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April 14, 2024

Alison Xie describes analgesic effects of glial-neuron interactions

By ABIGAIL TUSCHMAN | October 31, 2023

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COURTESY OF ABIGAIL TUSCHMAN

By activating Gq-GPCR signaling pathways, Xie was able to produce an analgesic effect in mice.

The Department of Biology hosted a seminar on Thursday, Oct. 26 featuring Alison Xie, an assistant research professor of surgery-urology at the University of Colorado Anschutz Medical Campus. The talk, titled “Glial-Neuron Interactions in Sensory Ganglia Alleviate the Symptoms of Chronic Pain in Mice,” discussed Xie’s research on how Gq-GPCR activation in satellite glial cells (SGCs) of the sensory ganglia can produce analgesic effects in mouse models. 

Xie and her team used injections of the bio-inert small molecule Clozapine N-oxide (CNO) to activate the Gq-GPCR signaling pathways in sensory ganglionic SGCs of mice. The specific glial cells tested were glial fibrillary acidic protein-positive (GFAP+). The first experiment Xie discussed, which was described in a 2020 paper published in Scientific Reports, used Complete Freund’s Adjuvant (CFA) to induce inflammatory pain in mice’s hind paws.

“This CFA is a pretty nasty molecule,” Xie said. “You inject it and start seeing redness and inflammation in the paw in 24 hours. Then the mouse will be so hypersensitive that they won’t even put their foot down. With this, we can quantify the sensitivity of those mouse hind paws by using [a] Von Frey assay.”

A Von Frey test measures mechanical sensitivity in mice. Researchers can use Von Frey filaments of different thicknesses and stiffness to measure a mouse’s threshold of hind paw retraction.

Xie described how a control mouse can typically withstand 0.4 grams of force without retracting. However, a mouse whose paw is red and inflamed can tolerate much less force before retracting its paw. A CFA-injected paw has around a 60% reduction in the threshold of retraction.

Xie explained the result of injecting CNO to combat the CFA injection. 

“After one CNO injection, within 15 minutes, you can see a pretty big difference in the threshold [of retraction]. The threshold basically just reverts back to the baseline,” she said. “The paw is still red and swollen, but the mouse just stands on it as if the CFA was not injected. This lasts about 90 minutes to up to two hours.”

The researchers demonstrated that CNO’s analgesic effect is independent of the paw inflammation produced by CFA injection. They discovered that even a paw initially injected with saline, rather than CFA, experiences about a 50% increase in the retraction threshold once administered CNO. 

Xie and her team found that central nervous system activation is not sufficient to induce this effect and that peripheral nervous system glial activation is necessary. They performed a variation of the experiment in which the mice were pre-treated with either trospium chloride or a saline control before the injection of CFA.

The drug trospium chloride is a peripheral muscarinic receptor blocker and is prescribed to patients with overactive bladder. According to Xie, the medication has minimal impact on the central nervous system because it does not cross the blood-brain barrier.

While pretreatment with a saline injection did not affect the CNO-induced reduction of mechanical allodynia, the injection of trospium chloride resulted in a complete block of CNO-induced analgesic effects. 

To further study how CNO induces an analgesic effect, Xie tested mice’s reaction to thermal pain rather than the inflammation caused by CFA. By measuring the latency with which a mouse moved its paw when exposed to a hot laser, Xie and her team could quantify the change in the mice’s pain threshold — the time of retraction nearly doubled for mice injected with CNO.

Xie conducted a similar experiment using histamine-induced itching. While control mice receiving an injection of histamine would scratch themselves about 100–120 times over the course of 30 minutes post-injection, mice treated with CNO would completely stop scratching.

“This [was] a pretty strong phenotype, and [it] gave us a lot of confidence,” Xie said.

Xie and her team determined that the CNO-induced analgesic effect is dependent on the activation of adenosine A1 receptors in the peripheral nervous system.

When Xie presented her research to her colleagues in the urology department, they asked her about CNO’s effect on visceral sensitivity rather than somatic sensitivity. 

“There are a lot of diseases that we treat in the patients in the urology department that actually have an underlying mechanism of hypersensitivity in the visceral sensory pathways,” she said.

To test how CNO affects visceral pain in mice, Xie and her team measured the effects of CNO injection in an overactive bladder mice model. Xie and her team created this model by using vascular endothelial growth factor A (VEGFA) to induce symptoms of urologic chronic pelvic pain syndrome in mice. Xie explained that VEGFA is a biomarker of overactive bladder. The severity of the mice’s symptoms was measured using spontaneous voiding spot assays and manual Von Frey tests. 

Xie highlighted how this experiment studying visceral pain, unlike those on somatic pain, demonstrated the need to separate male and female data due to a demonstrated difference in baseline sensitivity. 

“This is something we actually [have] never seen in the somatic activity,” she said. “In terms of visceral hypersensitivity, mechanically, male and female [mice] are very different. We think this is also a little bit behavior [related] because we have to put a filament against their lower abdomen in the vicinity of their bladder area. And [in] the males, that’s basically [in] the vicinity of their penis, and they hated to have anything just come at it. So they would just always retract.”

However, in both male and female mice, CNO injections reduced the hypersensitivity to even lower than baseline levels.

Xie wrapped up the seminar by discussing additional findings of her research. She found that peroxisome proliferator-activated receptor (PPAR) signaling pathways may contribute to visceral hypersensitivity in both males and females. Additionally, there may be sex-specific mechanisms during the VEGFA-induced pathogenesis of visceral hypersensitivity, as indicated by the observed differences in immuno- and nociceptive-signaling between males and females. 

In an interview with The News-Letter, Cameron Allen, a graduate student in the Doctoral Program in Cell, Molecular, Developmental Biology and Biophysics who attended the seminar, expressed his surprise at Xie’s discovery that CNO can be used to alleviate different kinds of pain.

“I think the most interesting part to me was the weird discovery that this was working with multiple different types of stimuli,” Allen said. “Actually being able to see both somatic and visceral [analgesic effects] was very interesting. It’s a very robust response to see from such a small change.”

Xie spoke about how the results of the experiment were a complete surprise in an interview with The News-Letter. Xie and her team had previously hypothesized that another type of glia was producing the analgesic effect of interest. 

“We accidentally found out that this population of glial cells was important, as opposed to another population we cared about,” she said. “It’s unexpected but exciting because the data kind of pushes you into a new territory that you never thought about before.”

Xie also discussed how the trial and error involved in research can lead to satisfying results.

“You’re trying to prove yourself wrong every step along the way,” she said. “But, in the end, you get there [and] are saying, ‘Oh, I’m right,’ or, ‘My initial hypothesis is right. These [glial cells] are indeed important. I can’t disprove myself.’ That’s kind of exciting.”


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