Experimental design
A total of 48 rats were divided into four groups at random: (i) control, (ii) sham, (iii) I/R + saline, and (iv) I/R + HFSCs. Rats in the control group were heathy and not subjected to an operation, whereas the I/R model was established via surgical MCA occlusion (MCAO). In the sham group, rats received a similar operation but without MCA occlusion. The I/R + HFSCs group underwent HFSC transplantation (1 × 106 cells dispersed in 1 mL saline) via tail vein injection 24 h after reperfusion, whereas animals in the I/R + saline group and sham group were similarly administered 1 mL saline.
Animals were maintained for 2 weeks after transplantation, and then, relevant tissue samples from half of the animals were analysed using TTC (Amresco, OH, USA) and the other half were used for histological staining (n = 6 each, Fig. 1). The 2-week survival point was chosen because it might be the shortest time required for the transplanted cells to migrate to the penumbra and exert putative protective effects on stroke recovery. Neurological scores (Table 1) were recorded daily after cell transplantation [19]. The score of each rat was estimated three times. A full score represents a normal neurological status, whereas lower scores are indicative of behavioural deficits.
Animals
Male Sprague-Dawley (S-D) rats weighing 280 ± 10 g were purchased from the animal centre of the Second Affiliated Hospital of Harbin Medical University. The animals were housed at 22 ± 2 °C with a humidity of 40 ± 5%, under a 12-h light/dark cycle, and fed a standard diet and water ad libitum. The rats were forbidden to eat 12 h before the experiments but were allowed free access to drinking water. All study design and experimental procedures were conducted in accordance with institutional guides for animal experiments approved by the Experimental Center of the Second Affiliated Hospital of Harbin Medical University.
Isolation, culture, labelling, and transplantation of rat-derived HFSCs
We harvested hair follicles via enzyme digestion following mechanical dissection [20]. The upper lips containing the vibrissa pad of 4-week-old male S-D rats were cut and digested with 0.1% collagenase in DMEM (both from Gibco, BRL, Gaithersburg, MD, USA). Then, the vibrissa follicles were gently plucked from the pad under a binocular microscope, placed in 24-well tissue-culture dishes (Corning, NY, USA) pre-treated with IV collagen, and cultured in DMEM containing 10% FBS (ScienCell, Santiago, CA) and 1% penicillin and streptomycin (Gibco, BRL, Gaithersburg, MD, USA) at 37 °C in an atmosphere of 95% air–5% CO2. All surgical procedures were conducted in accordance with aseptic principles, and the entire culture medium was first replaced with fresh medium 12 h later. The non-adherent cells were discarded with the waste culture medium, and thereafter the culture medium was replaced every 48 h. When the culture grew to approximately 80% confluency (~10 days), the new adherent cells were passaged using the same culture method. The prepared HFSCs were used at passage 3 in the experiments. To monitor the grafted cells in the brain, the HFSCs were pre-labelled by non-invasive membrane-labelling green fluorescent dye PKH 67. Two weeks after HFSC transplantation, the TTC and histological staining were performed.
Induction of focal cerebral I/R
The focal cerebral I/R model was induced by right MCAO for 1 h following reperfusion, as reported previously [21, 22]. Briefly, after intraperitoneal anesthetisation with compound anaesthetic consisting of 4.25% chloral hydrate and 0.886% pentobarbital sodium (0.3 mL/100 g), a midline ventral incision was made on the neck to expose the vessels. The right external carotid artery (ECA) was isolated and the branches were cauterised. The right MCA was occluded by gently inserting a monofilament nylon suture with a rounding tip through the right ECA. After a 60-min occlusion, the suture was slowly pulled back to achieve reperfusion. The rats in the sham group received the similar procedure except that the right MCA was not occluded. The body temperature was maintained at 37 ± 5 °C using a thermostat-controlled heating pad from the start of the operation until the animals recovered from anaesthesia.
TTC staining
To measure the infarct volume, TTC staining was carried out as described previously [23]. Briefly, brain tissue was cut into five coronal slices (2 mm thick) and incubated in 1% TTC dissolved in PBS for 15 min at 37 °C. The non-infarcted tissue was stained red, whereas the infarcted tissue area remained white. The infarct volume was calculated as follows: [(left hemisphere area − right non-infarcted area) / (left hemisphere area × 2)] to avoid the influence of oedema [24].
Nissl staining
Following anaesthesia, rats were trans-cardially perfused with 0.9% saline until no blood flowed out, followed by 4% paraformaldehyde in PBS (pH 7.4). Brain tissue was removed and kept in 4% paraformaldehyde for 48 h, cryoprotected in 30% sucrose in PBS for another 48 h at 4 °C, and then embedded in OCT compound (Sakura Finetek, Torrance, CA, USA). The brain specimens were cut into coronal slices at 10-μm thickness between the optic chiasma and the cerebral caudal end using a cryostat machine (Thermo Scientific Microm HM560, Waltham, MA, USA). The sections were then air-dried and processed for pathological experiments, including Nissl staining. Nissl bodies were stained using Cresyl violet acetate (Sigma-Aldrich, St Louis, MO, USA). Briefly, the brain tissue slices were immersed in the Cresyl violet acetate solution for 2 h at 37 °C, successively dehydrated and hyalinised, and observed under an optical microscope [21].
Immunofluorescence
Frozen sections were blocked with 5% goat serum (Cat. No. abs933, Absin, Shanghai, China) in PBS for 30 min and incubated with primary rabbit anti-doublecortin (Cat. No. ab18723, DCX, Abcam, Cambridge, UK), mouse anti-neuron-specific nuclear protein (Cat. No. MAB377, NeuN, Millipore Corp, Billerica, MA, USA), and rabbit anti-glial fibrillary acidic protein (Cat. No. ab7260, GFAP, Abcam, Cambridge, UK) antibodies overnight at 4 °C. After rinsing with PBS, the sections were incubated with rhodamine-conjugated anti- rabbit IgG (1:500, Cat. No. 4413S, Cell Signaling Technology, VT, USA) or anti- mouse IgG (1:500, Cat. No. 4409S, Cell Signaling Technology, VT, USA) for 90 min at 25 °C. The nuclei were stained with DAPI. PKH 67 (green), neuron-specific markers (red), and DAPI (blue) were observed using laser scanning confocal microscopy (Zeiss LSM800; Carl Zeiss, Jena, Germany) at wavelengths of 594 nm (red), 488 nm (green), and 405 nm (blue), respectively.
Immunohistochemistry
The brain sections were incubated with 0.3% H2O2 to eliminate endogenous peroxidase, blocked with 10% goat serum, and treated with 0.1% triton X-100 in PBS for 30 min at 25 °C. Then, the sections were incubated with rabbit anti-DCX, mouse anti-NeuN, and rabbit GFAP antibodies overnight at 4 °C. After washing with PBS, the sections were incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse IgG (1:500, Cell Signaling Technology, VT, USA) for 60 min at 25 °C, and stained with DAB (Cell Signaling Technology, VT, USA). The sections were rinsed with PBS, counterstained with haematoxylin, then dehydrated and observed using an optical microscope.
Statistical analysis
The statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Prism Software, San Diego, CA, USA). The data were analysed using one way-ANOVA, followed by a Tukey’s test for multiple comparisons and Student’s t-test for two group comparisons. Values with P < 0.05 were considered statistically significant. All data are expressed as means ± standard deviations (SDs).