Electroacupuncture facilitates the integration of a grafted TrkC ‐modified mesenchymal stem cell‐derived neural network into transected spinal cord in rats via increasing neurotrophin‐3

2.2. Isolation and culture of MSCs and SCs

Mesenchymal stem cells were isolated, as previously described, from green fluorescent protein (GFP) transgenic SD rats (Osaka University, Osaka, Japan), which ubiquitously express GFP in all tissues. Briefly, one‐week‐old rats ( n = 10) were sacrificed, their femurs were removed, and flushed of bone marrow, which was cultured in low‐glucose Dulbecco's modified Eagle medium (L‐DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, TBD Co, Tianjin, China) and 4 mM L‐glutamine (Invitrogen, USA), in a 5% CO ₂ incubator at 37°C. When adherent cells reached 80% confluence, they were passaged (1:3) into different culture flasks. MSCs from passages 3 to 5 were used for all experiments in this study.

To isolate Schwann cells (SCs), five‐day‐old neonatal SD rats (not GFP transgenic rats, n = 30) were decapitated and sterilized. The sciatic nerves and brachial plexus were dissected, and the adherent connective tissue and epineurium were removed under a dissecting microscope. The nerves were cut into small pieces (< 2 mm) and digested with 0.16% collagenase (Sigma‐Aldrich, St. Louis, MO) at 37°C for 15 min. The dissociated tissue was placed on culture dishes coated with poly‐L‐lysine and containing DMEM/F12 medium containing 10% FBS for 30 min at 37°C in a 5% CO ₂ humidified atmosphere. After 30 min, an additional 2 ml of medium was added to each culture dish. The medium was changed every 2 days. After 5–7 days, when the cells reached 80% confluency, they were subcultured and purified using differential velocity adherent methods. Based on our recent study, 95–96% of the cells were estimated to be SCs.

2.3. MSC and SC transfection and seeding on a 3D gelatin sponge scaffold

Briefly, in vitro , the MSCs and SCs were transduced with recombinant adenoviruses (Advs) containing the TrkC gene (Adv‐TrkC) and the NT ‐ 3 gene (Adv‐NT‐3), respectively, to induce the overexpression of TrkC and the oversecretion of NT‐3. After 3 h, Adv‐TrkC, administered at a multiplicity of infection (MOI) =300, yielded 81.32% transduced MSCs, whereas Adv‐NT‐3 administered at an MOI =100 yielded 78.19% transduced SCs, with excellent viability. When higher viral titers were used, the cells tended to show pathological changes. The medium was replaced with DMEM/F12 supplemented with 10% FBS, and the cells were incubated for 24 h at 37°C.

A three‐dimensional (3D) gelatin sponge scaffold with a 3‐mm diameter and a 2‐mm length was prepared, as previously described. Scaffolds were seeded with 1 × 10 ⁵ total cells (equal numbers of MSCs and SCs) to generate MNs and were cultured in 10 µl culture medium and incubated at 37°C for 14 days; the culture medium was changed every 2 days.

2.4. Spinal cord injury and transplantation

Three days before surgery, the rats received a subcutaneous cyclosporin A injection in the belly (1 mg/100 g per rat). They were anesthetized with 1% pentobarbital sodium (40 mg/kg, i.p.). A laminectomy was performed to expose the T9 and T10 spinal cord segments, and the dura was slit vertically with a pair of micro‐forceps and micro‐scissors. A pair of angled micro‐scissors was used to fully transect the spinal cord, and a 2‐mm segment of the spinal cord was removed at the T10 spinal cord level. Then, either the generated MNs (the MN group) or gelatin sponge scaffolds containing no cells (the GS group) were used to fill the spinal cord gap. After the surgical incisions were sutured, the rats received extensive postoperative care, including the intramuscular injection of penicillin (50,000 U/kg/day) for 3 days. The manual emiction was performed on the experimental rats twice daily until automatic micturition was re‐established. Cyclosporin A was administered once daily for 8 weeks.

