Transplanted GABAergic Cells Ease Pain
Gamma aminobutyric acid (GABA) is a key inhibitory neurotransmitter produced by interneurons of the human central nervous system. After nerve injury, a major path leading to neuropathic pain and the development of allodynia and hyperalgesia begins with the loss of GABA-mediated inhibition in the spinal cord. Some drugs used to treat chronic pain after nerve injury act to increase GABA signaling, whereas other, still experimental approaches, have used gene therapy or genetically engineered stem cells to boost levels of GABA in the spinal cord in animal models. However, none of these techniques accomplish permanent relief, and some carry adverse side effects. To date, there is no treatment that reliably reduces chronic pain for most patients following nerve injury.
In a new approach to restoring GABAergic function, João Bráz, Allan Basbaum and colleagues at University of California San Francisco, US, show that chronic pain can be substantially reduced by transplanting embryonic GABAergic cortical interneuron precursors into the spinal cord in a mouse model of neuropathic pain. The neuronal precursors integrated into their new environment, responded to afferent inputs, and appeared to restore GABA-mediated inhibition, resulting in a reversion of the mechanical hypersensitivity generated by nerve injury.
The results offer a novel and promising cell-based approach that may be useful for treating chronic pain “where loss of inhibition is a major contributor to the clinical condition, which includes many of the neuropathic pain conditions,” Bráz said.
The findings were published in the May 24th issue of Neuron.
The key accomplishment of this research is the successful integration of transplanted cortical cells into the novel environment of the spinal cord, and their differentiation into functional GABA-expressing interneurons. Manuel Dolado, Andalusian Center for Molecular Biology and Regenerative Medicine in Seville, Spain, who was not involved in this study, explains that the idea of using GABA producing cells for neuropathic pain is not new, but the functional integration of interneurons into the spinal cord is a major breakthrough. “What is really new is the integration of the grafted cells in the circuitry in a permanent way and the modulation of the circuits by the grafted cells, that leads to modification of the disease. This is really remarkable,” he wrote in an email to PRF.
At home in the spinal cord
For their transplantation strategy, the authors used precursors of cortical interneurons derived from the medial ganglionic eminence (MGE) of embronic day 13.5 mice that were engineered to express green fluorescent protein (GFP) to track the cells’ fate in the spinal cord, and a neuron-specific marker, NeuN, to confirm the differentiation of MGE cells into neurons. One month after transplantation into the dorsal horn of the spinal cord, the majority of the surviving MGE cells had acquired a GABAergic neuronal phenotype, the researchers showed.
Integration of the MGE cells into the host spinal cord circuitry was established using markers that move from cell to cell through synaptic connections. The authors transplanted MGE cells into transgenic mice (ZW mice) that express the transneuronal tracer protein wheat germ agglutinin (WGA) in sensory neurons of the dorsal root ganglion. The authors found transneuronal transfer of WGA from sensory neurons to the transplanted cells, indicating the presence of synaptic connections between the cells.
To test if the transplanted cells also communicate with spinal cord neurons, the authors genetically modified MGE cells to express the WGA tracer.The authors show that WGA is taken up by spinal cord neurons near the grafted cells. The authors subsequently used retrograde transneuronal spread of pseudorabies virus to show that transplanted cells also formed connections with spinal cord projection neurons, which normally carry inputs from nociceptors to the brain. Bráz and colleagues showed functionality of the connections by demonstrating that either nonnoxious stimulation (walking on a rotating rod) or noxious chemical stimuli could activate grafted neurons, as measured by expression of the marker protein c-Fos.
After establishing the functionality of the grafted neurons, the authors assessed the behavioral consequences of the transplants in adult mice with spared nerve injury (SNI), which results in prolonged mechanical hypersensitivity and is a model for neuropathic pain in people. One week after injury, SNI mice received either a suspension of MGE cells or medium with no cells (control). The results show that mechanical thresholds in the grafted mice returned to normal levels, while control mice displayed persistent mechanical hypersensitivity. The return to normal thresholds in the grafted mice began at two weeks after transplant, corresponding to the time when the cells take on neuronal properties. The transplants had no effect on baseline mechanical thresholds in non-injured mice.
Previous studies have shown that peripheral nerve injury results in a reduction of glutamic acid decarboxylase (GAD) (the biosynthetic enzyme for GABA) in the spinal cord, so the authors also asked if MGE transplants might be able to counteract the drop. Quantitative PCR performed one week after MGE transplantation showed that nerve-injured, transplanted mice had significantly higher levels of GAD compared to the nerve-injured, control groups. The levels of GAD mRNA were similar to naive animals, suggesting that MGE transplantation does not increase GAD levels beyond the normal range, but instead overcomes the decrease in GAD mRNA (and, presumably, GABAergic signaling) triggered by peripheral nerve injury.
