Bone marrow mesenchymal stem cells attenuate pain and modulate peripheral sodium channel activity in a rat model of complex regional pain syndrome type I

Establishment of the CRPS-I rat model

The Animal Ethics Committee of The Beijing Institute of Biotechnology, Beijing, China (No. AMMS-06-2016-003) approved all animal experiments.

As previously described, a chronic post-ischemia pain model was established to mimic CRPS-I by prolonged ischemia-reperfusion of the right hind limb ¹⁶ . Male Sprague-Dawley rats (250 ± 20 g) were obtained from the Experimental Animal Center of the PLA General Hospital. Animals were fasted for 12 h with free access to water before the experiment. Under anesthesia with 3% pentobarbital sodium (50 mg/kg, i.p.), a 3 ml syringe barrel with both ends removed was fitted around the right hind limb. A rubber O-ring (inner diameter: 5.0 mm) was placed 1.5 cm above the ankle to induce ischemia. After 3 h, the ring was removed to allow reperfusion. Rats recovered spontaneously from anesthesia.

Following reperfusion, the affected limbs exhibited erythema, edema, and plasma extravasation, which lasted approximately 2–4 h. To assess model validity, acute pain responses were measured 8 h post-reperfusion to evaluate early nociceptive changes due to inflammation ¹⁶ . Given the distinct mechanisms of acute and chronic pain ¹⁷ , behavioral assessments for chronic pain hypersensitivity were conducted on postoperative day 7 ¹ . Mechanical allodynia was assessed using von Frey filaments (Stoelting, Chicago, IL, USA) with an ascending stimulus paradigm ¹⁸ . Eight filaments (0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, and 15.0 g) were applied to the plantar surface of the hind paw through a wire mesh floor (mesh size: 10 × 10 mm) after 30 min of acclimatization. Each stimulus was applied perpendicularly for 2–3 s. Paw withdrawal, shaking, or licking was recorded as a positive response. Each filament was tested five times, and the 50% withdrawal threshold was calculated using Dixon’s up-down method. Cold allodynia was evaluated using a blunt metal probe (diameter: 1.0 mm) gently applied to the plantar surface for 2–3 s without penetrating the skin. Although this stimulus typically does not elicit acute mechanical pain, it may induce cold or mechanical hypersensitivity in the context of CRPS-I due to abnormal activation of C and Aδ fibers. Spontaneous pain behaviors, such as limb shaking, licking, and reduced weight-bearing, were also observed, indicating the successful establishment of the model. The defined time points for acute and chronic pain assessments ensured accurate characterization of pain states and were consistent with previous studies^1,16,17.

Isolation and characterization of primary rat BMSCs

Bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from 4- to 5-week-old male Sprague-Dawley rats (150–200 g; Laboratory Animal Center, Chinese PLA General Hospital) using the whole bone marrow adherence method. Under sterile conditions, rats were euthanized by cervical dislocation, and femurs and tibias were harvested. Bone marrow was flushed from the cavities using sterile PBS (pH 7.4) containing 1% penicillin/streptomycin with a 1 ml syringe and 18G needle. The cell suspension was centrifuged at 1500 rpm for 5 min, and the pellet was resuspended in low-glucose DMEM (LG-DMEM) at 1–3 × 10⁶ cells/ml. Cells were seeded into T-25 flasks and cultured at 37°C with 5% CO₂. Non-adherent cells were removed after 48 h, and the medium was changed every 3 days. Upon reaching 80% confluence, cells were passaged at a 1:3 ratio following digestion with 0.25% trypsin-EDTA.

Third-passage BMSCs were washed and resuspended in PBS containing 0.5% bovine serum albumin (BSA). Surface marker expression was analyzed by flow cytometry using FITC- or PE-conjugated antibodies against CD105, CD90, and CD44 (positive markers), and CD45 and CD11b (negative markers), following the manufacturer’s protocols. Cells were incubated for 30 min at room temperature in the dark and washed before analysis. To assess multipotency, adipogenic and osteogenic differentiation were induced. For adipogenesis, BMSCs were cultured in high-glucose DMEM supplemented with 10% FBS, 0.5 mM isobutylmethylxanthine (IBMX), 1 µM dexamethasone, 10 µg/ml insulin, 100 µM indomethacin, and 1% penicillin/streptomycin for 21 days. Lipid droplet formation was visualized by Oil Red O staining. For osteogenesis, cells were cultured in high-glucose DMEM containing 10% FBS, 0.05 µM ascorbic acid-2-phosphate, 100 nM dexamethasone, 10 mM β-glycerophosphate, and 1% penicillin/streptomycin. After 21 days, calcium deposition was evaluated using Alizarin Red S staining. All media components (LG-DMEM, trypsin, FBS, antibiotics) were obtained from Gibco (New York, USA). Antibodies for flow cytometry included FITC-CD44, PE-CD45, PE-CD90, PE-CD11b (eBioscience, CA, USA), and FITC-CD105 (Bio-Rad, CA, USA).

