Ethics statement

The animals were fed and maintained with free access to water until one day before the intervention. The treatment of animals adhered to the Council for International Organizations of Medical Sciences (CIOMS) International Guiding Principles for Biomedical Research Involving Animals. The protocol for the use of animals was reviewed and approved by the Institutional Ethics Committee for the Use of Animals of the Universidad Peruana Cayetano Heredia (SIDISI 201478). The animals were sedated using an intramuscular injection of ketamine (10 mg/kg) and xylazine (2 mg/kg). The intervention sites were shaved, followed by soap scrubs. Endotracheal intubation was achieved in all animals and kept sedated with intravenous doses of ketamine (5 mg/kg) every 20 minutes. The entire cerebrovascular surgical procedure was performed with microcatheters in a sterile environment.

Skeletal Muscle Biopsy Collection

In the two pigs chosen for mitochondria infusion, the area selected for biopsy collection was the hind leg. The zone was desinfected with a povidone-iodine solution (10%), and a small incision was made using a #11 scalpel blade (USA Medical, St. Louis, MO; USA). Three samples of the gracilis muscle were collected for each pig using a #6 biopsy punch (Fisher Scientific, Waltham, MA, USA) and transported in sterile 50 ml tubes with 20 ml of phosphate-buffered saline (PBS pH 7.4) inside a container with ice. Finally, manual pressure and sutures were applied to the biopsy site.

Isolation and purification of mitocondria

Isolation of mitochondria was performed in a laminar flow cabinet following the protocol of Preble ¹⁸. Sample were transfered to a 15 ml tube containing 10 ml of Homogenization Buffer (300 mM sucrose; 10 mM HEPES; 1 mM EDTA; all Sigma-Aldrich, ST. Louis, USA) and disintegrated using a homogenizer (Omni International, Kennesaw, GA, USA) in 4 cycles of 60 sec. 250 µl of a trypsin solution (4 mg of trypsin dissolved in 1 ml of Homogenization Buffer) was added and incubated on ice for 10 minutes. Each homogenate was filtered with a 40 µm filter (Falcon) pre-wet with Homogenization Buffer into a new 15 ml tube, then 250 µl of a BSA solution (20 mg BSA dissolved in 1 ml Homogenization Buffer) was added. The samples were filtered with 40 and 10 µm filter pre-wet with homogenization buffer. They were centrifuged at 9,000 G (9000 x g) for 10 min at 4°C, the supernatant was discarded, and the pellet was resuspended in 1 ml of Respiration Buffer (sucrose 250 mM; KH2PO4 2 mM; MgCl2 10 mM; HEPES 20 mM, EDTA 0.5 mM; all Sigma-Aldrich brand, ST. Louis, USA).

Viability and mitochondrial labeling

A 1 ml aliquot of the mitochondria suspension was taken and incubated with MitoView Green (Biotium, Fremont, CA, USA) (200 nM) for 30 min to determine the total number of mitochondria obtained, then incubated with Mitotracker Red CM-H₂Xros (Thermo Fisher Scientific, Waltham, MA, USA) (500 mM) for 30 min to determine the number of viable mitochondria. Counting was performed using a hemocytometer, and images were captured by confocal microscopy (Zeiss, LSM880, Oberkochen, Germany). The mean ± standard deviation of total and viable mitochondria count was obtained from the 3 muscle samples collected for each pig. Before proceeding with the infusion, the remaining reconstituted mitochondria were incubated with a mouse anti-mitochondrial monoclonal antibody (Abcam ab14730, Cambridge, UK) at a concentration of 1 µg/ml for 30 min at 4°C.

Vascular access and BBB disruption

Using the manubrium as an anatomical landmark and assisted by ultrasound guidance, a sterile #11 blade was used to incise the tissues of the ventral neck to expose the carotid artery. A Braun micro-introducer kit (Bethlehem, Pennsylvania USA), including a 21-gauge X 7 mm echogenic introducer needle was used to access the left common carotid artery. The needle was advanced until pulsatile flow was observed, followed by an 0.018 in X 40 cm stainless steel mandrel guidewire. Using the Seldinger technique ¹⁹, the needle was removed and replaced with a microcatheter and dilator. After microcatheter aspiration, an 0.9% intra-arterial saline drip line was attached. Through the intra-arterial saline drip line, osmotic blood-brain barrier (BBB) disruption ¹⁷ was performed using 0.25 mL/s per-kg infusion of mannitol over 20 seconds.

Microcatheter-based brain infusion of autologous mitochondria

At the five-minute peak of osmotic BBB disruption ²¹, the saline drip was disconnected from the microcatheter, and a sterile 10 ml syringe was used to deliver 5 mL of labeled mitochondria followed by a 10 mL flush of respiration buffer. The catheter was removed from the left carotid artery, and hemostasis was obtained by applying manual pressure for 15 minutes. This process was replicated in the contralateral control, minus the osmotic BBB disruption. The deep and superficial tissues of the ventral neck were closed with sutures. In the case of the control pig, it was infused only with respiration buffer. 

