Neurochem Res DOI 10.1007/s11064-016-2051-5

ORIGINAL PApER

Intrathecal Injection of Human Umbilical Cord-Derived Mesenchymal Stem Cells Ameliorates Neuropathic Pain in Rats Chunxiu Chen1 · Fengfeng Chen2 · Chengye Yao1 · Shaofang Shu1 · Juan Feng1,3 · Xiaoling Hu3 · Quan Hai4 · Shanglong Yao1 · Xiangdong Chen1

Received: 19 April 2016 / Revised: 23 August 2016 / Accepted: 27 August 2016 © Springer Science+Business Media New York 2016

Abstract  Neuropathic pain (NP) is a clinically incurable disease with miscellaneous causes, complicated mechanisms and available therapies show poor curative effect. Some recent studies have indicated that neuroinflammation plays a vital role in the occurrence and promotion of NP and anti-inflammatory therapy has the potential to relieve the pain. During the past decades, mesenchymal stem cells (MSCs) with properties of multipotentiality, low immunogenicity and anti-inflammatory activity have showed excellent therapeutic effects in cell therapy from animal models to clinical application, thus aroused great attention. However there are no reports about the effect of intrathecal human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) on NP which is induced by peripheral nerve injury. Therefore, in this study, intrathecally transplanted HUC-MSCs were utilized to examine the effect on neuropathic pain induced by a rat model with spinal nerve ligation (SNL), so as to explore the possible mechanism of those effects. As shown in the results, the HUC-MSCs transplantation obviously ameliorated SNL-induced mechanical allodynia and thermal

Xiangdong Chen [email protected] 1

Department of Anesthesiology, Institute of Anesthesiology and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

2

Huaxi MR Research Center, Department of Radiology, West China Hospital, Sichuan University, Chengdu, China

3

Department of Anesthesia, The First Affiliated Hospital of University of South China, Henyang, China

4

Sichuan Province Regenerative Medicine Engineering Technology Research Center, Chengdu, China





hyperalgesia, which was related to the inhibiting process of neuroinflammation, including the suppression of activated astrocytes and microglia, as well as the significant reduction of pro-inflammatory cytokines Interleukin-1β (IL-1β) and Interleukin −17A (IL-17A) and the up-regulation of anti-inflammatory cytokine Interleukin −10 (IL-10). Therefore, through the effect on glial cells, proinflammatory and anti-inflammatory cytokine, the targeting intrathecal HUC-MSCs may offer a novel treatment strategy for NP. Keywords  Neuropathic pain · Spinal nerve ligation · Mesenchymal stem cells · Astrocyte · Microglia

Introduction As a result of a noxious stimulus, neuropathic pain (NP) is an extremely complicated disease affecting the somatosensory system [1]. Moreover, its medical therapy can only relieve the pain partly while accumulating serious side effects in the long-term use, which can’t be tolerated by most patients [2, 3]. It is very significant to search for novel therapeutic approaches for NP so as to overcome the most limitations in the traditional treatment. A mass of researches have confirmed that NP is advanced by the interaction of cells in the neural network after noxious stimuli, and the over-activation of glial cells plays a critical role in the initiation and maintenance of pain hypersensitivity [4, 5]. In the rats’ model of neuropathic pain, it is observed that astrocytes and microglia in the ipsilateral dorsal horn of the spinal cord are obviously activated after nerve injury, accompanied by a wide cascaded release of pro-inflammatory cytokines and chemokines [6–8]. Therefore, blocking the activation of neuro-inflammatory