2.5. Electroacupuncture

Five days post‐surgery, the rats in the GS+EA and MN+EA groups were subjected to EA treatment every other day for 8 weeks. EA stimulation was applied at two pairs of Governing Vessel (GV) acupoints: 1.) GV9 (Zhiyang) and GV6 (Jizhong); and 2.) GV2 (Yaoshu) and GV1 (Changqiang) (Figure S1 ). The locations of the GV acupoints were determined as described previously. GV1 is located at the midpoint between the tip of the coccyx and the anus in the prone position. GV2 is located on the posterior midline, in the depression below the spinous process of the fourth sacrum. GV6 is located on the posterior midline, in the depression below the spinous process of the eleventh thoracic vertebra in the prone position. GV9 is located on the posterior midline, in the depression below the spinous process of the seventh thoracic vertebra in the prone position. GV6 and GV9 are located in the depressions below the rostral and caudal spinous processes of the transected spinal cord, respectively. EA applied to both GV6 and GV9 can be used directly to treat the injured spinal cord. EA applied to both the GV1 and GV2 can improve bowel and bladder emptying and reduce the paralysis of the lower or hindlimbs. The rats were positioned using a specially designed restrainer, without anesthesia, which maintained them in a recumbent position during the EA treatment. Stainless steel acupuncture needles (0.30 mm in diameter, 50 mm in length; Jiangsu Medical Instruments Inc., China) were inserted at a depth of 5 mm into the GV acupoints. The two pairs of needles were connected to the output terminals of an EA apparatus (model number G6805‐2A, Shanghai Medical Electronic Apparatus Company, China). EA was applied using alternating strings of dense sparse waves at alternating frequencies (60 Hz for 1.05 s and 2 Hz for 2.85 s, pulse width of 0.5 ms). The positive and negative electrodes used to connect the two pairs of needles were alternated with each treatment. During the EA process, the current intensity was tested in the animal's body between the acupoint pair across the graft location, which indicated a current intensity of approximately 5 µA.

2.6. Assessment of locomotor function

After surgery, the rat hindlimb function was assessed weekly, using the Basso, Beattie, and Bresnahan (BBB) open‐field locomotor test to evaluate voluntary movement and body weight support and a modified inclined‐grid climbing test to qualitatively assess the accuracy of foot placement and coordination, which differentiates local reflex activity from voluntary movement. Two independent investigators blinded to the experimental treatments determined the BBB scores.

2.7. Electrophysiology

Evoked potentials (EPs) were measured and recorded ( n = 5/group), as previously described, to evaluate motor axonal conduction. Under general anesthesia, the sensorimotor cortex (SMC) and sciatic nerve of rats were exposed. Stimulating electrodes (NeuroExam M‐800 Data Acquisition Analysis System, MEDCOM, Zhuhai, China) were placed in the SMC (located 2 mm lateral to the midline and 2 mm caudal to Bregma), and the recording electrodes were connected to the sciatic nerve. The amplitude of the cortical motor‐EP (CMEP) was calibrated and then recorded.

2.8. CTB retrograde tracing

Two months after surgery, the rats were anesthetized with 1% pentobarbital sodium (40 mg/kg, i.p.), and their sciatic nerves were exposed under sterile conditions. Using a dissecting stereomicroscope (Leica Microsystems, Inc., Wetzlar, Germany), the needle tip of a 30 g needle Hamilton syringe (Hamilton Co., Reno, USA) was inserted 10 mm into the sciatic nerve along its longitudinal axis and then withdrawn 2 mm to create a potential pool for injection. Then, 2 µl of 2% cholera toxin B (CTB) conjugated to Alexa Fluor 555 (CTB‐555, Life, USA) was slowly injected, as previously described. The sciatic nerve proximal to the injection site was lightly crushed with a pair of blunt forceps to facilitate CTB‐555 uptake by the nerve fibers. The injection site was thoroughly rinsed using a sterile saline‐soaked cotton‐tipped stick, and the wound was sutured. The rats were sacrificed 1 week after tracer injection.

2.9. Pseudorabies virus (PRV‐CMV‐mRFP) retrograde tracing

The same injection procedure described for CTB retrograde tracing was used to slowly inject 1 µl of pseudorabies virus (PRV, 2.5 × 10 ⁹ PFU/ml; Brainvta, China), after which the needle was maintained in place for 5 min. The sciatic nerve was lightly crushed with a pair of blunt forceps proximal to the injection site to maximize PRV contacts with the nerve fibers. The injection site was thoroughly rinsed with a sterile saline‐soaked cotton‐tipped stick, and the wound was sutured. Twenty rats ( n = 5/group) were sacrificed 6 days after injection.

2.10. Tissue processing

Eight weeks after spinal cord transection, 20 rats ( n = 5/group) were anesthetized and perfused transcardially with 200 ml of 0.1 M PBS. A 1‐cm long segment of the spinal cord, which included the injury/graft site, was mechanically homogenized in 0.1 M PBS. Homogenates were centrifuged for 10 min at 18800 g at 4°C and used for Western blot analysis. The relative expression of target protein was indicated by the target protein/GAPDH (loading control) intensity ratio as reported in a previous study.

The rats were sacrificed at the end of 2 or 8 weeks following scaffold transplantation. All rats were deeply anesthetized with 1% pentobarbital sodium (50 mg/kg, i.p.) and transcardially perfused with normal saline containing 0.002% NaNO ₂ and 0.002% heparin, followed by a fixative containing 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4). The spinal cord was dissected and post‐fixed for 24 h in the same fixative, followed by 30% phosphate‐buffered sucrose at 4°C for 48 h. Samples were frozen and embedded in OCT compound. The T8–T12 successive segments of the spinal cord were cut into longitudinal 25‐µm‐thick sections.