Immunofluorescence in brain biopsies

After 4 hours, the animals were euthanized with a lethal dose of pentobarbital (120 mg/kg). The brains were removed and cut into 10-mm slices. Samples were fixed with 10% neutral buffered formalin for 24 h then transferred to 70% ethanol and embedded in paraffin. The formalin-fixed paraffin-embedded pig brain samples were cut in sections of 4 µm and placed on poly-L-lysine coated slides. The sections were deparaffinized at 56°C, immersed in xylene and then rehydrated in solutions with decreasing proportion of ethanol (100% to 70%), then permeabilized by heating in citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) for 30 min at 95°C. To eliminate nonspecific staining, slides were blocked for 30 min in a humid chamber at room temperature with a blocking solution (PBS pH 7.2, 0.05% Tween 20, 0.1 % Triton X-100, 2% goat serum, 2% BSA). Sections were incubated overnight at 4°C with the fluorescein-labeling goat anti-mouse IgG polyclonal antibody (Abcam, Cambridge, UK) diluted 1/500 in PBS. The sections were washed three times for 2 min with washing solution (PBS pH 7.2, 0.05% Tween 20) and mounted with VectaShield mounting medium with DAPI (Vector, Laboratories, Burlingame, Ca). The images were captured by confocal microscopy using the 10X objective (Zeiss, LSM880, Oberkochen, Germany).

Mitochondrial viability and quantification

The quantification of total mitochondria isolated from muscle tissue using a hemocytometer and the green signal from Mitoview Green staining (Figure 1A) determined an average total concentration of 1.05 X 10⁷ ± 7.20 X 10⁵ mitochondria per ml. However, quantification based on the Mitotracker red signal (Figure 1B) corresponding to mitochondria with an intact membrane potential determined an approximate concentration of 63% of viable mitochondria (6.61 X 10⁶ ± 9.90 X 10⁵ mitochondria per ml).

Figure 1 Mitochondrial viability and yield. Representative images of double staining in mitochondria isolated from skeletal muscle biopsies. Total mitochondria were count using the green signal from Mitoview Green (1A), the percentage of viable mitochondria was obtained using the red signal from Mitotracker (1B), mitochondria were also observed with brightfield (1C), and the merge of green and red signals is shown in (1D). Images were captured with a confocal microscope using a 20X objective. Scale bar: 20 μm. 

Arterial access and infusion

Successful arterial access and subsequent carotid microcatheterization was performed in all three pigs. A total of 5.24 x 10⁷ ± 3.59 x 10⁶ mitochondria were reconstituted into the respiration buffer and injected into the carotid artery as a 5 mL infusate. Procedural duration, including tissue harvest, vascular access, and mitochondrial infusion was 48 and 42 minutes; approximately 40 minutes dedicated to mitochondrial isolation, quantification, and labeling. The time from sedation onset to extubation was about 90 minutes. No difficulty in microcatheterization was encountered. No other complications, including hemodynamic instability, pneumothorax, vascular perforation, stroke, or incision site hematoma were seen.

Post-infusion evaluations

Samples from all cortical regions were collected and evaluated in triplicate by immunofluorescence. Representative images of frontal lobe sections with and without BBB disruption are shown (Figure 2). In the absence of osmotic BBB disruption, few labeled mitochondria were observed only within the lumen of capillaries (Figure 2B-C). There were no clusters or focal areas of aggregation appreciated, and they were completely absent in the brain parenchyma. After BBB disruption using a hyperosmotic mannitol solution, an increased concentration of labeled mitochondria was observed in all regions of the cortex and throughout the parenchyma in small clusters in a diffuse and linear pattern (Figure 2D-F). The control sample infused with respiration buffer gave no signal with or without BBB disruption (Figure 2G-I).

Figure 2 Autologous mitochondria infused in pig brain. Representative images of brain tissue after mitochondria infusion. In brain samples with BBB integrity (2A-C), few mitochondria are observed in the lumen of the capillaries (white arrows). The brain parenchyma did not show a positive signal for mitochondria. In samples with osmotic BBB disruption (2D-F), a large number of mitochondria are present in capillaries (white arrowheads) and the form of clusters within the parenchyma (red arrowheads). In the control sample infused with respiration buffer (2G-I), no positive signal for mitochondria was observed. Histological sections of 4 μm were stained with DAPI (2A, D, G), treated with an anti-IgG antibody (2B, E, H). The merge of both signals with the brightfield is observed in (2C, F, I). Images were captured with confocal microscopy using a 10X objective. Scale bar: 100 μm.