A new finding could improve and personalize future therapies for chronic pain.  A collaborative Canadian study has discovered sex-specific mechanisms of chronic pain processing in neurons of the spinal cord that are conserved across species from rodents to humans.

Only recently have pain scientists begun to include both sexes in traditionally male-biased studies on how pain is processed (nociception). Earlier studies on male rodent models have implicated disbalances between neural excitation and inhibition in cells of the superficial dorsal horn (SDH) of the spinal cord, as a driver of pain signals. Studies on male rodent models have also shown a protein called BDNF (brain-derived neurotrophic factor) potentiates signaling through a class of excitatory neuroreceptors called NMDARs (N-methyl D-aspartate receptors).

In an established rat model of inflammatory pain, where both males and females display severe pain in response to light touch (tactile allodynia), the scientists of the new study showed a pathway (STEP61–pFyn–pGluN2B) that excessively excites neurons in the spine was activated in males, but not in females. The researchers also showed a pathological molecular coupling that abnormally increases excitatory NMDAR signals in spinal SDH neurons, occurs specifically in males and not females.

The authors noted, “Unlike males, BDNF failed to downregulate inhibitory signaling elements (KCC2 and STEP61) and upregulate excitatory elements (pFyn, GluN2B, and pGluN2B) in female rats, resulting in no effect of ex vivo BDNF on synaptic NMDAR responses in female lamina I [spinal] neurons.”

Through comparisons of the signaling components in the rat model and in human spinal tissue, the authors showed the sex difference in spinal pain processing was conserved from rodents to humans. Like their observations in the rat model, the authors found treatment of human spinal samples with BDNF ex vivo downregulated markers inhibition and upregulated markers of excitation in SDH neurons in males but not females.

To determine the effect of sex hormones on spinal pain processing, the team probed differences in spinal hyperexcitability in ovariectomized female rats and showed when ovaries were removed from female rats they processed pain in a manner that is similar to male rats.

The findings were published in the journal Brain, in an article titled, “A sexually dimorphic neuronal mechanism of spinal hyperexcitability in rodent and human pain models.” This study is the first to identify a sex-related difference in pain signaling in human spinal cord tissue bolsters future precision medicine approaches in the treatment of chronic pain.

Annemarie Dedek, PhD, Eve Tsai, MD, PhD, and Mike Hildebrand, PhD, (left to right) have shown for the first time that neurons in the spinal cord process pain signals differently in women and men. [Carleton University]

“Developing new pain drugs requires a detailed understanding of how pain is processed at the biological level,” said Annemarie Dedek, PhD, who is the first author of the study. “This discovery lays the foundation for the development of new treatments to help those suffering from chronic pain.”

The study was a collaboration among the laboratories of Mike Hildebrand, PhD, an associate professor at Carleton University and an affiliate investigator at the Ottawa Hospital, Eve Tsai, MD, PhD, a neurosurgeon at the Ottawa Hospital and associate professor at the University of Ottawa Brain and Mind Research Institute, Yves De Koninck, PhD, the director of the CERVO Brain Research Center at Laval University, and Jian Xu, PhD, an associate research scientist at Yale University.

Hildebrand, who is the corresponding author of the study said, “These findings have big implications for treating pain in the clinical population. They suggest that in the future, sex may play a role in deciding what analgesics will be prescribed to patients. They may also lay the foundation for discovering new pain therapeutics that will work specifically on the female pain pathway.”

“I think this paper will prove to be a landmark set of findings for the field because it is the first paper to demonstrate that a sex difference in a pain mechanism found in rodents is also found in the human spinal cord,” said Theodore Price, PhD, director of the center for advanced pain studies at the University of Texas, who was not involved in the current study.

Price added, “This demonstrates that an emphasis on only studying male rodents in so much of pain neuroscience has limited our knowledge of basic pain mechanisms in women and has likely hurt our ability to translate basic science into new treatments. We need more emphasis on understanding pain mechanisms in women so we can improve pain therapeutic discovery and development of clinical trial designs that will benefit everyone suffering from chronic pain.”