2.11. Immunofluorescence staining

Specific proteins in the obtained spinal cord tissue sections were detected by immunofluorescence staining (IFS). Sections were immersed in 0.01 M phosphate‐buffered saline (PBS) three times for 5 min and then blocked with 10% goat serum for 30 min at 37°C. The sections were incubated with primary antibodies in 0.01 M PBS containing 0.3% Triton X‐100 at 4°C for 12 h. The tissue sections were washed with PBS three times for 5 min and incubated with secondary antibodies for 1 h at 37°C. The slides were observed, and images were captured using a fluorescence microscope (Leica) or confocal microscope (Carl Zeiss). A summary of the antibodies used can be found in Table S1 .

2.12. In situ hybridization

Twenty‐five rats ( n = 5/group) were transcardially perfused 2 weeks after SCI. The spinal cord was dissected from the T8 to T12 successive segments, post‐fixed in 4% paraformaldehyde for 2–4 h and saturated in 30% sucrose overnight at 4°C; 1% diethylpyrocarbonate was added to the above solution to prevent mRNA degradation. The tissue samples were cryosectioned into 20‐μm‐thick sections and stored at −20°C. The following procedures were performed according to the manufacturer's instructions (NT‐3 mRNA in situ hybridization kit, Boster, Wuhan, China). The sections were thawed, washed three times with in situ hybridization PBS, and incubated in blocking buffer (without probes) for 2–3 h at 42°C to reduce nonspecific hybridization. Sections were incubated with 20 µl hybridization solution containing a digoxin‐labeled NT‐3 mRNA oligonucleotide probe (No. MK1641‐r, Boster, Wuhan, China): 5’‐TCAAA GGAGT TTGCC AGAAG ACTCG CTCAA‐3’. The sections were rinsed with 2× SSC (NaCl 17.6 g, C ₆ H ₅ O ₇ Na ₃ 8.8 g in 1000 ml ddH ₂ O) for 15 min, 0.5× SSC for 15 min, and 0.2× SSC for 15 min. The sections were incubated with biotin for 60 min at 37°C, followed by incubation with streptavidin‐biotin complex (SABC) conjugated with Alexa Fluor 555 (SABC‐Alex555) for 1 h. After brief staining with Hoechst33342, the sections were covered with coverslips and examined under a fluorescence microscope.

2.13. Immunoelectron microscopy

For immunoelectron microscopy (IEM) analysis, two rats each in the MN+EA and MN groups were transcardially perfused with 0.1 mol/L sodium phosphate buffer containing 187.5 units/100 ml heparin, followed by perfusion with 4% PFA containing 0.1% glutaraldehyde and 15% saturated picric acid. The spinal cord segment containing the injury/graft site was post‐fixed overnight at 4°C in fresh 4% PFA and subsequently cut into 50‐µm‐thick sagittal sections on a vibratome. To improve the penetration of antibodies, vibratome sections were transferred into a cryoprotectant solution containing 25% sucrose and 10% glycerol in 0.1 M PBS overnight at 4°C, followed by three rapid freeze–thaw cycles in liquid nitrogen. After washing with PBS, the sections were treated for 1 h with 20% goat serum (Tris buffer, pH 7.4) to block nonspecific binding. The sections were incubated with primary antibodies (anti‐GFP, anti‐5‐hydroxytryptamine [5‐HT] or anti‐choline acetyltransferase [ChAT]) in 2% normal goat serum solution at 4°C for 24 h, followed by incubation with secondary antibody (1:300, 1.4 nm nano‐gold‐conjugated anti‐rabbit IgG, NanoProbes, USA) overnight at 4°C. Sections were post‐fixed in 1% glutaraldehyde for 10 min and enhanced with Gold Enhance TM EM Plus Kit (NanoProbes). When double‐labeling IEM was performed, the tissue slices were treated with 0.3% H ₂ O ₂ to scavenge endogenous peroxidase before adding the blocking serum. The horseradish peroxidase (HRP)‐conjugated secondary antibodies (Vectastain ABC‐HRP Kits, Vector Laboratories, Burlingame, CA, USA) were visualized using 3, 3′‐diaminobenzidine (DAB, Vector Laboratories). The sections were osmicated with 2% OsO ₄ , dehydrated in a graded ethanol series, and embedded in Epon812 for ultrathin sectioning. After staining with uranyl acetate, the sections were examined under a transmission electron microscope (Philips CM 10).

2.14. Morphological quantification

To quantify immunopositive cells in vivo , 10 separate sections from each animal were selected ( n = 5/group). After IFS with respective antibodies, five areas (four corners and one center) were examined at 200× magnification. The percentage of immunopositive cells was obtained by dividing the total number of immunopositive, GFP, and Hoechst33342 triple‐positive cells by the total number of GFP and Hoechst33342 double‐positive cells.

