cbd mitochondriaDecember 15, 2021
Effects of ER- and mitochondrion-acting drugs on CBD responses. A , B , The role of mitochondria in CBD responses were confirmed in neurons ( A ) and glia ( B ). The uncoupler FCCP prevented neuronal CBD response and largely reduce glial responses while blockade of IP3 and ryanodine receptors [by 2-APB and dantrolene (Dant.), respectively] did not significantly alter CBD responses in neurons, a sample trace of which is also shown ( C ). In the presence of CGP 37157 (CGP), but not in the presence of the mPTP inhibitor cyclosporin A (CsA), CBD responses were also blocked in normal and high-excitability HBS; CBD responses under high-excitability conditions no longer differed from standard HBS responses. Data are presented as %Δ F / F + SEM; n.s., not statistically significant; ** p < 0.01, *** p < 0.001.
Mitochondrial components of Ca 2+ regulation and targets for drug action used to assess the action of CBD. Drugs/enzymes used to induce cell death in SH-SY5Y cells are italic and underlined, and those applied to identify possible mechanisms of protection are outlined. SOD2, Superoxide dismutase 2.
Cannabinoids and the endocannabinoid system have attracted considerable interest for therapeutic applications. Nevertheless, the mechanism of action of one of the main nonpsychoactive phytocannabinoids, cannabidiol (CBD), remains elusive despite potentially beneficial properties as an anti-convulsant and neuroprotectant. Here, we characterize the mechanisms by which CBD regulates Ca 2+ homeostasis and mediates neuroprotection in neuronal preparations. Imaging studies in hippocampal cultures using fura-2 AM suggested that CBD-mediated Ca 2+ regulation is bidirectional, depending on the excitability of cells. Under physiological K + /Ca 2+ levels, CBD caused a subtle rise in [Ca 2+ ] i , whereas CBD reduced [Ca 2+ ] i and prevented Ca 2+ oscillations under high-excitability conditions (high K + or exposure to the K + channel antagonist 4AP). Regulation of [Ca 2+ ] i was not primarily mediated by interactions with ryanodine or IP 3 receptors of the endoplasmic reticulum. Instead, dual-calcium imaging experiments with a cytosolic (fura-2 AM) and a mitochondrial (Rhod-FF, AM) fluorophore implied that mitochondria act as sinks and sources for CBD’s [Ca 2+ ] i regulation. Application of carbonylcyanide- p -trifluoromethoxyphenylhydrazone (FCCP) and the mitochondrial Na + /Ca 2+ exchange inhibitor, CGP 37157, but not the mitochondrial permeability transition pore inhibitor cyclosporin A, prevented subsequent CBD-induced Ca 2+ responses. In established human neuroblastoma cell lines (SH-SY5Y) treated with mitochondrial toxins, CBD (0.1 and 1 μ m ) was neuroprotective against the uncoupler FCCP (53% protection), and modestly protective against hydrogen peroxide- (16%) and oligomycin- (15%) mediated cell death, a pattern also confirmed in cultured hippocampal neurons. Thus, under pathological conditions involving mitochondrial dysfunction and Ca 2+ dysregulation, CBD may prove beneficial in preventing apoptotic signaling via a restoration of Ca 2+ homeostasis.
Bidirectional Ca 2+ responses to CBD in hippocampal cultures. A , B , Sample traces for CBD-mediated Ca 2+ responses in neurons (black traces) and glia (gray traces) in normal ( A ) and high-excitability ( B ) HBS (double K + concentration). NMDA applications at the end of each experiment were used to confirm intact signaling in neurons. C , Mean responses of CBD in normal (ctrl) and high (high ex)-excitability HBS. Data are presented as %Δ F / F + SEM. *** p < 0.001.
Overall, FCCP eradicated CBD responses in neurons [mean: −1 ± 6% Δ F / F ( n = 25), p < 0.001 compared with controls] and significantly reduced responses in glia [reduced by 61 ± 5% ( n = 8), p < 0.001]. As these data strongly suggested a mitochondrial site of action, we aimed to exclude the ER as the primary source of Ca 2+ for CBD responses by applying CBD in the presence of specific antagonists to the receptors linked to Ca 2+ release pathways from the ER (dantrolene and 2-APB, acting as ryanodine and IP3 receptor antagonists, respectively). The blockade of one of these receptors has been shown to upregulate the activity of the other, implying that both release mechanisms share a common pool of Ca 2+ (White and McGeown, 2003). Thus, both antagonists were coapplied to fully block ER receptor-mediated release. Such a blockade transiently altered baseline Ca 2+ levels, but longer duration of antagonist treatment (10 min) allowed a settled baseline to be established before CBD application. When CBD was coapplied with 2-APB and dantrolene, responses did not significantly differ from control values ( p > 0.05), with glial responses increased compared with controls ( p < 0.001) ( Fig. 6 ), further confirming that ER receptors are somewhat modulating, but not mediating CBD-induced responses.
