Dulbecco’s modified Eagle’s medium (DMEM)/F12, fetal bovine serum (FBS), 0.25% EDTA-trypsin, DMEM, and penicillin/streptomycin solution were purchased from Gibco (Carlsbad, CA, USA); LPS, CCM, poly-Lysine (PLL), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), 0.2% gelatin, and crystal violet dye were purchased from Sigma-Aldrich (St. Louis, MO, USA); Dispase (neutral protease, grade II) was purchased from Roche (Basel, Switzerland); Anti-p75 antibody, anti-CD31 antibody, anti-pan-Akt antibody, anti- PI3K p85 α antibody, anti-phospho-PI3 K p85α (Y607) antibody and 5-ethynyl-2-deoxyuridine (EdU) Proliferation Kit (iFluor 488) were purchased from Abcam (Cambridge, MA, USA); Anti-phospho-Akt (Ser473) antibodies were purchased from Cell Signaling Technology (CST; Danvers, MA, USA). Human umbilical vein endothelial cells (HUVECs), endothelial cell medium (ECM), and endothelial cell growth supplement (ECGS) were purchased from Sciencell Research Laboratories (Carlsbad, CA, USA); Red Fluorescent Probe M02 kit was purchased from Bestbio (Shanghai, China); MK-2206 was purchased from Selleck Chemicals (Houston, TX, USA); Cell counting kit-8 (CCK-8) proliferation assay, western blotting kit, and anti-β-actin antibody were purchased from Boster Biological Technology (Wuhan, Hubei, China); DAPI Kit, Fluor594-conjugated goat anti-rabbit IgG and Fluor594-conjugated goat anti-mouse were purchased from Molecular Probes (Eugene, OR, USA). Anti-mouse/rabbit immunohistochemistry detection kit was from Proteintech Group (Cambridge, MA, USA). MICROFIL® angiography kit was purchased from Flow Tech Inc. (Carver, MA, USA). 35 mm dishes, cell culture plates, plastic coverslips, and flasks were all purchased from Thermo Fisher (Shanghai, China). Matrigel basement membrane matrix and 24-well Transwell plates were purchased from Corning (Corning, NY, USA); 35 mm confocal dishes and centrifuge tubes were from NEST (Wuxi, Jiangsu, China). Microcentrifuge tubes (EP tubes) were purchased from Eppendorf (Hamburg, Germany).
Primary culture, purification, and identification of OECs
All experimental protocols involving experimental animals used in this study were reviewed and approved by the ethics committee at Honghui Hospital, affiliated with Xi’an Jiaotong University, and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. As previously described, OECs were isolated and primarily cultured from olfactory blubs in rats at 2–3 months of age (Additional file 1: Fig. S1) and further purified by the modified differential adherent velocity method as published by H. Nash et al. [15, 29]. Minor modifications to this protocol included efficient removal of the tractus olfactorius (inner layer), meanwhile maintaining the outer olfactory bulb layer (nerve layer and glomerulus layer) based on the distribution characteristics of OECs, and to eliminate interference from other glial cells . After purification, the OECs were reseeded onto 25-cm2 PLL-coated culture flasks and maintained in DMEM/F12 media with 10% FBS, cultured at 37 °C and 5% CO2 in an incubator (Thermo Fisher, Waltham, MA, US). Half of the total volume replaced every 3 days. Typically, OECs would reach about 80% confluence 10 days after isolation and seeding. OECs were then identified by immunofluorescence staining of anti-p75 antibody or activated by CCM & LPS . Enhanced green fluorescent protein (GFP) transgenic rats were selected as the olfactory bulb donor when we prepared OECs for in vitro co-culture and in vivo transplantation. This modification to the previous protocol allowed us to observe the distribution of OECs more intuitively .
Activation of OECs and conditional-media (CM) collection
When the OECs reached optimal confluence, cells were digested by 0.25% EDTA-trypsin and made into suspensions, divided equally into two groups, and then reseeded on 6-well plates (for experimental operation) or coverslips (for immunofluorescence identification). A same batch of OECs is usually divided equally, and one designated group of OECs is activated randomly as the activated group.One group was noted as Activated-OECs (Act-OECs), the media for this group contained 1 μg/ml LPS and 1 μM CCM dissolved in DMSO as previously described by Hao et al. . The other group was noted as Unactivated-OECs (Una-OECs), in which this group’s media contained equal amounts of DMSO as a control (the solvent used for CCM and LPS). To investigate whether the OECs could promote angiogenesis of vascular endothelial cells, we collected the conditioned media of OECs from cell culture, in which DMEM/F12 media with 10%FBS was used as the no-treatment control group. After one day of treatment, the media mixed with drugs or DMSO were removed and replaced with complete media. To ensure adequate nutrients, conditioned media were collected once every other day after the day of media replacement, a total of three times. Three days later, OEC supernatants were combined, mixed, and centrifuged at 14,000 × g for 15 min at 4 °C and then filtered with sterile 0.22-μm pore membranes to remove cell debris. Finally, these collected conditioned media isolates were kept at − 80 °C until used.