To observe the area the injury/graft site of the spinal cord, one in every nine longitudinal sections from each animal (a total of 10 sections per rat, n = 5/group) was stained with anti‐GFP antibodies and imaged at 100× magnification; the areas emitting detectable GFP‐positive signal at this magnification were outlined and measured using ImageJ software.

The quantitative analysis of 5‐HT‐positive axons was performed as described previously. A calibrated reticle eyepiece was used to delineate regions 300 µm rostral to the injury site, at the injury site, and 300 µm caudal to the injury site (see reference and Additional file 1: PDF1 for a sketch of the injured spinal cord that was used to clarify the quantification location). The 5‐HT‐positive axon profiles were quantified in all regions, at 200× magnification. The spinal cord was cut in longitudinal sections, and every fifth section was mounted on a gelatin‐coated slide. Ten sections per rat were analyzed, and the total number of 5‐HT‐positive nerve fibers in all regions of each experimental group was averaged. The nerve fibers exceeding 25 µm were counted as 5‐HT‐positive axons.

To analyze the PRV‐labeled cells in the T9, T10, and T11 segments of the spinal cord, one in every nine longitudinal sections from each animal (a total of 10 sections per rat, n = 5/group) was stained with anti‐microtubule‐associated protein 2 (Map2) antibody and imaged at 100× magnification. Positive cells were counted using ImageJ software.

For the quantification of NT‐3 mRNA or NT‐3 protein in vivo , positive cells were quantified using ImageJ, as described previously. Setting the epicenter of the SCI site as the origin, the injury site, and adjacent areas were separated into eight regions. NT‐3 mRNA positive cells were converted into an area of interest (AOI), and the pixel area of each AOI was automatically calculated by ImageJ. The staining density for each region, representing the total pixels in each AOI, was then calculated.

2.15. Transwell migration assay

A chemotaxis assay examining the effect of NT‐3 on TrkC‐MSCs and MSCs was performed in 24‐well plates containing 8‐μm porosity inserts (Corning, NY, USA). The cells were washed twice with serum‐free DMEM and suspended at 5 × 10 ⁵ /ml in DMEM containing 0.5% BSA. Cells (3.0 × 10 ⁴ ) in 100 µl were loaded onto the top well. Serum‐free DMEM containing 0, 20, 40, 60, or 80 ng/ml NT‐3 was added to the bottom chamber at a total volume of 0.6 ml. For the inhibition experiment, TrkC‐MSCs and MSCs were pre‐treated with 10 nM of the TrkC inhibitor K252a (Millipore, USA; K252a is an efficient serine/threonine‐protein kinase inhibitor). After 4 h incubation, the non‐migrating cells were completely removed from the top surface of the filters, and the migrating cells adhering to the undersurface of the filters were stained with Hoechst33342 and quantified with Image J.

3.2. Donor MSCs differentiate preferentially into neuron‐like cells after EA treatment

Eight weeks after implantation into the injured spinal cord, some donor MSCs expressed NSC (Nestin) and immature neuron markers (doublecortin [DCX] and β‐tubulin III, Figure S2 A–L). Occasional donor MSCs in the MN group exhibited expression of the mature neuron marker neurofilament‐h (NF‐H, Figure S2 M,N). Following EA treatment, more donor MSCs differentiated into neuron‐like cells expressing neuronal markers (NF‐H, Figure S2 O,P) than in the MN group; a small number of MSCs maintained Nestin (Figure S2 C,D) and DCX (Figure S2 G,H) expression. Some MSCs in the MN+EA group expressed β‐tubulin III (Figure S2 K,L).

The quantification of neuron markers showed that EA treatment resulted in significantly more NF‐H‐positive donor MSC cells (37.71 ± 4.13% vs . 10.01 ± 2.65%) and fewer cells expressing the immature neuron markers β‐tubulin III (61.27 ± 3.46% vs . 74.22 ± 5.84%) and DCX (19.72 ± 11.78% vs . 24.92 ± 5.98%) or the NSC marker Nestin (22.48 ± 2.01% vs . 31.20 ± 2.53%) compared with those in the MN group (Figure S2 G–Q).

3.3. Formation of synapse‐like structures between MSC‐derived neuron‐like cells

To assess whether EA could promote the de novo formation of synaptic contacts between the transplanted MSC‐derived neuron‐like cells, western blot, immunofluorescence staining, and immunoelectron microscopy assays were performed. The results showed that donor Map2‐positive neuron‐like cells displayed only moderate expression levels of synaptophysin (SYN) in the injury/graft site of the spinal cord in the MN group (Figures 2 A, a1‐3); however, in the MN+EA group, many donor Map2‐positive neuron‐like cells emitted intense SYN‐positive immunofluorescence (Figures 2 B, b1‐3). Similarly, compared with the MN group (Figures 2 C, c1‐3), more donor Map2‐positive neuron‐like cells expressing postsynaptic density protein 95 (PSD95) were observed in the injury/graft site in the MN+EA group (Figures 2 D, d1‐3). Western blot analysis showed that the expression levels of SYN and PSD95 proteins in the injury/graft site of the MN+EA group were significantly higher than those in the MN group (Figure 2E–G , * p < 0.05).