Finally, the authors asked whether transplantation of GABAergic neuronal precursor cells influences inflammation-related pain. To this end, they injected 1 percent formalin into the hindpaw of mice, 4 weeks after MGE transplantation. They found that MGE transplants had no effect on pain behaviors, which indicates that transplantation of GABAergic precursor cells does not reverse pain in a chemical nociception model associated with inflammation. These results suggest that the transplants address a problem specific to neuropathic pain, that is, the loss of GABAergic tone after nerve injury.
How does it work?
The new research leaves some open questions. A main one is whether the pain-relieving effect of the transplanted cells is indeed mediated by GABA. This is a big question, says Bráz. “In the paper we didn’t show that GABA mediated the effect. However, given that more than 75 percent of the cells become GABAergic and that the levels of the GAD enzymes are normalized after transplantation, we suggest that this is the case,” he wrote in an email to PRF.
Bráz wrote that his team is now tackling this very question. “We are trying to confirm directly this hypothesis using cells that express GABA but are no longer able to release it. If we see that the effect of the transplant is gone, then that will show undoubtedly that GABA is indeed underlying the effects we reported.”
But additional questions remain, such as what happens over longer time periods? Will there be an improvement in pain or deterioration?“ This is another aspect we are looking at but are not able to answer yet,” Bráz said.
Dolado also points out that one problem future work will need to address is the extremely low survival rate of the transplanted cells, which the work pegs at less than 2 percent. “You will need a large amount of cells, and as far as I know nobody has been able to culture these cells while keeping their properties. We are working on that in our lab. This is just a technical problem that I’m sure with time will be resolved,” Dolado said.
Bráz explains that the low survival rate may indeed be due to technical issues, such as cells remaining in the injection pipette, cell death in the course of injection, cells trapped in the pia, and other issues.There is also the possibility that the nerve-injury associated factors that produced dysfunction in the host GABAergic interneurons may continue to negatively influence transplanted neurons.
Another interesting finding is that the transplanted cells retained their cortical interneuron phenotype, even after integration into the spinal cord environment. Bráz thinks this may mean that the new environment does not affect their differentiation program. “We were surprised by this. It means that at E13.5 (the age at which cells are harvested), these cells are already predetermined to become inhibitory interneurons, with cortical properties, and that the environment in which they are placed does not affect their rather rigid differentiation program,” he wrote.
The road ahead
Bráz says that their current findings may have important implications in the way chronic pain is treated. Pharmacological approaches to increasing GABA signaling, using systemic drug administration, have adverse effects such as sedation, which limits their use. Transplantation, on the other hand, offers a localized treatment, which may bypass the side effects of systemically administered drugs.
Also, he points out, “Current treatments for neuropathic pain are targeted at alleviating the symptoms without acting on the cause. This makes the pain relief temporary, not permanent. Our approach in contrast is disease-modifying because it restores the inhibitory control that is ‘missing’ in the injured tissue. So by treating the underlying cause of the problem, we can expect much longer and conceivably permanent effects.”
Currently, Bráz is looking at the mechanisms underlying the transplant-induced reduction of the neuropathic pain. “We are looking at the effects of the transplantation on the expression levels of GABA and GABA receptors in both naive and injured mice; we are confirming the integration aspects at the electron microscopy level; we are looking – with electrophysiology – at the responses of spinal cord neurons after transplantation, et cetera. Our future goal will be to test the potential of these cells in other models of chronic pain.”
The longer term outlook, of course, is to determine whether this approach can be brought into clinical application. To this end, the authors are planning to look at transplantion of human MGE cells. The first studies will involve transfer into immunocompromised mice, to determine whether human cells will survive and integrate in the spinal cord like the mouse cells. If successful, that could lead one day to clinical trials in which patients received transplants into the spinal cord, but also into the trigeminal nucleus caudalis, in order to address very difficult neuropathic pain conditions of the orofacial region (such as trigeminal neuralgia). Another long-term approach is to use embryonic stem cells or induced pluripotent stem cells that have been modified so that they take on the properties of GABAergic inhibitory interneurons.
Read more about this work in the Forum interview Pain and Its Control: A Conversation with Allan Basbaum, and see Basbaum and Bráz talk about their work in a video from Cell Press.
Karl Gruber is a Ph.D. candidate in genetics at Heinrich Heine University in Düsseldorf, Germany, and a freelance science writer.
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