2.1 Ethics

All animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2020061706), followed the Guide for the Care and Use of Laboratory Animals, and housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved animal facility in our institutes with controlled temperature and light cycles (24°C and 12/12 light cycle).

2.2 Procedure and protocol of SCI in Sprague–Dawley rats and animal grouping

Pathogen-free, adult-male Sprague–Dawley rats (n = 32) weighing 300–325 g (Charles River Technology, BioLASCO Taiwan Co. Ltd) were used for the present study. Animals in the SC group underwent opening of the skin and muscle layers only, while acute traumatic SCI groups received a complete traumatic SCI procedure. For SCI induction, animals were anaesthetised with inhalational 2.0% isoflurane and placed prone position on a warming pad at 37°C for laminectomy at T10 to T12. A modified aneurysm clip was further used to create an impact-compression injury of the spinal cord, and then, it was left for compressing the spinal cord for 30 s before removal.

The animals were categorized into group 1 [sham-operated control (SC)], group 2 SCI only +200 μl culture medium by peri-spinal cord injury area injection and intravenous injection], group 3 [SCI + ADMSCs (1.5 × 10⁶ cells in total, categorized into 3.0 × 10⁵ cells by peri-spinal cord injury area injection and 1.2 × 10⁶ by intravenous injection 3 h after SCI procedure, respectively), and group 4 [SCI + PrPc^OE-ADMSCs (1.5 × 10⁶ cells in total, categorized into 3.0 × 10⁵ cells by peri-spinal cord injury area injection and 1.2 × 10⁶ by intravenous injection 3 h after SCI procedure, respectively)]. Neurological status [i.e. by Basso, Beattie, Bresnahan (BBB) functional scale] was assessed prior to and by Days 1, 3, 7, 14, 21, 35 and 42 after the traumatic SCI procedure.

2.3 Cell culture

Raw 264.7 (BCRC 60001) were cultured in Dulbecco's modified Eagle's medium with high glucose (Gibco) containing 10% Fetal Bovine Serum (Gibco), 100 μg/ml streptomycin and 100 units/ml penicillin (Gibco).

2.4 Isolation of adipose tissue for culturing ADMSCs

For the preparation of allogenic ADMSCs, additional 12 rats were utilized in the present study. The procedure and protocol for ADMSCs isolation and culturing have been described in our previous reports.^{22, 24-26} In detail, allogenic ADMSC donated rats were anaesthetised and then cut into <1 mm³ size pieces using a pair of sharp, sterile surgical scissors. After serial incubation and centrifugation, the cells were obtained and cultured in a 100 mm diameter dish with 10 ml DMEM culture medium containing 10% FBS for 14 days.

2.5 Transfection of ADMSCs with plasmids for cellular prion protein (PrPc) overexpression

The procedure and protocol have been reported in our recent study.³⁵ We purchased the pCS6-PRNP plasmid from Transomic Technologies . The plasmid transfection process was carried out with Lipofectamine 3000 according to the steps specified in the manual. The steps were briefly described as follows: 10 μg PRNP expression vector and 20 μl Lipofectamine 3000 were first incubated at room temperature for 15 minutes, followed by overnight incubation of cells at 37°C in a humidified atmosphere of 5% CO₂ and Lipofectamine (i.e. mixed them together), and relevant experiments were carried out.

2.6 MTT assay

5 × 10³ ADMSC cells were seeded in 96-well dishes. After the PRNP expression vector was transfected overnight, the cells were treated with 150 μM H₂O₂ or 50 ng/ml lipopolysaccharide (LPS) (Sigma). After 6 h incubation, the medium was replaced and replenished, followed by incubation for an additional 24–72 h. At the time point for detection, the medium was removed, and 200 μl MTT reagent was added to the cells for 30 min. Following the formed formazan was solubilized by DMSO in the cell, the optical density was detected at wavelength 595 nm with a spectrophotometer.

2.7 Transwell migration assay for assessing the migratory capacity of PrPc^OE-ADMSCs

The 3.5 × 10⁴ cells were then added to the Boyden chambers (8 μm pore size; Millipore) with 0.5% FBS containing medium, and assay media containing 10% FBS was added to the culture plates. After 24 h incubation, the nonmotile cells at the top of the filter were removed, and the motile cells at the bottom of the filter were fixed with methanol and stained with a one-tenth dilution of Giemsa (Sigma Corporation). The number of migrated cells in each chamber was counted in five randomly chosen fields under the microscope for three independent experiments.

2.8 Cell culturing for ADMSCs or Raw 264.7 cells

5 × 10⁵ ADMSCs were seeded overnight to the 12-well Boyden chambers (8 um pore size; Millipore). After the PRNP expression vector was transfected for 24 h, the ADMSCs were treated with 150 μM H₂O₂ or 50 ng/ml LPS (Sigma) for 6 hours. After the medium was exchanged and replenished, the cells were collected for western blotting and immunofluorescence stain following an additional 24 h incubation.