Open in a separate window

FIGURE 2

Mesenchymal stem cell (MSC)‐derived neuron‐like cells and synapse‐like structures in the graft site of the spinal cord at 8 wpi. (A–D) Representative images triple‐labeled with anti‐green fluorescent protein (GFP, green), microtubule‐associate protein (Map2, red; a2,b2,c2, and d2) and synapsin (SYN, white; a1 and b1) or postsynaptic density protein 95 (PSD95, white; c1 and d1) in the epicenter of the injury/graft site. The merged images show the GFP ⁺ cells that colocalize with Map2/SYN (arrows, a3 and b3) or Map2/PSD95 (arrows, c3 and d3). The cell nuclei were counterstained with Hoechst33342 (Hoe). (E–G) Western blot analysis of SYN and PSD95 expression in the injury/graft site of the spinal cord. Values represent the mean ± SD ( n = 5/group, * p < 0.05). (H) Bar chart showing the percentage of GFP ⁺ cells that differentiated into Map2 ⁺ neuron‐like cells. Values represent the mean ± SEM ( n = 5/group, * p < 0.05). (I, J) IEM revealed that a GFP ⁺ cell, labeled by silver‐enhanced nanogold particles (arrows), formed a synapse‐like structure with another GFP ⁺ cell. The structure was characterized by the presence of some synaptic vesicles (J, arrows) of varying diameters in the “presynaptic element,” electron dense material in the “postsynaptic element” (J, asterisk), and a “synaptic cleft” between the presynaptic and postsynaptic elements. Scale bars = 1 mm in (A–D); 20 µm in (a1)–(a3), (b1)–(b3), (c1)–(c3) and (d1)–(d3)

To detect the effects of EA on MSC differentiation in vivo , we quantified the percentage of cells expressing the mature neuron marker Map2. The results revealed that the percentage (34.30 ± 3.10%) of Map2 ⁺ GFP ⁺ cells in the MN+EA group was significantly higher than that (10.12 ± 2.40%) in the MN group (Figure 2H , * p < 0.05). Furthermore, MSC‐derived neuron‐like cells appeared to form synapse‐like structures with each other in the MN+EA group, as confirmed by IEM (Figure 2I and J ). These results suggested that EA may promote the formation of synapse‐like structures between transplanted MSC‐derived neuron‐like cells in vivo .

At 8 weeks after EA treatment, 19.70 ± 3.67% of transplanted Map2‐positive cells expressed gamma‐aminobutyric acid (GABA, Figure S3 A,B,G), 8.03 ± 3.09% of the cells expressed ChAT (Figure S3 C,D,G), and 67.73 ± 5.78% of the cells expressed calmodulin‐dependent protein kinase II (CaMKII, Figure S3 E,F,G). These findings indicated that the transplanted MSC‐derived neuron‐like cells possessed both excitatory and inhibitory characteristics.

3.4. MSC‐derived neuron‐like cells integrate into host neural circuits

After SCI, 5‐HT positive axons in the graft site of the spinal cord were interrupted. In the MN+EA group, GFP/5‐HT/Map2/SYN quadruple‐labeled immunostaining showed that a 5‐HT‐positive axon terminal (button‐like) formed synaptic‐like contacts with a GFP/Map2‐positive neuron‐like cell (Figure 3A–K ). By IEM, 5‐HT‐positive axons, stained with silver‐enhanced nanogold particles, appeared to form synapse‐like contacts with transplanted GFP‐positive cells (Figure 3L,M ). The synapse‐like structure was characterized by the existence of some synaptic vesicles of varying diameters in the axonal terminal (Figure 3M ). Vesicular glutamate transporter 2 (V‐glut2) is a descending excitatory nerve fiber marker, including in the rubrospinal and propriospinal tracts. V‐glut2‐positive axons also regenerated in the injury/graft site of the spinal cord and formed close contacts with GFP/Map2‐positive MSC‐derived neuron‐like cells in the MN+EA group (Figure 3N,O ).