CBD effects on epileptiform activity in cultured hippocampal neurons. A , Application of the K + channel antagonist 4AP to naive cultures induces spontaneous Ca 2+ oscillations. B , C , The presence of CBD following ( B ), or preceding ( C ), 4AP application dampened Ca 2+ oscillations. Data are presented as %Δ F / F .
Significance for all statistical analyses performed was set at p < 0.05 = significant; p < 0.01 = highly significant; p < 0.001 = very highly significant.
Determination of cell death in hippocampal cultured neurons (live–dead cell staining kit) by multichannel image capture in cells treated with 20 μ m oligomycin. A , Transmission image. B , Cells with compromised cell membranes (rhodamine filter). C , Healthy cells (FITC filter). D , Merged image. A dead sample neuron is circled in each image. For further details, see Materials and Methods.
Since an antioxidant capacity has been widely reported for CBD (Hampson et al., 1998; Chen and Buck, 2000), its neuroprotective properties were subsequently compared with the protective capabilities of the free radical scavenger butylated hydroxytoluene (BHT). Interestingly, coapplication of FCCP with BHT (at 3 and 10 μ m ; n values = 11 and 12, respectively) conferred no significant protection ( p values >0.05), yet joint application of CBD (1 μ m ) applied with the higher concentration of BHT (10 μ m ) provided a complete prevention of FCCP’s toxic effects [100 ± 7% protection ( n = 12), p < 0.001 compared with FCCP controls], significantly more potent than CBD alone ( p < 0.001) ( Fig. 9 B ). The superadditive nature of this protection strongly suggests independent but synergistic modes of action. Overall, our data suggest that CBD directly acts on mitochondria, and this action offers protection against toxins that directly target mitochondria.
Two fundamental determinants of neuronal survival and viability under pathological conditions are Ca 2+ homeostasis and metabolic activity, both reliant on mitochondrial function. Neurons have a particularly high energy demand and correspondingly high metabolic activity, alongside large fluctuations in [Ca 2+ ] i ; thus, mitochondria play a particularly important role in this cell type. Even subtle mitochondrial deficits can have deleterious effects that can ultimately result in degenerative processes (for review, see Kajta, 2004). Energy deficiencies are also associated with aging (Bowling et al., 1993) (for review, see Wiesner et al., 2006) and age-related disorders, e.g., Alzheimer’s disease (de la Monte and Wands, 2006), indicating a correlation with mitochondrial dysfunction, as also recently suggested by a corresponding treatment success in Alzheimer’s patients (Doody et al., 2008). Mitochondria are preferentially located in areas of highest [Ca 2+ ] i adjacent to the endoplasmic reticulum, essential for the functional coupling of these two organelles (Robb-Gaspers et al., 1998; Szabadkai et al., 2003; Saris and Carafoli, 2005). Moreover, mitochondria determine cellular survival by generation of reactive oxygen species (Lafon-Cazal et al., 1993) and apoptotic factors (Hong et al., 2004). This process involves an increased permeability of mitochondrial membranes [including opening of the mitochondrial permeability transition pore (mPTP) (Hunter et al., 1976)]. Therefore, identification of agents that can restore normal mitochondrial function is highly desirable.
We here report bidirectional regulation of [Ca 2+ ] i and protection provided by CBD. This was evident acutely as CBD reduced cytosolic Ca 2+ levels in high-K + solution, and also silenced and prevented epileptiform-like activity induced by 4AP. The latter experiments were performed in the absence of TTX (as sustained spontaneous firing and neurotransmitter release is fundamental for epileptiform activity), hence one possibility is that anticonvulsant activity could be mediated by actions on transmitter release, a property already identified for a number of cannabinoids with respect to glutamate (Szabo and Schlicker, 2005; Shen et al., 1996) and GABA (Katona et al., 1999; Köfalvi et al., 2005). Such actions can potentially alter excitability but would require CBD to act on the endocannabinoid system. While modulatory interactions between CBD and endocannabinoids were demonstrated in our previous work (Ryan et al., 2007), this did not involve agonism on known CB receptors, although an indirect action on these receptors via inhibition of endocannabinoid reuptake and hydrolysis remains a possibility (Bisogno et al., 2001; Ligresti et al., 2006). A number of other studies have identified CBD as an anti-epileptic agent both in vitro and in vivo (for review, see Pertwee, 2004), and our data imply that this can be achieved by a mitochondrial regulation of [Ca 2+ ] i . We also propose that this action would offer beneficial protection in disease states that involve hyperexcitability, as CBD’s mode of action may allow it to functioning as a Ca 2+ sensor and regulator.
