Power in Numbers

by Megan Talkington on 6/6/2011

A study published online May 9 in Pain takes a bird’s-eye view of the vast assortment of genes whose expression is altered in pain. In a meta-analysis of 20 published microarray gene expression experiments from rat and mouse models of neuropathic and inflammatory pain, researchers found 79 genes that were regulated in four or more independent studies. Follow-up analysis confirmed many of those and identified two, Ccl2 and Reg3b, that were regulated especially reliably across models and species. Ccl2 (monocyte chemoattractant protein-1, MCP-1) has already been extensively studied for its role in chronic pain, but Reg3b (pancreatitis-associated protein, PAP) is a newcomer. The analysis also indicated that gene regulation in neuropathic vs. inflammatory pain, and in rats vs. mice, may be profoundly different.

For basic biologists, these results offer tip-offs on genes that may be involved in chronic pain. For researchers focused on drug discovery, the meta-analysis could help in prioritizing targets. And for anyone who relies on data from animal models, the findings hint at important distinctions between the molecular mechanisms of pain in different species.

Jeffrey Mogil of McGill University in Montreal, who led the study, said that the concept for the meta-analysis occurred to him when he realized that between 2002 and 2008, the field had amassed 20 published microarray studies on pain. Despite their many differences, all of the studies compared rodents in chronic pain states with normal animals, and all together the work identified 2254 unique genes whose mRNA levels were significantly altered. However, Mogil was most interested in genes that were flagged in multiple studies. “If genes come up more often than would be predicted by chance, they ought to be taken very seriously,” he said.

When first author Michael LaCroix-Fralish and his coworkers compiled the gene lists, they found that most genes were identified in only one study. Some, however, cropped up again and again, and the researchers focused on 79 that were reported in four or more studies. Functional annotation showed that many of those genes were immune-related. Some of the genes had previous evidence for involvement in pain, but 38 had little or no prior validation.

To independently evaluate the expression of the 79 genes, the team performed quantitative real-time polymerase chain reaction (qPCR) in four animal pain models. They analyzed dorsal root ganglion (DRG) and spinal cord tissue from rats and mice with either sciatic chronic constriction injury (CCI, a neuropathic pain model) or injection of complete Freund’s adjuvant (a model of inflammation). In one condition (DRG/rat/CCI), many of the genes—43 of the 79 tested—were significantly regulated. That made sense, says Mogil, because most of the 20 original studies were done on rats with neuropathic pain. On the other hand, many of the 79 turned out not to be regulated in the inflammatory pain model. Particularly striking to Mogil was the fact that few of the genes were significantly regulated in mice. “I think everyone would expect that genes expressed in the rat for neuropathic pain should also be expressed in the mouse for neuropathic pain… We implicitly assume that these things work across species by using the mouse or rat to stand in for people,” Mogil said. “Things look very different in a mouse—which is pretty scary.”

Michael Costigan, Children’s Hospital Boston, Massachusetts, says he’s not surprised that mice and rats looked so dissimilar. (Costigan was not involved in the LaCroix-Fralish study, but two of the papers included in the meta-analysis came from his work). He is currently working on a study of gene expression following nerve injury in nine different strains of mice, and is seeing strain-specific patterns of gene expression. “If there are differences between strains of mice, you can imagine there are huge differences between mice and a different animal,” Costigan said. As a result, he told PRF, any protein that looks like a promising therapeutic target in one animal “should really be repeated in at least one other animal before you even attempt to go to human trials.”

In their qPCR experiments, the Mogil group found that two genes, Ccl2 and Reg3b, were regulated particularly consistently, showing significant regulation in rats in both the spinal cord and DRG in both pain states. There is already evidence that CCL2 (chemokine [C-C motif] ligand 2; also known as MCP-1) and its receptor, CCR2, are upregulated in neurons and glial cells in the DRG, spinal cord, and brain following nerve injury, and both proteins are actively being pursued as targets for neuropathic pain therapy (for a review, see Gao & Ji, 2010). A paper published April 13 in The Journal of Neuroscience indicates that CCL2 may act not only as a chemokine but as a neuromodulator similar to classical neuropeptides including substance P. In the study, Stéphane Mélik Parsadaniantz, INSERM, Paris, France, and colleagues found evidence that following peripheral nerve injury in rats, CCL2 was secreted from presynaptic terminals of sensory neurons in the dorsal horn of the spinal cord, and that this release led to inflammation and pain hypersensitivity.

Meanwhile, Mogil says, Reg3b (PAP) has not been directly implicated in pain, so his lab is hoping to test Reg3b knockout mice to see if their pain phenotype is altered. It is possible, he says, that Reg3b, Ccl2, and other genes whose expression reliably correlates with pain state could serve as clinical biomarkers for pain.

Mogil points out that while microarray studies, and his group’s meta-analysis, bring to light genes whose expression is affected in pain conditions, these experiments alone do not prove that those genes actually cause pain. “If, on the other hand, you just want some new ideas, microarray is really useful, ” he said.