Rat aortic ring assay (Neovascularization)
This operation is performed based on previously published protocols [30, 31]. Rat aortic rings were isolated from young rats (three-weeks-old, weighing 50 g ± 10 g) and incubated in serum-free DMEM overnight as starved pretreatment. Before the aortic ring sprouting assay, 100 μL of Matrigel was added to each well of a 96-well plate by precooled pipette tip, with one aortic ring per well embedded in the middle of the gel. Following this step, plates were incubated in a 37 ℃ cell incubator for half an hour to allow for the gel to set. (Additional file 2: Fig. S2b) Once the gel was set, 150 µL ACM, UCM, or control media with 10%FBS was added to each well according to their respective groups, in which each group had three replicates. These aortic rings were cultured at 37℃ and 5% CO2 for 7 days, and then the sprouting results were photographed under an inverted phase-contrast microscope (Leica DM IL; Leica Microsystems, Germany). Each test was repeated three times, and relevant sprouting indexes were digitally calculated using Image J software (National Institutes of Health, Rockville, MD, USA).
Endothelial wound healing (cell migration) assay
Migration of endothelial cells was determined using a two-dimensional wound-scratch assay on 24-well plates. HUVECs were seeded onto each well of 24-well plates (1 × 105 cells/well) and maintained in DMEM with 10% FBS for 24 h, followed by overnight starvation with serum-free media. At the bottom of each hole, adherent cells were scratched radially through the center by 10 μL pipette tips, and then each hole was washed with phosphate-buffered saline (PBS) three times to remove the floating cells, allowing for the wound to be immediately seen under a microscope. Next, the HUVECs were cultured in 500 μL ACM or UCM or control media, respectively. To examine the motility contributing to the healing and exclude components related to cell proliferation, HUVECs were incubated with the antimitotic agent mitomycin (Sigma-Aldrich, USA). The wounds were observed using an inverted phase-contrast microscope after 24 h. Photographs were taken at regular intervals over 24 h until wound closure was achieved. The operation was repeated three times, and the results were analyzed with Image J. Healing index = (initial area—final area)/ initial area × 100%.
CCK-8 proliferation assay
The cultured HUVECs were digested and centrifuged, and the liquid supernatant was removed. After collecting the supernatant, 100 μL was dispersed uniformly with respect to the experimental groups, and the HUVEC suspension was distributed and inoculated to each well (5000 cells/well) in a 96-well plate. The HUVEC distributed plates were incubated for 1 h (37 °C, 5% CO2) until cells were adherent. Following adhesion, 10 μL of CCK-8 solution was added to each well of the plate and then incubated for 6 h (37 °C, 5% CO2). The plates were measured for absorbance every hour at 450 nm throughout the incubation period using a microplate reader (Thermo Fisher, Waltham, MA, US), and the OD450 values were recorded.
HUVECs tube formation assay
Matrigel was coated in each well of a precooled 96-well plate (50 μL/well) and incubated at 37 °C for 0.5 h. Following Matrigel coating, 3 × 104 HUVEC cells/well were seeded within the designated media of preset groups on Matrigel as illustrated in Additional file 2: Fig. S2C and previously described. The tube formation ability of HUVECs was measured at 3, 6, 9 h. After incubation, images were captured by an inverted phase-contrast microscope. The capillary length and the number of nodes/junctions/meshes of the tubular structures were quantified by Image J, and these processes were independently repeated as triplicates.
