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Spontaneous Firing in DRG Neurons From Patients With Neuropathic Pain

Two studies of human transcriptome also bolster evidence for sex differences

by Stephani Sutherland


13 June 2019


PRF News

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Two studies of human transcriptome also bolster evidence for sex differences

Women suffer from chronic pain conditions at much higher rates than men do, and they report higher pain than men. Researchers are hard at work studying the biological underpinnings of such differences, and now two new studies of the human transcriptome in peripheral nerve tissues confirm findings from a growing number of animal studies indicating that males and females have very different gene expression profiles—and perhaps fundamentally different ways of developing chronic pain.

 

The first study, led by Patrick Dougherty, University of Texas MD Anderson Cancer Center, Houston, US, and Ted Price, University of Texas at Dallas, US, performed RNA sequencing of whole human dorsal root ganglia (DRG) from people being treated for spinal tumors. The researchers also made electrophysiological recordings from live, dissociated human DRG neurons. The recordings confirmed that neurons from ganglia that served dermatomes in which people experienced neuropathic pain—but not those in which they did not have pain—exhibited spontaneous ectopic firing. The transcriptome data revealed small differences in expression levels between men and women in hundreds of genes. Many of the genes were related to neuroimmune signaling, with different pathways reflected in the male and female transcriptomes. The study was published May 1, 2019, in Brain.

 

Franziska Denk, a pain neuroscientist at King’s College, London, UK, who was not involved in the new studies, said the use of human DRG is a highlight of the work. “That’s very difficult to do, and it’s rare to obtain” these cells from people, she said.

 

The second study, also led by Price, reanalyzed data from the Genotype-Tissue Expression (GTEx) Consortium project, which has collected RNA sequencing data from hundreds of human donors since its inception (Lonsdale et al., 2013). First author Pradipta Ray, also at University of Texas at Dallas, and colleagues focused on samples from the human tibial nerve (hTN), where they found 149 genes differentially expressed (DE) between males and females. Here, too, neuroimmune signaling genes accounted for many of the DE genes. The GTEx dataset did not include explicit information about pain conditions, so it points to baseline differences in expression between the sexes rather than pain-related genes. This study was published March 5, 2019, in Frontiers in Molecular Neuroscience.

 

Denk was keen on the study’s reanalysis of existing data. “That’s amazing, and we should do more of it. Lots of information out there gets wasted, because people don't think to look at it. And Dr. Price always tries really hard to make his data accessible, which I really appreciate.”

 

A unique opportunity reveals sex differences

In contrast to the Frontiers study, the DRG samples used for the Brain study came with a detailed medical history of each donor. The tissue was from patients undergoing surgery at MD Anderson Cancer Center for carcinomas impinging on the spine. Because patients were affected differently by the tumors, the researchers were able to compare electrophysiological and transcriptomic data from ganglia innervating dermatomes with and without neuropathic pain, sometimes even in a single patient.

 

“To have that unique opportunity to get a ganglion from one dermatome that’s affected and another that is not lets you cancel out all the rest of the patient’s medical history, which is pretty cool,” Dougherty said.

 

Six patients had localized spinal pain, but without radicular pain that shoots down the leg or other features of neuropathic pain; 15 patients had unilateral radicular/neuropathic pain; and another five patients had bilateral radicular/neuropathic pain. All the patients with neuropathic pain had it for at least a month, and most had pain for more than six months. Radiographic tests showed evidence of nerve root compression in ganglia affected by neuropathic pain.

 

Co-first authors Robert North, Baylor College of Medicine, Houston, US; Yan Li, MD Anderson; and Ray expected to see gene expression differences pop out when they compared the transcriptomes of ganglia from dermatomes with and without neuropathic pain, but only a few did.

 

“We ran the DRG in pain versus no pain, and we saw differences in just seven genes,” Ray said. “But when we separated the sexes, that number jumped to 150,” referring to the number of genes upregulated in the “female pain” ganglia. In “male pain” ganglia, 426 genes were upregulated. “I was not surprised that there were sex differences, but by the number of differences,” he said.

 

Price recalled struggling with the data. “We flew to Houston and had a powwow with Pat [Dougherty], and we were poring over the data and brainstorming. Then we separated the samples by sex and boom—the story was sitting in front of us.”

 

When comparing male and female “pain” DRG transcriptomes, the researchers found that, in general, Price said, “in males, it looks more macrophage-y,” suggesting a role for these infiltrating peripheral immune cells, “whereas in females, it’s very hard to put a finger on it.” Some of the upregulated genes in females included G protein-coupled receptors.

 

The researchers also turned to a machine learning approach to gain more insight. “We looked to see if we could predict sex from the gene expression pattern, and we could do that with over 90 percent accuracy, based on many predictors,” said Ray. Once the samples were separated by sex, the investigators could also predict whether a sample was from a ganglion affected by neuropathic pain or not, based on gene expression.

 

Ectopic activity as a driver of neuropathic pain?

When the researchers made electrophysiological recordings of DRG neurons from the patients, they saw spontaneously firing action potentials. “They were the first thing we looked for, and they jumped right out,” recalled Dougherty of the so-called ectopic activity. “It seems to be fundamentally important.”