BMSC transplantation

After weighing, the rats were anesthetized with an intraperitoneal injection of 3% pentobarbital sodium (50 mg/kg) and positioned in a prone posture with a cushion under the abdomen to arch the lower back. The L4/L5 region was shaved and sterilized. A microsyringe needle was inserted at a 30° angle and advanced horizontally along the interspinous ligament to puncture the dura mater and access the subarachnoid space. A total of 20 µL of cell suspension (1 × 10⁷ cells/ml) was injected. The experimental rats were randomly divided into four groups: Blank group (wild-type; WT rats with no intervention), Model group (CRPS-I rats with no intervention), Treatment group (CRPS-I rats with intrathecal BMSC transplantation), and Control group (WT rats with intrathecal injection of cell culture medium). Tissues were harvested on day 14 after BMSC transplantation, coinciding with the final behavioral assessment to align molecular and behavioral outcomes.

Behavioral assessment

To evaluate the analgesic effects of bone marrow mesenchymal stem cell (BMSC) transplantation in a CRPS-I rat model, four behavioral tests were conducted: thermal withdrawal latency (TWL), mechanical withdrawal latency (MWL), spontaneous pain scoring, and cold allodynia testing (acetone evaporation method). For TWL measurement, rats were placed on a transparent glass platform maintained at 37°C and allowed to acclimate for 30 min to ensure a relaxed state. A plantar thermal stimulator was then used to apply a focused heat stimulus to the hind paw. Before testing, the focal distance and light intensity were adjusted to ensure consistent stimulation at the same plantar region. TWL was recorded as the time from stimulus onset to paw withdrawal. Each rat underwent five trials per day with at least 10-min intervals between tests, and the average was calculated as the daily TWL.

For MWL measurement, rats were placed on a preheated metal mesh surface and acclimated for 30 min. Mechanical stimuli were applied to the plantar surface using von Frey filaments with gradually increasing force until a withdrawal response was elicited. A response was considered positive if paw withdrawal occurred during or immediately after filament removal. Each rat was tested five times daily, with a minimum 10-min interval between trials. The average of the five measurements was recorded as the daily MWL.

Spontaneous pain behavior was assessed on postoperative day 14 using video recording. Rats were individually placed in transparent observation chambers and allowed to acclimate for 15 min during a quiet period. Subsequently, spontaneous behaviors were recorded for 10 min. Observed pain-related behaviors included limping, paw lifting, paw licking, and reduced weight-bearing on the affected limb. Pain severity was scored on a 0–3 scale: 0 = normal behavior; 1 = mild pain (e.g. occasional paw lifting); 2 = moderate pain (e.g. sustained licking or weight avoidance); 3 = severe pain (e.g. frequent licking, prolonged lifting, or complete avoidance of weight-bearing) ¹⁶ .

Cold allodynia was evaluated using the acetone evaporation method on postoperative day 14. A 20 μl droplet of room-temperature acetone was gently applied to the mid-plantar surface of the affected hind paw using a micropipette, avoiding direct skin contact. Behavioral responses within 30 s were recorded and scored as follows: 0 = no response; 1 = slight paw withdrawal; 2 = obvious withdrawal or licking; 3 = intense response such as sustained lifting, rapid shaking, or vigorous licking ¹⁹ .

