Electroacupuncture enhances mesenchymal stem cell therapy via improved perfusion and inflammation modulation in peripheral nerve injury: an IVIM-MRI study in rats

Abstract

Background: Stem cells are widely applied in peripheral nerve repair; however, their therapeutic potential is constrained by immune rejection, inflammatory responses, and a poor regenerative microenvironment. Therefore, reducing the inflammatory response, improving the regenerative environment and dynamically monitoring these processes by imaging techniques are critical. This study examined the effectiveness of electroacupuncture (EA) and bone mesenchymal stem cells (BMSCs) on acute sciatic nerve injury in rats. By employing intravoxel incoherent motion (IVIM) MRI, the study monitored perfusion and explored how EA improves the regenerative environment to optimize stem cell transplantation outcomes.

Methods: Seventy-two rats were randomly assigned to four groups: EA, EA + BMSCs, BMSCs, and PBS. EA was applied at GB30 and ST36. IVIM-MRI (perfusion fraction f), T2WI, histological staining, immunostaining (CD31, IL-1α, IL-10, PPARγ), and SFI were used to evaluate treatment effects.

Results: At 2-4 weeks, the nerve perfusion fraction f in the EA group recovered faster than in the BMSCs group (p < 0.05). By week 4, the EA group showed the greatest myelin regeneration and nerve fiber restoration (p < 0.05). The expression of vascular marker CD31 and anti-inflammatory markers IL-10 and PPARγ increased (p < 0.05), while pro-inflammatory marker IL-1α decreased in the EA and EA + BMSCs groups (p < 0.05). Furthermore, f values were strongly correlated with histological and functional outcomes (p < 0.05).

Conclusion: EA is more effective than BMSCs alone in promoting nerve repair, enhancing blood flow, and reducing inflammation. Moreover, EA enhances the anti-inflammatory effects of BMSCs. The perfusion fraction (f) is a sensitive biomarker for evaluating nerve repair and perfusion restoration.

Keywords: electroacupuncture; magnetic resonance imaging; mesenchymal stem cells; microcirculation; nerve regeneration; peripheral nerve injuries.

Figure 1

Study timeline and flowchart. W: week; EA, electroacupuncture; BMSCs, bone mesenchymal stem cells; PBS, phosphate-buffered saline; f, perfusion fraction; D, diffusion coefficient; D*, pseudo-diffusion coefficient; SFI, sciatic nerve function index.

Figure 2

FS-T2WI of injured sciatic nerve and uninjured contralateral nerve. (A) At week 1 post-surgery, T2WI showed hyperintensity in the injured sciatic nerve across all groups due to edema, accompanied by nerve thickening and increased signal intensity. From weeks 2 to 4, nerve swelling (white arrows) and T2WI hyperintensity gradually decreased in all groups except the PBS group, returning to preoperative levels. Intergroup comparisons showed that at week 1, muscle and nerve edema were more pronounced in the BMSCs and PBS groups than in the EA and EA + BMSCs groups. By week 4, the PBS group still had mild residual edema, whereas the nerves in the other groups had largely returned to normal. (B) T2 values of the injured nerve peaked at week 1 and gradually returned to preoperative levels from weeks 2 to 4. Intergroup comparisons revealed that the EA (red) and EA + BMSCs (green) groups, both receiving electroacupuncture, consistently exhibited lower T2 values than the PBS group (gray). In the BMSCs group (blue), T2 values showed no significant difference from the PBS group in the early stages (weeks 1–2), but became lower than the PBS group at weeks 3–4. Table 2 shows the specific statistical analysis. Scale bar = 5 mm; W: week; EA, electroacupuncture; BMSCs, bone mesenchymal stem cells; PBS, phosphate-buffered saline.

Figure 3

IVIM of the injured sciatic nerve in rats. At 4 weeks post-surgery, the f, D, and D* values of all injured sciatic nerves were compared with their respective control values. (A) At week 1 post-surgery, the nerve f values in all groups decreased; from weeks 2 to 4, the nerve f values gradually recovered, with the EA group (red) demonstrating a faster recovery rate than the other groups, indicating that nerve perfusion in the EA group recovered most rapidly. (B,C) No distinct patterns were observed in the D and D* values across the groups. Table 2 shows the specific statistical analyses. W: week; IVIM, Intravoxel Incoherent Motion; EA, electroacupuncture; BMSCs, bone mesenchymal stem cells; PBS, phosphate-buffered saline; f, perfusion fraction; D, diffusion coefficient; D*, pseudo-diffusion coefficient.

Figure 4

IVIM of the surrounding muscles of the injured sciatic nerve in rats. Post-surgery, the f, D, and D* values of the muscles surrounding all injured nerves were compared with their respective control values. (A) The f values in the three treatment groups exhibited an overall trend of initial increase followed by a decline. In the EA group (red) and EA + BMSCs group (green), both receiving electroacupuncture, muscle f values peaked at week 1, whereas in the standalone BMSCs group (blue), the peak occurred at week 2. (B,C) No clear patterns were observed in the D and D* values across the groups. Table 3 shows the specific statistical analysis. W: week; IVIM, Intravoxel Incoherent Motion; EA, electroacupuncture; BMSCs, bone mesenchymal stem cells; PBS, phosphate-buffered saline; f, perfusion fraction; D, diffusion coefficient; D*, pseudo-diffusion coefficient.

