ABOVE: Intervertebral disc degeneration is a common cause of back pain. © iStock, andreswd
Three floors below Dmitriy Sheyn’s lab at Cedars-Sinai Medical Center, doctors remove degenerated intervertebral discs from patients with severe back pain. Instead of tossing the old discs, however, they sometimes give them to one of Sheyn’s lab members, who rushes them upstairs to harvest the fresh cells. The patients in these operating rooms accepted surgery because they were suffering, but many people with degenerated discs feel no pain at all. Sheyn wanted to know why.
In a new paper in Science Translational Medicine, stem cell biologist Sheyn and his colleagues began to answer that question: They reported that when aging or under degenerative stress, a subset of cells in the center of the disc can release a cry for help, a particular signal that causes outside neurons to extend their axons within, allowing the brain to feel the pain inside.1
"We’ve got lots of patients who have back pain that we don’t really know the cause," said Christopher Bono, an orthopedic surgeon at Massachusetts General Hospital who was not involved in the study. "One of the causes, we think, is a disc that degenerates, and a pain mechanism that hasn’t been exactly elucidated. This study is suggesting a likely mechanism for a disc to become painful.”
The center of a healthy intervertebral disc contains no blood vessels or neurons. This area is called the nucleus pulposus, and it’s like a jelly-filled shock absorber. But under conditions like low pH, low oxygen, low nutrients, or inflammation, discs can degenerate, and sometimes neurons innervate the disc, leading to pain.
Wensen Jiang, a postdoctoral scientist in Sheyn’s research team, wanted to determine which cells within the nucleus pulposus contributed to the development of this pain. He performed single cell transcriptomic analysis (RNA-seq) on cells from three degenerated discs surgically removed from people in pain and compared them to cells in degenerated, non-painful discs from four cadavers. The team found that a certain subtype of nucleus pulposus cells (NPC) called NPC1 were much more prevalent in discs from people in pain than in discs from people who hadn’t been in pain. The researchers then compared back pain NPC1 (bpNPC1) to asymptomatic NPC. They found that bpNPC1 showed increased expression of nerve growth factor and fibroblast growth factor 2, which help regulate neuron outgrowth. Interleukin-8 and nitric oxide synthase 2, both of which play a role in oxidative stress, also exhibited increased expression. bpNPC1 also had reduced expression of peroxiredoxin-1, an antioxidant enzyme.
Next, the team studied the molecular triggers that caused NPC to switch from a degenerated and painless state to a degenerated and painful state similar to the bpNPC1 subtype. They created a stressful environment by reducing pH and glucose levels and adding proinflammatory factor IL-1β. After exposure to this cocktail, the cells increased expression of degeneration and inflammatory markers, and their transcriptomes appeared comparable to the bpNPC1.
Sheyn’s group then put these stress-stimulated NPC into one side of a microfluidic organ-chip system. On the other side of the chip grew induced pluripotent stem cell derived nociceptor-like sensory neurons connected by tiny channels that allowed axons and secreted factors to cross, but not whole cell bodies. They saw that several axon-like structures crossed the microfluidic channel towards the stressed NPC, whereas no axons crossed to the side of the chip with the non-stressed NPC.
“That was really cool to see,” said Sheyn. “Now more questions are rising. So, for example, is it the stressed cells that attract sensory neurons, or maybe it’s the healthy cells that block that process? Maybe they secrete something that says, ‘don’t come near me!’ And then, when stressed, this signal is shut down.”
To determine the effects of these cells in vivo, the team injected the stressed NPC into rat intervertebral discs and compared them to rats injected with non-stressed NPC. They found elevated expression of genes associated with the stressful phenotype in the discs of rats injected with stressed NPC versus non-stressed NPC, including inflammatory markers, similar to the in vitro phenotype. In rat models, pain levels can be assessed by measuring how quickly the animal reacts when its paw is poked with a thin filament. In the present experiment, researchers found that rats injected with the stressed NPC reacted more quickly to paw pokes than rats injected with non-stressed NPC or saline, indicating higher levels of pain. While there was no visible disc degeneration in any of the treatment groups, there was an upregulation of pain-associated genes such as transient receptor potential vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP) in the discs and some evidence of neuronal innervation eight weeks after injection in the stressed NPC group.
Sheyn said that future work needs to be conducted on the outer ring of cells in the intervertebral disc to determine if they play a role in lower back pain. They also need to search for other cell types needed to elicit pain.
Bono said that the study has potential, but since the work was performed in an animal model, much more research needs to be conducted to understand the system fully before the results can be translated into humans. "In order to have a therapeutic that would actually treat the back pain in a degenerated disc, it would have to keep the disc from degenerating while also treating whatever the mechanism of back pain is. It becomes much more complicated. This is understanding one mechanism of how the pain might be induced.”
This work could inform future treatments for discogenic lower back pain. Researchers may someday be able to target the “bad” NPC subtype or supplement the “good” cell types. “The main strategy that we and others are pursuing now is injecting healthy, non-stressed, maybe premature cells that can differentiate to dilute the stressed cells, and in that way, kind of replace them,” said Sheyn.
Reference
- Jiang W, et al. Intervertebral disc human nucleus pulposus cells associated with back pain trigger neurite outgrowth in vitro and pain behavior in rats. Sci Transl Med. 2023(15):eadg7020.