Effects of CBD and other potential neuroprotectants against toxicity induced by the mitochondrial uncoupler FCCP in SH-SY5Y cells. A , The phytocannabinoid CBD is neuroprotective with and without preincubation (Pre-inc.), although protection was enhanced when CBD was present before FCCP was applied. Such neuroprotection was similarly observed in hippocampal cultures (HIPP.) Maximal protection with CBD was comparable to that observed with cyclosporin A (CsA). B , Comparison of antioxidant effects of CBD and butylated hydroxytoluene (BHT). Note the apparent synergism between these two compounds, achieving full (100%) protection. C , Confirmation of FCCP’s actions to disrupt mitochondrial potential was obtained using MitoCapture, demonstrating both a reduced number of healthy cells (red signal) and increased number of cells with disrupted mitochondrial potential (green signal) in the presence of FCCP. Data are presented relative to controls (+SEM). * p < 0.05, *** p < 0.001; n.s., not statistically significant.
The apparent mitochondrial site of action of CBD led to the hypothesis that CBD may act as a neuroprotectant against mitochondrially acting toxins, acting either directly on mitochondrial sites or downstream thereof ( Fig. 1 ). Initial tests used the mitochondria-reliant viability assay Alamar Blue in SH-SY5Y cells, with protective actions of CBD confirmed in hippocampal cultures using a live–dead stain ( Fig. 7 ).
Finally, as a continuation of our imaging data, CBD was again tested in combination with the uncoupler of ATP synthesis, FCCP (see above and Fig. 1 ). To further confirm the mitochondrial site of action of FCCP, SH-SY5Y cells were loaded with the MitoCapture fluorescent dye, also used as a marker for apoptosis. In healthy cells, the reagent congregates in the mitochondria and is detected as a red fluorescence signal. Conversely, in apoptotic cells, MitoCapture remains in the cell cytosol (due to the disrupted mitochondrial membrane potential) and can be monitored as a green fluorescent signal. Following FCCP incubation (20 μ m , overnight) green fluorescence was increased by 45 ± 8%, while red fluorescence was decreased by 51 ± 6% (in both cases n = 30 and p < 0.001 compared with controls) ( Fig. 9 C ). This indicates that FCCP is acting to primarily depolarize mitochondria.
SH-SY5Y cell preparation.
The imaging system, fitted onto an Olympus BX51WI fixed stage microscope, used the Improvision software package Openlab (version 4.03, Improvision) with a DG-4 illumination system (Sutter Instruments) and a Hamamatsu Orca-ER CCD camera for ratiometric imaging. After an appropriate field of cells was identified, a gray-scale transmission image was visualized and captured. Cells were excited with wavelengths of 340 and 380 nm, and the ratio of fluorescence emitted at 510 nm analyzed after subtraction of background fluorescence levels. As described in our previous publications, fields of cells and regions of interest (ROIs) were chosen based on homogenous and equal cell densities, with a neuronal population of 15–40 cells per field of view. ROIs were placed on all fura-2 AM-loaded neuronal cell bodies and large, star-shaped glia, confirmed to be astrocytes by GFAP staining, and based on an overlay of a transmission image (Koss et al., 2007). Following this, time courses were created for all cells (neurons and glia), with frames captured every 5 s.
The highly lipophilic nature of cannabinoids grants them access to intracellular sites of action, and a number of studies have suggested mitochondria as targets for cannabinoids (Bartova and Birmingham, 1976; Sarafian et al., 2003; Athanasiou et al., 2007). Modulation of [Ca 2+ ] i by CBD has also been observed in a variety of cell types (Ligresti et al., 2006; Giudice et al., 2007), including our previous work which demonstrated a CBD-induced non-CB 1 /TRPV 1 -receptor-mediated increase in [Ca 2+ ] i in hippocampal neurons (Drysdale et al., 2006). Subsequent studies showed CBD effects to be negatively modulated by the endocannabinoid system (Ryan et al., 2007), but the exact mechanisms remained to be fully characterized. Therefore, the present study investigated CBD actions upon mitochondria and Ca 2+ homeostasis as a potential basis for CBD’s neuroprotective properties.