2 × 10⁵ ADMSCs were seeded to the 6-well Boyden chambers (8 μm pore size; Millipore) overnight. Following the PRNP expression vector transfected for an additional 24 h, 8 × 10⁵ Raw 264.7 cells were seeded on the coverslips in a 6-well dish for 24 h. After 50 ng/ml LPS stimulation (Sigma) for 6 h, the medium was exchanged and covered with the Boyden chambers, which the PrPc protein was over-expressed in the ADMSC cells. After an additional incubation for 24 h, Raw 264.7 cells were analysed by immunofluorescence assay and western blot with indicated antibodies.

2.9 Procedure and protocol of flow cytometry and ELISA

After the cells were rinsed twice with PBS, they were incubated with a serum-free medium containing 10 μM H₂DCFDA in a 37°C incubator for 20 min. Following by rinsing twice with PBS to remove residue H₂DCFDA, the cells were incubated with a serum-containing culture medium for an additional 30 min. Then, the cells were analysed by flow cytometry. To analyse the content of superoxide radicals in the mitochondria, the cells were incubated in PBS containing 5 μM mitoSOX for 10 min and analysed the fluorescence intensity by flow cytometry.

To verify the circulating level of Ly6G+, CD11b/c + or myeloperoxidase (MPO) + cell populations, whole blood cells were stained and performed by flow cytometric analysis (FC500, Beckman) on Days 3 and 7 after SCI induction. The 1.0 × 10⁶ cells were stained with Ly6g-Alexa Fluor 488 (Abcam), CD11b/c-PE (BD bioscience), or MPO-PE (Abcam) antibodies to detect cell surface markers.

To analyse circulatory levels of inflammatory biomarkers, serum tumour necrosis factor (TNF)-α and interleukin (IL)-6 concentrations were assessed by 6H and 72 h after SCI induction in duplicate with a commercial ELISA kit (R&D Systems).

2.10 Basso, Beattie, Bresnahan (BBB) functional scale for the assessment of the locomotor capacity of rats after acute SCI

The animals were followed for 6 weeks (i.e. 42 days) after the acute SCI procedure. The procedure and protocol of the BBB scale were according to the previous reports.^{36, 37} Briefly, each rat was placed in an 80 × 80 × 30 cm³ clear box lined with a blue non-slippery material and stimulated to move freely. All movements were recorded by video. Identical copies of the edited videos were monitored by two independent evaluators blinded to the degree of injury severity. Each evaluator accessed the locomotor capacity of rats using the BBB functional scale. At the end of the experiments, the parameters in each rat were calculated and averaging scores from these two evaluators.

2.11 Western blot analysis

Western blot analysis was performed as described previously.^22-24 Equal amounts (50 μg) of protein extracts were loaded and separated by SDS-PAGE and transferred to PVDF membranes. Next, the membranes were incubated with the indicated primary antibodies as Table S1 for 1 h at room temperature. Horseradish peroxidase-conjugated anti-rabbit IgG (1:2000, Cell Signalling) was used as a secondary antibody. Immuno-reactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) and digitized using Labwork software (UVP).

2.12 Immunofluorescent (IF) staining

IF staining proceeded as we previously reported.^22-24 Rehydrated paraffin sections were first treated with 3% H₂O₂ for 30 min and then incubated with primary antibodies specifically against CD11b/c and CD68. Three sections of spinal-cord specimens were analysed in each rat. Three randomly selected high-power fields (HPFs) were analysed per section for quantification. Finally, the mean number of positively stained cells per HPF for each animal was determined across all nine HPFs.

2.13 Statistical analysis

Quantitative data are expressed as mean ± SD. The statistical analysis was performed by anova followed by Bonferroni multiple comparison post-hoc test. We utilized SAS statistical software for Windows version 8.2 (SAS Institute). A probability value <0.05 was considered statistically significant.

3.1 Impact of PrPc^OE-ADMSCs on time courses of cell viability and anti-oxidative stress underwent oxidative stress and inflammatory stimulation in vitro

First, to verify the protective capacity of PrPc^OE in ADMSCs (i.e. PrPc^OE-ADMSCs) on cell viability against oxidative stress or inflammatory stimulation, the in vitro study was categorized into G1 (ADMSCs only), G2 (ADMSCs + H₂O₂) and G3 (PrPc^OE-ADMSCs + H₂O₂), respectively. The result of the MTT assay demonstrated that as compared with G1, the cell viability at the time intervals of 24, 48 and 72 h was significantly lower in G2 and was significantly reversed in G3 (Figure 1A).

