Ethics
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2018032102) and performed in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition (NIH publication No. 85 − 23, National Academy Press, Washington, DC, USA, revised 2011). Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-approved animal facility in our hospital, with controlled temperature and light cycles (24 °C and 12/12 light/dark cycle).
Procedure and protocol of animal model of intracerebral hemorrhage (ICH) induced by type IV collagenase proteolytic enzyme
The procedure and protocol of ICH was based on previous report [29]. In details, the rats were anesthetized by inhalation of 2.0% isoflurane and then were placed on a warming pad at 37 °C, followed by securing the head and shaving the scalp hair. Under sterile conditions, 1 cm long midline incision of the scalp was carefully created to expose the perpendicular intersection point of the coronal and sagittal suture (i.e., bregma). The Hamilton syringe (250 µl) was then mounted onto the injection pump and stereotaxically guided the needle (26 Gauge) over bregma. The stereotactic manipulator arms were adjusted the position of the needle 1.4 mm anterior and 3.2 mm lateral to the right. A small cranial burr hole was then created by using a 1 mm drill bit. After this procedure, 1.0 µl collagenase type IV (0.25 IU/µl) was then carefully injected into corpus/dorsal striatum (5 mm below the skull) by Hamilton syringe 26 G at a rate of 0.2 µl/min. The syringe was removed slowly after the injection is completed and sterile bone wax is used to plug the hole quickly. The skin on the surface of head was then closed by using 4 − 0 prolene suture. Finally, the animals were cared for in a portable animal intensive care unit (ThermoCare®) with food and water for 24 hours.
Animal grouping
Pathogen-free, adult male Sprague-Dawley (SD) rats (n = 30) weighing 320–350 g (Charles River Technology, BioLASCO Taiwan Co. Ltd., Taiwan) were used in the present study. The animals were equally categorized into group 1 [sham-operated control (SC), i.e., only incision of the skin over scalp], group 2 [acute ICH induced by directly intracranial injection of collagenase (1.0 µl)], group 3 [ICH + HUCDMSCs (1.2 × 106 cells) by intravenous administration at 3 h and on days 1 and 2, respectively after ICH procedure, i.e., total amount was 3.6 × 106 cells], group 4 [ICH + hyperbaric oxygen therapy (HBO) (3 h duration for each time) at 3 h and at days 1 to 4 after ICH procedure] and group 5 (ICH + combined HUCDMSCs + HBO). Animals in each group were euthanized by day 28 after ICH induction and the brain specimen was harvested from each animal for individual study.
Hyperbaric oxygen therapy
The procedure and protocol of HBO therapy was based on a previous report [30]. Briefly, to induce tissue-level hyperoxia, SD rats were subjected to HBO administration in an animal tabletop chamber (Piersol-Dive, model 4934) with the animals exposed to 100% oxygen at 2.4 atmospheres absolute (ATA) for 90 minutes (3 h/one time) at 3 h and days 1 to 4 after ICH induction.
Corner test for assessment of neurological function prior to and after ICH induction
The sensorimotor functional test (corner test) was conducted for each rat of each group (i.e., n = 6 per group) at baseline and on days 1, 7, 14 and 28 after acute ICH induction as we previously described [28, 30, 31]. In detail, the rat could walk through a tunnel and then turn into a 60-degree corner. To exit the corner, the rat could turn either left or right. The results were recorded by a technician blinded to the study design. This test was repeated 10 to 15 times with at least 30 seconds between each trial. We recorded the number of right and left turns from 10 successful trials for each animal and used the results for statistical analysis.
Measurement of brain hemorrhagic area
To evaluate the impact of HUCDMSCs-HBO treatment on preserving the brain parenchyma, coronal sections of the brain were obtained from four extra animals in each group as 2 mm slices by day 14 after ICH induction. Each cross section of brain tissue was then stained with 2% 3,5-Triphenyl-2H-Tetrazolium Chloride (TTC) (Alfa Aesar) for BIA analysis. Briefly, all brain sections were placed on a tray with a scaled vertical bar to which a digital camera was attached. The sections were photographed from directly above at a fixed height. The images obtained were then be analyzed using Image Tool 3 (IT3) image analysis software (University of Texas, Health Science Center, San Antonio, UTHSCSA; Image Tool for Windows, Version 3.0, USA).
The hemorrhagic area was observed as either whitish or pale reddish regions (i.e., dis-colored region). Intracerebral hemorrhagic region was further confirmed by microscopic examination. The percentages of hemorrhagic area were then obtained by dividing the area with total cross-sectional area of the brain. The rest of the brain tissue was then cut into pieces for specific studies.
