Exogenous mitochondrial transplantation improves survival and ... - BMC Medicine

Cell culture

To determine whether exogenous donor mitochondria can be taken up into neurons growing in culture, we co-cultured exogenous mitochondria extracted from the brain or muscle tissues of donor rats with neural cell cultures. Both donor mitochondria and recipient neural cell cultures were derived from male Sprague–Dawley rats (12 weeks old; Charles River Laboratories, Wilmington, MA, USA). Neural cells were isolated using the Adult Brain Dissociation kit (Miltenyi Biotec, Inc., Somerville, MA, USA). Cells were seeded at a density of 1 × 10⁵ cells/cm² onto glass coverslips coated with poly-d-lysine (0.1 mg/mL). The endogenous (native) mitochondria of the cells were separately labeled 24 h before mitochondrial transfer with MitoTracker dye (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Briefly, the cells were suspended in a prewarmed (37 °C) staining solution containing the MitoTracker Green probe (300 nM) and incubated for 30 min in a standard medium under appropriate growth conditions. After staining, the cells were washed twice with phosphate-buffered saline (PBS) and resuspended in a fresh medium.

To visualize mitochondrial transfer, neural cells in which endogenous mitochondria were stained green (see above) were co-cultured with donated exogenous mitochondria (red) in a standard culture medium for 24 h, and cells were observed using an LSM 880 confocal imaging system (Carl Zeiss Meditec AG, Jena, Germany).

Mitochondrial isolation from brain tissue

For MTx experiments in the cell culture, brain tissues in mitochondrial isolation buffer [210 mM d-mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EGTA, and 0.5% (w/v) fatty acid-free bovine serum albumin (BSA), adjusted pH to 7.2 with KOH] were disrupted by 30 strokes at 500 rpm in a homogenizer, after which the homogenate was centrifuged at 800 g for 10 min at 4 °C in a swing-out rotor. The supernatant was then centrifuged at 12,000 g for 10 min at 4 °C to create a pellet containing the mitochondria. After removal of the supernatant, the pellet was washed twice with mitochondrial isolation buffer, and the pellet was resuspended in prewarmed (37 °C) staining solution with PBS containing the MitoTracker Deep Red probe (300 nM) and incubated for 30 min. After the removal of the staining solution, the labeled mitochondria were washed twice with PBS. Mitochondria were quantified by determining the protein concentration using the Bradford assay (Pierce, Rockford, IL, USA) and kept on ice until transplantation. Mitochondria (0.01 mg/mL, final concentration) resuspended in 500 μL of fresh prewarmed medium were immediately used for mitochondrial transfer.

Mitochondrial isolation from pectoral muscles

Muscle-derived mitochondria from rats were used both for MTx experiments (A) in cell culture and (B) as donor mitochondria used by infusion into an in vivo rat as outlined below in our CA model protocol. Mitochondria were isolated from a 6-mm piece of healthy pectoralis major muscle tissue from a rat using a rapid mitochondrial isolation method, as previously described [24]. This method using an automated homogenizer and different filtrations developed for clinical use when speed was essential as the full procedure may be completed in 30 min was recently reported by McCully et al. [8, 10, 24]. Briefly, immediately after obtaining the muscle using a 6-mm biopsy punch, the tissue was minced in cold homogenized buffer [300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA (pH 7.2)] at 4 °C and homogenized using an automated homogenizer (gentleMACS dissociator; Miltenyi Biotec Inc., San Diego, CA, USA). The homogenate was then subjected to digestion for 10 min with subtilisin A on ice (protease from Bacillus licheniformis; Sigma-Aldrich, St. Louis, MO, USA), and the digested homogenate with 0.49% fatty-acid free BSA was filtered through a series of disposable sterile mesh filters. The filtrate was centrifuged at 9000 g for 10 min at 4 °C, and the final pellet was resuspended in 0.5 mL of a cold respiration buffer [250 mM sucrose, 2 mM KH₂PO₄, 10 mM MgCl₂, 20 mM K-HEPES (pH 7.2), 0.5 mM K-EGTA (pH 8.0)]. The yield of mitochondrial particles obtained using a 6-mm biopsy tissue sample was reported to be approximately 1 × 10¹⁰ mitochondria, which provided sufficient mitochondria for infusion, as well as quality assurance and quality control assessment [8, 10, 24]. Previous studies have consistently demonstrated the viability and functionality of mitochondria isolated from the skeletal muscle using this method [10, 16, 24, 25]. Isolated mitochondria were used immediately for intravenous infusion as fresh donor mitochondria or were frozen and stored at − 80 °C for over 2 weeks until subsequent use as frozen-thawed mitochondria.