Isolating and primary culture of rat aortic endothelial cells (RAECs)
The aorta of young rats (three-week-olds, weighing 50 g ± 10 g) were isolated and obtained under aseptic conditions. Under an anatomical microscope (OPTON, German), the clot and connective tissue are stripped from the aorta using ophthalmic microscopic surgical instruments. After being washed by DMEM with 1% penicillin/streptomycin three times, a surgical suture was threaded through the active vessels, and the ends of the vessels were ligated. (Additional file 3: Fig. S3A–E). The intima of the aortas were turned over and exposed by suture-pulling. The unligated end was closed by a vascular clamp. (Additional file 3: Fig. S3F) The aorta was then incubated in 1 mg/ml Dispase for 60 min to digest the collagen in the cell–matrix in this inverted state of the intima. Next, the aorta was transferred to DMEM with 10% FBS and dissected into small 2 mm × 2 mm slices. These slices were placed at the bottom of the gelatin-coated 6-well plate (ensuring that the intima faces downward) with 1–2 samples per well, as shown in Additional file 3: Fig. S3G. During the time that the tissues were not adherent to the plate, the medium was maintained to moisten the bottom of the samples without flooding it. The amount of ECM with 5% FBS and 1% EGS added at each well during this process was about 800 μL. Following these steps, the 6-well plates were placed in a small wet box and cultured at 37 ℃, 5% CO2, and the media was changed every 48 h. 4–5 days after incubation, RAECs can be seen extending out from the tissue margins under a microscope. The tissue slices were removed at this stage, and 2 mL of media was added to each well, with media being replaced every other day. After day 10, the RAECs could be subcultured, cryopreserved, or used for further experiments. To validate these cell types, cultured cells were identified by immunofluorescence using an anti-CD31 antibody.
Co-culture of OECs and RAECs in vitro
After primary cell culturing, collections, and mixing, the RAECs were reseeded in each gelatin-coated well of a 24-well plate (1 × 105 cells/well) using DMEM/F12 media with 10% FBS for 2 h to allow the cells to adapt to the new environment. The original medium was then replaced with fresh media containing 0.5% red fluorescent probe M02. Two hours after medium replacement, the cells were labeled with fluorescence, washed with PBS three times, and replaced with fresh medium without a probe. Similar to the wound healing—cells migration assay, the wounds were generated by manually scratching the cell surface with 10 μL pipette tips, which can mimic the conditions of vascular endothelial injury in vivo. After that, suspension of pre-digested activated OECs or unactivated OECs were added into thewells of different groups, respectively. Wells containing medium without cells was set as a no-treatment control. All steps were repeated in triplicates. Similar to previous methods , before and after manual scratch, co-culture for 1 h and co-culture for 24 h was set as the shooting time, and the wound healing was analyzed using merged images of red fluorescence (M02: RAECs), green fluorescence (GFP: OECs) and bright-field (all cells) imaging. The healing index was calculated as described above.
Animal and experimental setting
Forty-eight female Sprague–Dawley rats weighing 200 ± 30 g (specific pathogen-free) were obtained from the Laboratory Animal Centre of Xi’an Jiaotong University. The rats were housed at constant temperature (23 ± 2 °C) and humidity of 50% ± 10% on a 12/12 h light/dark cycle with constant air renewal. The animals were divided into four groups at random as follows: (i) in the sham group, rats were subjected to laminectomy but not SCI; (ii) in the SCI group, rats were subjected to laminectomy and compressional SCI; (iii) in the activated-OECs transplantation group (AOT), a concentrated suspension of activated OECs were transplanted based on the SCI group; (iv) in the unactivated-OECs transplantation group (UOT), a concentrated suspension of unactivated OECs was transplanted based on the SCI group. Each group contained 12 experimental animals, and behavior and morphology were observed after the operations mentioned above.
Animal SCI model and OECs therapy
Twenty minutes before surgery, rats in each group were anesthetized using isoflurane and a veterinary anesthesia machine (RWD, Shenzhen, Guangdong, China). After isoflurane treatment, the rats were then anesthetized intraperitoneally with 1% sodium phenobarbital (4 mL/kg). Concomitantly, the rats were immobilized in the prone position. Vaseline oil was used on the eyes to prevent drying during surgery. After removing the hair of the animals, the operational locations were disinfected with medical iodine volts. The operational method for the compressional SCI model was performed as previously described . Briefly, for one case, a longitudinal incision was performed overlying the T7–11 area using operating scissors under aseptic conditions. After subperiosteal dissection of paraspinal muscles, a laminectomy was performed to expose the spinal cord from T9 or T10 (Additional file 4: Fig. S4a). In the exposed area of its spinal cord, a compressional lesion was produced using a calibrated compression method with microsurgical forceps (Yunkang, Jiangsu, China) for 20 s followed by removing the forceps carefully . The tip spacing of the forceps was 1 mm during compression. (Additional file 4: Fig. S4a–c).
OEC transplantation was performed with the rats positioned in a stereotaxic instrument (RWD). Either 5 μL of a cell suspension containing 1 × 105 cells or saline control (SCI group) was injected into the core site of the injured spinal cord using a 10 μL siliconized Hamilton syringe with a beveled glass pipette tip (80 × 90 mm inner diameter) (Fig. 4a). Injection speed was uniform and slow (0.2 µ l/min), and the glass pipette was left in position for an additional 5 min to prevent leakage before the withdrawal. The whole-cell transplant process took half an hour .