RT-PCR

DRG tissues from rats were collected and lysed in TRIzol reagent to extract total RNA. The integrity of the RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, California, USA). RNA concentration and purity were measured using a NanoDrop® ND-1000 spectrophotometer (NanoDrop, USA). For reverse transcription, the reaction mixture included total RNA (40 μg), 10× DNase I buffer (5 μl), RNase inhibitor (20 U), DNase I (RNase-free, 10 U/2 μl), and RNase-free dH₂O, with a total volume of 50 μl. Reverse transcription was performed using the TIANScript RT Kit (TIANGEN, Beijing, China) at 42°C for 50 min, followed by inactivation of reverse transcriptase at 95°C for 5 min. The RT-PCR reaction mixture (20 μl) included 1 μl of reverse transcription product, 1 μl each of 10 μM forward and reverse primers, 10 μl of 2× master mix, and 7 μl of nuclease-free water. The RT-PCR was conducted under the following conditions: initial denaturation at 96°C for 4 min, followed by 40 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s. Fluorescence signals were analyzed using the 2^−ΔΔCT method, with β-actin as the internal control. The primer sequences are listed in Table S1. TRIzol was purchased from Invitrogen (California, USA), the TIANScript RT Kit from TIANGEN (Beijing, China), and the HotStart Fluorescent PCR Core Reagent Kit (SYBR Green I) from BBI (Italy). The RT-PCR instrument (Prism® 7300) was obtained from ABI (USA).

Western blot

DRG tissues were collected from rats and lysed using RIPA buffer (IBL, USA) to extract total protein. Protein concentrations were determined using a BCA assay kit (ThermoFisher, China). Equal amounts of protein were mixed with loading buffer and denatured by boiling for 5 min. Samples were then separated on 10% SDS-PAGE gels (electrophoresis buffer from BOSTER, Wuhan, China) and transferred onto PVDF membranes using a wet transfer system. Membranes were blocked with 5% non-fat milk in TBST at room temperature for 1 h and incubated overnight at 4°C with primary antibodies against Nav1.7, Nav1.8, and Nav1.9 (rabbit polyclonal; ThermoFisher, China). After washing with PBS or TBST, membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody for 1 h at room temperature. Following additional washes, protein bands were visualized using an ECL detection kit (ThermoFisher, China). Band intensities were analyzed using Image Studio software, and the relative expression levels were calculated as the ratio of the target protein to the internal control (β-actin or GAPDH).

DRG neuron electrophysiology

Male CRPS-I model SD rats (250 ± 20 g) were used. L4-L6 DRGs were dissected under sterile conditions and connective tissue and nerve roots were removed. The tissue was digested with 0.25% trypsin/EDTA (ThermoFisher Scientific, 25200056, USA) at 37°C for 15–20 min, and the reaction was terminated with DMEM (Gibco, 11995065, USA) containing 10% FBS (ThermoFisher Scientific, A5670701, USA). Single-cell suspensions were obtained by gentle trituration and filtration through a 70 µm cell strainer, followed by centrifugation at 1000 rpm for 5 min. The pellet was resuspended in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (ThermoFisher Scientific, 15140122, USA) ²⁰ . Cells were plated on poly-l-lysine-coated coverslips (0.1 mg/ml; Sigma, P3150-10MG, USA) and maintained at 37°C in 5% CO₂. The culture medium consisted of Neurobasal-A (Gibco, 10888-022, USA) supplemented with 2% B27 (Gibco, 17504-044, USA), 10 ng/ml NGF (CUSABIO, CSB-EP016120HU, China), and 1% penicillin-streptomycin. Whole-cell patch-clamp recordings were performed after 2 h of culture, targeting medium-sized DRG neurons with diameters of 25–40 µm. Previous studies have shown that Nav1.7, Nav1.8, and Nav1.9 are the predominant sodium channel subtypes in small to medium-diameter C-type neurons ²¹ , while Nav1.8 and Nav1.9 are also expressed in subsets of medium to large neurons ²² . By contrast, Nav1.7 expression is relatively limited in medium-diameter A-fiber neurons (~15%) ²³ .

