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, 2018, 8179013
eCollection

Conditioned Medium of Bone Marrow-Derived Mesenchymal Stromal Cells as a Therapeutic Approach to Neuropathic Pain: A Preclinical Evaluation

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Conditioned Medium of Bone Marrow-Derived Mesenchymal Stromal Cells as a Therapeutic Approach to Neuropathic Pain: A Preclinical Evaluation

Kelly Barbosa Gama et al. Stem Cells Int.

Abstract

Neuropathic pain is a type of chronic pain caused by injury or dysfunction of the nervous system, without effective therapeutic approaches. Mesenchymal stromal cells (MSCs), through their paracrine action, have great potential in the treatment of this syndrome. In the present study, the therapeutic potential of MSC-derived conditioned medium (CM) was investigated in a mouse model of neuropathic pain induced by partial sciatic nerve ligation (PSL). PSL mice were treated by endovenous route with bone marrow-derived MSCs (1 × 106), CM, or vehicle. Gabapentin was the reference drug. Twelve hours after administration, neuropathic mice treated with CM exhibited an antinociceptive effect that was maintained throughout the evaluation period. MSCs also induced nonreversed antinociception, while gabapentin induced short-lasting antinociception. The levels of IL-1β, TNF-α, and IL-6 were reduced, while IL-10 was enhanced on sciatic nerve and spinal cord by treatment with CM and MSCs. Preliminary analysis of the CM secretome revealed the presence of growth factors and cytokines likely involved in the antinociception. In conclusion, the CM, similar to injection of live cells, produces a powerful and long-lasting antinociceptive effect on neuropathic pain, which is related with modulatory properties on peripheral and central levels of cytokines involved with the maintenance of this syndrome.

Figures

Figure 1
Figure 1
Effect of the conditioned medium from MSCs on PSL-induced neuropathic pain-like behaviors. The nociceptive thresholds were assessed in the ipsilateral paw of each mouse before (b) and after the PSL surgery, performed at time zero. (a) Thermal nociceptive threshold: the axis of ordinates represents the time (seconds) the animal takes to withdraw its paw. (b) Mechanical nociceptive thresholds: ordinates represent the filament weight (g) in which the animal responds in 50% of presentations. Sham group represents mice without neuropathy, in which the sciatic nerve was exposed but left intact. Seven days after PSL mice were treated (arrow) by endovenous route with bone marrow-derived mesenchymal cells (MSCs; 1 × 106/100 μl), conditioned medium from MSCs (CM; 100 μl) or vehicle (control medium; 100 μl). Gabapentin (70 mg/kg), the reference drug, was administered twice daily by oral route for six consecutive days (7 to 12 day). For gabapentin group, nociceptive threshold evaluations were made one hour before and one hour after the first treatment day. Data are expressed as means ± SEM; n = 6 mice per group. Significantly different from the vehicle-treated group (p < 0.05); #significantly different from the MSC and CM groups (p < 0.05). Two-way ANOVA followed by the Bonferroni's test.
Figure 2
Figure 2
Effects of the conditioned medium from MSCs on motor function and body weight of neuropathic mice. Bar graphs representing (a) the body weight variation of mice from different experimental groups at the end of the experimental period (60 days) and (b) the run time on the rotarod 24 h after treatments. Sham group represents mice without neuropathy, in which the sciatic nerve was exposed but left intact. Seven days after PSL mice were treated by endovenous route with bone marrow-derived mesenchymal cells (MSCs; 1 × 106/100 μl), conditioned medium from MSCs (CM; 100 μl) or vehicle (control medium; 100 μl). Gabapentin (70 mg/kg), the reference drug, was administered twice daily by oral route for six consecutive days. Mice treated with diazepam (10 mg/kg), the reference drug of the rotarod test, were tested 1 h after treatment. Data are reported as means ± SEM; n = 6 mice per group. Significantly different from the sham group (p < 0.01); #significantly different from the vehicle-treated group (p < 0.05); +significantly different from the remaining groups (p < 0.001). ANOVA followed by Tukey's multiple comparison test.
Figure 3
Figure 3
Modulatory effects of the conditioned medium from MSCs on cytokines sciatic nerve levels of neuropathic mice. The naïve group consists of mice that did not receive any experimental manipulation, while the sham group represents mice in which the sciatic nerve was exposed but left intact. Seven days after PSL, mice were treated by endovenous route with bone marrow-derived mesenchymal cells (MSCs; 1 × 106/100 μl), conditioned medium from MSCs (CM; 100 μl), or vehicle (control medium; 100 μl). Panels shows the sciatic nerve levels of (a) interleukin-1β (IL-1β), (b) tumor necrosis factor-α (TNF-α), (c) interleukin-6 (IL-6), and (d) interleukin-10 (IL-10), determined in ipsilateral sciatic nerve samples by ELISA at day 21 after the PSL surgery. The results are expressed as picograms of cytokine per milligram of protein. Data are expressed as means ± SEM; n = 6 mice per group. Significantly different from the vehicle group (p < 0.05). ANOVA followed by Tukey's multiple comparison test.
Figure 4
Figure 4
Modulatory effects of the conditioned medium from MSCs on cytokines spinal cord levels of neuropathic mice. The naïve group consists of mice that did not receive any experimental manipulation, while the sham group represents mice in which the sciatic nerve was exposed but left intact. Seven days after PSL mice were treated by endovenous route with bone marrow-derived mesenchymal cells (MSCs; 1 × 106/100 μl), conditioned medium from MSCs (CM; 100 μl), or vehicle (control medium; 100 μl). Panels shows the spinal cord levels of (a) interleukin-1β (IL-1β), (b) tumor necrosis factor-α (TNF-α), (c) interleukin-6 (IL-6), and (d) interleukin-10 (IL-10), determined in L4-L5 spinal segments by ELISA at day 21 after the PSL surgery. The results are expressed as picograms of cytokine per milligram of protein. Data are expressed as means ± SEM; n = 6 mice per group. Significantly different from the vehicle group (p < 0.05). ANOVA followed by Tukey's multiple comparison test.
Figure 5
Figure 5
Factors detected in conditioned medium from MSCs. Antibody arrays against 111 specified proteins were performed on 3 different samples of CM and revealed the presence of chemokines, cytokines, binding proteins, enzymes, and growth factors. Bar graph representing the densitometry of spotted antibody array results. Data are represented as the mean spot pixel density subtracted from the averaged background signal.

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