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cascades is considered as one of the most important therapeutic strategies for NP. In recent years, studies have revealed that mesenchymal stem cells (MSCs) of different origins play critical roles in the treatment of multitudinous diseases [9], including the relief of chronic pain [10–12]. As the undifferentiated cells with strong immunosuppressive properties, MSCs can be transplanted to different individuals without accepting pharmacological immunosuppression and this unique characteristic render them more suitable and useful for transplantations than other cells [13, 14]. During the past decades, researchers have focused on their multipotent differentiation capacity, but now more and more researchers show a growing interest in their strong antiinflammatory and immunomodulatory ability [5, 15, 16]. A variety of animal models with inflammatory diseases and some clinical trials have demonstrated that MSCs can regulate inflammation subside [9, 15]. Studies proved that MSCs which were derived from bone marrow could alleviate the pain behavior induced by sciatic nerve injury, although its explicit mechanism of action still remains ambiguous [17, 18]. Recently, human umbilical cord has been identified as a promising source of MSCs thus arousing researchers’ attention. Though human umbilical cord (HUC-MSCs) have similar biological characteristics to MSCs derived from bone marrow, they are unrestricted by ethics and lower in immunogenicity, which make them more suitable for laboratory investigations and clinical trials [19, 20]. Moreover, studies showed whether MSCs derived from HUC-MSCs contributed to the relief of neuropathic pain induced by spinal nerve injury have not been presented yet. Therefore, this research was designed to investigate if intrathecally transplanted HUC-MSCs could ameliorate neuropathic pain induced by peripheral nerve injury, and the effect of HUC-MSCs on the inflammation subside, especially on the effects of astrocytes and microglia, proinflammatory and anti-inflammatory cytokine.

Materials and Methods Experimental Animals Male Sprague–Dawley rats (250 ± 20 g) were bought from the Animal Experimental Center of Wuhan University, Wuhan, China. The rats were fed at the animal center of Tongji Medical College under the standard conditions with adequate food and water. All experiments of this study were approved by Animal Care Committee of Tongji Medical College, Wuhan, China and were performed according to the guide for the care and use of laboratory animals from the National Institute of Health.

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Animal Model of Neuropathic Pain Induced by Spinal Nerve Ligation (SNL) Neuropathic pain was induced as described previously [21]. Briefly, the rats were anesthetized with 2 % sodium pentobarbital intraperitoneally (50  mg/kg body weight) and a midline incision was made along the skin of L4–L6. After separating the left paraspinal muscles and removing the L6 transverse process, the left L5 spinal nerve was exposed. Subsequently, the L5 spinal nerve was separated carefully and ligated tightly with a 4-0 silk thread. The rats of Sham group received the same operation except for nerve ligation. Intrathecal Catheter Implantation After the nerve ligation was completed, the L5–L6 intervertebral space was exposed and punctured. Subsequently, a PE-10 catheter (Smith medical, UK) was inserted rostrally into the lumbar subarachnoid space until it reached the lumbar enlargement segments of spinal cord (about 3.5 cm) as previously mentioned [22]. Subsequently, the catheter was fastened to para-spinal muscles and the other side, which passed through subcutaneous tissues, and further fastened to the hands of the rats. After the operation, the wounds were washed with saline and then sutured with 4-0 silk thread. To verify whether the catheter was inserted successfully, 2 % lidocaine (20 μl) was given intrathecally. Treatment and groups The day of surgery was recorded as day 0 and the behavioral tests were performed on day 1, 2, 3, 5, 7, 9, 11 and 14 after surgery. The rats were randomly divided into four groups: (1) Sham group; (2) SNL group; (3) SNL + PBS group; (4) SNL + HUC-MSC group. Sham animals: only to find out the L5 nerve without making ligation; SNL animals: rats received the surgery of L5 nerve ligation. Then 20 μl PBS or HUC-MSCs (total 1 × 106 cells) of intrathecal injection was implemented on day 3 after SNL in SNL + PBS group and SNL + HUC-MSC group. The Culture of Human Umbilical Cord-Derived Mesenchymal Stem Cells (HUC-MSCs) The collection of umbilical cord was permitted by the Clinical Research Committee of the Wuhan Union Hospital and performed according to the amended Declaration of Helsinki. The HUC-MSCs were cultured as described previously [23]. Briefly, human umbilical cords collected from healthy mothers were cleaned in Hanks’ balanced salt solution (HBSS, Gibco, USA), sterilized by 75 % ethanol for 30 s, and then stripped off their vessels carefully in HBSS.