Electrophysiological

Whole-cell patch-clamp recordings were performed on DRG neurons using an EPC-10 USB amplifier (HEKA Elektronik, Lambrecht, Germany) controlled by PatchMaster software at room temperature (25°C ± 2°C). Recording pipettes were pulled from borosilicate glass capillaries (1.5 mm) using a P-97 puller (Sutter, Novato, CA), fire-polished, and had a resistance of 2.0–2.5 MΩ. All salts were purchased from Sigma. The internal solution (mmol/L) contained 140 CsCl, 2 MgCl₂, 10 HEPES, 10 EGTA, and 2 Na₂ATP (pH adjusted to 7.2 with KOH), and the external solution (mmol/L) contained 140 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, 1 CdCl₂, 10 TEA-Cl, and 10 glucose (pH adjusted to 7.4 with NaOH). A continuous perfusion system (1–2 ml/min) was used to maintain stable extracellular conditions. To pharmacologically isolate sodium channel subtypes, tetrodotoxin (TTX, 1 μmol/L; MCE, HY-P5868, USA) was applied to block TTX-sensitive currents (Nav1.7), and A-803467 (100 nmol/L; MCE, HY-11079, USA) was used to selectively inhibit Nav1.8 ²⁴ . As no selective blocker is available for Nav1.9, only Nav1.7- and Nav1.8-mediated currents were analyzed. After achieving a gigaohm seal, whole-cell configuration was obtained by gentle suction. Cells were held at −80 mV, and depolarizing steps were applied from −80 to +60 mV in 10 mV increments (200 ms duration) to evoke inward sodium currents. Capacitive transients were canceled, and series resistance was compensated by 80% to minimize voltage errors; liquid junction potentials were corrected prior to seal formation. Currents were sampled at 30 kHz and filtered at 2.9 kHz ²⁵ . For each group, 15 DRG neurons were recorded randomly, and mean values were used to construct current-voltage (I-V) relationships and to analyze activation threshold (defined as the voltage at 10% of peak current), peak current density (pA/pF), and inactivation time constant (τ) ²⁵ .

Data were low-pass filtered at 1 kHz and analyzed using pCLAMP 12.0 (Molecular Devices). I-V relationships were normalized and fitted using a Boltzmann function: I/Imax=1/(1+e(V1/2−V)/k) where V1/2 is the half-activation voltage and k is the slope factor. Inactivation kinetics were fitted with a single-exponential function: I(t)=Ipeak.e−t/τinactivation+Isteady.

Statistical analysis

All data are expressed as mean ± standard deviation (SD). Normality was assessed using the Shapiro–Wilk test, and Levene’s test was used to evaluate homogeneity of variance. Two-group comparisons were performed using independent samples t-tests. For multiple group comparisons, one-way ANOVA was used, followed by Tukey’s post hoc test when significant differences were detected (P < 0.05). The Kruskal–Wallis test was applied for non-normally distributed data, followed by Dunn’s multiple comparison test. Correlation analysis was performed using the Pearson or Spearman methods, depending on the data distribution. Statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA) or SPSS 19.0 (IBM, USA), and a P-value < 0.05 was considered statistically significant.

Results of isolation and identification of primary rat BMSCs

Primary rat bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and cultured using the whole bone marrow adherence method. Under light microscopy, the cells exhibited a typical spindle-shaped or fibroblast-like morphology and proliferated gradually over time. A confluent monolayer was observed when the cell density reached approximately 80% (Fig. 1a). Third-passage BMSCs were used for surface marker analysis. Flow cytometry revealed that the positive expression rates of CD105, CD90, and CD44 were 98.01%, 99.93%, and 87.10%, respectively (Fig. 1b, c, and d). The hematopoietic and immune cell markers CD45 and CD11b showed low expression levels, with positive rates of 1.46% and 0.28%, respectively (Fig. 1e and f). To assess multipotent differentiation potential, adipogenic and osteogenic induction were performed. After 2–3 weeks of adipogenic induction, Oil Red O staining revealed intracellular lipid droplet formation (Fig. 1g). Following 3 weeks of osteogenic induction, Alizarin Red S staining demonstrated calcium nodule deposition (Fig. 1h).

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Figure 1.

Morphological observation, surface marker characterization, and multipotent differentiation potential of primary rat BMSCs. (a) Morphology of primary rat BMSCs observed under an inverted microscope. (b) Flow cytometry analysis of the surface marker CD105 in third-generation BMSCs using FITC-conjugated anti-CD105 antibody. (c) Flow cytometry analysis of the surface marker CD90 in third-generation BMSCs using PE-conjugated anti-CD90 antibody. (d) Flow cytometry analysis of the surface marker CD44 in third-generation BMSCs using FITC-conjugated anti-CD44 antibody. (e) Flow cytometry analysis of the surface marker CD45 in third-generation BMSCs using PE-conjugated anti-CD45 antibody. (f) Flow cytometry analysis of the surface marker CD11b in third-generation BMSCs using PE-conjugated anti-CD11b antibody. (g) Oil Red O staining for adipogenic differentiation of BMSCs, showing lipid droplet formation. (h) Alizarin Red staining for osteogenic differentiation of BMSCs, showing calcium nodule formation (n = 3).