Second, to further verify whether PrPc^OE-ADMSCs also had the capacity to protect the cells against the inflammatory stress, the in vitro study was categorized into G4 (ADMSCs only), G5 [ADMSCs + lipopolysaccharide (LPS)] and G6 (PrPc^OE-ADMSCs + LPS), respectively. The MTT assay result showed that compared with G4 the cell viability at the time points of 24, 48 and 72 h was significantly lower in G5 than was significantly reversed in G6 (Figure 1B).

Accordingly, the results of these in vitro studies implicated that PrPc^OE-ADMSCs offered much more excellent benefits than the ADMSCs-only counterpart in maintaining the cell proliferation against oxidative stress or inflammatory inhibition of cell growth.

To verify these issues of oxidative stress and inflammation, the in vitro study was categorized into six groups as shown in Figure 1A,B, that is G1 to G6 and the flow cytometric analysis was utilized in the present study. The result showed that the fluorescent intensity of total intracellular and mitochondrial ROS under H₂O₂ treatment were significantly increased in G2 than in G1 and G3 and significantly increased in G3 than in G1 (Figure 1C). Additionally, under LPS treatment these parameters were significantly increased in G5 than in G4 and G6, and significantly increased in G6 than in G4 (Figure 1D). Our findings supported that PrPc gene overexpression in ADMSCs enhanced the resistant capacity against the generation of ROS resulted from oxidative stress and inflammatory stimulation.

3.2 Impact of PrPc^OE-ADMSCs on cell migration under oxidative stress and inflammatory stimulations

To elucidate whether PrPc^OE-ADMSC treatment would preserve the ADMSCs migratory ability with respect to oxidative stress and inflammatory stimulations, the in vitro study was categorized into six groups as shown in Figure 1.

Under the oxidative stress, that is treated by H₂O₂, the ADMSCs migratory ability was significantly lower in G2 than in G1 and G3 and significantly lower in G3 than in G1 (Figure 2A–G). Additionally, under the inflammatory stimulation, that is treated by LPS, the ADMSCs migratory ability also exhibited an identical pattern of oxidative stress among groups G4 to G6 (Figure 2H–N). These findings implied that PrPc gene overexpression in ADMSCs offered much more excellent benefits than the ADMSC-only counterpart in maintaining the migratory cell ability against oxidative stress or inflammatory stimulation.

3.3 Impact of PrPc^OE-ADMSCs on suppressing the inflammatory expression of macrophage cell line and the circulatory level of proinflammatory cytokines

In the in vitro study, we utilized the Raw 264.7 cells (i.e. a macrophage cell line) to assess the therapeutic impact of PrPc^OE-ADMSCs on suppressing the cellular level of inflammatory expression and the cells, therefore, were categorized into R1 (Raw 264.7), R2 (Raw 264.7 + LPS/50 ng/ml for 6 h treatment), R3 (Raw 264.7 + LPS + ADMSCs), and R4 (Raw 264.7 + LPS + PrPc^OE-ADMSC). The IF microscopic finding demonstrated that the surface marker expressions of CD11b and CD68 in Raw 264.7 cells, two indicators of inflammation, were highest in R2, lowest in R1 and significantly higher in R3 than R4 (Figure 3A–D).

Next, we collected the circulatory blood sample from post SCI rats and utilized the ELISA to determine the circulating levels of TNF-α and IL-6. The results showed that these two parameters were highest in group 2 (SCI), lowest in group 1 (SC), and significantly higher in group 3 (SCI + ADMSCs) than in group 4 (SCI + PrPc^OE-ADMSCs) at time points of 6 h and 72 h after SCI induction in rodent (Figure 3E,F). Our in vitro and in vivo studies indicated that PrPc^OE-ADMSCs were better than merely ADMSCs alone at attenuating the inflammatory reaction.

3.4 The time courses of neurological function after acute SCI procedure

The BBB functional scale for assessing the locomotor capacity of rats was performed for each rat on Days 1, 3, 7, 14, 21, 35 and 42 after acute SCI induction (Figure 4A). By Days 1 and 3, the BBB score of the left or right lower limb was significantly higher in group 1 than in groups 2, 3 and 4, but it did not differ among groups 2–4 at these time intervals. Additionally, by days 7–42, this parameter remained persistently higher in group 1 than that of groups 2–4 with respect to either left or right lower limb. However, as compared with group 2, this parameter of left or right lower limb was significantly higher in groups 3 and 4 and significantly higher in group 4 than in group 3 at the time points of Days 7–35, whereas this parameter did not differ between groups 3 and 4 by the time point of day 42 (i.e., the end of the study period). These findings implicated the attainment of a steady-state of neurological functional improvement after Days 7–42 following SCI procedure between groups 3 and 4 animals as compared with group 2 animals. Additionally, by the time point of Day 42, the ADMSCs were non-inferior to PrPc^OE-ADMSCs for improving the BBB score, suggesting that it could be attributed to the intrinsic capacity of recovery in neurological function in rodent. Taken together, both ADMSCs and PrPc^OE-ADMSCs significantly improved neurological functions compared to SCI group 2.

