Isolation and culture of canine AD-MSCs
MSCs derived from the adipose tissue of canine hip fat were isolated and characterized [11]. Specifically, adipose tissue was collected aseptically from the subcutaneous fat of a 2-year-old beagle dog under anesthesia. Tissues were washed with phosphate-buffered saline (PBS), minced, and digested with collagenase type I (1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 30–60 minutes with intermittent shaking. The suspension was filtered through a 100 μm nylon mesh and centrifuged to separate floating adipocytes from stromal cells. Preadipocytes in the stromal vascular fraction were plated at 8000–10,000 cells/cm2 in T175 culture flasks containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 3.7 g/l sodium bicarbonate, 1 % penicillin and streptomycin, 1.7 mM l-glutamine, 0.1 mM β-mercaptoethanol, and 10 % fetal bovine serum (FBS). The cells were incubated in a humidified atmosphere at 37 °C with 5 % CO2. Unattached cells and residual nonadherent red blood cells were removed after 24 hours using a PBS wash, and the cell medium was replaced every 2 days. At passage 3, the cells were used for the following experiments.
Animals
Seventeen healthy 2–3-year-old beagle dogs weighing 8.5 ± 2.2 kg were used in the study. All dogs were clinically judged to be in good health and to have a normal neurological status. During the experiment, all dogs were cared for in accordance with the animal care guidelines of the Institute of Laboratory Animal Resources at Seoul National University, Korea. Sixteen dogs were assigned to four groups based on the treatments: control (no treatment after SCI; n = 4), MPSS (administration of MPSS after SCI; n = 4), AD-MSCs (administration of AD-MSCs; n = 4), and AD-MSCs + MPSS (administration of both AD-MSCs and MPSS; n = 4). One additional dog was used to evaluate distribution of MSCs in the body after injection. The Institutional Animal Care and Use Committee of Seoul National University approved the experimental design (SNU-111102-8).
Induction of SCI
SCI was induced using a previously described balloon compression method [10]. Briefly, the dogs were medicated and anesthetized with tramadol (4 mg/kg intravenously, Toranzin; Samsung Pharm. Ind. Co., Seoul, Korea), propofol (6 mg/kg intravenously, Provive 1 %; Claris Lifesciences, Ahmedabad, India), and atropine sulfate (0.05 mg/kg subcutaneously, Atropine; Jeil Pharm., Yongin, Korea). Anesthesia was maintained with isoflurane (Forane solution; Choongwae Pharm. Co., Seoul, Korea) at a minimum alveolar concentration (MAC) of 1.5 throughout the procedure. Electrocardiography, pulse oximetry, respiratory gas analysis, and rectal temperature measurement were performed using an anesthetic monitoring system (Datex-Ohmeda S/5; GE Healthcare, Little Chalfont, UK). The dogs were suspended in a ventral recumbent position, and hemilaminectomy was performed through a left paramedian approach at the fourth lumbar segment (L4). A hole of 3–5 mm was made in the left vertebral arch at L4 using a high-speed pneumatic burr, and a 4-French embolectomy catheter (Edwards Lifesciences, Irvine, CA, USA) was inserted into the hole. Under fluoroscopic guidance, the balloon catheter was advanced until the tip was positioned at the cranial margin of the first lumbar segment (L1) vertebral body. The balloon was then inflated by injecting 50 μl/kg contrast agent (Omnipaque; GE Healthcare) diluted with saline in a 50:50 proportion. The balloon’s positioning was confirmed using fluoroscopy. According to a previous study [9], this SCI model occludes more than 85 % of the spinal canal, as confirmed by computed tomography. Following induction of the injury, the soft tissue and skin were closed using standard methods. The balloon was fixed with a Chinese finger-type suture and removed after 6 hours. After the operation, the dogs were monitored in an ICU, and manual bladder expression was performed at least three times daily if needed.
Administration of MPSS and AD-MSCs
Administration of MPSS and AD-MSCs was initiated following removal of the embolectomy catheter. MPSS (30 mg/kg, Methysol; Kunhwa Pharmaceutical Co., Seoul, Korea) was bolus injected into the cephalic vein and followed by continuous rate infusion (5.4 mg/kg/hour) for the next 47 hours [6]. A suspension of 1 × 107 allogenic AD-MSCs in 10 ml lactated Ringer’s solution was administered intravenously once a day for 3 successive days.
Labeling and tracking of AD-MSCs
Twenty-four hours before transfection, 4 × 106 HEK293 cells were seeded into a 100 mm dish. The following day, a lentiviral packaging mix (System Biosciences, San Diego, CA, USA) encoding viral proteins Gag-Pol, Rev, and VSV-G and lentiviral transgene plasmids were transfected into each well for lentivirus production using Turbofect (Thermo Scientific, Waltham, MA, USA). Green fluorescent protein (GFP)-expressing virus particles were collected and transduced into AD-MSCs at passage 1. After the AD-MSCs reached 90 % confluence, the cells were selected by puromycin (3 μg/ml; Gibco-BRL). Approximately 30 % of cells were successfully transduced after puromycin selection. The AD-MSCs were subcultured, and passage 3 cells were used for the following experiments. The GFP-labeled AD-MSCs were intravenously injected after SCI as already described. The dog was sacrificed at 7 days after transplantation and tissue samples from the lung, liver, spleen, normal spinal cord, and injured spinal cord were collected. GFP protein was identified in each specimen using western blot analysis. The injured spinal cord was fixed in a 10 % formalin solution and embedded in paraffin. Longitudinal sections were made and the tissue was stained with 4′,6-diamidino-2-phenylindole (DAPI, 1:100; Sigma-Aldrich) to identify nuclei. Slides were observed using a fluorescent microscope.