Measurements of ATP content in isolated mitochondria

ATP contents were determined in (a) respiration buffer as a negative control, (b) frozen-thawed, and (c) freshly isolated mitochondria using a luminescent assay kit (ATPlite, PerkinElmer, MA) according to the manufacturer's instructions. A total of 10 µL of mitochondrial particles from the prepared samples or respiration buffer were added to each well of a white, opaque bottom, 96-well plate. After measuring luminescence, ATP concentration in each well was calculated using the standard curve obtained from ATP standard stock solution.

Flow cytometry analysis of JC1 assay for isolated mitochondria

The mitochondrial membrane potential (ΔψM) was evaluated by MitoProbe™ JC1 (5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) Assay Kit (Thermo Fisher Scientific, Waltham, MA) using a BD FAC Symphony flow cytometer (BD Biosciences, San Jose, CA). JC1 exhibits potential-dependent accumulation (J-aggregates) in mitochondria, indicated by a fluorescence emission shift from green (~ 529 nm) to red (~ 590 nm). Therefore, ΔψM can be assessed by an increase in the red fluorescence J-aggregates. After isolation, mitochondrial suspension in 1 mL respiration buffer was mixed with 10 μL of 200 μM JC1 (2 μM final concentration) with or without 1 μL of 50 mM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 50 μM final concentration). CCCP is a well-established mitochondrial membrane potential disrupter. After incubation at 37 °C for 30 min, the suspension was centrifuged at 9000 g for 10 min at 4 °C. The pellets were washed once by adding 1 mL PBS and centrifuged. The pellets were resuspended in 500 µL fresh respiration buffer. Unstained samples or size reference beads (Spherotech, Inc., Lake Forest, IL) were used to establish a proper mitochondrial size and voltage setting. The acquisition for JC1 Red-positive events was performed on 100,000 events. The percentages of the JC1 Red fluorescence J-aggregates were measured as ΔψM in the freshly isolated mitochondria, frozen-thawed mitochondria, and a subgroup of frozen-thawed mitochondria which was treated with a membrane potential disrupter CCCP as the lowest ΔψM control. Unstained mitochondria were used as a negative control. The data were analyzed with the FlowJo software (Tree Star, Ashland, OR, USA).

Animal care and surgical preparation

Adult male Sprague–Dawley rats (400‒545 g, 12‒16 weeks old; Charles River Laboratories) were used in this in vivo study. The animals were housed in a rodent facility under a 12:12-h light/dark cycle with ad libitum access to food and water. The rats were intubated with a 14-gauge plastic catheter (Surflo; Terumo Medical Corporation, Somerset, NJ, USA) under anesthesia with 4% isoflurane (Isosthesia; Butler–Schein AHS, Dublin, OH, USA), mechanically ventilated, and surgically prepared under anesthesia (2% isoflurane). Before the surgical procedure, surgical sites were cleaned with povidone-iodine and then covered with a sterile, self-adhesive, transparent, povidone-treated surgical blanket. All surgical procedures were conducted using sterile equipment and performed by the investigators blinded to the experimental groups. End-tidal carbon dioxide was maintained at 40 ± 5 mmHg during the experiment by adjusting the respiratory rate (RR) and tidal volume (TV), with these settings adjusted within the range of 40/min to 50/min for the RR and 3.5 to 5.0 mL of TV. Microcatheters (PE-50; Becton Dickinson, Franklin Lakes, NJ, USA) were inserted into the left femoral artery and left femoral vein to monitor blood pressure and infuse drugs and donor mitochondria, respectively. Heparin (300 U) was injected into the femoral vein. The esophageal temperature was maintained at 37.0 ± 0.5 °C using a thermostatically regulated heating pad and heating lamp during the experiment. Blood pressure and needle-probe electrocardiogram-monitoring data were recorded and analyzed using a personal computer-based data-acquisition system.

