Animals and Experimental Protocol

All experimental protocols involved in this study were reviewed and approved by the Ethics Committee for the Experimental Animals of Hong Hui Hospital, Xi’an Jiaotong University. All animals used in this study were supplied by the Laboratory Animal Center of Xi’an Jiaotong University. In total, 60 Sprague Dawley (SD) female rats, weighing approximately 220 g, were randomly allocated to the following four experimental groups: (i) in the sham group, rats only suffered laminectomy only at the T8-10; (ii) in the control group, rats suffered from SCI surgery and were injected with the same volume saline; (iii) in the OECs group, rats suffered from SCI surgery and were transplanted with OECs; and (iv) in the aOECs group, rats suffered from SCI surgery and were transplanted with aOECs.

Isolation, Culture and Identification of Primary OECs

Primary OECs were obtained from adult SD rat olfactory bulb (OB) at 2–3 months of age and were further purified by differential cell adhesiveness according to previous reports (Hao et al. 2017; Nash et al. 2001). In brief, the olfactory bulb was dissected and the superficial meninges and blood vessels were removed (Supplementary Fig. 1a). After removal of OB inner layer, the outer layer including olfactory nerve and glomerular layer was collected. Concomitantly, the tissues were rinsed thrice in HBSS, minced with iris scissor, and digested through 0.25% trypsin (Gibco, Carlsbad, CA, USA) at 37 °C for 25 min. When the trypsinization was terminated, the digested tissue was triturated and centrifuged at 1000 rpm/min for 5 min. Cells were resuspended in the Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (Gibco, Carlsbad, CA, USA) containing 20% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA), seeded into flasks coated with poly-lysine (PLL; Sigma-Aldrich, St. Louis, MO, USA) and cultured at 37 °C incubator at 5% CO2. Culture medium was refreshed every three-four days. As for cell purification, the procedure was conducted according to previous reports (Hao et al. 2017). Lastly, the purity of OECs were determined by immunofluorescence staining with anti-p75 antibody and DAPI.

Activation and Collection of Conditional Medium of OECs

To conduct the following experiments, OECs were reseeded on the 6-well plates or coverslips, and divided into two groups including the activated and control groups. For activated group, OECs were treated with 1µM CCM (Sigma-Aldrich, St. Louis, MO, USA). For control group, OECs were treated with equal volume of DMSO vehicle (Sigma-Aldrich, St. Louis, MO, USA). As for collection of conditioned medium, in order to eliminate the nonspecific effects of CCM or DMSO itself, the culture supernatant of both groups was collected at 24, 48 and 72 h, respectively after culture medium was replaced with the fresh medium at 3 days post treatment with CCM. Lastly, the harvested conditional media were mixed, centrifuged to eliminate cell debris and stored at -80℃ for further experiments.

Culture and Purification of Primary Microglia

Primary microglia were obtained from 3-day-old SD rat brain as described previously (Ji et al. 2019). Briefly, the cortices were dissected, removed the superficial meninges and blood vessels and thoroughly rinsed with ice-cold HBSS (Supplementary Fig. 1b, c). Concomitantly the tissues were minced and digested with 0.25% trypsin for 20 min. After the digestion was terminated, the digested tissues were triturated and centrifuged at 1000 rpm for 5 min. The dissociated cells were resuspended with DMEM (Gibco, Carlsbad, CA, USA) containing 10% FBS and 1% penicillin-streptomycin. The cells were seeded into PLL-coated flask and cultured in an incubator at 37 °C in a humidified atmosphere of 5% CO2. Half of medium was replaced every three days until cells reached over 85% confluence. The cell-grown flask was fixed on a shaker and continuously shaken for 5 h at 260 rpm to harvest microglia. The purity of microglia was identified by immunostaining with anti-Iba1 antibody.

SCI Model and Cell Transplantation

The rat SCI models were developed according to previous publication (Fan et al. 2019). In brief, the adult SD rats weighing 220 g were firstly anesthetized by intraperitoneal injection of 1% sodium pentobarbital (60 mg/kg). After hair removal and disinfection, a laminectomy was performed at T8-10 to expose the underlying spinal cord the tissues. The spinal cord at T9 was then clamped with forceps (53327T, 66 Vision-Tech Co., Ltd.) for 20 s. Notably, the parallel distance of forceps tip was kept at 0.5 mm width when the spinal cord was crushed.

For cell transplantation, 2 µL cell suspension containing 1 × 105 OECs or aOECs were immediately injected into the core site of the injured spinal cord through 10 µL siliconized Hamilton syringe post-injury. The syringe was fixed on the stereotaxic instrument to maintain a uniform rate of 0.2 µL/min. In control group, the equal volume saline was injected using the same method. Notably, in order to trace the growth and migration of OECs in vivo, OECs obtained from enhanced green fluorescent protein (EGFP) transgenic rats, treated with CCM, and transplanted into SCI models (n = 3/group), respectively. As for other experiments, OECs/aOECs were derived from normal SD rats.

After surgery, antibiotics, penicillin (1 × 104 U) and gentamicin (8 × 104 U), were administered subcutaneously to prevent infection (Guo et al. 2020). Meanwhile, manual-assisted urination was performed until the recovery of micturition function.