Western blot analysis
The procedure and protocol for Western blot analysis were based on our recent reports [28, 30–33]. Briefly, equal amounts (50 µg) of protein extracts were loaded and separated by SDS-PAGE using acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (GE, UK). Nonspecific sites were blocked by incubation of the membrane in blocking buffer [5% nonfat dry milk in T-TBS (TBS containing 0.05% Tween 20)] overnight. The membranes were incubated with the indicated primary antibodies [Caspase 3 (1: 1000, Cell Signaling), Poly (ADP-ribose) polymerase (PARP) (1: 1000, Cell Signaling), high mobility group box 1 (HMGB1) (1: 1000, Cell Signaling), toll-like receptor (TLR)-2 (1: 1000, Abcam), TLR-4 (1: 1000, Abcam), myeloid differentiation primary response 88 (MyD88) (1: 1000, Abcam), TNF receptor associated factor 6 (TRAF6) (1: 1000, Abcam), IκB-ß (1: 1000, Abcam), nuclear factor (NF)-κB (1: 600, Abcam), interferon (INF)- γ (1: 1000, Abcam), LC3B-I (1: 1000, Cell Signaling), LC3B-II (1: 1000, Cell Signaling), oxidized protein (1:200, Millipore), tumor necrosis factor (TNF)-α (1: 1000, Cell Signaling), interleukin (IL)-1ß (1: 1000, Cell Signaling), NOX-1 (1: 1500, Sigma), NOX-2 (1: 750, Sigma), and Actin (1: 1000, Millipore)] for 1 hour at room temperature. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1:2000, Cell Signaling, Danvers, MA, USA) was used as a secondary antibody for one-hour incubation at room temperature. The washing procedure was repeated eight times within one hour. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Amersham, UK) and exposed to Biomax L film (Kodak, Rochester, NY, USA). For the purpose of quantification, ECL signals were digitized using Labwork software (UVP, Waltham, MA, USA).
Immunofluorescent (IF) staining of brain specimens
The procedure and protocol of IF staining were based on our previous reports [28, 30–33]. In detail, frozen sections (4 µm thick) was obtained from the brain hemorrhagic area/at risk area of each animal, permeated with 0.5% Triton X-100, and incubated with antibodies against NueN (1:100, Merck), CD31 (1:100, Abcam), von Willebrand factor (vWF) (1:200, Abcam), CXCR4 (1:100, Invitrogen), CD14 (1:200, Proteintech), and F4/80 (1:100, Santa cruz) at 4 ºC overnight. Alexa Fluor488, Alexa Fluor568, or Alexa Fluor594-conjugated goat anti-mouse or rabbit IgG was used to localize signals. Sections were finally counterstained with DAPI and observed with a fluorescent microscope equipped with epifluorescence (Olympus IX-40). Three brain sections were analyzed for each rat. For quantification, three randomly selected high-power fields (HPFs; 400x for IF study) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was then be determined by summation of all numbers divided by 9.
Vessel density in brain infarct zone
The procedure and protocol for identifying number of small vessels in the brain hemorrhagic area/at risk area were based on our previous reports [31–33]. In detail, staining of small blood vessels was performed with alpha smooth muscle actin (α-SMA) (1:400) as primary antibody at room temperature for 1 hour, followed by washing with PBS thrice. Ten minutes after the addition of anti-mouse-HRP conjugated secondary antibody, the tissue sections were washed with PBS thrice. Then 3,3’ diaminobenzidine (DAB) (0.7 gm/tablet) (Sigma) will be added, followed by washing with PBS thrice after one minute. Finally, hematoxylin was added as a counter-stain for nuclei, followed by washing twice with PBS after one minute. Three brain sections were analyzed in each rat. For quantification, three randomly selected HPFs (100x) were analyzed in each section. The mean number per HPF for each animal was then be determined by summation of all numbers divided by 9.
Procedure and protocol of brain magnetic resonance imaging (MRI) for determining the brain hemorrhagic volume (BHV)
The procedure and protocol for brain magnetic resonance imaging (MRI) study were based on our previous report [28]. The MRI was performed at day 28 after IS induction. Briefly, during MRI measurements, mice were anesthetized by 2% inhalational isoflurane with room air and placed in an MRI-compatible holder (Biospec 94/20, Bruker, Ettingen, Germany). Rectal temperature and respiration were monitored throughout the procedure to ensure normal physiological conditions were maintained. MRI data were collected using a Varian 9.4T animal scanner (Biospec 94/20, Bruker, Ettingen, Germany) with a rat surface array. The MRI protocol consisted of 40 T2-weighted images. Forty continuous slice locations were imaged with a field-of-view of 30 mm x 30 mm, an acquisition matrix dimension of 256 × 256 and slice thickness of 0.5 mm. The repetition time (TR) and echo time (TE) for each fast spin-echo volume were 4200 ms and 30 ms, respectively. Custom software, ImageJ (1.43i, NIH, USA), was used to process the region of interest (ROI). Planimetric measurements of images from MRI T2 were performed to calculate the stroke volumes of cortex. Collectively, the BHV was calculated by summation of total coronal sections and then divided the numbers of coronal sections to obtain the means of infarct areas. Additionally, the height of the infarct zone was calculated by summation of the thickness of each coronal sections. Finally, the BHV was obtained by mean of infarct area x height.
Statistical analysis
Quantitative data were expressed as means ± SD. Statistical analysis was adequately performed by ANOVA, followed by Bonferroni multiple-comparison post hoc test. SAS statistical software for Windows version 8.2 (SAS institute, Cary, NC) was utilized. A P value of less than 0.05 was considered statistically significant.