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Afterwards, the Wharton’s jelly containing mesenchymal stem cells was retained and cut into cubes of approximately 0.2–0.5 cm3. After washing twice with serum free Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA), the tissue blocks were seeded in a 175 cm2 cell culture flask with DMEM containing 10 % Fetal bovine serum (FBS, Gibco, USA) at 37 °C in an incubator with 5 % CO2. When 80 % of the cells were covered in the cell culture flask, 0.25 % trypsin was then applied for the passages. The third generation of mesenchymal stem cells was identified by flow cytometry and adopted for treating the rats. Flow Cytometry MSCs were detected as immune-phenotypes through flow cytometry analysis. In brief cells were digested with 0.25 % trypsin washed with PBS for three times (pH = 7.4) and incubated for 30 min with the antibodies of CD90-PC5, CD105-PE, CD73-PE, CD45-PC7, CD11b-FITC, CD34PE, CD19-ECD and HLA-DR-PC7 (all antibodies from Beckman, USA) under ordinary temperature and in darkness. Then the cells were washed with PBS for another time and resuspended with PBS. Consequently the specific fluorescence of 20,000 cells was analyzed on FC500 (Beckman, USA) with CXP software. Behavioral Tests Paw withdrawal mechanical threshold (PWMT) was considered as mechanical allodynia and while paw withdrawal thermal latency (PWTL) was considered as thermal hyperalgesia, and these behavioral tests were performed in the laboratory accordingly on day 0 (before surgery) and day 1, 2, 3, 5, 7, 9, 11 and 14 after SNL as mentioned above [24]. Before being tested, rats were habituated to the plastic cages with metal mesh floor (for Paw withdrawal mechanical threshold test) or glass floor (for Paw withdrawal thermal latency test) for at least 1 h until they became quiet (neither walking around nor or licking their paws). The behavioral tests were induced by stimulating the mid-plantar surface of the ipsilateral hind paws and performed by the same observer. PMWT was assessed by an electronic Von Frey device (IITC Life Science Inc, USA), to stimulate the midplantar surface of the hind paws and corresponding readout was recorded until a withdrawal reflex was observed. The measurement was repeated for three times on the same paw with a 5 min interval and the average was considered as PWMT. PWTL was determined by employing Hargreaves Test (Type 7370, Planter Test Instrument, UgoBasile, Italy). A plantar test apparatus was applied to produce a beam of radiant heat to stimulate the mid-plantar surface of the hind paws and record the readout until the rats lifted the paws away. 20 s was set as the cut-off time to prevent damage on

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their paws. The measurement was repeated for three times on the same paw with a 5 min interval and the average was considered as PWTL. Immunofluorescence Staining and Quantification Rats were circulatively perfused with 0.9 % normal saline of 150 ml and subsequently with 4 % paraformaldehyde (dissolved in PBS) of 250 ml after anesthesia. The L4–L5 spinal cords were collected and steeped in 4 % paraformaldehyde at 4 °C overnight and then paraffin-embedded and serial sectioned (4 μm thick). Subsequently, the spinal cord sections were deparaffinized in xylene and rehydrated in a gradient concentration of ethanol. After antigen retrieval and blocking the endogenous peroxidase activity, sections were incubated with a mouse monoclonal antibody anti-human nuclei (1:100, Millipore, USA); a rabbit monoclonal antibody antiglial fibrillary acidic protein (GFAP, 1:200, Proteintech, China) and a rabbit monoclonal antibody anti-ionized calcium-binding adapter molecule 1 (IBA-1, 1:50, Proteintech, China) overnight at 4 °C. Then incubated with the FITCconjugated goat anti-rabbit IgG (1:300; Boster, China) for 1 h at room temperature, followed by DAPI (Sigma, USA) for 10 min. Fluorescent imaging was observed through an Olympus microscope (BX51, Japan). The fluorescent imaging of GFAP and IBA-1was analyzed with the Image J Software (version 1.45, NIH, USA). Western Blot Analysis A protein extraction reagent kit (Keygen Biotech, China) was utilized to extract the total protein in the ipsilateral L4– L5 spinal cords according to manufacturer’s instructions. BCA protein assay kit (Keygen Biotech, China) was used to detect protein concentrations. Equal proteins were separated by electrophoresis within SDS-PAGE and transferred onto the polyvinyllidenedifluoride (PVDF) membranes (Millipore,USA).The membranes were blocked with 5 % non-fat milk for 60 min at room temperature and then incubated with the following primary antibodies of anti-GFAP (1:500), anti-IBA-1 (1:500), mouse monoclonal anti-β-actin antibody (1:5000, Abcam, UK) overnight at 4 °C. Then the membranes were incubated with goat-anti-mouse or goat-anti-rabbit antibody (1:4000, Antgene Biotechnology) for 60 min at room temperature. The labeled protein was detected with the chemiluminescence system (UVP LabWorks, Upland, CA). Signal intensity was then analyzed with the Image J Software. Enzyme-Linked Immunosorbent Assay (ELISA) The protein of IL-1β, IL-17A, and IL-10 of the L4–L5 spinal cords and serum were measured by respective ELISA

