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-1
Neuropathic pain is a progressive nervous system disease initiated by a primary lesion or dysfunction in the nervous system, commonly caused by a trauma, infection, or ischemia. This chronic syndrome is characterized by abnormal sensory symptoms, such as spontaneous pain and an increased response to painful (hyperalgesia) and innocuous (allodynia) stimuli [
Thus, the successful control of neuropathic pain is linked to the establishment of new disease-modifying therapeutic approaches. In this context, cell-based therapies represent a promising alternative. Recently, cellular therapies have been considered as a potential successful approach in treating diseases and injuries of the nervous system, for which currently no effective treatment strategies are available [
Similar to other diseases of the nervous system, neuropathic pain appears to be amenable to stem cell therapy [
Mesenchymal stem cells were obtained from the bone marrow of femurs and tibias of mice. Bone marrow collected samples were diluted in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA), and the fraction with mononuclear cells was obtained by Ficoll-Hypaque gradient (Sigma, St. Louis, MO, USA), after centrifugation at 400 ×g for 30 minutes at 20°C. The interface containing mononuclear cells was collected in individualized tubes and washed twice in incomplete DMEM. Mononuclear cells were resuspended in DMEM medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50
Conditioned medium (CM) was obtained from MSC cultures (5 passage), as previously described [
Aiming to obtain a broad view of molecules present in CM, it was analyzed for a panel of specified proteins using an antibody array. The relative expression levels of 111 soluble mouse proteins was determined in CM using the Proteome Profiler Mouse XL Cytokine Array (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Spot pixel densities on developed X-ray film was collected and analyzed using a transmission-mode scanner and image analysis software. Data are represented as the mean spot pixel density subtracted from the averaged background signal. Antibody arrays were performed on 3 distinct CM samples.
Experiments were performed on male C57Bl/6 mice (20–25 g) obtained from the Animal Facilities of Instituto Gonçalo Moniz/FIOCRUZ (Brazil). Animals were housed in temperature-controlled rooms (22–25°C), under a 12 : 12 h light-dark cycle, with access to water and food ad libitum. All behavioral tests were performed between 8:00 a.m. and 5:00 p.m., and animals were only tested once. Animal care and handling procedures were in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH, 8023) and the Institutional Animal Care and Use Committee FIOCRUZ (CPqGM 025/2011). Every effort was made to minimize the number of animals used and to avoid any discomfort [
Under deep anesthesia with 2,2,2-tribromoethanol (Sigma Chemical Company, St. Louis, MO, USA) and aseptic conditions, the left sciatic nerve was exposed at high-thigh level and partially ligated as previously described [
Pain-like behaviors were assessed for examination of the neuropathic pain state in mice, before (baseline) and daily after PSL surgery. Behavioral tests were done without knowing to which experimental group each mouse belonged.
Withdrawal threshold to mechanical stimulation was measured with von Frey filaments (Stoelting; Chicago, IL, USA). In a quiet room, mice were placed in acrylic cages (12 × 10 × 17 cm) with wire grid floor which allowed full access to the ventral aspect of the hindpaws, 40 min before the beginning of the test. A logarithmic series of 9 filaments were applied to the plantar surface of the ipsilateral hindpaw to determine the threshold stiffness required for 50% paw withdrawal according to the nonparametric method of Dixon, as described by Chaplan and collaborators [
Withdrawal threshold to heat stimulation was determined using the Plantar Test (Hargreaves Apparatus, Ugo Basile Biological Instruments, Gemonio, Italy) as previously described [
To evaluate the motor performance, mice were submitted to the rotarod test, as previously described [
Mice were divided into the following groups (
Nociceptive tests (von Frey and plantar test) were performed at baseline and daily after the PSL surgical procedure. Seven days after PSL, and after the establishment of behavioral neuropathic pain as assessed by nociceptive tests, the animals received the treatments. Mice from MSCs group were transplanted by tail vein injection with 1 × 106 cells/mouse in a final volume of 100
Twenty-one days after PSL surgery, 6 mice from sham, vehicle, MSCs, and CM groups were sacrificed for biological sampling. Naïve group was included for biological sampling aiming to show the cytokine profile of mice without any manipulation. Motor performance and body weight was recorded once weekly for general toxicity assessment, starting from the baseline behavioral tests through the end of the experimental period.