Assessment of Behavior

Functional assessment was performed with the Basso, Beattie, and Bresnahan (BBB) locomotor scale reported by Basso et al. (1995). The BBB scores of each rat were recorded at preoperative 1 day and 1, 3, 7, 14, 21 and 28 days after transplantation. The scores were observed and recorded independently by two trainees blinded to the allocation of experimental groups.

Treatment of Primary Microglia

Microglia were reseeded on the 6-well plates or coverslips, and divided into the following groups: control, LPS, LPS + OECs-CM, and LPS + aOECs-CM groups. Except for the control group, the other groups were treated with medium containing 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) for 24 h and the medium was replaced with normal medium, OECs-CM and aOECs-CM, respectively. Forty-eight hours later, cells were collected to perform immunofluorescence and extract total protein for western blot.

Small Interfering RNA (siRNA) Transfection of Microglia

To examine the involvement of TREM2 in microglial polarization, microglia grown in the 6 well plates were transfected with siRNA (GenenPharma, Shanghai, China) targeting TREM2 using Lipofectamine® 3000 Transfection Reagent (Invitrogen, NY, USA) according to the instruction of manufacturer. The sequences are displayed in the Supplementary Table 1. Forty-eight hours later, the total proteins were extracted to assess the efficiency of siRNA knockdown by western blot analysis.

Western Blot Analysis

At the termination of different treatments, cells or spinal cord tissues (2.0 mm above and below the lesion, Supplementary Fig. 2) were collected to extract total proteins for western blot as the previously described (Wang et al. 2022; Guo et al. 2020). In this study, the following specific antibodies were used: TG2 (3557, 1:1000, Cell Signaling Technology), PSR(PAB916Hu01, 1:1000, Cloud-Clone Corp.), iNOS (ab49999, 1:2000, Abcam), Arg-1 (ab60176, 1:3000, Abcam), CD86 (91882, 1:2000, Cell Signaling Technology), CD206 (60143-1-Ig, 1:2000, Proteintech), TREM2 (ab95470, 1:1500, Abcam), pNF-κB (3033, 1:2000, Cell Signaling Technology), APOE (ab183597, 1:2000, Abcam) and β-actin (T0022, 1:5000, Affinity). After primary antibodies overnight at 4 °C, the corresponding secondary antibodies were incubated based on the manufacturer’s instructions. The immunoblots were visualized using ECL kit and scanned by ChemiDoc XRS (Bio-Rad, CA, USA). Image J software was used to analyze the intensity of the bands based on the β-actin level.

Immunocytofluorescence Staining

OECs and microglia grown on the coverslips were treated according to the experimental protocols. Then the immunofluorescence was performed following previously described methods (Wang et al. 2022; Guo et al. 2020). The primary antibodies were used to stain cells, including anti-p75 (ab245134, 1:300, Abcam), Iba1 (019-19741, 1:500, Wako), iNOS (ab49999, 1:300, Abcam), Arg-1 (ab60176, 1:300, Abcam), TREM2 (ab95470, 1:500, Abcam), F4/80(ab6640, 1:200, Abcam), pNF-κB (3033, 1:200, Cell Signaling Technology), APOE (ab183597, 1:200, Abcam) and DAPI (ab228549, 1:1000, Abcam). After incubation with the corresponding secondary antibodies, coverslips were washed and mounted on the slides. All images were captured by fluorescence microscope (Leica Microsystems, Germany).

Immunohistofluorescence Staining

For immunohistofluorescence staining, rats were anesthetized and transcardially perfused with 200 mL 0.9% saline, followed by 400 mL 4% paraformaldehyde at seven days post-transplantation (Supplementary Fig. 3a). Thereafter, the spinal cord was dissected (1.0 cm above and below the injured site, Supplementary Fig. 3b) and cryoprotected in 30% sucrose in 0.1 M PBS dehydrate at 4 °C. The tissues were sliced at 10 mm thickness by cryostat and fixed on the PLL-coated slides. The details of immunohistofluorescence staining were consistent with the above-mentioned description (Fan et al. 2019). The primary antibodies included anti-p75 (ab245134, 1:200, Abcam), GFP (ab6673,1:200, Abcam), Iba1 (019-19741, 1:500, Wako), iNOS (ab49999, 1:200, Abcam), Arg-1 (ab60176, 1:200, Abcam), NF68 (2835s, 1:50, Cell Signaling Technology) and DAPI (ab228549, 1:1000, Abcam). Other following procedures were same as the immunocytofluorescence.

Enzyme-linked Immuno Sorbent Assay (ELISA)

To explore the potential mechanism, ELISA was used to detect the APOE concentration of conditional medium from culture of OECs and aOECs. The procedures were performed in accordance with the guidelines of ELISA kit (Elabscience®, E-EL-R1230c, Wuhan, China). After reaction termination, the optical density (OD) was measured in a microplate reader at 450 nm wavelength. Standard curve was established according to OD value of standard samples and then calculated the concentration of APOE based on OD of samples.

Statistical Analysis

All experimental data were obtained from at least three independent repetitions and presented as the mean ± standard deviation (SD). SPSS 23.0 software (IBM, NY, USA) was used to perform statistical analysis. Student’s t-test were used to compare the difference between the two groups. The significance of multiple groups was determined by analysis of variance. Statistical significance was defined as P value<0.05. Graph Prism 8.0 (GraphPad Software, CA, USA) was used to prepare graphs based on the results of statistical analysis.