Establishment of the CRPS-I rat model

MWL and TWL were measured to verify the successful induction of the CRPS-I model. One-way ANOVA showed significant differences among time points for both MWL (F(3,56) = 161.23, P < 0.0001) and TWL (F(3,56) = 109.78, P < 0.0001). Compared with baseline (MWL: 11.87 ± 1.31 s; TWL: 3.29 ± 0.36 s), the model group exhibited significantly reduced latencies on postoperative days 7, 10, and 14 (MWL: 2.26 ± 0.30 s, 2.73 v 0.35 s, 2.86 ± 0.36 s; TWL: 0.95 ± 0.34 s, 1.06 ± 0.32 s, 1.10 ± 0.31 s; all P < 0.0001 vs baseline and blank). Tukey’s post hoc analysis confirmed significant reductions at all time points (Fig. 2a, b). These results indicate the successful induction of mechanical and thermal hyperalgesia, validating the CRPS-I rat model.

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Figure 2.

Assessment of MWL and TWL in CRPS-I rat models. (a) MWL measurements of rats in different experimental groups at preoperative and on postoperative days 7, 10, and 14. Each group included n = 15. Data are presented as Mean ± SD. ****P < 0.0001 compared to the blank group (b) TWL measurements of rats in different experimental groups at preoperative and postoperative days 7, 10, and 14. Each group included n = 15. Data are presented as Mean ± SD. ****P < 0.0001 compared to the blank group.

Effect of BMSC transplantation on pain behavior in CRPS-I rats

BMSC-induced analgesia was assessed in CRPS-I rats through TWL, MWL, spontaneous pain scoring, and cold allodynia testing. One-way ANOVA showed a significant group effect for TWL on postoperative day 7 (F(3,68) = 5.29, P = 0.0024) while no significant differences were observed on days 10 and 14 (Fig. 3a). In contrast, MWL showed highly significant group differences at all time points (day 7: F(3,65) = 132.66; day 10: F(3,65) = 120.39; day 14: F(3,65) = 109.59; all P < 0.0001; Fig. 3a).

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Figure 3.

Effects of BMSC transplantation on TWL and MWL in CRPS-I rat models. (a) Comparison of TWL (in seconds) among different groups of rats at specified time points (Mean ± SD, n = 15). ****P < 0.0001 compared to the blank group; ####P < 0.0001 compared to the model group. (b) Comparison of MWL (in seconds) among different groups of rats at specified time points (Mean ± SD, n = 15). ****P < 0.0001 compared to the blank group; ###P < 0.001, ####P < 0.0001 compared to the model group. (c) Spontaneous pain behavior scores of rats on day 14. (Mean ± SD, n = 15). ****P < 0.0001 compared to the blank group; ##P < 0.0001 compared to the model group. (d) Cold allodynia scores measured by acetone test. ****P < 0.0001 compared to the blank group; ##P < 0.0001 compared to the model group.

Tukey’s post hoc test revealed that TWL and MWL in the model group were significantly reduced compared to baseline and the blank group, indicating the successful induction of thermal and mechanical hyperalgesia (TWL: 0.95 ± 0.10, 1.07 ± 0.13, 1.13 ± 0.09 s; MWL: 2.26 ± 0.12, 2.73 ± 0.19, 2.86 ± 0.12 s; all P < 0.05). In the BMSC-treated group, both TWL and MWL were significantly improved at each time point (TWL: 1.27 ± 0.17, 1.49 ± 0.19, 1.52 ± 0.21 s; MWL: 3.54 ± 0.34, 5.26 ± 0.55, 6.44 ± 0.55 s; all P < 0.05). No significant changes were observed in the control group, and its values were not significantly different from the model group (P > 0.05), confirming that the analgesic effects were specific to BMSC treatment.

During the spontaneous pain behavior assessment conducted during a quiet period, rats in the Model group exhibited pronounced pain-related behaviors such as paw lifting, licking, and reduced weight-bearing, with a mean score of 2.60 ± 0.34. This was significantly higher than that of the Blank group (0.23 ± 0.15, P < 0.0001). The Control group showed a similar score to the Model group (2.51 ± 0.30, P > 0.05), while the BMSC-treated group exhibited a significantly lower score (1.04 ± 0.32, P < 0.01), indicating that BMSC transplantation effectively alleviated spontaneous pain behavior (Fig. 3c).

