Stem Cells Quell Neuropathic Pain in Mice
Preclinical evidence that bone marrow stromal cells (BMSCs) are a promising treatment for neuropathic and inflammatory pain continues to accumulate. A new study led by Ru-Rong Ji and Gang Chen, Duke University, Durham, North Carolina, US, reports that a single intrathecal injection of BMSCs can quickly relieve mechanical hypersensitivity and heat hyperalgesia for several weeks in mice, and without adverse consequences. The researchers further demonstrate that this effect is mediated by the release of transforming growth factor (TGF)-β1 from BMSCs in response to secretion of the chemokine CXCL12 by dorsal root ganglion (DRG) neurons following nerve injury.
“It’s very nice to see that BMSCs can reduce pain without side effects,” said Ke Ren, University of Maryland, Baltimore, US, who was not involved in the new work. “These cells can really produce very long-lasting relief,” he added.
The new findings were published August 3 in the Journal of Clinical Investigation.
Pain relief from BMSCs
BMSCs, connective tissue cells derived from bone marrow, are a kind of mesenchymal stem cell that can give rise to multiple cell types. They have been studied for more than a decade to treat conditions like brain/spinal cord injury and graft-versus-host disease, but have only recently been investigated as a treatment for pain, said Ren. A number of preclinical studies, including one from Ren’s group, have demonstrated that BMSCs can produce long-term pain relief (for example, see Guo et al., 2011).
“The treatment of pain with BMSCs is very feasible,” said Ren, citing a recent clinical trial demonstrating reduced pain and disability for two years following a single injection of BMSCs into the symptomatic discs of patients with disc-related back pain (Pettine et al., 2015).
However, a number of important factors concerning the BMSC approach for pain relief had remained unknown, said Ji. For example, the molecular mechanism by which BMSCs produce analgesia is unknown, as are the most effective injection route and final destination of the injected stem cells, he explained.
Ru-Rong Ji and Gang Chen
In the new study, first author Chen and colleagues set out to address these questions, using chronic constriction injury (CCI) to induce neuropathic pain lasting 5 to 8 weeks. As expected, CCI mice displayed mechanical allodynia and heat hyperalgesia. The investigators then administered a single intrathecal injection of BMSCs four days following CCI, which is after the onset but prior to the peak of neuropathic pain.
Within one day, both low (100,000 cells) and high (250,000 cells) doses of BMSCs significantly decreased the CCI-induced mechanical allodynia and thermal hyperalgesia, an effect that lasted for more than five weeks. Similar analgesic effects were also observed when BMSCs were administered 14 days post-CCI; here the effect of low-dose BMSCs lasted for one week, while the effect of the higher dose persisted for at least four weeks. A subsequent experiment using a conditioned place preference test revealed that BMSCs were also effective in reducing ongoing pain from CCI. Importantly, injection of the cells did not affect baseline mechanical or thermal pain thresholds in healthy mice, suggesting that the treatment did not disturb the normal physiological pain response.
Injection of BMSCs also had a protective effect on DRG neurons by preventing axonal injury usually seen after CCI. The stem cells not only blocked the CCI-induced increase in the DRG injury marker activating transcription factor 3 (ATF3), but also reversed CCI-induced downregulation of calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4), two markers that are associated with nerve injury, in DRG neurons and in the spinal cord dorsal horn. Additional experiments found that BMSCs could also quell neuroinflammation in DRGs and the dorsal horn, a process characterized by increased macrophage numbers, glial cell activation, and upregulation of pro-inflammatory cytokines, and one that has been linked to the generation of neuropathic pain (for review see Ji and Xu, 2014).
Turning to TGF-β1
Based on the rapid analgesic effect of the BMSCs, Chen and colleagues hypothesized that the cells themselves were secreting pain-relieving factors rather than differentiating into new cells. They zeroed in on the anti-inflammatory cytokines TGF-β and interleukin (IL)-10, which have been linked to beneficial effects of BMSCs on asthma and sepsis in animal studies (Nemeth et al., 2010; Nemeth et al., 2009), as well as to inhibition of neuroinflammation and neuropathic pain following nerve injury (Echeverry et al., 2009; Milligan et al., 2006).