Rat cardiac arrest protocol

All animal studies were performed using protocols approved by the Institutional Animal Care and Use Committee at our institution and in accordance with National Institutes of Health guidelines. Rats were subjected to CA and cardiopulmonary resuscitation, as previously described [26, 27], with minor modifications. Briefly, prior to the induction of asphyxia, rats were mechanically ventilated with a fraction of inspired O₂ (F_IO₂; 0.3), and anesthesia was maintained with 2% isoflurane during surgical procedures. Asphyxia was induced by intravenous vecuronium bromide (2 mg/kg), followed by switching off the ventilator and discontinuation of isoflurane. CA was defined as a mean arterial pressure of < 20 mmHg. At 10 min after induction of asphyxia, mechanical ventilation was restarted at an F_IO₂ of 1.0, and manual chest compressions were performed at a rate of 300/min by a single investigator blinded to the experimental groups. At 30 s after beginning chest compressions, a 20-μg/kg bolus of epinephrine was administered, and chest compressions were continued until successful resuscitation, which was defined as the return of supraventricular rhythm with a mean arterial pressure of > 60 mmHg for 10 s. Rats were mechanically ventilated with an F_IO₂ of 1.0 for the first 10 min after resuscitation, after which F_IO₂ was reduced to 0.3, followed by disconnection from the mechanical ventilator and extubation at 2 h post-CA. Arterial blood pressure, electrocardiogram recordings, and esophageal temperature were monitored for 2 h. Arterial blood samples for blood gas and lactate analyses were obtained at baseline and 15- and 120-min post-CA. No additional inotropic agent was administered. After a recovery period of 2 h, the animals were weaned from the ventilator, all vascular catheters and tracheal tubes were removed, and surgical wounds were sutured. The rats were then returned to their cages with easily accessible food and water, and observed in a rodent facility with a controlled room temperature of 22 °C. Buprenorphine XR (0.2 mL; 0.26 mg) was subcutaneously injected once to relieve any pain due to incisions in all animals during the recovery period. The survival time after CA was recorded for up to 72 h.

Assessment of neurological function

Neurological function score (NFS) was evaluated by a blinded investigator at 24, 48, and 72 h post-CA using a previously reported neurofunctional scoring system [27]. With this score, neurologically normal animals would receive a score of 500, while dead or brain-dead rats were scored at 0 points.

Echocardiography

We assessed left ventricular ejection fraction (LVEF) at baseline and 2 h post-CA using echocardiography. Transthoracic closed-chest echocardiography was performed by a single blinded investigator using a 12–4 MHz probe (S12-4 sector array transducer; Philips, Amsterdam, The Netherlands), and all measurements were averaged over three cardiac cycles.

Assessment of 72-h lung injury

The lung wet-to-dry weight ratio (W/D) was used as an index of pulmonary edema formation. The left lower lobe was removed at 72 h post-CA, weighed immediately after removal (wet weight) and again after drying in an oven at 37 °C for 7 days (dry weight). Lung W/D was calculated as the ratio of wet weight to dry weight.

Mitochondrial infusion immediately after CA

Animals subjected to CA were block-randomized into one of three groups of interventions that were administered immediately after animals achieved successful resuscitation: (a) infusion of the respiration buffer with 0.49% BSA (vehicle group; n = 11), (b) infusion of nonfunctional frozen-thawed mitochondria (frozen-thawed-mito group; n = 11), or (c) infusion of fresh viable mitochondria (fresh-mito group; n = 11). The process of freezing and thawing leads to widespread disruption of mitochondrial outer membrane integrity and suppresses electron-transport chain activity through the loss of cytochrome c from the inter-membrane space [28]. For that reason, we used frozen-thawed mitochondria as an additional control group to maintain similar amounts of mitochondrial protein, lipids, DNA, RNA, and other macromolecules, as infused into the animals treated with freshly isolated mitochondria. These disrupted frozen-thawed mitochondria contain similar quantities of biological molecules but do not have viability and respiratory competence.

