Chronic opioid treatment leads to neuroinflammation, hyperalgesia, and anhedonia. Recent studies in both humans and rodents show that disruption of the gut microbiota is also a consequence of opioid use. Now, a new study led by Anna Taylor, University of Alberta, Edmonton, Canada, shows a causal link between morphine-induced alterations in the gut microbiota and the symptoms of opioid dependence, and suggests that targeting those changes may help alleviate the adverse side effects of opioids.
Taylor and colleagues show that both sustained and intermittent morphine treatment altered the gut microbiota in mice. However, only intermittent morphine, which was interspaced by daily periods of morphine withdrawal, produced the microglia-driven neuroinflammation, hyperalgesia, and impaired reward processing that are associated with opioid use. Microbiota transfer from intermittent morphine-treated mice to opioid-naïve mice recapitulated those outcomes, suggesting a causal connection between an altered gut microbiota resulting from morphine use and the negative effects of the drug.
“I thought it was a nice paper bringing in a new component for us to consider,” said Peter Grace, University of Texas MD Anderson Cancer Center, Houston, US, referring to the authors’ newly identified role for the gut microbiota. Grace was also particularly interested in the differential effects found between intermittent versus sustained morphine administration. “It is a good lesson for all of us who work on the neuroimmunology of opioids, as it suggests that what we might actually be studying is the withdrawal and the effects related to that [rather than the effects of the drug itself]. That is an important consideration for future study design,” according to Grace, who was not involved with the new work.
The study was published online September 10 in Neuropsychopharmacology.
Suspecting a role for the gut microbiome
Microglia-driven neuroinflammation contributes to opioid-induced hyperalgesia and tolerance (see PRF related news here and here), as well as opioid withdrawal (see PRF related news). Taylor’s previous work showed that, after morphine withdrawal in rodents, microglia activation in the ventral tegmental area (VTA), a key region in the brain’s reward system, led to decreased dopamine signaling and impaired reward behavior that is typically seen with chronic opioid administration (Taylor et al., 2016).
As Taylor further pursued the mechanisms of opioid-driven neuroinflammation, she noticed the growing list of behavioral disorders, including autism, schizophrenia, and depression, in which the gut microbiome was implicated. She noted that, much like opioids, changes in the microbiome influence neuronal signaling as well as immune cell activation in the brain. This raised the possibility that the microbiome could have a role in the adverse effects of opioids.
The researchers in Taylor’s group were also encouraged to investigate the gut microbiome because opioids have profound effects on gut motility, including constipation during prolonged opioid use, and diarrhea during opioid withdrawal; such changes in motility are known to impact microbiota composition. “Given this evidence, we thought it was a pretty sure-fire thing that opioids are influencing the gut microbiome,” Taylor said. And first author Kevin Lee, University of California, Los Angeles, US, added, “We thought that perhaps the microbiome might have something to do with the negative effects of opioid treatment.”
When Taylor’s group began the experiments in the current study, there were no published reports of opioid-induced changes in gut microbiota composition. Since then, several researchers have shown these results in humans (Acharya et al., 2017) and in rodent models (Banerjee et al., 2016; Wang et at., 2018).These studies all showed similar broad changes in microbiota composition but differed in their quantification of individual bacterial species. Taylor attributes these differences to variability in the RNA sequencing technique used to quantify bacterial gene expression, but also noted that the results depend on the type of opioid used as well as the route of administration and length of treatment.
Testing the hypothesis
Using both sustained and intermittent morphine treatment regimens, the team assessed three major opioid side effects: tolerance, hyperalgesia, and altered reward processing. In the sustained treatment group, morphine was delivered via a subcutaneous pellet, and in the intermittent treatment group, the researchers gave intraperitoneal morphine injections twice a day for four days. According to tail flick testing, both treatment regimens resulted in morphine tolerance, but only the intermittent group exhibited hyperalgesia. The sustained group developed hyperalgesia only after the morphine pellet was removed and the mice underwent 12 hours of withdrawal.