In the current study, one particular TGF-β family member, TGF-β1, but not IL-10, was present in large amounts in BMSC culture medium. In addition, when the researchers challenged the cells using the pro-inflammatory molecules tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS), they observed an increase in TGF-β1, but not IL-10 levels. Levels of TGF-β1 also increased in the cerebrospinal fluid and then DRGs of CCI mice following BMSC injection. In further support for a role for TGF-β, the analgesic effect of BMSCs was reversed by a neutralizing TGF-β (but not IL-10) antibody administered either one, three, or 24 days post-injection of BMSCs. Knockdown of TGF-β1 in BMSCs using small interfering RNA (siRNA) produced similar findings. Conversely, intrathecal injection of exogenous TGF-β1 five days after CCI mimicked the analgesic effects of BMSCs, reducing both mechanical allodynia and heat hyperalgesia, via TGF-β receptor 1.
“We think this is a very unique TGF-β-mediated mechanism, but not an IL-10-mediated one,” Ji said.
Next, Chen and colleagues turned to the impact of TGF-β1 on neuronal function. Patch clamp recordings from lamina IIo neurons in spinal cord slices revealed that TGF-β1 modulated CCI-induced synaptic transmission, but not basal synaptic transmission, through TGF-β receptor 1. Additional experiments examining the firing frequency of evoked action potentials in DRG neurons implicated TGF-β1 and its receptor in the modulation of neuronal excitability following CCI. Thus, in addition to its well-known anti-inflammatory actions, TGF-β1 can also directly modulate neuronal plasticity in the pain pathway.
CXCL12/CXCR4 axis controls BMSC migration to lumbar DRGs and mediates the antiallodynic effect of BMSCs in CCI mice. Migration of [fluorescently]–labeled BMSCs [red] to ipsilateral L5 DRGs 7 days after i.t. injection (day 21 after CCI). Note that this migration was blocked by Cxcr4 siRNA. Left panels: i.t. injection of NT [control] siRNA-treated BMSCs. Right panels: i.t. injection of Cxcr4 siRNA-treated BMSCs. NT: non-targeting. Scale bar: 50 μm. Image and caption from Chen et al. Reproduced with permission of the American Society for Clinical Investigation.
The researchers then examined the destination of the BMSCs. Fluorescent dye labeling revealed that the majority of cells ended up in lumbar L4-L6 DRG neurons that project to the sciatic nerve. Following CCI, injured L4-L6 neurons displayed elevated levels of the chemokine CXCL12, which the researchers thought may act as a “homing” signal for the BMSCs. Indeed, a cell migration assay revealed that CXCL12 did induce the migration of BMSCs in vitro, via the CXCL12 receptor CXCR4. Furthermore, in vivo, an siRNA targeting CXCR4 gradually reduced the migration and analgesic effect of the BMSCs following CCI. These results suggest that the BMSCs inhibited pain through a paracrine mechanism, rather than by differentiating into new cells, according to Ji. “These stem cells act like little drug factories, producing TGF-β1,” he said.
Collection of L5 DRG neurons across time revealed that the number of fluorescently-labeled BMSCs peaked three days post-injection. The majority of BMSCs were still present after 56 days, suggesting long-term survival of the cells. However, because the number of BMSCs declined drastically by day 70, the researchers concluded that the risk of tumor formation, one common concern about stem cell treatments to treat disease, was unlikely.
Finally, to confirm their findings, Chen and colleagues tested the efficacy of BMSCs in the spared nerve injury (SNI) model of neuropathic pain, where pain is present for several months. Similar to results from the CCI model, BMSCs relieved SNI-induced mechanical allodynia via TGF-β1.
Taken together, the results demonstrate a major advantage of the BMSCs: they act very precisely, both at the right place (the site of injury), the right time (once CXCL12 has been produced), and for the right duration (a few months), said Ji.
Going forward, Ji said the next steps are to validate the current findings in other pain models, such as chemotherapy-induced neuropathy, arthritis, and cancer pain models. Another goal is to try to use human stem cell lines and stem cells from other species to relieve pain.
“I’m sure that BMSCs also produce many other beneficial factors besides TGF-β, such as additional growth factors, that will not only inhibit pain but also help the tissue recover,” Ji said. Thus, a third line of research will be to identify these additional players, he added.
Allison Marin (Curley), PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, Pennsylvania, US.
Article Contributors:
