Rat SCI model
The animal study was approved by the Animal Care and Use Committee of the Second Affiliated Hospital of Fujian Medical University and Shenzhen Pingle Orthopedic Hospital. The surgical procedures were conducted in compliance with the institutional guidelines for laboratory animal usage in neuroscience and behavioral research. Male Sprague-Dawley rats (10-week old, 250~300g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were housed in the pathogen-free condition.
The SCI model was established by the following steps. Rats were anesthetized via inhaling 3% of isoflurane at the flow rate of 1 L/min. Midline skin incisions were cut and the T12 spinous processes were exposed. A laminectomy was performed at T12. The compression was conducted by placing the base of a compression platform (area 2×5 mm2) onto the exposed spinal cord. A 50-g weight was then placed steadily to the platform for 5 min. The platform was then removed and the muscles and skins were sutured. Rats were transferred to the cages after they regained the righting reflex. The urinary retention was relieved by twice-daily bladder expressions. The sham-operated rats received every surgical step except for the spinal cord compression.
Immune cell enrichment from spinal cords
Rats were anesthetized by inhalation of 3% isoflurane. Each rat was transcardially perfused with 200 ml of ice-cold phosphate-buffered saline (PBS). The spinal cord was taken, minced into approximately 1-mm3 pieces, and treated with RPMI1640 supplemented with 2 mg/ml collagenase IV (Thermo Fisher Scientific), 200 U/ml DNase I (Sigma-Aldrich), 10% fetal bovine serum (FBS) and 2.0 mM CaCl2 for 30 min at 37 oC in an incubator. Digested spinal cord tissues were then filtered through 70-μm cell strainers and overlaid onto 20% Percoll (GE Healthcare), followed by centrifugation at 250g for 10 min. The cell pellet was resuspended in PBS or culture medium before further experiments. In some experiments, spinal cords of 3 to 5 rats were pooled to collect enough cells.
Flow Cytometry and cell sorting
The following fluorophore-conjugated antibodies were purchased from Biolegend: APC anti-Granulocyte (RP-1), PE anti-CD45 (OX-1), APC anti-TCRαβ (R73), PE anti-TCRγδ (V65) and PE-Cy7 anti-CD11b (OX-42). Polyclonal APC anti-Arginase-1 antibody (IC5868A) was purchased from Novus Biologicals. Unconjugated polyclonal anti-inducible nitric oxide synthase (iNOS) antibody (ab15323) was purchased from Abcam. For cell surface marker staining, cells were stained with 2 µg/ml each antibody on ice for 30 minutes. Dead cells were excluded by staining with 1 µg/ml propidium iodide (PI, ebioscience). For intracellular staining, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 90% ice-cold methanol for 30 minutes, followed by incubation with 1:100 diluted anti-Arginase-1 antibody and 10 µg/ml anti-iNOS antibody for 1 hour at room temperature. After three washes with PBS, cells were incubated with 1 µg/ml Alexa Fluor® 594 goat anti-rabbit IgG (ab150080, Abcam) for 30 minutes. For apoptosis assay, cells were stained with PI and Annexin V following the instructions of the APC Annexin V Apoptosis Detection Kit with PI (Biolegend). Cells were then washed with PBS twice and loaded either onto either a BD LSR-II flow cytometer (BD Biosciences) for analysis or a BD FACSAria II sorter (BD Biosciences) for sorting.
Quantitative RT-PCR (qRT-PCR)
Cellular RNAs were purified using the ARCTURUS PicoPure RNA Isolation Kit (Thermo Fisher Scientific). Tissue RNAs were extracted using the Trizol reagent (Thermo Fisher Scientific) following the vendor’s manual. SuperScript® III First-Strand Synthesis Kit (Thermo Fisher Scientific) was used to prepare cDNA. On a 7300 qPCR thermocycler (Invitrogen), quantitative PCR was achieved using Fast SYBR®Green Master Mix (Thermo Fisher Scientific) at the following steps: 50oC for 2 min, then 94oC for 10 min, and then 40 cycles of 30 sec at 94oC and 1 min at 60oC. The data were analyzed using the 2-ΔΔCt method. The primers are listed in Supplementary Table 1.
Primary rat microglia culture
Primary microglia were obtained by isolation from mixed glial cell cultures of 1–day-old neonatal rat brains, according to previous publications [15-17]. Briefly, after removing the meninges, the cerebral cortices were incubated in 0.25% trypsin-EDTA (Invitrogen) for 30 minutes at 37 °C while shaking at 50 rpm on an orbital shaker. The cortices were then mechanically dissociated in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 200 U/ml DNase I until no tissue clump was seen. The whole cortical cells were passed through a 70-μm cell strainer and washed with DMEM once. The cells were cultured in DMEM supplemented with 10% FBS and the medium was replaced every 3 days. Two weeks later, the culture was mildly trypsinized with 1:4 diluted 0.25% trypsin-EDTA for 20 minutes at room temperature. The floating cells were carefully aspirated and microglia which remained attaching to the bottom were kept for further experiments.
Lentiviral packaging and transduction
Lrch1-set siRNA/shRNA/RNAi Lentivector (i050632) and the corresponding control lentivector piLenti-siRNA-GFP were purchased from abmgood Inc. The packaging was conducted using the Ecotropic Lentiviral Packaging System (VPK-205, Cell Biolabs, Inc.) according to the vendor’s protocol. The lentiviruses were purified with Lenti-X™ Maxi Purification Kit (Clontech). The viral titer was determined by Neuronbiotech Company. The lentivirus containing the LRCH1 shRNA sequence was termed “LL” while the control virus was termed “LC”.