Cannabinoids as potential neuroprotectants against mitochondrial stressors in SH-SY5Y cells. Data are expressed as percentage protection (+SEM) relative to within-experiment controls and shown for peroxide (0.1 m m ) ( A ) and oligomycin (20 μ m ) ( B ) toxicity. CBD conferred protection against oligomycin toxicity in both SH-SY5Y cells and hippocampal cultures (HIPP.). Pre-Inc., Following 1 h preincubation; * p < 0.05, *** p < 0.001.
The established human neuroblastoma cell line, SHSY-5Y (SH), was grown in 30 ml flasks in MEM-based medium supplemented with growth factor F12, 10% fetal bovine serum, 2 m m l -glutamine, and 50 μg/ml antibiotic. Cells were maintained at 37°C at 5% CO 2 . Medium was replaced every 2–4 d, after washing with PBS (1 m m phosphate). Once cells proliferated to ≥80% confluency they were passaged or transferred to a 96-well plate (Greiner) for experimental treatment (final volume in medium: 150 μl of cell suspension per well). Plates were used for experimentation when ≥80% confluency was achieved (typically taking 4 d).
Dual-loading of hippocampal cultures with fura-2 AM and Rhod-FF, AM. A , Typical transmission image shows clearly defined neuronal appearance. B , C , Rhod-FF fluorescence ( B ) demonstrates a clear compartmentalization into mitochondria, a pattern disrupted by FCCP application ( Ci , Cii ). The corresponding cytosolic Ca 2+ alterations are monitored using fura-2 AM ( D ) with responses shown in both compartments to the mitochondrial uncoupler FCCP and NMDA ( B , Di–Div ). E , The raw values (OD, optical density) for each channel are plotted.
Previous work from our laboratory indicated a link between CBD-induced Ca 2+ responses and intracellular Ca 2+ stores (Drysdale et al., 2006), rather than extracellular Ca 2+ sources. Thus, we next investigated a potential role of mitochondria, fundamental players in cellular Ca 2+ homeostasis, in CBD’s action. To simultaneously study mitochondrial signaling together with cytosolic Ca 2+ responses, cultures were preloaded with the mitochondrion-specific Ca 2+ -sensitive fluorescent marker, Rhod-FF, AM, followed by fura-2 AM loading ( Fig. 4 ). The fluorescence pattern and responses to FCCP (10 μ m ), an uncoupler of ATP synthesis due to its action as a protonophore, confirmed the specificity of this protocol, causing leakage of mitochondrial Ca 2+ from mitochondria accompanied by an increased cytosolic Ca 2+ concentration ( Fig. 4 ). Application of CBD (1 μ m ) resulted in an increase in cytosolic Ca 2+ , preceded by a response in the Rhod-FF fluorescence ( Fig. 5 ). Two Rhod-FF response patterns were observed, biphasic (an initial rise followed by a decrease) or a continuous decline (see sample traces given in Fig. 5 A , B ). Subsequently, we confirmed that the pattern observed with CBD in this dual-fluorescence model genuinely represents a release from mitochondrial Ca 2+ stores by preapplication of FCCP (1 μ m ), applied to dual-loaded cultures (see Fig. 1 for the sites of action for this and other mitochondrion-acting compounds). At this concentration, FCCP induced an immediate reduction in Rhod-FF fluorescence in the mitochondrial compartment, and somewhat delayed in onset and progression, an increase in cytosolic Ca 2+ levels was observed. More importantly, no further responses to CBD could be induced in mitochondria ( Fig. 5 C ), while raised cytosolic Ca 2+ levels recovered partially, in agreement with our previous experiments in high-K + HBS and 4AP.
Hippocampal cultures were preincubated with CBD for 1 h before coapplication of CBD with mitochondrion-acting toxins overnight, following which cell death was quantified using a Live-Dead staining kit (Sigma) (modified from our previous publications) (Platt et al., 2007). Briefly, 10 μl of solution A and 4 μl of solution B were diluted in 5 ml of HBS (at room temperature). Each dish was washed in HBS three times and 500 μl of the staining solution added and incubated for 20 min (in the dark, at room temperature). After a further wash with HBS, live images were capture in HBS with a 40× phase-contrast water-immersion objective [brightfield, FITC (live cells) and rhodamine filters (dead cells)] using an Axioskop 2 plus microscope (Carl Zeiss) fitted with an AxioCam HRc camera, with AxioVision software (version 3.1). Three images were taken from each dish and each experiment performed on at least two dishes from three different cultures.