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systems (Boster, China) according to the manufacturer’s instructions. Absorbance was detected at 450 nm with the Sunrise Enzyme Standard Instrument (TECAN, Austria). Statistical Analyses All data were presented as mean ± SD. GraphPad Prism 5.0 (San Diego, CA, USA) software was used for all analysis. The differences of the behavioral test,the results of ELISA, and the immunoreactivity were analyzed by one-way or two-way analysis of variance (ANOVA), followed by Tukey post hoc test. A value of P < 0.05 was considered statistically significant.

Results The Identification of HUC-MSCs HUC-MSCs were analyzed for expressing the cell protein markers of CD105 (Fig. 1a), CD90 (Fig.  1b), and CD73 (Fig. 1c) which were widely regarded as markers of mesenchymal stem cell, as well as CD45 (Fig. 1d), CD34 (Fig. 1e), CD11b (Fig.  1f), CD19 (Fig.  1g) and HLA-DR (Fig. 1h) which were utilized to exclude the cells of others by flow cytometry. The high expression of protein markers CD105 (95.62  ± 1.62 %), CD90 (95.58  ± 1.78 %) and CD73 (94.95 ± 2.14 %) and the lack expression of CD34 (0.49  ± 0.19 %), CD45 (1.87 ± 0.68 %), CD19 (0.33 ± 0.11 %), CD11b (0.10 ± 0.03 %) and HLA-DR (0.18 ± 0.07 %) in the total isolated cells proved that the cells were HUC-MSCs and the culture was homogeneous. Intrathecally HUC-MSCs Ameliorated the Mechanical Allodynia and Thermal Hyperalgesia Induced by SNL The mechanical allodynia and thermal hyperalgesia were identified by the decrease of the PWMT (Fig. 2a) and PWTL (Fig. 2b). In our results,As the results revealed, there was a sharp decline at the first 3 days of ipsilateral PWMT from 29.64 ± 1.63 g to 12.27 ± 1.78 g (P < 0.05) and PWTL from 16.91 ± 1.93 s to 7.05 ± 1.09 s (P < 0.05), and then persistently maintained till day 14 after SNL. Vehicle PBS alone did not produce any beneficial effects on neuropathic pain induced by SNL (Fig. 2a, b). In order to evaluate the therapeutic effect of HUC-MSCs on neuropathic pain, rats were treated with HUC-MSCs on day 3 after SNL, when mechanical allodynia and thermal hyperalgesia appeared most obvious. After intrathecal injection of 1 × 106 HUC-MSCs into the SNL-rats, a significant amelioration of mechanical hyperalgesia was presented from day 7 (12.30 ± 1.37  g vs. 17.14 ± 1.04  g, P < 0.05) to day 14 (12.94 ± 1.61 g vs.