Open in a separate window

FIGURE 3

Mesenchyma stem cell (MSC)‐derived neuron‐like cells integrated into host neural circuits. (A, B) A few 5‐hydroxytryptamine (5‐HT) ⁺ nerve fibers regenerated into the injury/graft site of the spinal cord. (C–K) Green fluorescent protein (GFP)/5‐HT/microtubule‐associated protein 2 (Map2)/synaptophysin (SYN) quadruple‐labeled immunostaining showing 5‐HT ⁺ axon terminals in close apposition to GFP/Map2‐positive neuron‐like cells. (G–K) Representative magnified images of the boxed area in (F), quadruple‐labeled with anti‐5‐HT (red, H), GFP (green, I), Map2 (blue, J), and SYN (white, H and K), and their merged image (H). (L) Immunoelectron microscopy (IEM) revealed that 5‐HT ⁺ axons stained with silver‐enhanced nanogold particles (arrows) formed synapse‐like structures with transplanted GFP‐positive cells (stained by 3, 3′‐diaminobenzidine [DAB]). The synapse‐like structures are characterized by the presence of some “synaptic vesicles” (arrows) of varying diameters in the cytoplasm of one of the processes in (M), electro‐dense material in the “postsynaptic element” (asterisk) in (M), and a narrow intercellular space between these elements. (N) and (O) Some vesicular glutamine transporter 2 (V‐Glut2) ⁺ boutons (R, arrows) made contacts with GFP ⁺ /Map2 ⁺ neuron‐like cells in the injury/graft site of the spinal cord. (P–R) CTB ⁺ ascending nerve fibers (Q) contacted GFP ⁺ cells, featuring bouton‐like terminals (R). (S, T) IEM revealed that GFP ⁺ cells stained with silver‐enhanced nanogold particles (S, arrows) could form synapse‐like structures with host axons (DAB ⁺ , arrows, T). Scale bars = 1 mm in (A), (N), and (P); 100 µm in (B); 50 µm in (Q); 20 µm in (C)–(F), (O), and (R); 1 µm in (G)–(K)

To ascertain whether the ascending regenerating axons could form contacts with transplanted MSC‐derived cells, CTB‐Alex555 was injected bilaterally into the sciatic nerve to retrogradely trace the ascending sensory nerve bundle. CTB/SYN‐positive axons and their boutons were in close association with Map2/GFP‐positive neuron‐like cells in the injury/graft site (Figure 3P–R ). By IEM, in the area caudal to the graft site, a host axon (labeled by DAB) was found to form a synapse‐like structure with the grafted GFP‐positive neuron‐like cell (labeled by silver‐enhanced nanogold particles, Figure 3S ). The synapse‐like structure contained some spherical vesicles in the presynaptic element formed by the host axon (Figure 3T ). Compared with the MN group, the MN+EA group showed a larger number of CTB‐positive axons, many of which were distributed in the caudal lesion margin (Figure S4 A–G). At 8 wpi in the MN+EA group, many MSC‐derived cells had migrated and made contacts with the CTB‐positive axons, which occurred more frequently than in the MN group (Figure S4 C,F). Moreover, in the MN+EA group, the host nerve fibers had extended and were localized in the area caudal to the injury site (Figure S4 H), where they often appeared to converge onto MSC‐derived neuron‐like cells. Very strikingly, many of these fibers converged on the processes of the MSC‐derived neuron‐like cells forming synaptic like contacts that were SYN‐positive (Figure S4 H–M).

3.5. Pseudorabies virus tracing

To explore whether EA promoted the integration of transplanted MSC‐derived neuron‐like cells with the host spinal cord neural circuit, PRV was injected into the sciatic nerves bilaterally, allowing for retrograde trans‐synaptic tracing (Figure 4A ). The distribution and frequency of PRV‐infected neurons at the injury/graft site (T10) and the areas rostral (T9) and caudal (T11) to the injury/graft site 8 weeks after implantation were examined (Figure 4B ). PRV‐labeled neurons were not detected in the rostral (T9) areas of the GS (Figure 4B1 and B2) or GS+EA (Figure 4 B7,B8) groups. However, the number of PRV‐labeled neuron‐like cells at the injury/graft (T9) site in the MN+EA group (Figure 4 B21,B22,C; 6.80 ± 1.20) was significantly increased compared with that in the MN group (Figure 4 B15,B16,C; 2.50 ± 1.20, p < 0.05). PRV‐labeled neuron‐like cells were absent or not detected at the injury/graft (T9) sites of the GS (Figure 4 B3,B4) and GS+EA (Figure 4 B9,B10) groups. In the area rostral (T9) to the graft region, more PRV‐labeled neurons were identified in the MN+EA group (Figure 4 B19,B20,C; 7.10 ± 0.50) than in the MN group (Figure 4 B13,B14,C; 4.30 ± 0.60, p < 0.05). In areas caudal (T11) to the injury/graft site, the number of PRV‐labeled neurons in the MN+EA group (Figure 4 B23,B24,C; 27.10 ± 1.10) was significantly increased compared with those in the GS (Figure 4 B5,B6,C; 19.03 ± 2.54, p < 0.05) and GS + EA (Figure 4 B11,B12,C; 22.80 ± 1.60, p < 0.05) groups. However, the number of PRV‐labeled neurons in areas caudal (T11) to the injury/graft site did not differ significantly between the MN+EA and MN (Figure 4 B17,B18,C; 26.40 ± 0.80, p > 0.05) groups. These results indicated that EA treatment facilitated the integration of transplanted MSC‐derived neuron‐like cells into the host spinal cord neural circuit, which may ultimately serve as a neuronal relay able to transmit neural signals from the rostral host neurons to the grafted MN, allowing signals to reach the area caudal to the graft region of the spinal cord.