Clinical assessment
Behavioral assessments were performed on days 2, 4, and 7 after the operation to evaluate functional recovery of the hind limbs. Each dog was videotaped from both sides and behind during the neurologic examination. Using video footage and the revised Tarlov scale [12], the dogs’ gaits were scored independently by two individuals blinded to the experimental condition. The animal’s vital signs and operation site were also checked. A fecal occult blood test (Cell Biolabs, Inc., San Diego, CA, USA) was performed to evaluate any adverse gastrointestinal effects of intravenous administration of AD-MSCs and/or MPSS.
Histopathological assessment
The dogs were sacrificed through intravenous injection of potassium chloride under general anesthesia at 7 days after transplantation. The spinal cord from the 12th thoracic segment (T12) to the third lumbar segment (L3) was extracted by dissection. Each section was then placed in 10 % sucrose/PBS at 4 °C for 12 hours and subsequently immersed in 20 % sucrose solution overnight at 4 °C. The sample was divided into two parts longitudinally. One-half of each section was immediately frozen with liquid nitrogen for western blot analysis and enzyme-linked immunosorbent assay (ELISA) tests. The other half was embedded in optimal cutting temperature compound (Surgipath®; Leica Biosystems Richmond, Inc., Richmond, IL, USA), frozen, and cut longitudinally into 12 μm sections with a cryomicrotome. These sections were mounted on silane-coated glass slides and stained with hematoxylin and eosin (H&E) or Luxol fast blue stain. The hemorrhagic area was measured by tracing the hemorrhagic margin and calculated using an image analysis program (ImageJ; NIH, Bethesda, MD, USA). The number of microglia was counted manually in the high-powered field from five randomly selected areas of the injured area margin.
Oxidant metabolite assessment
The peroxynitrite (PN) formation of 3-nitrotyrosine (3-NT), the LP product 4-hydroxynenonal (4-HNE), and the protein oxidation-derived protein carbonyls (PC) were used as markers for oxidative damage. The level of each oxidation product (3-NT, 4-HNE, and PC) was assessed using ELISA kits (Cell Biolabs, Inc.). The samples were placed into the wells of the 3-NT, 4-HNE, and PC conjugate-coated plates and incubated at room temperature for 10 minutes. After incubation, the secondary antibody was added to each of the wells, incubated at room temperature for 1 hour, washed three times with washing buffer, and incubated with secondary antibody–horseradish peroxidase conjugate for 1 hour. The wells were washed, the substrate solution added for color change, and absorbance measured at 450 nm.
Western blot analysis
The organ tissues and the frozen half of each injured spinal cord specimen were used for western blot analysis. Briefly, the tissue was washed twice with PBS, and then homogenized with a sonicator in lysis buffer (20 mM Tris at pH 7.5, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 1 % Triton X-100, 1 mg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, 0.5 mM sodium orthovanadate) on ice for 30 minutes. Lysates were cleared by centrifugation (10 minutes at 15,000 rpm, 4 °C), and protein concentrations were determined using the Bradford method [13]. Equal amounts of protein (20 μg) were resolved by electrophoresis on 10 % sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membrane blots were washed with TBST (10 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.05 % Tween-20), blocked with 5 % skimmed milk for 1 hour, and incubated with the appropriate primary antibodies at the recommended dilutions. The antibodies used included antibodies against actin (A3853; Sigma-Aldrich), GFP (MA515256; Thermo Scientific), interleukin (IL)-6 (ab6672; Abcam, Cambridge, UK), and cyclooxygenase-2 (COX-2, sc-7951), tumor necrosis factor alpha (TNFα, sc-1350), phosphorylated signal transducer and activator of transcription 3 (pSTAT3, sc-8001-R), β3-tubulin (sc-69966), glial fibrillary acidic protein (GFAP, sc-65343), and galactosylceramidase (GalC, sc-67352) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibodies (1:1000) were diluted in TBST. The membrane was then washed, and the primary antibodies were detected with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (1:5000; Invitrogen, Waltham, MA, USA). Bands were visualized using enhanced chemiluminescence (Invitrogen).
Statistical analysis
All results are expressed as mean ± standard deviation (SD). Statistical analysis was performed using a commercially available statistical software program (SPSS Statistics, version 21.0; IBM Corp., Armonk, NY, USA). In all experiments, Kruskal–Wallis tests were followed by Mann–Whitney U tests to compare between groups. P <0.05 was considered significant.
