Nearly half of chronic low back pain stems from degeneration of the intervertebral discs that sit between each of the spine’s vertebra. To study this phenomenon in animals, researchers can surgically injure lumbar discs in rats, but the procedure is invasive and causes significant tissue damage. Instead, some investigators injure a disc at the base of the rat tail, which is surgically more accessible, but have done so only to study the degeneration of the disc itself, and not the pain. Now, researchers have characterized the pain phenotype of this rat tail model—and found a way to relieve it.
A collaborative effort led by Abhay Pandit, a biomaterials scientist, and David Finn, a pain researcher, both at the National University of Ireland, Galway, reveals that the rats develop mechanical allodynia and thermal hyperalgesia near the injury, and thermal hypoalgesia at a site on the tail more distal to the injury. Implantation of a hyaluronic acid (HA) hydrogel biomaterial into the disc at the time of injury prevented these behavioral changes; HA is a key component of the extracellular matrix within the disc. The study highlights both the potential of this rat model for the study of low back pain as well as the therapeutic promise of HA hydrogels.
“If you look at the proportion of people with back pain versus other chronic pain and compare that to the minimal amount of preclinical work being done, there’s an immense mismatch,” said Laura Stone, McGill University, Montreal, Canada, who was not involved in the study. “A big reason for this is that we need to develop more animal models of low back pain, and so this paper nicely addresses that.”
The new research was published online April 4 in Science Advances.
Looking to the tail to study back pain
Low back pain is the most common form of chronic pain in people. The leading cause of this condition is the age-related degeneration of the intervertebral discs that provide support and flexibility to the spine.
The spine is formed of a repeating pattern of vertebrae and intervertebral discs. The discs consist of two areas: an inner gel-like center called the nucleus pulposus (NP), which is devoid of sensory neurons, and an outer fibrous ring called the annulus fibrosus (AF). When a disc degenerates, neurogenic and inflammatory substances leak out of the NP and sensitize nociceptors in the AF.
“Even in healthy discs, if you suck out the NP’s inner contents and drop that onto the sciatic nerve, you cause immense neuronal activation because of all the nasty stuff in the NP,” explained Stone.
Meanwhile, because of the neurogenic substances leaking out of the NP, sensory fibers grow into the NP in a phenomenon known as hyperinnervation.
“Then you have a situation where the nerves that get into the disc are further exposed to all that nasty stuff,” said Stone. “Plus, they’re also exposed to the mechanical forces that discs normally experience during movement.” All of this leads to a perfect storm that causes chronic low back pain.
Orthopedic researchers have studied the molecular and cellular events of this process in rodents by surgically puncturing the disc to cause degeneration (Shi et al., 2018). They do this in the lumbar region since that is where low back pain occurs in people, but extensive surgery is necessary to gain access to the discs in this area, which causes widespread tissue damage. To avoid this outcome, some investigators have adjusted their sights down the spine and into the tail.
“It’s important to acknowledge the limitation of studying low back pain in the tail, but this model is attractive because it’s minimally invasive and focused on a site that is readily accessible, which ensures minimal damage to the surrounding tissue,” said Finn. “The rat and human disc anatomy and fundamental nociceptive mechanisms share considerable similarity on an anatomical and physiological level.”
Numerous groups have used the tail model but only in the context of exploring how the discs degenerate. “Nobody has looked at pain following the injury,” said Finn. This is a glaring deficiency, since, as Stone explained, “patients don’t really care if their discs are degenerating. They care if they have pain.”
When tail discs degenerate, pain emerges
So first author Isma Liza Mohd Isa and colleagues sought to identify a pain phenotype in their model. They anesthetized rats and performed surgery at the base of the tail to gain access to the intervertebral discs in the coccyx region. They then used a tissue punch to puncture a hole in one or two intervertebral discs. They included two sham groups as controls. The first underwent anesthesia but not surgery, while the second underwent surgery but not a disc puncture.
After the animals recovered for two days, the group tested them for thermal hyperalgesia and mechanical allodynia once a week for a month, on the ventral side of the tail opposite the surgery. “It’s a relatively straightforward surgery, yet it results in quite a persistent pain-like phenotype,” explained Finn. “We find both mechanical allodynia and thermal hyperalgesia that goes out at least a month.”