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21.73 ± 2.37  g, P < 0.05), and thermal allodynia was alleviated from day 5 (7.24 ± 1.26 s vs. 8.97 ± 1.64  s, P < 0.05) to day 14 (7.29 ± 1.04 s vs. 12.55 ± 1.36  s, P < 0.05). The results indicated that the HUC-MSCs have had therapeutic effect on neuropathic pain. Intrathecal HUC-MSCs Transplantation Migrated to the Spinal Cord in SNL-Rats Since the experiment was xenotransplantation, specific antibody of anti-human nuclei (Fig. 3) was used to trace the survival and distribution of HUC-MSCs. The immunofluorescence staining detected the cells which expressed antihuman nuclei monoclonal antibody, indicating the survival of HUC-MSCs. Most of them were distributed in the left side of the spinal dorsal horn in rats of SNL + HUC-MSC group on day 7 after SNL (Fig. 3), which demonstrated that HUC-MSCs could migrate to and survive in the spinal cord after SNL. Administration of HUC-MSCs Attenuated the Production of IL-1β and IL-17A and Upregulated the Level of IL-10 in Ipsilateral Spinal Cord but not in Serum After SNL As inflammatory factors were very important in the progress of NP, the levels of IL-1β (Fig. 4a, d), IL-17A (Fig. 4b, e) and IL-10 (Fig.  4c, f) were examined by ELISA in the ipsilateral spinal cord and serum on day 14 after SNL. Compared with the Sham group, SNL induced an obvious upregulation of IL-1β (P < 0.01), and IL-17A (P < 0.01) in ipsilateral spinal cord but not in serum (P > 0.05), while the level of IL-10 had not changed in ipsilateral spinal cord or serum (P > 0.05), suggesting that SNL induced local inflammation but not systemic inflammation. After the treatment of HUC-MSCs, compared with the SNL group, HUC-MSCs significantly attenuated the production of IL-1β, IL-17A and upregulated the levels of IL-10 on day 14 after SNL (P < 0.01) but had not influence on the level of IL-1β, IL17A and IL-10 in serum (P > 0.05). The empirical result confirmed that the inflammation induced by SNL was mainly involved in spinal cord and HUC-MSCs significantly attenuated the expression of pro-inflammatory factor IL-1β and IL-17A while increasing the expression of antiinflammatory factor IL-10. Intrathecal Injection of HUC-MSCs Significantly Suppressed Astrocytes and Microglia Which were Activated by SNL The activation of glial cells was analyzed by western blotting and immunofluorescence staining. It was observed that GFAP (marker of activated astrocyte) detected by

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Fig. 1 Flow cytometry detected the immunophenotype of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs). The high expression of protein markers CD105 (a), CD90 (b) and CD73

(c) and lack expression of CD34 (d), CD45 (e), CD19 (f), CD11b (g) and HLA-DR (h) in the total isolated cells proved that the cells were HUC-MSCs and the culture was homogeneous

immunofluorescence staining was highly expressed after SNL and was decreased after the treatment of HUC-MSCs (Fig. 5a–i, c′, f′, i′, j). GFAP protein was upregulated from 0.57 ± 0.05 in sham group to 1.59 ± 0.39 (P < 0.05) in SNLtreated rats, and HUC-MSCs decreased the expression of GFAP after SNL from 1.59 ± 0.39 to 0.94 ± 0.22 (P < 0.01) (Fig. 5k, l). It was also shown that IBA-1 (marker of activated microglia) examined by immunofluorescence staining was obviously increased after SNL but reduced sharply

after the treatment of HUC-MSCs (Fig. 6a–i, c′, f′, i′, j). IBA-1 protein examined by western blotting was increased from 0.21 ± 0.06 in sham group to 0.97 ± 0.31 (P < 0.05) in SNL-treated rats, while HUC-MSCs reduced the level of IBA-1 after SNL from 0.97 ± 0.31 to 0.51 ± 0.17 (P < 0.01) (Fig. 6d, k). The result verified that intrathecal injection of HUC-MSCs could significantly suppress the activation of astrocytes and microglia which were obviously activated after SNL.

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Fig. 2  HUC-MSCs attenatued the mechanical allodynia and thermal hyperalgesia induced by spinal nerve ligation (SNL) in ipsilateral spinal cord. Paw withdrawal mechanical threshold (PWMT, a) and paw withdrawal thermal latency (PWTL, b) were reduced after SNL in rats,

and intrathecal injection of HUC-MSCs recovered the mechanical allodynia and thermal hyperalgesia induced by SNL. *P < 0.05 vs. the Sham group, #P < 0. 05 vs. the SNL + PBS group. Data are expressed as mean ± SD (n = 10 per group)

Fig. 3  HUC-MSCs migrated to and survived in the spinal cord on day 7 after SNL. Representative immunohistological staining of the human nuclei (a, d) and the nucleus marker DAPI (b, e) and their merged (c,

f). d, e, f (scale bar 50 μm) were the enlargement of spinal dorsal horn from a, b, c (scale bar 200 μm)

Discussion

It is found that numerous factors affect the efficacy of MSC transplantation on neuropathic pain, such as different animal models of NP (central or peripheral), the route and time point of transplantation, the source and the number of transplanted MSCs [28, 29]. This is the reason why MSCs transplantation can relieve the neuropathic pain symptoms in some studies [25–27, 30, 31], but not in others [29, 32]. Though the MSCs from bone marrow were the earliest and the most commonly cells used for cell therapy, however, Schäfer et al. found that pain hypersensitivity was not affected by bone marrow-derived mesenchymal stem cell transplantation and the cytokine levels in the spinal cord were unchanged in a female rat’s model of partial sciatic