Measurements of gene expressions

RNA isolation, reverse transcription, and real-time PCR analysis were performed on brain and spleen tissues harvested at 72 h post-CA resuscitation and MTx according to the manufacturer's instructions. Total RNA was extracted using TRIzol Reagent (Sigma-Aldrich, USA) and reverse transcribed using SuperScript IV VILO™ Master Mix with ezDNase Enzyme (Thermo Fisher, USA). Real-time PCR was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher, USA) on the LightCycler 480 system (Roche Diagnostics). The primers used are dynamin-related protein 1 (Drp1, Rn00586466_m1), mitochondrial fission 1 protein (Fis1, Rn01480911_m1), optic atrophy-1 (Opa1, Rn00592200_m1), Mitofusin-1 (Mfn-1, Rn00594496_m1), and Mitofusin-2 (Mfn-2, Rn00500120_m1).

Measurement of cytochrome c oxidase activity

To measure cytochrome c oxidase (COX) activity in tissues, the brain and spleen were obtained from sham-operated rats and surviving rats at 72 h post-CA in the vehicle, frozen-thawed, or fresh-mito groups. The COX activity in tissue homogenates was measured using the Cytochrome C oxidase Kit (Abcam, ab239711) according to the manufacturer's instructions.

Brain perfusion measured with laser speckle flowmetry and image processing

In a separate set of experiments aimed at determining the impacts of MTx on brain perfusion during the acute phase post-CA, we monitored relative cerebral blood flow (rCBF) for the first 2 h after CA. Laser speckle imaging of the brain was performed using the full-field laser perfusion imager RFLS III system according to the manufacturer's instructions (RWD Life Science Co., Ltd., Guangdong, China), as previously described [29]. A midline scalp incision was made to expose the skull for imaging. The skull over the left cortical surface was thinned using a dental drill, ensuring that the dura remained intact. The imager was positioned directly above the surface of the thinned skull. Continuous image acquisition (time constant, 1 s; camera exposure time, 5 ms; laser intensity, 100 mA; resolution, 2048 × 2048) started at the pre-CA baseline and continued until 2 h post-CA. The vessels were recognized according to anatomical characteristics, and then three regions of interest (ROIs) were selected at the pre-CA baseline. The ROIs included the area over the left superior cerebral vein and two capillary areas of the left cortical surface between the superior cerebral veins. The signal intensities of perfusion were calculated at each time point using the laser speckle imaging system software and normalized against the baseline [29]. The means of rCBF values at the three ROIs were compared between the groups.

Confocal fluorescence microscopy

In a separate set of experiments, rats subjected to CA were used to ascertain the uptake and persistence after 1 and 24 h of labeled mitochondrial in vital organs using a confocal microscope. The freshly isolated mitochondria were labeled with MitoTracker Deep Red immediately after isolation, and the vehicle or the labeled mitochondria were infused upon resuscitation from CA. At 1 or 24 h post-CA, animals were euthanized, and the brain, heart, lung, kidney, liver, and spleen were harvested and fixed with 4% paraformaldehyde solution. Sections were mounted with mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) and observed using an LSM 880 confocal imaging system (Carl Zeiss Meditec AG, Jena, Germany).

Statistical analysis

Data represent the mean ± standard deviation. Neurological function scores were compared using a Kruskal–Wallis test, followed by Dunn's multiple comparison test. Continuous data were analyzed by one-way analysis of variance (ANOVA) with Šidák's correction for post hoc comparisons between multiple experimental groups. Hemodynamic, body weight changes, laboratory, and rCBF data were examined using a mixed-effects model for repeated-measures analyses, followed by ANOVA with Šidák's correction for post hoc comparisons. For the in vivo survival study, we performed a power analysis to calculate the sample size necessary to achieve a reliable measurement of the effect. As the mean survival rate at 3 days after CA was expected as 40% in the vehicle group and 85% in the freshly isolated mitochondria group, we anticipated that 11 rats per group were required in each survival study (α = 0.05, β = 0.2 [power = 80%], two-sided). All data are included (no outlier values or animals were excluded from the study). Kaplan–Meier analysis using the Gehan–Breslow–Wilcoxon test was used to calculate the survival rates between groups. A P < 0.05 was considered statistically significant. GraphPad Prism (v.9.2.0; GraphPad Software Inc., La Jolla, CA, USA) was used for all statistical analyses.

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