Open in a separate window

FIGURE 4

Pseudorabies virus (PRV) retrograde transsynaptic labeling confirmed the integration of transplanted mesenchymal stem cell (MSC)‐derived neuron‐like cells into the host spinal cord neuronal circuit. (A) A schematic diagram showing that PRV that was injected into the sciatic nerve was transported from the caudal area to the rostral area through the injury/graft site of the spinal cord. (B) Representative images showing the host neurons or MSC‐derived neuron‐like cells retrogradely labeled with PRV (red, arrowheads) in the rostral and caudal regions relative to the graft tissue of spinal cord in the GS group (B1–B6), GS+EA group (B7–B12), MN group (B13–B18) and MN+EA group (B19–B24). The cell nuclei were counterstained with Hoechst33342 (Hoe). (C) Bar chart showing the number of PRV ⁺ neurons in the T9, T10, and T11 areas of the four groups. Values represent the mean ±SD. n = 5/group. * p < 0.05, compared with the GS group, ^# p < 0.05, compared with the GS+EA group, and ^& p < 0.05, compared with the MN group by one‐way ANOVA with LSD‐t. Green fluorescent protein (GFP, green), PRV (red), microtubule‐associated protein (Map2, white), and Hoe (blue). Scale bars =50 µm in (B1)–(B14), (B17)–(B20), (B23), and (B24); 10 µm in (B15) and (B16), (B21), and (B22). GS: gelatin sponge scaffold with no cells; GS+EA: GS combined electroacupuncture; MN: MSC‐derived neural network; MN+EA: MN combined with electroacupuncture

3.6. MN implantation combined with EA attenuates local inflammation

Inflammatory reactions commonly result in a cascade of secondary injuries after SCI. To assess inflammation 8 weeks after SCI, CD68‐positive macrophage/microglia were assessed (Figure S5 ). Immunofluorescence staining revealed fewer CD68‐reactive cells in the injury/graft site of the spinal cord in the MN+EA group compared with those in the MN, GS+EA, and GS groups (Figure S5 A–D), which is consistent with the results of Western blot analysis (Figure S5 E,F). These results suggested that implantation of MN, combined with EA, may have beneficial effects associated with the suppression of local inflammatory reactions following SCI.

3.7. Regeneration of 5‐Hydroxytryptamine (5‐HT)‐positive axons

Axonal regeneration was assessed by immunohistochemistry. Although 5‐HT‐positive axons reached the rostral host/injury interface (Figure S6 A,A1), none of them crossed the rostral host/injury interface into the injury/graft site in the GS group (Figure S6 A,A2). However, in the GS+EA and MN groups, some 5‐HT positive axons appeared to extend into the graft site but did not reach the caudal injury/host interface (Figure S6 B,B3,C,C3). Remarkably, in the MN+EA group, significantly more 5‐HT positive axons regenerated into the injury/graft site (Figure S6 D2 compared with those in the other groups (Figure S6 E, p < 0.05). These results suggested that MN+EA could promote 5‐HT‐positive axon regeneration into the graft site.

3.8. Improvements in paralyzed hindlimb motor function

Behavioral observations, electrophysiological detection, the BBB open‐field motor assessment, and modified grid climbing tests were then performed to assess the functional recovery of rats subjected to the various treatments. In normal animals, stimulation at certain points of the sensorimotor cortex could evoke CMEPs with short latencies and large response amplitudes (Figure 5A–C ). Small CMEPs could be evoked in the GS group (Figure 5A ). In the GS+EA, MN, and MN+EA groups, EA treatment or MN transplantation markedly improved CMEP performance compared with that in the GS group (Figure 5 B,C, p < 0.05), and MN+EA treatment further shortened the latency and increased the amplitudes of CMEPs (Figure 5 B,C, p < 0.05). BBB open‐field motor assessments showed that the hindlimbs of all rats were completely paralyzed following T10 spinal cord transection. Over 8 weeks after operation and transplantation, hindlimb locomotor performance showed progressive improvements in all groups. At 8 wpi, the mean BBB score in the MN+EA group was 8.92 ± 0.81. Some animals in the MN+EA group showed the extensive movement of all three hindlimb joints and interval plantar support of the paw, with BBB scores as high as 10 (Figure 5D ). At 8 wpi, the 45° sloping grid climbing method was used to evaluate the spontaneous placement reflex triggered by direct touch (Figure 5E ; Video S1 shows the grid climb test in the GS, GS+EA, MN, and MN+EA groups). The hindlimbs of rats in the GS group were dragged behind when rats climbed the sloping grid with their forelimbs. In the GS+EA and MN groups, the rats climbed up the grid using the backs of their paws. The rats in the MN+EA group, however, stepped on the grid with the plantar surfaces of their hind feet and displayed the coordinated movement of their forelegs and hindlegs.