The mitochondrial and cytosolic Ca 2+ compartments were visualized simultaneously by preloading cultures with the mitochondrion-specific Ca 2+ sensor Rhod-FF, AM (Invitrogen). Culture dishes were incubated with Rhod-FF, AM (5 μ m , in standard HBS) for 15 min on the day before experimentation to allow compartmentalization of the marker (specificity of this marker was confirmed by the abolition of compartmentalization by FCCP application) (see Fig. 4 Ci , Cii ). HBS was replaced with fresh Neurobasal medium and returned to the incubator overnight. The following day, cells were loaded with fura-2 AM as described above. Dual imaging was performed with alternating wavelengths relevant to Rhod-FF (excitation: 550 nm; emission: 580 nm) and fura-2 AM (as above) delivered at intervals of 3 s. Both images were background subtracted, and separate graphs were plotted on-line (see Fig. 4 ). For off-line analysis of mitochondrial responses, data were imported into the Volocity analysis program (version 4.02, Improvision). Areas of most intense Rhod-FF mitochondrial fluorescence within a single neuron were allocated ROIs.
Thus, our data strongly suggested that [Ca 2+ ] i regulation via CBD is achieved via mitochondrial uptake and release, which could potentially be achieved via either the mPTP or the mitochondrial Na + /Ca 2+ -exchanger (NCX) (Griffiths, 1999). Experiments with the mPTP inhibitor CsA showed no difference to control CBD responses, implying that the mPTP is not the principal mechanism of CBD’s actions ( Fig. 6 ). When the role of the NCX in CBD-mediated responses was investigated using the specific antagonist CGP 37157 (CGP) (Chiesi et al., 1988; Medvedeva et al., 2008), preapplied and coapplied (10 μ m ), CBD (1 μ m ) responses were abolished [remaining response: neurons: 10 ± 10% Δ F / F ( n = 8), glia: 3 ± 7% Δ F / F ( n = 14), p values <0.001] ( Fig. 6 ). To confirm that NCX was also fundamental to [Ca 2+ ] i reducing CBD responses, the experiment was repeated in the presence of elevated [K + ] e (as above). The reversal of neuronal CBD responses normally seen under these conditions was no longer observed. Accordingly, the CBD response in CGP no longer differed between high-K + and standard HBS in both neurons and glia ( p values >0.05) ( Fig. 6 ). Therefore, we conclude that CBD is acting via the mitochondrial NCX to elevate or decrease cytosolic Ca 2+ levels, dependent on resting [Ca 2+ ] i .
The plant Cannabis sativa has for many centuries been reputed to possess therapeutically relevant properties. Its most widely studied and characterized component, Δ 9 -tetrahydrocannabinol (THC), is one of 60+ compounds from Cannabis sativa , collectively known as phytocannabinoids. However, THC may have a limited usefulness due to psychoactivity, dependence, and tolerance (Sim-Selley and Martin, 2002); therefore, attention has turned to some of the nonpsychoactive phytocannabinoids, most notably cannabidiol (CBD). CBD has little agonistic activity at the known cannabinoid receptors (CB 1 and CB 2 ) (Pertwee, 2004), and may possess therapeutic potential, e.g., anti-epileptic (Cunha et al., 1980), anxiolytic (Guimarães et al., 1994), anti-inflammatory (Carrier et al., 2006), and even anti-psychotic properties (Leweke et al., 2000) [for review, see Pertwee (2004) and Drysdale and Platt (2003)]. In addition, CBD has shown neuroprotection in a range of in vivo (Lastres-Becker et al., 2005) and in vitro models (Esposito et al., 2006), some in association with a reduction in [Ca 2+ ] i (Iuvone et al., 2004).
CGP may act not only upon mitochondrial NCX, but also as an inhibitor of VGCCs in dorsal root ganglion neurons (Baron and Thayer, 1997). However, our previous data with VGCC blockers (Drysdale et al., 2006) are not consistent with an effect of CBD on this target. Others have found CGP to inhibit the NCX in the plasma membrane of cerebellar granule cells (Czyz and Kiedrowski, 2003), although with an IC 50 of 13 μ m , a concentration higher than that used here, and higher than CGP’s IC 50 (4 μ m ) for the mitochondrial NCX in cultured rat DRGs (Baron and Thayer, 1997). Indeed, the concentration of CGP used here is in keeping with recent work by others in cultured neurons (Medvedeva et al., 2008).