They saw no thermal sensitivity in either control group, but did note significant mechanical allodynia in animals that received surgery but no puncture. This suggested that at least some mechanical allodynia resulted from the incision itself.
The team also tested whether other parts of the tail away from the injury had also been sensitized. Surprisingly, they observed thermal hypoalgesia—less sensitivity to heat—upon stimulation at a tail site more distal to the injury. This could be due to a diffuse noxious inhibitory control process, often described as "pain inhibiting pain," the authors speculate (van Wijk and Veldhuijzen, 2010).
Additional experiments showed that morphine reversed the changes in pain sensation.
“This was an important part of the model development. We needed to confirm that the injury-induced behavioral alterations could be attenuated by a well-described analgesic,” explained Finn.
A breakdown of the discs
The focus then turned to the disc injury itself. Through histological analysis performed at the end of the month of sensory testing, the authors confirmed that the puncture caused structural changes, with accompanying increases of nociceptive markers, within the disc. This suggested the presence of hyperinnervation.
Also observed were alterations in the discs’ glycosignature, which refers to the pattern of glycosylation (the addition of sugar moieties to proteins). Changes in protein glycosylation occur in numerous pathological cellular processes, including inflammation. And, indeed, results showed increases in markers of proinflammatory cytokines in the plasma and discs of injured rats.
Finally, a proteomic analysis identified nearly 400 dysregulated proteins in the discs, while pathway modeling indicated that these proteins play a role in multiple cellular processes, again including inflammation. All of these findings suggested a degenerative, inflammatory state within the disc.
“There really are four pillars here that we demonstrate: 1) inflammation; 2) proteomic changes; 3) modification of the glycosignature; and 4) anatomical disruption of the disc itself,” said Finn.
A hyaluronic acid hydrogel biomaterial to the rescue
When discs degenerate, the extracellular matrix component hyaluronic acid (HA) is broken down. HA is a carbohydrate polymer involved in cellular events ranging from cancer to inflammation. As HA degrades, it depolymerizes from a high-molecular weight to a low-molecular weight compound.
“We hypothesized that putting HA back into the disc could stop degeneration and reduce pain since we know that HA does so in other model systems, such as arthritis,” explained Finn.
So Pandit formulated high-molecular weight HA into a small, spherical hydrogel resembling a tablet. Implanting the hydrogel into the disc at the time of the puncture surgery prevented the development of all the puncture-induced changes in pain sensitivity.
“It was the month-long duration that was quite surprising,” said Finn. “It was a very pronounced and sustained effect.”
These beneficial effects on pain sensitivity in injured animals were associated with decreased levels of pro-nociceptive markers in the AF and NP, along with decreases in the hyperinnervation of those tissues.
Further, HA hydrogel treatment prevented the “four pillars” of molecular and cellular changes the investigators had observed in the discs of the injured rats.
“So we had this effect on the behavior as well as associated effects on the injury-induced tissue and molecular changes,” said Finn.
A spur to basic research on back pain?
The study stands as the first to explore pain in the rat tail model of disc degeneration. And, the HA hydrogel, which can be adapted for injectable delivery, could be a less invasive treatment option than surgery for those with discogenic lower back pain, according to Pandit.
But the team only tested whether the hydrogel could prevent pain, not reverse it.
“They gave this treatment at the time of injury, which is a common first step to test therapies, but it’s not what happens clinically where patients experience degeneration before they see a physician,” explained Stone.
Finn agrees. “We see this as the first steps in fully fleshing out this model and treatment approach,” he said. “In future studies, we will explore implanting or injecting the hydrogel weeks after the injury and see if it can reverse degradation and pain.”
Still, discogenic low back pain afflicts a large percentage of the chronic pain population, so further development of animal models to study this condition is essential for the field to move forward.
“Given the scale of the clinical problem,” said Finn, “more basic research on low back pain would be very welcome, and so perhaps this model will facilitate that.”
Nathan Fried is a postdoctoral fellow at the University of Pennsylvania, Philadelphia, US.
Image credit: Mohd Isa et al., 2018
