Ethics statement
All methods have been reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (https://arriveguidelines.org) and the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). All study protocols were approved by the Animal Testing Committee Guidelines of Hiroshima University (A19-78).
Isolation and culture of rcMSCs and rbMSCs
rcMSCs and rbMSCs were isolated and cultured as previously described [8, 11]. Briefly, rcMSCs were isolated from the cranial bone of 4–5-week-old female Sprague-Dawley (SD) rats weighing 250–300 g. The periosteum, muscle, dura mater, and olfactory nerves were completely removed from the cranial bones. Cranial bone samples were seeded in culture dishes (Sumitomo Bakelite Co., Tokyo, Japan) containing low glucose Dulbecco’s Modified Eagle Medium (Sigma-Aldrich Co., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific HyClone, South Logan, UT, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL; both from Sigma-Aldrich). The dishes were incubated at 37 ℃ under 5% CO2. The adherent cells were detached 7–14 days after seeding the cranial bone samples. The culture medium was changed 14 days after seeding to eliminate floating debris and bone particles. The adherent cells were identified as rcMSCs and passaged several times. Meanwhile, rbMSCs were isolated from the femur and tibia bone marrow of 4–5-week-old female SD rats weighing 250–300 g. Bone marrow samples were seeded in culture dishes containing the same growth medium described above, incubated at 37 ℃ under 5% CO2, and the culture medium was changed every three days. The adherent cells were identified as rbMSCs and passaged several times.
C5 hemicontusion SCI model and cell transplantation
The surgical procedure for C5 hemicontusion SCI was performed on 45 adult female SD rats weighing 250–300 g under isoflurane anesthesia, as previously described [36]. Briefly, a midline dorsal skin incision was made between the spinous processes of C2 and T2, and the underlying paravertebral muscles of C4-6 were coagulated and detached from the laminae. Bilateral laminectomy was performed at the fifth cervical vertebra to expose the dorsal aspect of the spinal cord. The spinal column was stabilized by clamping the vertebral body of C2 and spinous process of T2 using toothed Adson forceps connected to the supporting arms and platform. The spinal cord was aligned under the 0.8-mm impactor tip so that the tip was entirely confined to the left side. Hemicontusion SCI was performed using an Infinite Horizon (IH) impactor (Precision Systems and Instrumentation, Lexington, KY, USA). Cervical hemicontusion injury was induced with an intended force of 100 kdyn. Following cSCI, the musculature was sutured in layers using Vicryl® absorbable sutures (Vedco Inc., St. Joseph, MO, USA), and the skin was sutured using nylon nonabsorbable sutures (C.P. Medical, Portland, OR, USA).
The rats were divided into three groups according to the type of cell transplantation: rcMSC group (n = 15), rbMSC group (n = 15), and control group (n = 15). Rats in the rbMSC and rcMSC groups were injected with MSCs (1.0 × 106 cells in 300 µL phosphate-buffered saline (PBS]) via the tail vein 24 h after undergoing cSCI, while those in the control group were administered only PBS [11].
Behavioral testing
Behavioral testing was performed 1, 4, 7, 14, 21, and 28 days after cSCI in the open field. The functional recovery of each fore- and hindlimb ipsilateral to the cSCI was evaluated using a previously reported 20-point scoring system [37]. The scoring system evaluated six factors, including fore- and hindlimb articular movement, weight support, digit position, stepping, fore- and hindlimb coordination, and tail position.
Fore- and hindlimb articular movement. The movements of the wrist, elbow, shoulder, ankle, knee, and hip were assessed. Joint movements were categorized as absent (score = 0), slight (less than 50% of the maximum range of joint motion; score = 1), and normal (> 50% of the maximum articular amplitude; score = 2).
Weight support. Weight support was judged as present (score = 1) or absent (score = 0) during stance and locomotion.
Digit position. Digit position was evaluated while the rat was stationary and during displacement in the open field as fully extended (score = 2), atonic (score = 1), and flexed (score = 0).
Stepping. Stepping assessment included three factors: paw placement at initial contact, paw orientation during lift-off, and movement. During locomotion, the paw’s initial contact with the ground was evaluated as dorsal (score = 0) or plantar. If the paw placement was plantar, the paw orientation relative to the body axis was evaluated as either parallel (score = 2) or deviated internally or externally to the body axis (score = 1). Similarly, the paw orientation to the body axis at lift-off was assessed as parallel (score = 2) or rotated internally or externally (score = 1). Harmonious movement of each of the three main joints in the fore- and hindlimbs was evaluated as regular (score = 2) or irregular (score = 1).
Fore- and hindlimb coordination. Fore- and hindlimb coordination was described as absent (score = 0), occasional (< 50% of the time; score = 1), frequent (50–90% of the time; score = 2), or consistent (> 90% of the time; score = 3).
Tail position. The tail position was evaluated as elevated (score = 1) or lowered (score = 0).
