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Hippocampal Extracellular Matrix Modulates Chronic Pain and Related Memory Deficits

Mouse study suggests matrix metalloproteinase 8 as potential drug target

by Stephani Sutherland


14 January 2019


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Mouse study suggests matrix metalloproteinase 8 as potential drug target

The vast majority of pain research focuses on neurons, and to a much lesser extent glia, but new research now shows that the extracellular matrix (ECM), the supportive milieu outside of cells, plays an active role in shaping the experience of chronic pain.

 

The study reveals that the ECM surrounding inhibitory hippocampal neurons deteriorated following chronic pain from tibia fracture in mice. At the same time, hippocampal long-term potentiation (LTP) increased, and, behaviorally, the animals showed impaired performance on memory tasks. The authors propose that pain-related upregulation of matrix metalloproteinase 8 (MMP8) was responsible for these changes.

 

The work from first author Maral Tajerian, J. David Clark, and colleagues at Stanford University, US, was published online September 26, 2018, in Molecular Psychiatry. Tajerian is now at Queens College, City University of New York, Flushing, US.

 

“This study adds to the growing body of literature indicating that the extracellular matrix, particularly specialized structures known as perineuronal nets, are involved in structural and synaptic plasticity,” wrote Amy Lasek, University of Illinois at Chicago, US, in an email to PRF. “Novel aspects of this study are the demonstration of a role for the ECM in the hippocampus in chronic pain [and in] the memory impairment that develops during chronic pain, and a demonstration that upregulation of the matrix metalloproteinase MMP8 contributes to the chronic pain state,” according to Lasek, who studies the role of the ECM in addiction but was not involved in the current study.

 

Ru-Rong Ji, Duke University, Durham, US, who has studied the role of the ECM in pain but also was not involved in the new research, agrees. “This study is interesting in that it shows how the ECM in the hippocampus regulates postoperative pain after orthopedic surgery. In particular, the authors identified MMP8 as a trigger of ECM dysregulation. They also demonstrated that knocking down MMP8 can reverse surgery-induced adverse effects,” wrote Ji in an email to PRF.

 

Memory impaired by pain

Tajerian and colleagues used a mouse model of chronic pain in which the tibia bone is fractured and allowed to heal with a cast that is removed after three weeks. After seven weeks, the mice still displayed tactile allodynia and memory deficits.

 

The researchers used two tests to assess pain-related memory deficits. In one test, on day one, male mice were placed in an arena containing a small cage in each corner, one of which contained a novel female mouse—a memorable experience. On day two, the mice were placed in an identical arena that never held a female mouse (and therefore had no odor).

 

Time spent exploring the cage in the position where the female had previously been held indicated how well the male recalled her location. Injured mice spent less time at the female’s cage on day two, compared to uninjured animals, indicative of a memory impairment.

 

On a second task, the investigators placed mice in a Y-shaped maze and allowed the animals to explore the three arms, each marked with a unique visual cue. Usually mice enter the three arms alternately, favoring a novel arm rather than immediately re-entering a given arm, but injured mice entered the same arm repeatedly more often than controls.

 

More things in heaven and earth than are dreamt of in the neuron philosophy

To find the root of the memory problems, Tajerian turned her attention to the hippocampus. But in addition to looking at neural activity there, she also wanted to probe the hippocampal ECM. The ECM is a matrix of proteins that surrounds and connects cells, including all the cells of the brain. A graduate course on the history of neuroscience had sparked the idea.

 

In the late nineteenth century, Tajerian said, “scientists thought that the brain worked as a mesh,” referring to the reticular theory, which imagined the nervous system as a continuous network, based on Camillo Golgi’s pioneering microscopy. About the turn of the twentieth century, though, the neuron doctrine crowned neurons as independent functional units—a hypothesis driven by the work of Santiago Ramón y Cajal.

 

“Because of them, everyone studies neurons, which is great. But at the same time, I blame them a bit, because we are not studying anything else. Only recently are neuroscientists looking at glia, and even that is still considered heresy,” Tajerian said.

