BALB/c mice at 8 to 10 weeks of age, either sex, weighing 20 to 22 grams, were purchased from the Animal Center of Second Affiliated Hospital of Harbin Medical University. Animals were housed at constant temperature and humidity, with a 12/12 hr light/dark illumination cycle and free access to food chow and water. All experimental procedures used in the present study were approved by the Harbin Medical University Administrative Panel on Laboratory Animal Care, which is compatible with the NIH guidelines for use and care of laboratory animals.
The primary antibodies and detecting reagents included mouse anti-PCNA antibody (BM0104, Boster company, Santa Cruz Biotechnology), goat-anti-doublecortin (SC-8006, Boster company, Santa Cruz Biotechnology), Alexa Fluor® 488 and 594 conjugated donkey anti-mouse and anti-goat IgGs (Invitrogen, Carlsbad, CA). The fluorescent dye PKH26 was obtained from Sigma-Aldrich (MINI26, Saint louis, Missouri, USA). All fluorescent cytoflow markers were purchased from Biolegend (San Diego, USA), including (FITC rat IgG 2 Isotype ctrl. Clone: RT K 2758 (Cat. No. 400505), APC rat IgG 2 Isotype ctrl. Clone: RT K 2758 (400511), Percp rat IgG 2b Isotype ctrl. Clone: RT K 2758 (118419), PE rat IgG 2b Isotype ctrl. Clone: RT K 2758 (119316), APC anti-mouse CD34 Clone: MEC 14.7 (119309), FITC anti-mouse LY-6A/EC Sca-1 Clone: E13-16.1.7 (122505), PE anti-mouse/human CD44 Clone: IM 7 (103007), Percp anti-mouse CD45 Clone: 30-F11 (103129).
Isolation and lipophilic fluorescent labeling of bone marrow cells
BALB/c mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.). Femoral bones were removed under sterile condition, with the medullary cavity bathed by heparin (50 U/mL) in normal saline [41, 42]. Bone marrow was aspirated and suspended in a lymphocyte isolation medium, followed by centrifuge at 2000 rpm for 20 minutes. The cell pellets were subsequently diluted with DMEM/F12 medium (DMEM/F12, 15% FBS, 100000 U/L penicillin, pH = 7.4) to yield a density of more than 5×107 cells/mL. The cells were then labeled with the red-fluorescent lipophilic tracer PKH26 according to the manufacturer's instruction . The density of PKH26-labeled cell suspension was adjusted to approximately 3×107 cells/mL. Cells with viability greater than 95% as measured by trypan blue exclusion were used for the subsequent transplant studies.
Focal cerebral ischemia and bone marrow cell transfusion
An acute ischemic stroke model was established by coagulation of the middle cerebral artery in experimental mice. In brief, mice were anesthetized with pentobarbital (50 mg/kg, i.p.), and a craniotomy was carried out by maintaining the animals over a heating pad (with rectal temperature at 36.5 to 37.5°C). The left middle cerebral artery (MCA) was exposed, and occluded with a short period of coagulation using a general metallic heat applicator. The cranial hole was sealed with dental cement, and skin sutured before the mice were returned to their cages. After the brain surgery, the experimental animals were infused via tail vein either with PKH26 pre-labeled bone marrow cells (n = 16) or phosphate buffered solution (PBS) as control (n = 16). Animals were allowed to survive for 6 hours (n = 4), and 7 (n = 4), 14 (n = 4) and 21 (n = 4) days post bone marrow cell transplantation.
Fluorescence-activated cell sorting analysis
To evaluate the purity of the isolated bone marrow cells as putative bone marrow mononuclear cells (BMMCs) or bone marrow stem cells, part of the cell pellet was subject to fluorescence-activated cell sorting (FACS) analysis for the expression of various signature antigen markers of mesenchymal stem cells. Thus, approximately 1×106 cells were incubated in 2% fetal bovine serum in PBS at 4°C for 30 minutes with 1 μl of monoclonal antibody specific for CD34, CD44, CD45, Sca-1 (all from BioLegend 11080 Roselle Street, San Diego, CA 92121). Negative control was processed by incubating the cells in buffer without primary antibodies. The immunofluorescent signal was analyzed using the FACS Calibur with CellQuest software (Becton Dickinson, USA).
Except for the short time surviving group (6 hrs post surgery), all other animals were perfused transcardially with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, USA) in 0.01 M phosphate-buffered saline (pH 7.4, PBS) under overdose anesthesia (sodium pentobarbital 100 mg/kg, i.p.). Brains were removed from the skull, postfixed in the perfusion fixative overnight, and cryoprotected in 30% sucrose at 4°C. Brains were cut at the coronal plane in a cryostat at 6 μm, and sections passing through the infarct areas were collected alternatively (every 20 sections) by thaw-mounting on gelatin-coated microslides. Sections were stored at -70°C for a few weeks before histological (H.E stain) and immunohistochemical examinations.
For immunolabeling with the peroxidase method, sections were first treated 1% H2O2 in PBS for 30 minutes, and then pre-incubated in 5% normal horse or rabbit serum in PBS with 0.3% Triton X-100 for 1 hour. Subsequently, sections were incubated with mouse anti-PCNA (1:400) or goat anti-DCX (1:100) antibodies in PBS containing the blocking serum and Triton X-100 at 4°C overnight. Sections were further reacted with biotinylated horse anti-mouse or rabbit anti-goat secondary antibodies at 1:400 (BM0104 Boster company, Santa Cruz Biotechnology, CA) for 2 hours, and finally with the avidin-biotin complex (ABC) reagents (1:400) (Vector Laboratories, Burlingame, CA) for another 1 hour. Immunoreation product was visualized in 0.003% H2O2, 0.05% diaminobenzidine (DAB; Sigma, St, Louis, MO). Three 10-minute washes were used between all incubations. Sections were allowed to air-dry, dehydrated through ascending ethanol, cleared with xylene and coverslipped. Some of the above immunohistochemically prepared sections were counterstained with haematoxylin before dehydration.
To determine the colocalization of PCNA and DCX expression with transplanted BMMCs, additional sections were subject to double fluorescent labeling for PCNA or DCX with PKH26. Sections were incubated in PBS containing 5% normal donkey serum and mouse anti-PCNA antibody (1:400) or goat anti-DCX antibody (1:200) at 4°C overnight, followed by a 2 hours reaction with Alexa Fluor® 488 conjugated donkey anti-mouse or goat IgGs (1:200, A21206 Invitrogen, Carlsbad, CA). Sections were then washed and mounted with anti-fading medium before microscopic examination. Sections from animals received tail vein infusion of the PBS vehicle were processed in parallel as negative control for fluorescent labeling.