Morphological assessment of the spinal cord lesion
Twenty-eight days after transplantation, the rats were transcardially perfused with 4% paraformaldehyde in phosphate buffer under isoflurane anesthesia. A 1-cm-long spinal cord segment was symmetrically separated at the central injury site and embedded in paraffin. The spinal cord was cut into 30-µm transverse sections every 1 mm over a distance of 6 mm using a cryostat (Leica Microsystems GmbH, Wetzlar, Germany). The segments were mounted on microscope slides for hematoxylin and eosin (H&E) staining. The H&E-stained segments were examined using a BZ-9000 fluorescence microscope (Keyence Corp., Osaka, Japan) and cavity areas were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The cavity ratio was calculated for each segment by dividing the cavity area by the total tissue area [38].
Spinal cord lesion mRNA analysis
The rats were anesthetized 7 days after transplantation. A 5-mm-long spinal cord segment was removed at the central injury site, and total RNA was extracted using the ISOGEN RNA Extraction Kit (Nippon Gene, Tokyo, Japan). Reverse transcription was performed using the ReverTra Ace-α kit (Toyobo Co. Ltd., Osaka, Japan). Using cDNA as the template, RT-PCR was performed using the 7900 HT RT-PCR system (Applied Biosystems, Carlsbad, CA, USA) with Fast Start Universal Probe Master Mix (Roche, Basel, Switzerland) and TaqMan Gene Expression Assays for rat TGF-β, IL-6, VEGF, TNF-α, NOS2, and IL-10 (Life Technologies, Carlsbad, CA, USA). β-Actin (Actb) was used as an internal endogenous control. The assay IDs for the TaqMan Gene Expression Assays used in this study are listed in Table 1.
Table 1
TaqMan assays used for RT-PCR analysis of spinal cord tissue lesions
Gene symbol | Gene name | Assay ID |
TBG-β | Transforming growth factor beta | Rn01440674_m1 |
IL-6 | Interleukin-6 | Rn01489669_m1 |
VEGF | Vascular endothelial growth factor | Rn01511602_m1 |
TNF-α | Tumor necrosis factor alpha | Rn01525859_g1 |
NOS2 | Nitric oxide synthase 2 | Rn00561646_m1 |
IL-10 | Interleukin-10 | Rn01483988_g1 |
Actb | Actin beta | Rn00667869_m1 |
rcMSC and rbMSC RNA analysis
RNA extraction and quality control. Passage three rcMSCs and rbMSCs were collected using TrypLE™ Select (Thermo Fisher Scientific) and centrifuged at 500 × g for 5 min. Total RNA was extracted using the NucleoSpin™ RNA mini kit (MACHEREY-NAGEL GmbH & Co. KG, Duren, Germany) according to the manufacturer’s protocol. The quality of the total RNA was assessed using a Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) to ensure that the RNA integrity number was > 7.0. After poly (A) + RNA enrichment using the NEBNext Poly (A) mRNA Magnetic Isolation Module (New England Biolabs Inc., Ipswich, MA, USA), double-stranded cDNA libraries (RNA-seq libraries) were prepared using the SMARTer Stranded Total RNA Sample Prep Kit - Hl Mammalian (Clontech Laboratories, Mountain View, CA, USA) and MGIEasy Universal Library Conversion Kit (App-A; MGI Tech Co., Ltd., Wuhan, China), according to the manufacturers’ instructions.
RNA library building and sequencing. RNA-seq libraries were sequenced using paired-end reads (150 nt of reads 1 and 2) on a DNBSEQ-G400RS instrument (MGI Tech Co., Ltd.). The obtained raw reads were trimmed and quality-filtered using the Trim Galore! (version 0.6.7) and Trimmomatic (version 0.39) tools. The trimmed reads were then mapped to the Rat mRatBN7.2 genome using STAR (version 2.7.9a) software. Reads of the annotated genes were counted using the featureCounts program in the “subReads” package (version 2.0.1) in R software.
DEG analysis. Fragments per kilobase million values were calculated from the mapped reads by normalizing to total counts and transcripts. DEGs were detected using the “DESeq2” package (version 1.20.0) in R software. The thresholds of significant DEGs were set at |log2fold-change (FC)| >1.5 and P value < 0.05. Significant DEGs corresponding to log2FC > 1.5 and log2FC < -1.5 were categorized as upregulated and downregulated DEGs, respectively.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Term Pathway Enrichment Analysis. GO term and KEGG pathway enrichment analyses were performed using the iDEP (version 0.96) tool (http://bioinformatics.sdstate.edu/idep96/). The GO enrichment analysis included the following three domains: biological processes, cellular components, and molecular functions. KEGG pathway enrichment analysis was performed using the parametric gene set enrichment analysis (PGSEA) method. Statistical significance was defined as a P-value < 0.05.
Statistical analysis
All statistical inferences were performed using a two-sided test at the 5% significance level. All analyses were performed using the ‘‘rms’’ package in R software version 4.2.0 (https://cran.r-project.org/). Student’s t-test or Fisher’s exact test was used to assess univariate differences between groups for continuous variables or categorical variables, as appropriate. One-way analysis of variance (ANOVA) with Tukey’s correction was used for multiple comparisons. Mixed-effects models were used to assess the longitudinal change in fore- and hindlimb functional recovery and serial changes in the lesion cavity ratio in the cephalocaudal direction.