Inclusive measures

Hildebrand said, “In the shadow of the opioid epidemic, the need to find new therapeutics for pain has never been more urgent. There is, however, a problem: almost all baseline knowledge on mechanisms of pain processing come from investigations of male rodent models, but most chronic pain patients are human females,” said Hildebrand.

Although recent pain studies have indicated differences in pain processing between the sexes, it is currently held that variations in immune responses underlie these differences while neural processing of pain is believed to be the same across males and females. “The shocking part of this assumption is that our understanding of the regulation of neuronal plasticity and excitability is based almost exclusively on studies using male or unsexed rodents,” said Hildebrand.

The current findings point to hitherto unappreciated complexities of pain physiology in the spinal cord. Hildebrand said, “Since their discovery, NMDA receptors have been known to play critical roles in mediating plasticity, including in mediating the plastic changes that can result in chronic pain. Indeed, many clinically available analgesics, like amantadine, ketamine, and magnesium, target and inhibit NMDA receptors. The finding that the role of these NMDA receptors in spinal cord pain processing neurons differs between sexes in both our rat models and in human tissue fundamentally changes how we will look at modulation of excitability and plasticity.”

Bridging translational divides

Hildebrand’s collaborative team undertook this study to patch the gap in knowledge on pain processing in the spinal cord and adopt a more inclusive approach that is relevant to the patient population.

“Hildebrand and colleagues offer an alternative possibility to potentially de-risk translational failures by testing mechanisms leading to pain in live spinal cord slices from human organ donors. Until now, performing studies in the human spinal cord has been challenging,” said Rajesh Khanna, PhD, director of the pain research center at New York University, who was not involved in the current study. “This rigorous study may promote wide-spread adoption and utilization of spinal cord slices from human donors as a key preclinical step in the evaluation of novel drugs for pain.”

The authors systematically investigated molecular mechanisms of neural disinhibition and NMDAR potentiation at SDH synapses in male and female rat models by measuring behavioral pain responses, changes in the distribution and abundance of synaptic proteins, and synaptic NMDAR responses in SDH neurons. To bridge the translational divide, the authors then paired ex vivo BDNF and in vivo inflammatory rodent models of pain with studies on human spinal cord tissue that were generously donated by deceased individuals and their families.

“We have therefore been able to successfully develop assays to isolate, section, stain, sequence, and record from viable human spinal cord neurons under conditions that parallel our preclinical work in rodent pain models,” said Hildebrand.

Since the tissue was collected within one to three hours of aortic cross-clamping under neuroprotective conditions, the quality of the tissue collected was far superior to conventional methods of collecting tissue samples hours after death which reduces cell viability and the integrity of molecular components.

It is critical that scientists use animal models as the foundation for their research. But there is a huge gap that exists when findings from preclinical animal studies are applied to humans. Under the leadership and vision of Eve Tsai, our team has been bridging this translational divide by implementing the use of human spinal cord tissue collected from consenting organ donors, thus allowing us to validate targets in human tissues at early stages of discovery,” Hildebrand said.

In an earlier study published in Brain in 2019, Hildebrand’s team had reported the ex vivo human spinal pain model from donor tissue and the conservation of the pain signaling pathway across male rats and humans. “We have now used this model to conclude that this pathway is sexually dimorphic, and that the sexual dimorphism is conserved across species as well,” said Hildebrand.

Next steps

The team intends to continue to explore the biological differences in pain perception between men and women using new tools and approaches and high-quality human spinal tissues. The team is conducting some of the first electrophysiological studies on human spinal cord pain processing neurons and partnering with international collaborators to develop a genetic expression map of each cell in the human spinal cord.

“Our future studies will, amongst other things, look at circuit-wide changes in spinal cord molecular signaling and excitability in rats and humans of both sexes,” said Hildebrand. “We hope these investigations will continue to bridge the gap between basic science rodent models and treating human patients in the clinic and highlight the importance of conducting inclusive research on both sexes.”

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