Our study uncovers a new intracellular, and potentially direct, target for CBD, which has so far been largely elusive despite the wide-ranging use of this phytocannabinoid in diverse preparations and applications. Evidence for actions of CBD on mitochondria was strongly supported by cell death models with mitochondrial toxins. The most potent CBD protection was seen against FCCP toxicity (in SH cells and reproduced in cultured hippocampal neurons), a mitochondrial uncoupler, causing the collapse of the mitochondrial membrane potential and the release of Ca 2+ into the cytosol. A more modest protection by CBD was observed against other oxidative stress related agents, hydrogen peroxide and oligomycin. FCCP causes the accumulation of protons into mitochondria leading to uncoupling of the mitochondrial potential (ΔΨ m ), ultimately causing a loss of ATP production (for review, see Wallace and Starkov, 2000), and has previously been demonstrated to cause apoptosis in PC12 cells (Dispersyn et al., 1999) and primary neurons (Moon et al., 2005). The loss of ΔΨ m (confirmed by MitoCapture) and resultant cell death is also assumed to involve mPTP formation (Marques-Santos et al., 2006), in keeping with our finding that CsA can protect against FCCP toxicity in a dose-dependent manner.
CBD, obtained from GW Pharmaceuticals, and AM281 (Tocris Bioscience) were stored in ethanol (1 mg/ml) at −20°C. For use in experiments, the ethanol was evaporated and the cannabinoid resuspended in dimethyl sulfoxide (DMSO) at 1 m m (control experiments confirmed that 0.1% DMSO did not alter basal Ca 2+ levels or NMDA-induced Ca 2+ responses, data not shown). The toxins tested in the SH-SY5Y model were as follows: hydrogen peroxide (H 2 O 2 , Sigma-Aldrich) at 0.1 and 0.5 m m , 3 h application; oligomycin (20 μ m , Sigma-Aldrich) applied overnight (used in the same manner in hippocampal culture cell death models also); FCCP (Sigma-Aldrich) was also applied overnight at 20 μ m (also applied in the same concentration and duration in hippocampal culture cell death models). In each case, pilot experiments were performed to determine suitable concentrations resulting in a degree of cell death that leaves capacity for either a reduction or increase in cell viability (targeted reduction in cell viability: 40–70%). The sites of action of these toxins can be seen in Figure 1 . Other compounds tested to elucidate CBD’s mechanisms of action were (with final concentrations and stock solvents listed) as follows: catalase (500 and 1000 U/ml; MEM), cyclosporin A (CsA; 1–20 μ m ; DMSO), butylated hydroxytoluene (BHT; 3 and 10 μ m ; DMSO), and dantrolene (10 μ m ; H 2 O), all from Sigma-Aldrich. Additionally, 4-aminopyridine (4AP; 50 μ m ; DMSO), 2-aminoethoxydiphenyl borate (2-APB; 100 μ m ; DMSO), and CGP 37157 (10 μ m ; DMSO) were obtained from Tocris Bioscience. For all compounds tested, drug-only controls were performed.
Reactive oxygen species production from mitochondria and cytosol cytochrome c levels in reperfused CBD and DCD hearts.
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(A.) H 2 O 2 production in CBD and DCD hearts subjected to 60 minutes of reperfusion (RPF), n = 8, each. *p<0.05 vs CBD group, using two tailed non-paired t-test. (B.) Upper panel shows representative blot for immunoblotting of cytosolic cytochrome c with anti-cytochrome c antibody. Lower panel graph represents ratio of cytochrome c to GAPDH, the loading control. Data are expressed as mean ±SEM. *p<0.05 vs. CBD group; ǂ p<0.05 vs. DCD group; Ŧ p<0.05 vs. CBD with reperfusion group, using one-way ANOVA. n = 4 in each group. (C). Total infarct size as measured by triphenyl tetrazolium chloride (TTC) staining, in CBD and DCD hearts subjected to reperfusion (RPF), n = 8, each. *p<0.05 vs CBD group, using two tailed non-paired t-test. (D.) Coronary flow from CBD and DCD hearts following 60 minutes of reperfusion. *p<0.05 vs CBD group, using two tailed non-paired t-test.