Following these steps, we used sterile normal saline to wash out the incisal opening and sew up each tissue layer. Three days after the operation, the rats were fasted but received an appropriate amount of glucose to their water source for energy. Furthermore, they were given daily intraperitoneal injections of cefuroxime to prevent infection. Urination was artificially assisted by massaging the bladders every day, and feeding quantity was gradually adjusted to their behavior and recovery.
Basso, beattie, and bresnahan (BBB) locomotor scale
The model’s establishment effect and treatment effect were evaluated using the BBB locomotor scale using the methods described in the published literature . The scores for each group were recorded 1 d, 3 d, 7 d, 14 d, and 21d post-injury. The BBB scores for each group were observed, averaged, and recorded by two trainees who were blinded to the groups. The scoring criteria included joint movement, paw placement, and coordination. Two trainees recorded these animal data in a noise-free, open field arena for 5 min at least. The rats with normal motor function obtained a BBB score of 21points, while the rats losing complete motor function were scored 0 points. Finally, the data was compiled and analyzed using a two-sample t-test and linear mixed-effects model analysis.
Immunofluorescence and immunohistochemistry
OECs and RAECs on plastic coverslips of all groups were fixed with 4% paraformaldehyde (Sigma) for 30 min, and then treated with 3% BSA in 0.01 M PBS as a nonspecific blocker for 30 min. Coverslips were incubated with primary rabbit monoclonal antibodies against p75 (1:500, for OECs) or mouse monoclonal antibodies against CD31 (1:1000, for RAECs), at 4 °C overnight, washed in PBS three times and incubated with the corresponding fluorescence conjugated secondary antibody (Alexa Fluor® 594 goat anti-rabbit IgG and Alexa Fluor® 594 goat anti-mouse IgG antibody, 1:1000 dilution) for 2 h followed by DAPI nuclear staining (1:1000) at room temperature (RT) for 10 min. Coverslips were then inverted onto the glass slides and sealed by antifading mounting medium (Boster) after rinsing in PBS. All cells were primarily cultured independently for three times.
For animal models 7-day post-surgery, after being anesthetized with 1% sodium phenobarbital (4 mL/kg), their left ventricle was washed in saline and perfusion fixed by 4% paraformaldehyde. Spinal cords were isolated from the vertebral canal and fixed in 4% paraformaldehyde at 4 °C for three days, cryoprotected in 30% sucrose in 0.1 M PBS for 1 week, cut into segments, embedded in optimum cutting temperature compound (SAKURA Tissue-Tek, CA, USA), sectioned at 10 mm thickness, and pasted into PLL coated slides for immunofluorescent and histochemical staining analysis.
Immunofluorescence of histological sections was performed following previously described methods of immunofluorescence staining of cells. An additional step was added in which the sections were incubated with 0.01% triton-X100 (Sigma-Aldrich, USA) for 1 h at RT before BSA incubation.
One week after injury, the immunohistochemistry method identified CD31-positive vascular endothelial cells were quantified at the spinal lesion site. Briefly, at RT, the histological sections were incubated with 3% H2O2 for 10 min to inactivate endogenous enzymes, incubated in BSA for 1 h, incubated overnight at 4 °C with anti-CD31 antibody, and finally washed three times by PBS. After being combined with goat anti-Mouse/Rabbit Poly- horseradish peroxidase (HRP) secondary antibody, 3,3’ Diaminobenzidine Tetrahydrochloride was added to the tissue to produce a chromogenic reaction. After washing, it was redyed with hematoxylin for 2–3 min, then rinsed with distilled water, followed by gradient dehydration with alcohol (60%,75%,100%) for 5 min each. After removal, it was placed in xylene twice for 5 min, sealed with neutral balsam (Bestbio, China), and observed.
All slides were observed under a Leica DM6 B microscope (Leica Microsystems, Germany). All images were captured using the Leica LAS X software (Leica Microsystems, Diegem, Belgium). Results were measured by Image J software.
Angiography of spinal cord
Paraformaldehyde perfusion fixation was performed on animals as previously described. After that, the circulatory systems of rats were perfused with the Microfil® MV-122 (Yellow) silicone rubber contrast agents. A sign of successful infusion is yellow-dying of the microvessels in the sclera or liver of the animal. Following infusion, the sacrificed animals were stored at 4 °C to promote Microfil in the blood vessels to fully polymerize. The spinal cords were extracted and cut to remain 1.5 cm (centered around the lesion). (Additional file 5: Fig. S5).