 

She wanted to measure the effects of chronic pain on the hippocampal ECM. Because the proteins of the ECM are mainly insoluble, the typical biochemistry technique of extracting proteins would not work.

 

“If we can’t extract components of the matrix, we can do the opposite and leave the matrix intact,” she said. So Tajerian “decellularized” the hippocampus using a previously published technique so that only the matrix remained behind (De Waele et al., 2015).

 

The researchers then used surface-scanning electron microscopy to show that fibers of the ECM appeared thinner and were fewer in number in injured mice compared to control animals. The geometry of the ECM was also altered.

 

 

Collaborator Andrey Malkovskiy, Stanford University, employed atomic force microscopy, a technique used in cancer biology to measure tumor rigidity. He found that ECM rigidity was significantly reduced in mice with the tibial fracture compared to control mice.

 

“It seems that chronic pain literally made them soft in the head,” Tajerian said.

 

When the researchers quantified specific components of the ECM, they saw reduced levels of hapln1 and aggrecan. The brains of injured mice had increased levels of MMP8, an enzyme that breaks down aggrecan and hapln1, whereas levels of TIMP2, an endogenous MMP8 inhibitor, were reduced compared to control animals.

 

A protective jacket

The proteins affected—hapln1 and aggrecan—are important components of the perineuronal net (PNN), a specialized type of extracellular matrix that wraps around neurons and provides them with structural support.

 

“It literally looks like a little jacket around the neurons. It hugs them and provides stability,” said Tajerian.

 

The PNN also influences neuronal activity, by protecting synapses, for instance, according to Tajerian. “It provides a physical barrier that prevents certain neurites from getting phagocytosed by microglia, for example.”

 

Ji was not surprised that the ECM was involved in remodeling the brain after chronic pain. “The ECM can modulate the number and function of synapses,” he wrote in an email.

 

To assess the effects of ECM changes on neural activity, co-author Bende Zou made extracellular recordings from hippocampal brain slices. “We saw increased, aberrant LTP,” Tajerian said, referring to an electrophysiological signature thought to be the molecular basis for encoding memory in the hippocampus.

 

Staining for parvalbumin (PV)-positive, inhibitory interneurons showed that injured mice had fewer PV neurons surrounded by a PNN. “We think that because interneurons are not stabilized enough, the excitatory drive increased,” she said.

 

That makes sense, noted Ji. “Changes in synaptic function and loss of inhibition will lead to more LTP.”

 

Lasek wrote PRF that while “this study showed that a decrease in PNNs during a chronic pain state is associated with memory impairment, others have published studies showing that digestion of PNNs can improve cognitive performance in memory tasks.” (For example, see Riga et al., 2017, who used a mouse model of depression).

 

That finding, Tajerian noted, “further highlights the requisite for a matrix that is ‘just right,’ and that either increased rigidity, for example, with age, or decreased rigidity following injury, can lead to memory dysfunction.”

 

Reversing the effects of MMP8

The researchers then tested whether they could reverse the memory deficits and pain-related behaviors by manipulating MMP8 levels. They used a short hairpin RNA (shRNA), which they introduced with a lentivirus stereotactically injected directly into the hippocampi, to downregulate MMP8.

 

Mice treated with the shRNA showed improvements in tactile allodynia and on both memory tasks. In addition, the number of PV neurons surrounded by a PNN was normalized.

 

“The therapeutic implications of this study are that targeting MMP8 with an inhibitor could be a novel way to treat chronic pain, particularly since MMPs are considered ‘druggable’ targets,” according to Lasek.

 

The ECM regulates neuronal structure and function in multiple ways, Tajerian said. The ECM and neurons “do talk to each other, just not by using action potentials.”

 

Instead, the ECM can make neuronal dendrites more or less likely to branch, it stores glia-secreted substances and cytokines, it provides the rigidity neurons need to grow properly, and it forms a physical barrier—"protection from microglia who might eat them up,” said Tajerian. “It’s a completely different way of communication.”

 

In the future, Tajerian hopes that others will join in studying the role of the ECM in pain.

 

“I want to call it the neo-reticularist movement,” she said with a laugh.

 

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

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