Primary microglia were cultured at the density of 2×106 cells/ml in DMEM supplemented with 10% FBS, 4 mM L-glutamine, 50 µg/ml penicillin/streptomycin, and in the presence of 6 μg/ml polybrene (Thermo Fisher Scientific) in 48-well plates. Lentiviral particles were added into microglia culture at the MOI of 20 and incubated overnight. The next morning the medium was then replaced with fresh medium. Cells were incubated in fresh medium for 2 days, followed by incubation with 2 μg/ml puromycin (Sigma-Aldrich) for 4 days.
In vitro treatment of microglia
1×106/ml lentivirus-infected microglia were primed with 20 ng/ml lipopolysaccharide (LPS, Sigma-Aldrich) for 6 hours (or 1 hour if specified) followed by treatment with 5 mM adenosine triphosphate (ATP) for additional 1 hour.
The N27 rat dopaminergic neural cell line was purchased from Sigma-Aldrich. N27 cells were cultured in RPMI 1640 medium supplemented with 10% FBS in a humidified atmosphere of 5% CO2 at 37°C. N27 cells were differentiated with 2 mM dibutyryl cAMP (D0260, Sigma-Aldrich) for 5 days and then used for the experiment described below. To test microglia-mediate neuron death, 5×104 N27 cells were cultured in each well of a Transwell 96-well plate (Corning). 1×105 unprimed or primed primary microglia were seeded into the Transwell 0.4-µm pore membrane insert. N27 cells and microglia were then co-cultured without direct contact for 24 hours. N27 cells were then lifted by 5-min trypsinization at 37oC with 0.25% trypsin-EDTA. N27 cell apoptosis and necrosis were then determined using the APC Annexin V Apoptosis Detection Kit with PI (Biolegend).
Primary rat spinal cord neurons were purchased from Guangzhou Ubigene Biosciences and cultured in Neurobasal-A medium supplemented with the B27 supplement. 1×104 neurons were seeded in each well of a Transwell 96-well plate, and were labeled with 5 µM carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher) following the vendor’s instructions. 5×104 unprimed or primed primary microglia were then seeded into the Transwell 0.4-µm pore membrane insert. Neurons and microglia were co-cultured without direct contact for 24 hours. After that, the insert was removed, neurons were rinsed with 100 µl of PBS twice before CFSE intensity was read on a Tecan Infinite 200 PRO plate reader.
The culture supernatants were collected and centrifuged at 250g for 5 minutes before storage at -80oC. Microglia were lysed using the denaturing M-PER Mammalian Protein Extraction Reagent (78503, Thermo Fisher Scientific) supplemented with the protease inhibitor cocktail (S8820, Sigma-Aldrich) for 5 minutes on ice. The cell lysates were then centrifuged at 10000g for 5 minutes before storage at -80oC. The indicated cytokines were measured with the Rat IL-6 ELISA Set (550319, BD Biosciences), Rat TNF ELISA Set (558535, BD Biosciences), and Rat IL-1 beta ELISA Kit (ab100768, Abcam), respectively.
Rabbit anti-LRCH1 antibody (bs-9327R) was purchased from Beijing Bioss Antibodies Inc. The following antibodies were purchased from Cell Signaling Technology: beta-actin antibody (3700), phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182) antibody (9216), phospho-SAPK/Jun N-terminal kinase (JNK) (Thr183/Tyr185) antibody (9255), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (9101), p44/42 MAPK (Erk1/2) antibody (4695), p38 MAPK antibody (8690), SAPK/JNK Antibody (9252).
Adoptive transfer of microglia
The adoptive transfer procedure was conducted based on established approaches with several modifications [18-20]. Briefly, immediately before SCI, 1×106 lentivirus-infected microglia in 5 μl of 0.9% NaCl solution was injected into the SCI area (epicenter) at a depth of 1 mm at the rate of 0.2 μl/min, using a 5-μl micro-syringe with a 33-gauge Hamilton needle. Rats in the vehicle group received the same amount of saline. After injection, the injectors were removed and the compression of the spinal cord was conducted to induce SCI as described above. After that, muscles and skins were sutured in separate layers. Hematoxylin and eosin (H&E) staining and immunofluorescent stainingRats were transcardially perfused with ice-cold PBS followed by cold 4% paraformaldehyde (PFA). Spinal cords were then fixed in 4% PFA for 16 hours. The next day, spinal cords were immersed in 30% sucrose-PBS for three days. Spinal cords were then embedded in paraffin. Five-micron thick cross-sections were prepared and stained following the standard H&E staining protocol.To detect the transferred microglia and endogenous microglia, the spinal cord sections were stained with the GFP antibody (1:1000, Abcam ab13970) and Iba1 antibody (10 µg/ml, Abcam ab5076) at 4oC overnight. 5 µg/ml Alexa Fluor® 488-conjugated donkey-anti rabbit IgG (5 µg/ml, Abcam ab150073) and Alexa Fluor® 594-conjugated donkey anti-goat IgG (5 µg/ml, Abcam ab150132) were used as secondary antibodies to incubate the sections for 1 hour at room temperature.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
The spinal cord sections were labeled with the DeadEnd™ Fluorometric TUNEL System (Promega) following the manufacturer’s manual.
The hindlimb locomotor function was evaluated before and 1, 3, 7, 14, and 21 days after SCI using the BBB locomotor test developed by Basso et al. . The hindlimb movements during locomotion were quantified using a scale ranging from 0 to 21. The rats were observed for 5 minutes at each time point by two observers who were blinded to the study.
Experiments were independently conducted three times unless specified, with 6 to 10 different samples in each group. Data were shown as mean ± standard deviation and were analyzed by GraphPad Prism 7.0. Student’s t-test or one-way ANOVA was used to compare the mean values among the groups. P-value < 0.05 was considered significant.