Investigators at Washington University School of Medicine in St. Louis have found that a protein that allows nerve cells to communicate may enhance perceptions of chronic and persistent pain. In the February issue of Nature Neuroscience, they report the protein, called NR2B, makes mice more aware of minor pain for longer periods of time.
"That sustained response appears to mimic what happens in people who experience pain long after the painful stimulus has disappeared," says principal investigator Min Zhuo, Ph.D., an associate professor of anesthesiology and of anatomy and neurobiology. "So interfering with NR2B in humans might be a strategy for treating chronic pain."
NR2B is one of the mix-and-match building blocks of important cellular proteins called NMDA receptors. Like radio receivers, these receptors sit on the cell surface, tuned in to messages sent from neighboring cells and carried by the chemical messenger glutamate. More importantly, NMDA receptors are a neuron's activity detectors, and they function only when the number and intensity of neuronal messages reach a prescribed, threshold level.
Because NMDA receptors only become activated at these threshold levels, they tend to be the primary neuronal receptors involved in important brain functions such as learning and memory and in injurious brain processes such as stroke, head injury and drug abuse. They also are the primary receptors involved in persistent pain.
The researchers studied a strain of genetically altered mice that was created by Joe Tsien, Ph.D. and colleagues at Princeton University to make extra NR2B in forebrain areas. The Washington University group found that these mice reacted to acute pain in the same way as normal mice. But the NR2B mice seemed to have stronger or longer periods of behavioral responses to two different models of more persistent, inflammatory pain. None of the mice demonstrated pain symptoms in the absence of an injury.
"We believe that when an injury occurs, NMDA receptors in regions of the forebrain may play an important role in processing discomfort from that pain, and they could be a potentially important target for treating chronic pain," Zhuo explains.
Past research with mice has shown that abnormally high NR2B levels are related to other behavioral changes and biological changes in the brain. In 1999, Zhuo was part of a research team that reported in Nature that NR2B mice performed better in behavioral tests of learning and memory.
Long-term potentiation (LTP), a physiological change thought to be important for learning and memory, also was enhanced in the NR2B mice. Like oil that lubricates pistons to help an engine work, LTP lowers the threshold that neurons must reach before firing off a message. That makes it easier for them to send messages back and forth along sensory pathways.
Back in 1999, Zhuo detected the LTP enhancement by studying slices of NR2B mouse brain. When the brain slices were exposed to a weak electrical stimulation, transmissions between neurons at junctions called synapses were significantly strengthened (potentiated) for a long period of time.
"In these experiments, we wanted to learn if strengthened connections in forebrain areas effect animals' responses to pain," Zhuo says. The genetically modified mice made excess NR2B in the forebrain. Brain imaging studies in humans have shown that forebrain structures called the cingulate cortex and the insular cortex help perceive pain. But how cells in those structures perform that function was not known.
When a mouse or human encounters a painful event, receptors on the skin, muscle or internal organs trigger an electrical impulse that travels along a nerve fiber to the dorsal horn of the spinal cord. There, the fiber connects with a nerve cell, which relays the pain signal up the spinal cord to the brain.
Under normal conditions, these synaptic transmissions are handled by neuronal receptors called glutamate AMPA and kainate receptors. However, when excessive information reaches these synapses between nerve cells, normally-dormant NMDA receptors can be activated, and a series of intracellular signaling molecules can be triggered. This cascade of events leads to long-lasting changes in the way neurons communicate and can lead to changes in how the human or the animal perceives external pain signals.
The genetically altered mouse that overexpresses NR2B, consequently enhancing the activity of NMDA receptors, provided Zhuo and colleagues with a ready-made model for exploring NR2B's role in the forebrain.
The researchers prepared brain slices from the anterior cingulate cortex and the insular cortex of both normal and NR2B mice. They also examined spinal cord slices from both strains. The spinal cord slices responded similarly to a small electrical stimulus. But the NMDA receptor-mediated responses in the forebrain slices from the NR2B mice were enhanced when compared with the normal mice.
In other experiments, the research team -- which included anesthesiology fellows Feng Wei, Ph.D., and Guo-Du Wang, Ph.D., M.D./Ph.D. student Geoffrey Kerchner, and Zhou-Feng Chen, Ph.D., assistant professor of anesthesiology -- observed the behavior of mice subjected to mildly painful stimuli.
There was no observable difference in behavior between the standard and NR2B mice. The differences that did occur were evident only hours or days following injections that caused an inflammatory response at the injection site.
The mice were anesthetized and then given an injection in the hind paw with either formalin or a chemical irritant called complete Freund's adjuvant (CFA). In the formalin test, when the anesthesia wore off, both normal and NR2B mice licked and bit the injection site.
But the NR2B mice who got formalin injections continued licking long after the other mice had stopped. And in response to contact from a small filament that normally causes no response, the NR2B mice injected with CFA would withdraw their paw and escape.
The results can be interpreted in several ways. "Perhaps the animals with more NR2B can detect pain sooner in case of injury," says Zhuo. "That could help them avoid more serious injury. But if an injury is not avoidable, the enhanced NMDA receptor activity in the forebrain would make them more likely to feel persistent pain."
Zhuo believes that deactivating the NR2B protein might help people with chronic pain. In particular, such a strategy may help patients with allodynia -- pain induced by a stimulus that is not normally painful, such as a gentle touch. Many current drugs that target NMDA receptor activity work by interfering with all NMDA receptors. Therefore, they also block acute pain, which can be protective.
"You want to be able to feel painful heat on your skin when you're cooking, so you can quickly withdraw your hand if you need to," Zhuo says. "Our study has provided a target for the development of drugs that would be highly selective for persistent pain. They would allow people to ignore chronic pain while leaving the rest of the pain system intact."
Min Zhuo's research is supported by grants from the National Institutes of Health.
For more information, refer to Wei, F, Wang G-D, Kerchner GA, Kim SJ, Chen Z-F, Zhuo M. Genetic enhancement of inflammatory pain by forebrain NR2B overexpression. Nature Neuroscience 164-169, Feb. 1, 2001.