be consistent with the majority of in vitro experiments amplitudes of the largest four peaks in each spectrum were previously published. Also, the in vivo ischemic penumbra is a computed to minimize the random noise effects on the indi- heterogeneous milieu in which ionic, pH, metabolic substrate, vidual peaks. Computer simulation was carried out to identify and gaseous derangements vary considerably depending on the radical species enveloped in the signals using published the severity of insult and the time since insult-onset. There- hyperfine coupling constants ( fore, this region is difficult to exactly duplicate in vitro. IS hyperfine parameters of DEPMPO-OOH and DEPMPO-OH radi- Protein extraction and Western blotting cal adducts are inserted into the WinSim 2002 program Samples grown in 150 cm2 culture flasks were treated as indi- ) including initial guess of the percentage contribu- cated in the results section, and then rinsed twice with PBS tion from each species, and the program is allowed to produce and detached from the matrix with a cell scrapper into ice- the resultant spectrum, or to fit the experimental spectrum cold PBS. The resulting cell suspensions were centrifuged at with the calculated one. This procedure was used to confirm 250 ×g for 5 min at 4 °C, the supernatant was aspirated away, the assignment of the radical species observed via representa- and cells were re-suspended in cell lysis buffer. Samples tive spectra.
were then homogenized by vortexing for 60 s and proteinswere extracted by incubation in lysis buffer with mixing at DIDS permeation imaging 4 °C for 45 min, followed by centrifugation for 10 min at14,000 ×g at 4 °C. Supernatants were taken as whole cell ly- Samples were grown in glass-bottom 35-mm culture dishes.
sates and protein concentration was measured using a Prior to experimentation, cells were rinsed once in serum- bicinchoninic acid kit, according to the manufacturer's in- free DMEM and then incubated for 2 h with 0.2% DMSO or structions (Sigma).
DIDS in DMEM or IS, and then rinsed once with DMEM or Equal amounts of protein (40 μg/well) were separated on IS. DIDS fluorescence (Ex/Em: 342/418 nm in water, with an 4–12% precast NuPAGE bis-Tris SDS-PAGE gels (Invitrogen) emission Red-shift to > 450 nM when bound to protein and transferred to polyvinylidene difluoride membranes (was visualized by a Zeiss LSM510 (Immobilin-P; Millipore, Bedford, MA). Western blots were per- META confocal laser scanning 2-photon microscope using a formed with antibodies against α actin and cleaved Lamin A 40 X C-Apochromat (NA 1.2) water immersion objective. Exci- (1:2000, Cell Signaling, Danvers, MA). Specific bands were vi- tation was achieved by a Coherent Mira 900 laser tuning to sualized after incubation with the appropriate secondary anti- 800 nm. Fluorescence shorter than 490 nm was reflected by bodies (Invitrogen) using enhanced chemiluminescence (GE a long pass filter (NFT 490), and then filtered by a short Healthcare/Amersham Biosciences, Buckinghamshire, UK).
pass filter with cutoff at 685 nm (KP685). After taking DIDS Densitometry of Western blots from each experimental and DIC images, 10 μg/ml of propidium iodide (PI, Sigma, group was obtained (n = 3 for each), and absolute values were Ex/Em: 514/590 nm) was added, and images from the same normalized to α-actin. Results were analyzed in arbitrary view were taken 5 to 8 min afterwards (excited by laser line units, comparing each value with that obtained from each re- 514 nm, and filtered by a long pass filter (LP560). Z-stacks spective α-actin measurement on each blot.
were taken at 0.4 μm intervals. Z-projections from 4 to 5 op-tical sections were created by averaging pixel intensity at Vital dye exclusion membrane viability assay each pixel position using Image J (NIH). Composite pictureswere generated using Image J from DIC (grey), DIDS (blue), Membrane viability was assessed as the ability of cells to ex- and PI (red).
clude the vital dye propidium iodide (PI). The dose-dependent response of DIDS on IS-induced PI uptake was ATP luciferase assay assessed using a high-throughput 96-well microplate-basedassay. PI uptake was assessed immediately following experi- Total ATP content [ATP] was assessed in solid-bottom, black mental treatment on a Bio-Tek PowerWave 340 microplate 96-well microplates (Corning) using PerkinElmer ATPlite Lu- spectrophotometer (Bio-Tek, Winooski, VT, Ex/Em: 485/ minescence Assay System kits as specified by the manufac- 630 nm), and analyzed using Gen 5 software (Bio-Tek). Micro- turers protocol (PerkinElmer, MA, USA) and a Bio-Tek plate PI experiments were repeated 3–5 times and each plate PowerWave 340 microplate spectrophotometer (Bio-Tek, Wi- contained 16 replicate wells each of treatment groups. Blank nooski, VT). Equal numbers of cells were seeded into each wells and cell-free wells containing each treatment perfusate well and standard curves were generated using serial dilu- with PI were also included on each plate, and the final data is tions of a known ATP standard provided in each kit. The corrected for these factors.
sensitivity of the detector was calibrated to the lumines-cence of the highest [ATP] standard in each experiment.
DIDS quenched luminescence in a dose-dependent fashionand this effect was quantified in a separate experiment by Data were analyzed using one-way analysis of variance adding serial dilutions of DIDS to serial dilutions of the (ANOVA), followed by Dunnet's post-test. Significances were ATP standard and subtracting the resulting luminescence indicated if P < 0.05 assuming two groups had an equal vari- from ATP standard luminescence measurements in the ab- ance. Statistical analysis was performed using Prism software sence of DIDS on the same plate. Results were corrected (GraphPad, San Diego, CA, USA).
for this factor and then normalized to ATP luminescencerecorded from control cells assayed at t = 0 h. MicroplateATP luciferase experiments were repeated 10 times and each plate contained 16 replicate wells of each treatmentgroup. Blank wells and cell-free wells containing each treat- This work was supported in part by NIH 5P01HD032573 to ment perfusate were also included on each plate, and the GGH, NIA 1K25AG026379 to SSA, and a National Sciences and final data is corrected for these factors.
Engineering Research Council of Canada fellowship to MEP.
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