For the measurement of cytokine levels, the spinal cord, and sciatic nerve were collected at day 21 after PSL, in mice terminally anesthetized with halothane from each experimental group. The L4-L5 spinal segments and 1 cm sciatic nerve sample containing the lesion site (or comparable region of sham-operated mouse) were removed and rapidly frozen and stored at −80°C. Frozen tissues were later homogenized in ice cold phosphate-buffered saline (PBS; 100 mg tissue/ml) to which 0.4 M NaCl, 0.05% Tween 20, and protease inhibitors (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 KI aprotinin A/100 ml) were added (Sigma). The samples were centrifuged for 10 minutes at 3000
All data are presented as means ± standard error of the mean (SEM) of measurements made on six animals in each group. Behavioral data were analyzed using two-way ANOVA (group and time) followed by Bonferroni’s multiple comparisons. Remaining data were analyzed using one-way ANOVA followed by Tukey’s posttest. All data were analyzed using the Prism 5 computer software (GraphPad, San Diego, CA, USA). Statistical differences were considered to be significant at
The therapeutic potential of the CM was evaluated in an established PSL-induced painful neuropathy model. Behavioral testing was performed at baseline and daily after the PSL surgical procedure, and the antinociceptive activity was expressed as reduction of pain-like behaviors. Gabapentin was used as the gold standard drug. PSL surgery induced sensorial neuropathy associated with thermal hyperalgesia and mechanical allodynia in mice without causing motor impairment (Figures
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
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
In order to monitor well-being, mice were daily observed and once weekly weighed throughout the experiment. All mice survived until the end of study. The assessment of body weight changes showed that vehicle-treated mice submitted to PSL exhibited less body weight gain through the experimental period relative to sham mice (Figure
The dysregulation of cytokines at sites of both the central and peripheral nervous systems is a key event in the development and maintenance of neuropathic pain. Considering that a single CM administration induced complete reversion of pain-like behaviors in neuropathic mice, a possible modulatory action of CM on cytokine production during neuropathy was next evaluated. The levels of cytokines were evaluated 21 days after sciatic nerve surgery, that is, 14 days after treatments. This time point was chosen based on behavioral results, indicating a slight maximum antinociceptive effect of CM at this time. Data obtained by ELISA analysis shows that vehicle-treated PSL mice exhibited upregulation of IL-1
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
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
A preliminary analysis of the CM secretome was performed using a high-density protein array. Antibody arrays was performed on 3 distinct CM samples. CM contained 21 of the 111 proteins assayed (Figure
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.
The present study demonstrated that a single CM treatment was able to reverse the behavioral neuropathic pain and to modify cytokine signaling associated with its maintenance. Importantly, the profile and magnitude of the CM-induced beneficial effects were similar to those induced by MSCs transplantation, highlighting the potential of this cell-free therapeutic approach for the treatment of neuropathic pain.
The MSCs transplantation induced a robust and long-lasting antinociceptive effect in neuropathic mice. In fact, the consistent antinociceptive properties of mesenchymal stem cells on neuropathic conditions have been demonstrated [
Since experimental studies have shown that the paracrine action of stem cells, rather than their transdifferentiation, accounts for the functional restoration of damaged tissues, the development of cell-free therapeutics based on secretome from stem cells has been explored. Over the last years, a growing number of studies have demonstrated that the conditioned medium from mesenchymal cell cultures contains a large variety of cytokines, enzymes, and growth factors, promoting beneficial effects in different experimental conditions. The therapeutic efficacy of conditioned medium of MSCs has also been successfully demonstrated in animal models of neurological disorders [
Interestingly, the antinociceptive effect induced by single CM injection was as long-lasting as that induced by MSCs. Brini and coworkers described a similar phenomenon in diabetic neuropathy [
While several mechanisms have been implicated in the development of neuropathic pain, it is now clear that the neuroimmune response triggered by the neuronal damage is a key event of this painful syndrome [
Upregulation of IL-1
Aiming to understand the molecular mediators of the CM-induced antinociceptive effect, a preliminary analysis of the CM secretome was performed. Interestingly, although the antibody array revealed the presence of several cytokines in CM, IL10 was not detected. This result indicates that CM is not the primary source of IL-10 but stimulates the local release of this cytokine in the nervous system during neuropathy. Among the 21 proteins presented on CM, some of them, such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), chemerin, and angiopoietin-1, are potential mediators of the antinociceptive effects exerted by the CM on neuropathic conditions. Besides its prominent role in angiogenesis, VEGF also exerts important neuroprotective effect during neuropathic conditions [
Moreover, chemerin and angiopoetin-1 should also be highlighted, considering their beneficial effects on experimental neuropathic pain. The antinociceptive effect of chemerin was previously demonstrated in a mouse model of neuropathic pain [
HGF is also a good candidate to be one of the factors facilitating CM-induced therapeutic effects on neuropathy because of its powerful angiogenetic and neurotrophic actions [
The strategy that supports the proposal of replacement of the current pharmacological treatments by cellular therapies in neuropathic pain is a possible modulatory action on the pathophysiology of neuropathy. This property may confer to the cell therapy longer lasting, or even curative, effects in detriment to the palliative effects of the available analgesics. In the present study, the CM treatment induced a long-lasting antinociceptive effect with a disease-modifying profile similar to that showed by MSCs. This comparative study opens a new perspective for the treatment of neuropathic pain since it demonstrates that it is possible to develop cell-free treatments that are able to retain the benefits of cell therapy for neuropathic pain without exhibiting the inherent difficulties of cell-based therapy.
The authors declare that there is no conflict of interest regarding the publication of this article.
This work was supported by FAPESB (Grant no. DTE 0046/2011) and CNPq (Grant no. 445547/2014-6).