3.5 Flow cytometric analysis of circulatory inflammatory cells by Days 3 and 7 after acute SCI procedure

To elucidate the circulatory levels of inflammatory cells at the acute phase of SCI, the flow cytometric analysis was utilized in the present study. Consistent with in vitro study, the circulatory levels of CD11b/c+, Ly6G+ and MPO+ cells, three indicators of inflammation, were lowest in group 1, highest in group 2 and significantly higher in group 3 than in group 4 at the time points of Days 3 and 7 after acute SCI procedure (Figure 4B,C), suggesting that PrPc^OE-ADMSCs might have better ability than ADMSCs on suppressing the acute inflammatory reaction.

3.6 Role of NLRP3 inflammasome signalling in the acute phase (i.e. by Day 3) of SCI

To delve into the essential role of NLRP3 inflammasome signalling on neurological dysfunction in the setting of SCI, six animals in each group were euthanized and the Western blot analysis was utilized by Day 3 after the SCI procedure. The result demonstrated that the protein expressions of HGB1, TLR4, MyD88, TRIF, FADD, cleaved caspase8 and p-NF-κB (Figure 5), seven biomarkers of upstream inflammasome signalling, and the protein expressions of NEK7, NLRP3, ASC, cleaved caspase1 and IL-1ß (Figure 6), five indicators of downstream inflammasome signalling, were lowest in group 1, highest in group 2 and significantly higher in group 3 than in group 4. Additionally, the protein expression of PrPc was highest in group 1, lowest in group 2 and significantly higher in group 4 than in group 3. These findings suggested that intrinsic PrPc would be suppressed in the spinal cord in the acute phase of SCI. The PrPc^OE-ADMSCs were better than ADMSCs only for inhibiting the activation of NLRP3 inflammasome signalling. Based on our findings, we schematically proposed the underlying mechanism of the PrPc^OE-ADMSCs might be superior to ADMSCs therapy in attenuating the expression of NLRP3 inflammasome signalling in the acute phase of SCI in rodents (Figure 7).

3.7 Role of DAMP-signalling inflammatory pathway in chronic phase (i.e. by Day 42) of SCI

To deeply uncover the crucial role of the DAMP signalling pathway on neurological damage in the setting of SCI, six animals in each group were euthanized and the Western blot analysis was utilized again by Day 42 after the SCI procedure. The result showed that the protein expressions of HGMB1, TLR4, MyD88, TRIF, TRAF6 and p-NF-κB, six indicators of upstream DAMP-inflammatory signalling, and the protein expressions of TNF-α, MMP-9 and IL-1ß, three indicators of downstream inflammatory DAMP signalling, were highest in group 2, lowest in group 1 and significantly higher in group 3 than in group 4 (Figure S1). Additionally, the protein expression of PrPc was highest in group 1, lowest in group 2 and significantly higher in group 4 than in group 3. These finding implicated that the PrPc^OE-ADMSCs was better than ADMSCs merely for ameliorating the activation of DAMP-inflammatory signalling. Based on these findings, we schematically proposed the underlying mechanism of the PrPc^OE-ADMSCs might be superior to ADMSCs therapy in attenuating the expression of the DAMP-inflammatory signalling pathway in the chronic phase of SCI in rodent (Figure 7).

3.8 Gene and protein expressions of PrP^C in ADMSC and the expression of MSCs surface markers

The relative gene expression and protein expression of PrP^C in PrPc^OE-ADMSCs was significantly increased than in ADMSCs, suggesting that PrP^C overexpression in ADMSCs was successful conducted (Figure S2A,B). Additionally, the flow cytometric analysis showed that the ADMSCs surface markers, including CD29, CD73 and CD99 were highly populated by day-14 cell culture (Figure S2C–E).

4.1 Study limitation

This study has limitations. First, although the study period was 42 days, we remain concerned about whether such a time interval was long enough to define a true ‘chronic phase’ of SCI. Second, we did not test whether two doses of cell therapy would be better than a single dose for improving the outcome in the setting of SCI. Third, we remain uncertain whether two doses of PrPc^OE in ADMSCs would be superior to two doses of ADMSCs for augmenting the recovery of neurological function. Finally, we did not utilize agonists or antagonists (i.e. such as drugs, specific inhibitors, or gene manipulations) to prove or disprove the proposed signalling pathways of NLRP3 inflammasome and DAMP-inflammation involved in SCI. Thus, we cannot rule out there may also have other signalling pathways to participate in the damage of the injured spinal cord.