Ethical statement
Protocols for animal care and experimental management were approved by the Xi'an Jiaotong University Animal Experimentation Committee. Principles of laboratory animal care were followed, and all experimental procedures were conducted according to guidelines established by the National Institutes of Health. All efforts were made to minimize the number of animals used and their suffering.
Animals
Postnatal Sprague-Dawley 5-7 days old rats (n = 36) weighed 22-26 g and neonatal Sprague-Dawley 1 day old rats (n = 6) weighed 5-6 g were supplied by the Center of Experimental Animals at College of Medicine, Xi'an Jiaotong University. The 5-7 days old rats were used for OGD organotypic spinal cord slices, which were divided into transplantation group and non-transplantation group. In the transplantation group, the choroidal plexus epithelial cells which were cultured for 6-7 days and were labeled by 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanineperchlorate (CM-Dil), were transplanted to the OGD organotypic spinal cord slices, then the slices were normally cultured for 3 days (NT 3 d group), 7 days (NT 7 d group) and 14 days (NT 14 d group) respectively. In the non-transplantation group, the same amount of basal medium was dripped to the OGD organotypic spinal cord slices, then the slices were normally cultured for 3 days (N 3 d group), 7 days (N 7 d group) and 14 days (N 14 d group). The 1-day old rats were used for dissociation and primary culture of choroidal plexus epithelial cells.
Materials and reagents
Vibratome (ZQP-86; Shanghai Zhisun Equipment Co. Ltd, Shanghai, China), razor blades , ophthalmic scissors, corneal scissors, microforceps and operating knife blades were used in this experiment. Culture dishes of 35mm, six-well plates, and 0.4-μm-pore polyester membrane inserts (Transwell 3450) were provided by Corning Costar (New York City, New York, USA). Dulbecco's modified Eagle's medium (DMEM/ F12, DMEM/ low glucose, DMEM/ free from glucose), 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanineperchlorate (CM-Dil) and fetal bovine serum were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Lactate Dehydrogenase (LDH) Kit was provided by Ray Biotech (Norcross, GA, USA). Rabbit monoclonal anti-GFAP, anti-beta III tubulin (TUB-III), anti-synaptophysin (SYN) and anti-prealbumin (TTR) were purchased from Abcam (Cambridgeshire, UK). Rabbit polyclonal or monoclonal anti-caspase3, anti-caspase3 active, anti-ERK, anti-p-ERK, anti-P38, anti-p-P38, anti-JNK and anti-p-JNK were purchased from Cell Signaling Technology (Danvers, MA, USA). 4',6-diamino-2-phenylindole (DAPI) was purchased from Roche (Basel, Switzerland). Anti-rabbit HRP were purchased from KPL (Milford, MA, USA). RIPA buffer and protease inhibitor cocktail were provided by Cell Signaling Technology (Danvers, MA, USA). BCA assay and ECL were purchased from Pierce Chemical (Dallas, Texas, USA). Fluorescein-conjugated goat anti-rabbit IgG, biotin-conjugated goat anti-rabbit IgG and peroxidase streptavidin were purchased from CWBIO (Beijing, China). Recombinant rat epidermal growth factor (EGF) was obtained from PeproTech (Rocky Hill, NJ, USA).
Primary culture of choroidal plexus epithelial cells
The neonatal 1-day-old rats were used. Primary cultures of choroidal plexus epithelial cells were prepared using the procedures described in our previous study (Huang et al. 2013a). Briefly, 6 rat brains were removed and kept in chilled DMEM/ low glucose medium. The choroidal plexus tissues were extracted from both lateral ventricles, transferred into a beaker containing chilled DMEM/ low glucose medium. The tissue pieces were mechanically triturated by repeated passages through a 1 ml pipette. After centrifugation, the growth medium (DMEM/ low glucose supplemented with 10% FBS and 10 ng/mL EGF) was added. Then, cells suspended homogeneously in the growth medium were seeded in Petri dishes. They were then cultured in a 5% humidified CO2 incubator at 37°C. The growth medium was changed 48–72h later, then changed every 3 days.
Preparation of organotypic spinal cord slices
5-7 days old rats were used. The organotypic spinal cord slices were prepared according to the following method (Liu et al. 2017b). Lumbar spinal cord was extracted and the meninges were removed. The spinal cord was embedded by two agarose blocks. Then they were transverse sectioned into 350-μm slices. The slices were placed on the surface of the membrane insert and cultured for 14 days at 37 °C in an incubator with 5% humidified carbon dioxide. The growth medium (DMEM/ F12 supplemented with 10% FBS) was changed the day after plating, and then was changed every 3 days.
Preparation of OGD organotypic spinal cord slices
OGD organotypic spinal cord slices were prepared according to the following method (Liu et al. 2017a). Organotypic spinal cord slices, which were normally cultured for 14 days, were washed by PBS twice and placed into a 6-well plate with 1 ml of glucose-free DMEM medium in each well. The slices were then cultured in an anaerobic incubator (N2 90%, H2 5%, CO2 5%) at 37 °C for 60 min. The obtained OGD organotypic spinal cord slices were used in subsequent experimental procedures.