More and more evidence have demonstrated that MSCs transplantation is considered to own the better potential in the administration of chronic pain than other cells.It is also reported that some origins of human MSCs have been applied in several clinical trials, despite their relevant mechanisms for treatment remaining unclear [25–27]. In our present study, we demonstrated that the single intrathecally transplanted human umbilical cord-derived MSCs (HUC-MSCs) could exert significant effect in the therapy of neuropathic pain (NP) induced by peripheral nerve injury.

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Fig. 4  Expression of Interleukin-1β (IL-1β), Interleukin −17A (IL-17A) and Interleukin-10 (IL-10) was measured by ELISA with or without intrathecal injection of HUC-MSCs in the spinal cord and serum on day 14 after SNL. SNL induced an increase of IL-1β (a) and IL-17A (b), without changing IL-10 (c), while HUC-MSCs recovered the change of

IL-1β and IL-17A, and also increased IL-10 in the spinal cord. There is no difference in the level of IL-1β (d), IL-17A (e) and IL-10 (f) in serum. *P < 0.05 and **P < 0.01 vs. the sham group, #P < 0.05 and ##P < 0. 01 vs. the SNL + PBS group, there weren’t statistically significant of each group in serum, P > 0.05. Data are expressed as mean ± SD (n = 6 per group)

nerve ligation [32]. Although beneficial effects of intrathecal BM-MSC on pain hypersensitivity were not discovered by Schäfer et al. [32], our study revealed that intrathecal HUC-MSCs did alleviate neuropathic pain, except for the reason of different animal models used by the two studies, another important reason could be because that mesenchymal cells derived from umbilical cord (UC-MSCs) had stronger proliferation ability and anti-inflammatory effects than MSCs derived from bone marrow (BM-MSCs) [33]. Yousefifard et al. recently found that stem cells derived from both bone marrow (BM-MSC) and umbilical cord (UCMSC) were directly transplanted into the dorsal horn of the injured spinal cord alleviated the symptoms of neuropathic pain induced by spinal cord injuries (SCIs) [34]. Although Yousefifard et al. demonstrated that the direct transplantation of both BM-MSC and UC-MSC into the injured spinal cord could alleviate neuropathic pain, the direct application of stem cells required surgery, which would not be the best choice for clinical treatment. Yousefifard et al. also showed that the survival rate of UC-MSCs was significantly higher than that of BM-MSCs in SCI-rats [34]. These results suggest that cells derived from umbilical cord have advantages including anti-inflammatory effects over those derived from bone marrow and thus they can be used as an efficient source of cells in the clinical treatment. Indeed, the MSCs-mediated secretion of a series of bioactive cytokines plays a more significant biological role

than their ability of differentiation [15, 16]. With the main advantage of strong anti-inflammatory and immunomodulatory ability, MSCs play the role of regulating the process of inflammatory diseases [35–37]. It is reported that MSCs transplantation exerts their biological effects under injury conditions in the model of sciatic nerve injury [8, 16, 38, 39]. Recently, the increasing evidence have revealed that the inflammatory cytokines and chemokines are critical in the regulation of neuro-inflammation following different injuries in the nervous system [40, 41]. Pro-inflammatory cytokines such as IL-1β and IL-17A in the spinal cord after nerve injury are involved in the initiation and maintenance of central sensitization as well as chronic neuropathic pain [42]. Moreover, anti-inflammatory cytokine IL-10 can block the process of neuro-inflammation via inhibiting the synthesis of pro-inflammatory cytokines which are induced by the activated macrophages [43]. In order to explore the effect of HUC-MSCs on inflammatory cytokines, we tested the expression of IL-1β, IL-17A and IL-10 on day 14 after SNL (11 days after HUC-MSCs transplantation). Consequently, it is confirmed that intrathecal HUC-MSCs are able to down-regulate the expression of SNL-induced IL-1β and IL-17A in the ipsilateral spinal cord of NP rats on day 14 after SNL, while upregulating IL-10 (Fig. 4). More and more evidences prove that astrocytes and microglia are crucial in the exacerbation of chronic neuropathic pain and pain hypersensitivity [44–46], and the anti-inflammatory effect

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Fig. 5  Effect of HUC-MSCs on the expression of glial fibrillary acidic protein (GFAP) in the spinal cord on day 14 after SNL. Representative immunohistological staining of the astrocytic marker GFAP (a, d, g) and the nucleus marker DAPI (b, e, h) on the ipsilateral side of the lumbar spinal cord (scale bar 100  μm) on day 14 after SNL with or without intrathecal injection of HUC-MSCs were shown. c′, f′, i′ (Scale bar 20 μm) were the enlargement of the box from c, f, i.