Micro-computed tomography (micro-CT) images were obtained using eXplore Locus SP system (General Electric, Milwaukee, WI, USA), and the resolution was set at 5 microns. The angiographic images were reconstructed to 3D vascular models and analyzed by VGStudio MAX (Volume Graphics, Heidelberg, Germany).
Growth factor assay
The growth factors in the CM of OECs were semi-quantitatively evaluated using a multiplex growth factor array system (Rat Growth Factor Array 1; RayBiotech, Norcross, GA, USA). CM dilution was not necessary. However, the collected CM was calibrated according to cell numbers. All processes were operated according to the user manual. The chemiluminescence signal of each membrane dot was imaged using a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) and quantified using Quantity One software (Bio-Rad) which allowed for the assessment of growth factor content in the CM. Tests were repeated as triplicates. Collected CM came from different OEC-culture batches, and results were measured using the Image J software.
Based on the result of the growth factor assays, intervention was performed on three groups that had been set up for in vitro experiments. In each group, MK2206 was dissolved into a DMSO solution and added into media to achieve a final concentration of 5 μM. Three groups received MK2206, and three control groups received equal amounts of DMSO as a control for culturing HUVECs, respectively. HUVECs were mixed with grouping media and inoculated into wells corresponding to the group of 6-well plates (105 cells/ well). (Additional file 6: Fig. S6d) After 24 h, Akt and PI3K changes in phosphorylation were analyzed. Total cellular extracts were prepared as follows: HUVECs were carefully and briefly rinsed with saline buffer and extracted in ice-cold RIPA as described by Yang et al. . Notably, phenylmethanesulfonyl fluoride and phosphatase inhibitor (Beyotime, Jiangsu, China) was added into the PIPA (1:50) to prevent protein degradation and dephosphorylation . The protein concentrations of cell lysates were measured using the BCA protein assay (Qiagen, Germany), using BSA as a reference. The following antibodies were used: pan-Akt (1:500), phospho-Akt (1:2000), PI3K p85 α (1:1000), and Phospho-PI3K p85α (1:500). All protein lysates were run on 10% gradient SDS-PAGE for 1.5 h, transferred onto polyvinylidene fluoride (PVDF) membranes for 50 min, blocked with 5% fat-free milk for 1 h at RT for 30 min, washed with Tris-buffered saline-Tween20 (TBST), and probed with four kinds of primary antibodies at 4 °C overnight. β-actin was included as an internal loading control. After washing, the membrane was incubated with HRP-conjugated secondary antibody at RT for 1 h and washed again with TBST. The immunocomplex bands at PVDF membranes were detected using a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA), and all Western blotting experiments were repeated three times. The intensity of density value (IDV) was analyzed using ImageJ software.
Cell transwell migration assay
For evaluating the PI3K/Akt pathway involved in mediating the endothelial cell migration by activated OECs, the chemotactic motility of HUVECs was determined using Transwell migration chambers with 6.5 mm-diameter polycarbonate filters (8-µm pore size). As shown in Additional file 2: Fig. S2d, the lower chambers were filled with 600 µL of grouping media containing 5 μM MK 2206 or DMSO controls. HUVECs (3 × 104 / well) were seeded in upper chambers in 100 µL serum-free DMEM. Cells were allowed to migrate for 8 h. Non-migrated cells were removed with cotton swabs, and migrated cells were fixed with ice-cold methanol and stained with 0.1% crystal violet. Using a Leica DM IL, cells were inspected under inverted light microscopy (20 ×). Images were captured by Leica LAS AF Lite software (Leica Microsystems, Germany) and quantified by Image J software.
EdU incorporation assay
All operations were carried out according to the kit instructions. Briefly, HUVECs were seeded in the coverglass bottoms of 35 mm confocal dishes at a density of 1.5 × 104 cells per dish along with EdU at a working concentration of 10 μM in 200 μL grouping media for 1 h. After being labeled by EdU, cells were fixed and permeabilized by fixative solution and permeabilization buffer at RT. The mixed reaction solution is prepared by mixing copper sulfate, iFour 488 Azide, and TBS. The reaction mixture was then added into each dish for 30 min at RT, followed by washing with PBS and staining with DAPI (1:1000) for 10 min at RT. After the staining, the cells were imaged under a fluorescent microscope, Leica DM IL, and quantified by Image J software. All assays were repeated three times.
All data are presented as mean ± standard deviation (SD), and the statistical analyses were undertaken using GraphPad Prism7 software (La Jolla, USA). Statistical significance between groups was determined by analysis of variance (ANOVA). A Student’s t-test (normal distribution) was applied to compare the control and experimental groups. Statistical significance was defined as P < 0.05.