Figure 5

Toluidine blue staining of the injured and uninjured contralateral nerves. (A) Toluidine blue staining of injured and contralateral uninjured sciatic nerves. At week 1 post-surgery, all groups exhibited extensive macrophage infiltration engulfing myelin debris (triangular arrows). By week 2, myelin debris was mostly cleared in the EA group, while the EA + BMSCs group exhibited a few residual debris and macrophages. In contrast, the BMSCs and PBS groups retained more myelin debris and macrophages. Meanwhile, newly formed myelin sheaths with small thin-walled rings can be seen (dovetail arrows). At week 3, regenerated myelin area was significantly greater in the EA and EA + BMSCs groups than in the BMSCs and PBS groups. By week 4, myelin regeneration in the EA group were nearly complete, with increased myelin diameter and thickened walls. (B) Schematic representation of semi-quantitative analysis for myelin area and myelin debris area. (C,D) Comparison of the percentage of myelin area and myelin debris area in the injured nerves of each group with the contralateral uninjured nerve (control) post-surgery. At week 3, the myelin debris area was lower in the EA group (red) than in the other groups (D). At weeks 3 and 4, the regenerated myelin area in the EA group (red) surpassed that of the other groups (C). Table 4 shows the specific statistical analysis. Scale bar = 20 μm; W: week; EA, electroacupuncture; BMSCs, bone mesenchymal stem cells; PBS, phosphate-buffered saline.

Figure 6

Transmission electron microscopic images of the growth process of the injured sciatic nerve. (A) In the first week post-surgery, nerve injury leads to myelin sheath splitting, wrinkling, and fragmentation, with debris phagocytosed by macrophages for degradation. (B–D) By week 2, myelin debris is largely cleared in the EA (B) and EA + BMSCs (C) groups, where Schwann cells proliferate actively, wrapping axons to form thin, small-diameter myelin sheaths (arrows). In contrast, the BMSCs group (D) shows fewer newly formed sheaths, with macrophages still containing incompletely degraded myelin fragments. (E–G) At week 3, myelin sheaths in the EA (E) and EA + BMSCs (F) groups continue to grow, displaying a regular morphology and thickened walls. In contrast, in the BMSCs group (G), fewer myelin sheaths form, and their walls remain thin. (H,I) By week 4, axons and myelin sheaths in the EA (H) and EA + BMSCs (I) groups have nearly recovered, with structure and density approaching normal nerve fibers. Scale bars = 2 μm; W: week; EA, electroacupuncture; BMSCs, bone mesenchymal stem cells.

Figure 7

SPRR1A staining of the injured sciatic nerve. SPRR1A immunofluorescence staining, observed under a fluorescence microscope (400×), revealed the continuity of recovery in the injured sciatic nerve. Nerve regeneration and reconnection were significantly better in the EA group than in the PBS and BMSCs groups. Scale bar = 50 μm; W: week; EA, electroacupuncture; BMSCs, bone mesenchymal stem cells; PBS, phosphate-buffered saline.

Figure 8

Expression of CD31 and inflammatory factors in the injured sciatic nerve of rats. (A) The temporal trends of vascular endothelial cell marker CD31 across groups were as follows: the expression of CD31 decreased in week 1 and gradually recovered in weeks 2–4. CD31 expression in the EA group was consistently higher than that in the BMSCs and PBS groups during weeks 1–4. CD31 expression in the EA + BMSCs group was higher than that in the BMSCs group and the PBS group during weeks 1–2. (B-D) The temporal trends of inflammatory cytokines across groups were as follows: IL-1α (B) and PPARγ (C) peaked at week 1 and subsequently declined, while IL-10 (D) peaked at week 2 before gradually decreasing. By week 4, all three inflammatory markers had returned to normal levels. At weeks 2 and 3, IL-1α (B) expression was lower in the EA + BMSCs group (green) than in the other groups, while PPARγ (C) and IL-10 (D) expression was higher. Table 5 shows the specific statistical analysis. W: week; EA, electroacupuncture; BMSCs, bone mesenchymal stem cells; PBS, phosphate-buffered saline.

Figure 9

Comparison of the SFI among the four subgroups post-surgery. At week 1 post-surgery, the SFI was impaired in all groups, yet the EA group (red) exhibited the fastest recovery rate. From weeks 2 to 4, the SFI recovery rate in the EA group (red) surpassed that of the other groups, indicating that electroacupuncture (EA) treatment was the most effective in restoring nerve function. Table 6 shows the specific statistical analysis. W: week; SFI, sciatic nerve function index; EA, electroacupuncture; BMSCs, bone mesenchymal stem cells; PBS, phosphate-buffered saline.