Mitochondrial labelling and isolation
Allogeneic mitochondria were isolated from rat livers. For tracking of mitochondria after delivery, mitochondrial DNA (mtDNA) in hepatocytes was labeled with 5-Bromodeoxyuridine (BrdU) (Sigma-Aldrich, Burlington, MA, USA) prior to isolation. Rats were sacrificed 36 h after a single intraperitoneal (ip) injection of BrdU (50 mg/kg). Livers were homogenized in ice cold buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl and 1 mM EDTA, pH 7.4) using a Precellys homogenizer (Bertin Technologies, MD, USA). Through a series of filtration and centrifugation steps, as described previously , isolated mitochondria were re-suspended in ice-cold MiR05 respiration buffer (0.5 mM EGTA, 3 mM MgCl2, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM D-sucrose, 0.1% w/v fatty acid-free BSA) and prepared for use. BrdU immunofluorescence was used to track internalized mitochondria in sample tissue.
Mitochondria conjugated with Pep-1
Detailed procedures for Pep-1 conjugation have been described previously . 200 μg allogeneic mitochondria suspended in respiration buffer were conjugated with 0.11 mg Pep-1 diluted with sterile water (Anaspec, San Jose, CA, USA). Incubation for 10 min at room temperature was performed to ensure complex assembly.
Animals and intranasal administration of mitochondria
Adult female Sprague-Dawley rats (8 weeks old, weighing 250-300xg) were purchased from BioLASCO (Taipei, Taiwan) and were maintained under standard laboratory conditions with free access to food and tap water in the Laboratory Animal Center, Changhua Christian Hospital, Changhua, Taiwan. All experimental procedures of animals were approved by the Animal Experiments and Ethics Committee of Changhua Christian Hospital (approval number CCH-AE-105-018). A detailed procedure for creating a neurotoxic rat model of Parkinson's disease using a unilateral injection of 6-OHDA into the medial forebrain bundle (MFB) has been described previously . Successful induction of PD rats after three weeks was validated by a rotational behavior test, described below. PD rats were assigned randomly to five groups of six rats: 1) control group receiving no treatment (WT), 2) intranasal infusion of vehicle only (Sham), 3) mitochondria alone (Mito), 4) Pep-1-labelled mitochondria (P-Mito) and 5) Pep-1 alone (Pep-1) . Rats received a unilateral, intranasal infusion (ipsilateral side relative to the lesion) of 200 μg Mito or P-Mito in a total of 50 µL MiR05 respiration buffer, and treatment once a week for three months. Experiments were designed to minimize the number of animals used and their suffering.
Rotational behavior test
Motor imbalance of 6-OHDA-lesioned animals was assayed by methamphetamine (3 mg/kg, i.p.)-induced rotation. Rotational asymmetry was assessed using an automated rotometer system (AccuScan Instruments, Columbus, OH, USA) based on the design of Ungerstedt & Arbuthnott  to record the counterclockwise rotation with full-body turns. Rats that exhibited more than 360 rotations within 60 min were randomly assigned for experimental use. During the period of the experiment, the rotating test was reperformed for 3 or 4 weeks post-treatment.
Open field test
To assess general motor behavior, an open field test was employed 3 months after mitochondrial transplantation. Animals were placed in a 50 cm x 50 cm white plexiglass box and allowed an adaptation period of 30 min prior to being analyzed. Activity was recorded for two consecutive sessions, each lasting 15 min, using a ceiling-mounted video camera. Ethovision software (Noldus, Leesburg, VA, USA) was used to measure distance, velocity, total number of zone boundaries crossed, and duration of movement (seconds). Locomotion frequencies were assessed by dividing the floor of the box into four quadrats and by counting the number of quadrats entered. Entry was counted when a rat entered a new quadrat with all four paws. The apparatus was washed with 5% ethanol between tests to eliminate possible bias due to odors left by previous rats.