Transplantation of choroidal plexus epithelial cells into OGD organotypic spinal cord slices
Choroidal plexus epithelial cells cultured for 6-7 days were applied. The medium in Petri dishes was discarded, and CM-Dil (1 μg/ml) was added. The choroidal plexus epithelial cells and CM-Dil were incubated together at 37 °C for 30 minutes, then at 4 °C for 15 minutes. The choroidal plexus epithelial cells were washed by phosphate-buffered saline (PBS) for two times, then were mechanically dissociated into cells suspension. Next, they were centrifuged at 2000 ×g for 5 min. The supernatant was discarded, and PBS was added to modulate cells density to 0.5-1×107/ml. 2-μl cell suspension was dripped to the OGD organotypic spinal cord slices in transplantation group. Then the slices were normally cultured for 3 days (NT 3 d group), 7 days (NT 7 d group) and 14 days (NT 14 d group) respectively. Accordingly, 2-μl basal medium was dripped to the OGD organotypic spinal cord slices in non-transplantation group, then they were normally cultured for 3 days (N 3 d group), 7 days (N 7 d group) and 14 days (N 14 d group).
LDH quantification
The culture medium in the N 3 d, NT 3 d, N 7 d and NT 7 d groups was collected. The medium in different groups was screened for LDH level with MultiAnalyte ELISArray kits according to the manufacturer's protocol. Briefly, medium was directly added to the ELISArray plates for 2-h binding incubation. After three washes, the plates were incubated for 1 h with detection antibody. After an additional three washes, bound secondary antibody was detected using streptavidin-HRP and quantified with a spectrometer (Thermo, USA) at a 450-nm wave length.
Immunocytochemistry
The choroidal plexus epithelial cells cultured for 6-7 days were fixed by 4% paraformaldehyde for 30 min, incubated with 0.5% Triton X for 15 min, blocked with 0.3% H2O2 for 15 min, and 10% normal goat serum for 40 min. They were subsequently incubated with rabbit monoclonal anti-Prealbumin (1:1000) at 4 °C overnight. After 3 washes in PBS, they were incubated in the biotin-labeled secondary antibody followed by a further treatment of avidin-biotin-peroxidase complex. The nuclei were counterstained with Mayer's hematoxylin. Microscopy was performed (Olympus, Japan). Negative controls were performed, with primary antibodies omitted.
Immunofluorescence histochemistry
The OGD organotypic spinal cord slices in NT 14 d group were fixed with 4% paraformaldehyde for 30 min, incubated with 0.5% Triton X for 30 min and 10% normal goat serum for 40 min. Then they were incubated with rabbit monoclonal anti-GFAP (1:300), anti-beta III Tubulin (1:500), and anti-Synaptophysin (1:100), respectively, at 4 °C overnight. Subsequently, they were incubated with fluorescein-conjugated goat anti-rabbit IgG (1:200) for 2 h. Cell nuclei were counterstained using DAPI. The slices were then rinsed and cover-slipped with fluorescent mounting media. Laser confocal microscopy (Leica, Wetzlar, Germany) was performed. Negative controls were performed, with primary antibodies omitted.
Western blotting
There were 6 samples in each group. Samples in N 3 d, N 7 d, N 14 d, NT 3 d, NT 7 d and NT 14 d groups were collected and centrifuged at 2000 ×g. Then they were homogenized in ice-cold RIPA lysis buffer supplemented with protease inhibitors cocktail. Homogenates were centrifuged at 12,000 × g, and the supernatants were collected and measured by BCA protein assay. Protein samples were normalized and loaded for SDS-PAGE, then transferred to a PVDF membrane. Membranes were then incubated for 2 h in blocking buffer (5% skim milk, tris-buffered saline, 0.1% Tween 20). Subsequently, they were incubated in primary antibodies over night at 4°C: rabbit monoclonal anti-GFAP (1:5000), anti-beta III Tubulin (1:5000), anti-Synaptophysin (1:2000), anti-ERK (1:3000), anti-p-ERK (1:2000), anti-p-JNK (1:1000), anti-P38 (1:2000), and rabbit polyclonal anti-caspase3 (1:3000), anti-caspase 3 active (1:1000), anti-JNK (1:1000) and anti-p-P38 (1:1000) respectively, followed by incubation with HRP coupled anti-rabbit IgG for 1 h. Then they were incubated in ECL solution. The images were taken using Epson V300 camera system (Epson, Japan), and the immunoreactive bands were measured using Alpha view software (Alpha Innotech, San Leandro, CA).
Statistics
SPSS 26.0 software package was used. All data were reported as means ± SD. Group data were compared by Independent sample T-test. Statistical significance was assessed at P< 0.05.