Open in a separate window

FIGURE 5

Outcomes of the BBB locomotion assessment, grid climb test, and electrophysiology. (A) Cortical motor‐evoked potentials (CMEPs) were obtained by electrophysiological analysis in the GS, GS+EA, MN, and MN+EA groups. (B, C) Bar charts of the amplitude (B) and latency (C) of CMEPs, showing the higher amplitudes and shorter latencies of the CMEPs in the MN+EA group compared with the GS (* p < 0.05), GS+EA ( ^# p < 0.05), and MN ( ^& p < 0.05) groups. Values represent the mean ±SD ( n = 5/group; one‐way ANOVA with LSD‐ t ). (D) Comparison of Basso, Beattie, and Bresnahan (BBB) score for hindlimb locomotor function in the GS, GS+EA, MN, and MN+EA groups. Values represent the mean ± SD ( n = 10/group; one‐way ANOVA). * p < 0.05 compared with the GS group, ^# p < 0.05 compared with the GS+EA group, and ^& p < 0.05 compared with the MN group, by one‐way ANOVA. (E) Grid climb test was performed in the GS, GS+EA, MN, and MN+EA groups at 8 wpi. GS: gelatin sponge scaffold with no cells; GS+EA: GS combined electroacupuncture; MN: MSC‐derived neural network; MN+EA: MN combined with electroacupuncture

3.9. EA increases NT‐3 levels in the injured spinal cord

To evaluate NT‐3 expression in the injured spinal cord following various treatments, the injury/graft site and adjacent segment tissues were processed for immunofluorescence and in situ hybridization stainings. NT‐3 levels were calculated by measuring the standardized density of NT‐3 immunofluorescence and in situ hybridization stainings within the defined anatomical regions sampled from the Sham, GS, GS+EA, MN, and MN+EA groups. In situ hybridization staining showed that the relative density of NT‐3 mRNA‐positive staining among eight regions of the injured spinal cord in the Sham group at 2 wpi was not significantly different comparing with each other (Figures 6A and S7 A,A1–A3). Compared with the Sham group, the relative density of NT‐3 mRNA‐positive declined in all eight regions, especially within the injury/graft site, in the GS and MN groups (Figures 6A and S7 B,B1–B3,D,D1–D3). However, the relative density of NT‐3 mRNA‐positive staining in the GS+EA and MN+EA groups significantly increased compared with corresponding part in the GS and MN groups (Figures 6A and S7 C,C1–C3,E and E1–E3). The GS+EA group frequently exhibited dense clusters of NT‐3‐mRNA‐positive staining in regions (0–0.5 mm, 0.5–1.0 mm, 1.0–1.3 mm, and >1.3 mm) adjacent to the caudal injury epicenter (Figures 6A and S7 C), whereas the MN+EA group only exhibited clusters in areas (1.0–1.3 mm and >1.3 mm) adjacent to the caudal injury site (Figures 6A and S7 E). NT‐3 immunofluorescence staining showed that the relative densities of NT‐3 immunofluorescence in the MN+EA and GS+EA groups were significantly increased compared with those in the GS and MN groups in the regions 1–1.3 mm and >1.3 mm adjacent to the caudal injury site (Figures 6B and S8 ). The increase in the relative density of NT‐3‐positive staining in the caudal regions 1–1.3 mm relative to the epicenter was the most pronounced in the GS+EA group. In the MN+EA group, the density of NT‐3‐positive staining in the regions 1–1.3 mm and >1.3 mm caudal to the epicenter was significantly higher than that in the regions −1.3–1 mm and < −1.3 mm adjacent to the rostral injury site (Figures 6B and S8 E,E1–E3).

Open in a separate window

FIGURE 6

The density of neurotrophin‐3 (NT‐3) mRNA and protein in the injured spinal cord. (A) Bar charts displaying the relative densities of NT‐3 mRNA in eight regions in the rostral and caudal areas relative to the injury site of the spinal cord for five groups. The density of NT‐3 mRNA was significantly increased in the caudal site compared with the rostral area in the GS+EA and MN+EA groups. Values represent the mean ±SD ( n = 5/group, * p < 0.05). (B) Bar charts showing the relative densities of NT‐3 protein expression in eight regions in the rostral and caudal areas relative to the injury site of the spinal cord for five groups. The density of NT‐3 protein was significantly increased in the caudal area compared with the rostral area in the GS+EA and MN+EA groups. Values represent the mean ±SD ( n = 5/group, * p < 0.05). GS: gelatin sponge scaffold with no cells; GS+EA: GS combined electroacupuncture; MN: MSC‐derived neural network; MN+EA: MN combined with electroacupuncture