Quantification of integrated density of GFAP in the dorsal horn was shown in j. Representative immunoblots of GFAP was shown in k, the level of GFAP related to β-actin calculated by densitometric analysis was shown in l. *P < 0.05 and **P < 0.01 vs. the sham group, #P < 0.05 and ##P < 0.01 vs. the SNL + PBS group. Values are expressed as mean ± SD (n = 6 per group)

of HUC-MSCs can be mediated through suppressing the activation of glial cells including astrocytes and microglia. The activation of astrocytes and microglia within the ipsilateral dorsal horn of the spinal cord is concurrent with the release of a mass of inflammatory mediators, mostly including pro-inflammatory cytokines after nerve injury [47, 48]. The present results indicate that astrocytes and microglial cells may contribute to the process of NP and the therapeutic effect of HUC-MSCs is associated with the suppression on the activated astrocytes and microglial. In this experiment, a rat model of L5 spinal nerve ligation (SNL) was used to induce neuropathic pain for simulating

the pain symptoms of patients. Rats were treated with 1 × 106 HUC-MSCs on day 3 after SNL, when mechanical allodynia and thermal hyperalgesia appeared most obviously. Specifically, this time point was selected for the delivery of HUC-MSCs also attributed to the previous study of our laboratory which found the expression of IL-17A was the most obvious on day 3 after SNL [24]. It was also found that the higher cell numbers ranging from 104 to 106 are associated with the better benefit, but it had been described that the high amounts of cells could cause cell damage and decreased viability due to cluster formation in the microinjection cannula [49, 50].

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Fig. 6  Effect of HUC-MSCs on the expression of ionized calciumbinding adapter molecule 1 (IBA-1) in the ipsilateral spinal cord on day 14 after SNL. Representative immunohistological staining of the microglial marker IBA-1 (a, d, g) and the nucleus marker DAPI (b, e, h) on day 14 after SNL on the ipsilateral side of the lumbar spinal cord (scale bar 100 μm) with or without intrathecal injection of HUCMSCs were shown. c′, f′, i′(scale bar 20 μm) were the enlargement of

the box from c, f, i. Quantification of integrated density of IBA-1 in the dorsal horn was shown in b. Representative western blot analysis of IBA-1 was shown in k, the level of IBA-1 related to β-actin calculated by densitometric analysis was shown in l. *P < 0.05, **P < 0.01 vs. the sham group, #P < 0.05 and ##P < 0.01 vs. the SNL + PBS group. Values are expressed as mean ± SD (n = 6 per group)

There are still some deficiencies which need to be improved in the present study. First, the usage of fetal calf serum that contains many growth factors is not presented in the adult human, but it can affect the results and is difficult to translate for clinical application on patients. As a consequence of this the cell cultures have to be repeated with human serum before valid data regarding human cells and their behavior can be obtained. Second, in this study, we only focus on the effect of HUC-MSCs on neuro-inflammation, while the effects on neurons and other biomolecules need a long-term research.

In conclusion, our study demonstrates for the first time that intrathecal human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) are able to alleviate mechanical allodynia and thermal hyperalgesia which appear after spinal nerve ligation (SNL). This may be attributed to a decrease of the expression of pro-inflammatory interleukins IL-1β and IL-17A and an increase of the level of anti-inflammatory interleukin IL-10, together with the inhibition of the activated astrocytes and microglia. Therefore HUC-MSCs can be an important treatment strategy for neuropathic pain.

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10 Acknowledgments  Support of this project was provided by Grant 81171274 and 81571075 (to Dr. Xiangdong Chen) from the National Natural Science Foundation of China (Beijing, China). Compliance with Ethical Standards Conflict of Interest  We declare that there have been no conflicts of interests.

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