Histological and immunohistochemical staining
Rats that survived 3 months after transplantation were sacrificed with an overdose of chloral hydrate (800 mg/kg i.p.) and fixed by intracardiac perfusion with 300 mL of saline followed by 300 mL of 4% paraformaldehyde (Sigma-Aldrich). Brains were removed, postfixed in 4% paraformaldehyde for 4 h, and cryoprotected in 30% w/v sucrose in PBS for 20 h. Brains were then frozen and embedded in OCT medium (Tissue-Tek, Sakura Finetek, USA) and sectioned at 5-10-μm. For Nissl staining, sections mounted on glass slides were dried overnight. Slides were immersed in 0.025% cresyl violet (Sigma-Aldrich) in 90 mM acetic acid (Merck, Darmstadt, Germany) and 10 mM sodium acetate (Sigma-Aldrich) for 3 h, followed by dehydration in ascending ethanol and xylene series. Slides were then coverslipped with Histochoice mounting media (AMRESCO). For immunohistochemical staining (IHC), fixed sections were subjected to heat-induced epitope retrieval in 10mM citrate buffer pH 6.0 for 25 min at 100°C . Sections stained with BrdU were additionally treated with 2 N HCl for 30 min at 37°C. After washing and blocking non-specific sites with blocking buffer (5% BSA and 0.5% Tween-20 in PBS, pH7.4) for 30 min at room temperature, sections were incubated at 4°C overnight with a primary antibody, anti-Tyrosine hydroxylase (TH) (1:200 dilution, Novus Biologicals, Littleton, CO, USA), anti-8-hydroxydeoxyguanosine (8-OHdG) (1:100 dilution, Japan Institute for the Control of Aging NIKKEN SEIL. Co., Ltd., Shizuoka, Japan), anti-BrdU (1:200 dilution, Abcam, Cambridge, MA, USA), or anti-Doublecortin (DCX) (Abcam) containing 1% BSA and 0.1% NaN3 (Sigma-Aldrich). Samples were then washed to remove excess first antibody and incubated for 30 min at room temperature with HRP-conjugated secondary antibody (1:200 dilution, Millipore) at a 1:200 dilution in PBS. After being washed three times in PBS to remove excess secondary antibody, chromogenic detection of immunoreactivity was performed using a DAKO 3,3'-diaminobenzidine (DAB) kit (DakoCytomation, Carpinteria, CA, USA) according to the manufacturer’s protocol. A counterstain, Mayer's hematoxylin (Sigma-Aldrich), was then added for 1 min. Color development was terminated by washing, and slides were visualized using light microscopy and image analyzed with Image J Software (NIH, Bethesda, MD, USA). For fluorescent detection in IHC, fluorophore-conjugated secondary antibodies (1:500 dilution in 0.5% BSA/PBS, Jackson ImmunoResearch, West Grove, PA, USA,) were used. Fluorescent signals were detected and Z-stacks were acquired and analyzed with a confocal microscope (Olympus Fluoview FV1200, Olympus, Tokyo, Japan).
Western blot analysis
An 30-μg aliquot of whole-cell lysate was fractionated on a 12% SDS-polyacrylamide gel (Bio-Rad Laboratories, Richmond, CA, USA) and blotted onto a polyvinylidene difluoride membrane (Amersham Biosciences, Buckinghamshire, UK). Nonspecific binding was blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 buffer (Sigma-Aldrich), and the membrane was incubated with a cocktail of monoclonal antibodies (1:800 dilution) purchased from Mitosciences (Eugene, OR, USA) for evaluation of oxidative phosphorylation complexes, including complex I (CI)-20 kDa subunit (NDUFB8), complex II (CII)-30 kDa subunit (SDHB), complex III-core 2 subunit (CIII)-48 kDa, complex IV-subunit I (CIV-I)-40 kDa, and complex V-subunit alpha (CV)-55 kDa. After incubation with a horseradish peroxidase-conjugated secondary antibody (1:50,000 dilution; Jackson ImmunoResearch), protein intensity was determined using an enhanced chemiluminescence reagent (Immobilon Western, Millipore). Bands were digitally scanned, and intensities were quantified using Gel-PRO Analyzer software (Media Cybernetics, Rockville, MD, USA). Immunoreactivity of rabbit anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (GADPH) (1:1000 dilution, Santa Cruz Biotechnology) was analyzed simultaneously as an internal control.
Multiplex cytokine assay
Rat blood plasma was collected after 3 months of treatment and after addition of 1:100 diluted proteinase inhibitor cocktail (Millipore #539134), samples were frozen at -80°C until testing. Multiplex cytokines in rat plasma were measured in duplicate using a Luminex platform (MAGPIX, Millipore, St. Charles, MO, USA) with the customized panel, MILLIPLEX MAP rat Cytokine/Chemokine Magnetic Bead (MILLIPLEX MAP kits, EMD Millipore, Billerica, MA, USA) and analyzed with MILLIPLEX analyst software (ViageneTech, Carlisle, MA, USA) according to the manufacturer's instructions. Plasma levels of granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-1alpha (IL-1α), IL-4, IL-1beta (IL-β), IL10, IL-12(p70), interferon gamma (IFNγ) and IL17A were selected on the basis of significant upregulation resulting from mitochondrial transplantation .
All analyses were performed in triplicate or quadruplicate in each group of experiments. Biochemical data are presented as means ± standard deviations, except results of the animal behavior test, presented as means ± standard errors of the means. There were six animals per group. The two mitochondrial treatments were evaluated using paired Student’s t-tests and differences with p < 0.05 were considered statistically significant.