 

But only neurons from ganglia taken from people with neuropathic pain showed the spiking. “The activity just didn’t appear when there was no pain” associated with the dermatome, Dougherty said. “We do all these studies blind, but you can almost tell right away what dermatome they came from” based on their activity, he said. About one in five neurons with corresponding dermatomal pain or associated nerve root compression displayed spontaneous firing, whereas fewer than 5 percent of neurons without dermatomal pain did.

 

The idea that ectopic activity in sensory neurons in the DRG might underlie neuropathic pain arose from animal studies and was first put forth three decades ago by Patrick Wall and Marshall Devor (Wall and Devor, 1983Devor, 2009).

 

“The word 'ectopic'—it means ‘coming from the wrong place,’ so instead of originating at sensory endings, signals arise from a nerve injury site or from within the DRG itself,” said Devor, Hebrew University of Jerusalem, Israel, who was not involved in the current studies.

 

In 2014, Devor and colleagues confirmed the hypothesis in an experiment with patients suffering from phantom limb pain (PLP) after amputation, a condition thought to be driven by neuronal reorganization in the cortex. Devor showed, however, that intrathecal or intraforaminal—directly onto the DRG—application of lidocaine halted PLP in the patients, indicating the peripheral nervous system, and likely the DRG, was driving the neuropathic pain (Vaso et al., 2014). But they still didn’t know how.

 

Now, the current study has shown with single-cell recordings that human DRG neurons from people affected by chronic pain are spontaneously active. “We think that our paper, the electrophysiology, is the first evidence that this aberrant firing definitively happens in people. Now we need to know what drives it,” said Price.

 

Devor said while he was not surprised by the finding of ectopic activity in DRG neurons, “I’m pleased, of course, to know the same thing happens in humans as happens in rats. The ectopic firing that we’re familiar with—its presence in ganglia taken from people with neuropathic pain and not in ganglia from people without pain or whose pain is not neuropathic—that’s impressive.”

 

Denk thinks the observations should be exciting to patients because “we can actually observe the dysfunction. This hypersensitivity driving pain—we can measure it. You can see that the neuron behaves differently. It’s a real thing in these neurons, and it’s a real thing in people with chronic pain as well.”

 

Interestingly, there were no sex differences when it came to spontaneous activity, Dougherty said. “That’s conserved across all samples. Both sexes develop spontaneous activity, but the implication of the transcriptome data is that the mechanisms are likely quite distinct,” he added. “That says the therapeutics to treat men and women may be different.”

 

A drug like lidocaine, however, which blocks sodium channels and neuronal firing, could quell ectopic DRG activity—and pain—in both sexes. “There’s every reason to believe, if one did the things to stop the ectopic firing that they’re showing now in humans, that these drugs would work,” Devor said.

 

GTEx

The Frontiers study using the GTEx dataset identified 149 DE genes in the transcriptome of the tibial nerve of 248 men and women. Some genes had previously been associated with pain signaling, such as SP4, a transcription factor known to regulate pain genes that was more highly expressed in females. Ray said, “A lot of genes that were differentially expressed are genes in the inflammasome—they’re related to immune signaling or required for inflammation. We know that many inflammatory diseases, and those that cause inflammation and pain, have a different prevalence in males and females.”

 

Denk said the findings of sex differences in immune genes should not be unexpected. “Literature from the immunology field is very clear that there are differences in immune function between men and women, and these new findings suggest that the differences extend to pain. It’s nice to remind people of that, because we have not imported it into our consciousness in our field.” (See, for example, Klein and Flanagan, 2016.)

 

Together, Price said, “The papers highlight that there are sex differences in the human peripheral nervous system, and it looks like most sex differences are somehow related to neuroimmune genes.”

 

Only the beginning

Denk is cautious about putting too much stock in the specific genes identified in the study as targets for pain therapeutics, because of the small sample size, and Dougherty agreed. “The number is still very small,” he said, but the researchers are continuing to record from more samples. “As we continue to power up the studies, it’s likely that other players will emerge from the noisy signal we have now. With clinical work, we are always at the mercy of who happens to present for surgery, and there are so many variables affecting patients.”

 

The team also plans to collect RNA data from single cells after making electrophysiological recordings to compare gene expression between cells with and without spontaneous activity. “We expect the findings to become much more robust,” Dougherty said. Ultimately, by studying the neurons, he hopes to address the question, How do you figure out, for a given patient, which cells are spontaneously active and causing pain, and what’s driving that activity?

 

As to the findings in men versus women, Price says that recognizing sex differences will have multiple implications for developing pain therapeutics.

 

“I am increasingly thinking that failed clinical trials may give hints of efficacy if we stratified subjects by sex. Most therapies going to trials in the past 20 years came from information gleaned from male animals. And a lot of it—really basic stuff—doesn’t hold up in females. So it’s possible you’d want completely different therapeutics to treat males and females in pain, and I think there are some screaming flags of that in these studies.”

 

Stephani Sutherland, PhD, is a neuroscientist and freelance journalist in Southern California. Follow her on Twitter @SutherlandPhD.

 

Image credit: Griffith et al./Wikimedia Commons/Creative Commons Attribution 2.5 Generic license.

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