To assess reward behavior, the researchers employed a cocaine conditioned place preference assay. The sustained group, along with control groups, demonstrated a preference for cocaine, indicating normal reward processing. But the intermittent group lacked this preference, indicating an impaired reward response, which is thought to be associated with the anhedonia seen in opioid dependence.
After these behavioral tests, the team looked for signs of inflammation in the spinal cord and VTA by assessing microglia morphology. They found that, consistent with the behavioral results, only the intermittent group displayed significant increases in microglia cell size, in both the spinal cord dorsal horn and the VTA. The sustained group showed no difference in microglia cell size, unless the morphine pellet was removed 12 hours prior to analysis.
A causal relationship
In tandem with the behavioral studies, the team collected fecal pellets for microbiota analysis. Within each treatment group, the researchers investigated bacterial diversity and relative abundances of specific taxa. Both the intermittent and sustained groups differed from control groups in terms of diversity measures. However, the treatment groups differed in microbiota composition. The intermittent group showed a decrease in the Lactobacillus genus and an increase in the Ruminococcus genus. The sustained group did not exhibit these changes and instead showed increases in Clostridium and Rikenellaceae genera.
The defining task of the study was to determine whether disruption of the microbiota by intermittent morphine treatment was sufficient to induce an opioid dependence phenotype. To show this, opioid-naïve mice were colonized with microbiota samples from the intermittent morphine-treated group or a control saline-treated group; before the experiment, the recipient mice underwent antibiotic treatment to deplete their native microbiota. Unexpectedly, the antibiotics alone resulted in hyperalgesia, impaired reward processing, and inflammation in the VTA and dorsal horn, mirroring the intermittent morphine treatment side effects.
“We thought it was very surprising that just by treating with antibiotics, without treatment with opioids, we would be able to see that kind of behavior and neuroinflammation,” said Lee. Recolonization with healthy microbiota from the saline-treated group reversed these symptoms. In contrast, recolonization with intermittent morphine-associated microbiota perpetuated the symptoms.
Taylor acknowledged that the unanticipated negative effects of antibiotic treatment are controversial. “Other studies have found that antibiotic treatment has either no effect on pain or it alleviates pain,” she noted (see PRF related news story). The abnormal phenotype seen in the current study may be explained by the high doses of broad-spectrum antibiotics the researchers used in their experiments. Taylor said that they chose this cocktail because it is effective in depleting the microbiota and closely mimics mice raised in germ-free colonies. “I think we are causing a lot of effects outside of the gut in response to the strong antibiotic cocktail. It’s probably a stressful experience and may be mimicking a sickness behavior,” she explained.
Questions for the future
The study suggests that an opioid-disrupted microbiota may drive the symptoms of opioid dependence, and that there is potential to alleviate opioid side effects via gut microbiota manipulation. But many questions remain.
“The biggest unanswered questions are, Which specific bacteria work together to result in these effects and how can we target those bacteria?” Lee said. Taylor doubts that any one species is responsible for the effects seen in their study. “These bacteria exist within a population, and my feeling is that it’s the ecosystem of bacteria that’s important,” she noted.
In future studies, instead of narrowing down individual populations, Taylor aims to prevent microbiota disruption in the first place. Her strategy is to administer a locally acting opioid antagonist such as methylnaltrexone along with morphine in order to prevent opioid effects on intestinal motility. She hopes that this will “retain the analgesic and rewarding properties of opioids without causing changes in gut motility and changes in gut microbiome associated with chronic opioid use.”
Understanding the gut microbiota in the context of pain disorders and opioid dependence may lead to better-informed treatments. The current study provides insight into why the antibiotic minocycline did not succeed in clinical trials for pain treatment, according to Grace. “It could be a tug-of-war between direct microglial inhibition by minocycline as well as indirect activation by causing dysbiosis of the microbiome. This is clearly another system that we’ve been ignoring for a while,” he said.
Sarah Najjar is a PhD candidate at the University of Pittsburgh, US.
Image credit: illustrator/123RF Stock Photo.