In the cold allodynia test, rats in the Model group displayed intense responses such as strong paw withdrawal and persistent licking, with a mean acetone response score of 2.82 ± 0.28, which was significantly higher than that of the Blank group (0.41 ± 0.22, P < 0.0001). The Control group (2.75 ± 0.31) showed no significant difference from the Model group (P > 0.05). In contrast, the Treatment group showed a significantly reduced response score (1.18 ± 0.35, P < 0.01), suggesting that BMSC administration effectively attenuated cold hypersensitivity (Fig. 3d).

Regulatory effects of BMSC transplantation on Nav1.7, Nav1.8, and Nav1.9 sodium channel mRNA expression

RT-PCR analysis revealed that mRNA expression levels of Nav1.7, Nav1.8, and Nav1.9 in the DRG of CRPS-I model rats were significantly higher than those in the blank group (P < 0.05, Fig. 4). One-way ANOVA further confirmed significant differences among groups for each sodium channel: Nav1.7 (F(3,8) = 74.14, P < 0.0001), Nav1.8 (F(3,8) = 49.80, P < 0.0001), and Nav1.9 (F(3,8) = 32.67, P < 0.0001) supporting a pronounced upregulation of these channels in the CRPS-I pathological state. Following BMSC transplantation, the expression levels of Nav1.7, Nav1.8, and Nav1.9 were significantly reduced compared to the model group (P < 0.05), suggesting that BMSCs may exert analgesic effects by downregulating sodium channel expression and thereby reducing neuronal hyperexcitability. However, the mRNA levels did not fully return to baseline, indicating that further studies are needed to determine whether such molecular changes are sufficient to translate into behavioral analgesia. Additionally, there were no significant differences between the control and model groups (P > 0.05), indicating that the observed changes in gene expression were specific to the CRPS-I model and not attributable to procedural artifacts.

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Figure 4.

Effects of BMSC transplantation on Nav1.7, Nav1.8, and Nav1.9 mRNA levels in rat DRG tissue.

The regulatory effects of BMSC transplantation on the sodium channel proteins Nav1.7, Nav1.8, and Nav1.9

Western blot analysis showed that the protein expression levels of Nav1.7, Nav1.8, and Nav1.9 in the DRG of CRPS-I model rats were significantly higher than those in the blank group (Fig. 5a and b). One-way ANOVA confirmed significant differences among the groups for all three channels: Nav1.7 (F(3,8) = 63.57, P < 0.0001) Nav1.8 (F(3,8) = 47.59, P < 0.0001), and Nav1.9 (F(3,8) = 19.20, P = 0.001). These protein-level changes were consistent with the mRNA expression trends, suggesting that the upregulation of sodium channels may contribute to neuronal hyperexcitability and pain sensitization in CRPS-I. After BMSC transplantation, the expression levels of Nav1.7, Nav1.8, and Nav1.9 were significantly reduced compared to the model group and approached the levels observed in the blank group. It indicates that BMSCs may exert analgesic effects by modulating Nav channel expression and reducing sustained neuronal excitability. No significant differences were observed between the control and model groups (P > 0.05), further supporting that the changes in protein expression were attributable to CRPS-I pathology and BMSC treatment rather than procedural artifacts.

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Figure 5.

Effects of BMSC transplantation on Nav1.7, Nav1.8, and Nav1.9 protein levels in rat DRG tissue. (a) The expression levels of Nav1.7, Nav1.8, and Nav1.9 proteins in the blank group, model group, control group, and treatment group; (b) Statistical analysis of the relative expression levels of Nav1.7, Nav1.8, and Nav1.9 proteins in the blank group, model group, control group, and treatment group. (c) Current-voltage (I-V) relationships recorded from DRG neurons in the four groups.

Additionally, sodium I-V relationships were recorded from DRG neurons in all four groups. In the Model group, upregulation of Nav1.7, Nav1.8, and Nav1.9 was associated with increased current density, a leftward shift in the I-V curve, enhanced peak currents, and delayed inactivation, indicating heightened neuronal excitability. The Control group showed similar IV features to the Model group. In contrast, BMSC-treated rats exhibited reduced Nav channel expression, a rightward shift of the IV curve, decreased peak current density, and accelerated inactivation, approaching the profile of the Blank group, suggesting attenuation of DRG hyperexcitability (Fig. 5c).