Ketamine interactions with gut-microbiota in rats: relevance to its antidepressant and anti-inflammatory properties

This animal study investigated ketamine (2.5 mg/kg) interactions with gut-microbiota in rats to understand its antidepressant and anti-inflammatory properties. The data concluded that there are some antidepressant and anti-inflammatory effects of ketamine treatment through its interaction with specific gut bacteria such as Lactobacillus, Turicibacter, and Sarcina and confirmed the usefulness of microbiome as a target for therapy using ketamine for some of its anti-inflammatory effects for specific inflammatory diseases including Irritable bowel syndrome (IBS). The study called for more detailed investigations of the interaction of microbiome with central mediators of mood and/or inflammatory disorders.

Abstract

“Background Appreciable evidence suggest that dysbiosis in microbiota, reflected in gut microbial imbalance plays a key role in the pathogenesis of neuropsychiatric disorders including depression and inflammatory diseases. Recently, the antidepressant properties of ketamine have gained prominence due to its fast and long lasting effects. Additional uses for ketamine in inflammatory disorders such as irritable bowel syndrome have been suggested. However, ketamine’s exact mechanism of action and potential effects on microbiome is not known. Here, we examined the effects of low dose ketamine, known to induce antidepressant effects, on stool microbiome profile in adult male Wistar rats. Animals (5/group) were injected intraperitoneally with ketamine (2.5 mg/kg) or saline, daily for 7 days and sacrificed on day 8 when intestinal stools were collected and stored at − 80 °C. DNA was extracted from the samples and the 16 S rRNA gene-based microbiota analysis was performed using 16S Metagenomics application.

Results At genus–level, ketamine strikingly amplified Lactobacillus, Turicibacter and Sarcina by 3.3, 26 and 42 fold, respectively. Conversely, opportunistic pathogens Mucispirillum and Ruminococcus were reduced by approximately 2.6 and 26 fold, respectively, in ketamine group. Low levels of Lactobacillus and Turicibacter are associated with various disorders including depression and administration of certain species of Lactobacillus ameliorates depressive-like behavior in animal models. Hence, some of the antidepressant effects of ketamine might be mediated through its interaction with these gut bacteria. Additionally, high level of Ruminococcus is positively associated with the severity of irritable bowel syndrome (IBS), and some species of Mucispirillum have been associated with intestinal inflammation. Indirect evidence of anti-inflammatory role of Sarcina has been documented. Hence, some of the anti-inflammatory effects of ketamine and its usefulness in specific inflammatory diseases including IBS may be mediated through its interaction with these latter bacteria.

Conclusion Our data suggest that at least some of the antidepressant and anti-inflammatory effects of daily ketamine treatment for 7 days may be mediated via its interaction with specific gut bacteria. These findings further validate the usefulness of microbiome as a target for therapeutic intervention and call for more detailed investigation of microbiome interaction with central mediators of mood and/or inflammatory disorders.“

Authors: Bruk Getachew, Joseph I. Aubee, Richard S. Schottenfeld, Antonei B. Csoka, Karl M. Thompson & Yousef Tizabi

Summary

Abstract

Rats were injected with ketamine or saline daily for 7 days and their stools were collected on day 8. DNA was extracted and 16S rRNA gene-based microbiota analysis was performed using 16S Metagenomics application.

Ketamine increased the levels of Lactobacillus, Turicibacter and Sarcina in the gut and reduced the levels of opportunistic pathogens Mucispirillum and Ruminococcus. These effects may explain some of the antidepressant effects of ketamine and its usefulness in specific inflammatory diseases including IBS.

Our data suggest that some of the antidepressant and anti-inflammatory effects of ketamine may be mediated via its interaction with specific gut bacteria.

Background

Evidence suggests that the brain and the gut microbiota are in bidirectional communication with each other and also with inflammatory processes. This interaction may be involved in mood dysregulation. Depressive symptoms are often co-morbid with gastrointestinal disorders, and treatment of one condition can reverse the risk for the other. Moreover, some antidepressants may also possess antimicrobial properties, and a single bacterium such as Bifidobacterium infantis can reverse depressive-like behavior.

Ketamine has been used off-label for depression due to its prompt and sustained antidepressant effects. The sustained effectiveness of acute ketamine is likely mediated by additional mechanisms, including interaction of acute ketamine with gut microbiota.

Ketamine has been advocated for use in inflammatory diseases such as ulcerative colitis, but no studies have been carried out on interaction between ketamine and gut microbiota implicated in inflammatory diseases.

Animals

Age matched adult male Wistar rats were housed in a designated room at 24 – 26 °C at 51 – 66% relative humidity on a 12-h light/dark cycle.

Experimental design

The animals were randomly divided into two groups, control and experimental, and were housed in separate cages. The number of animals used in each group was based on behavioral observations seen using similar number of animals.

The experimental group received 2.5 mg/kg of ketamine intraperitoneally (i.p.) daily for 7 consecutive days, and the volume of injection was 1 ml/kg.

Sample collection

On day 8, animals were sacrificed and stools were collected. They were quick-frozen on dry ice and stored at 80 °C.

Stool DNA extraction

Total DNA was isolated from stool samples using Norgen’s Stool DNA Isolation Kit and the Precellys Dual-24 Homogenizer. The DNA was purified using spin column chromatography and analyzed using the 16S rRNA gene.

16S rRNA gene sequencing and analysis

A 16S rRNA gene was amplified from stool DNA and sequenced on the Illumina MiSeq platform. The data was analyzed using the Illumina 16S Metagenomics app and a high-performance implementation of the Ribosomal Database Project (RDP) Classifier.

Diversity and richness

A total of 1121 different bacterial species were identified in both saline and ketamine groups, but no significant difference was found in either diversity or richness.

Phylum-level effects

Ketamine reduced abundances of two low-abundance phyla, Deferibacteres and Tenericutes, by approximately 22 and 2 fold, respectively, compared to saline control group.

Order-level effects

Ketamine increased the abundance of Turicibacterales order by 28 fold and reduced the abundance of four other orders.

Family-level effects

Ketamine increased the number of Tuberibacteraceae, Clostridiaceae and Lactobacillaceae in the gut by 98 fold, 89 fold and 1.5 fold, respectively, compared to the saline control group.

Genus-level effects

Ketamine significantly enriched abundances of genera Sarcina, Turicibater, Lactobacillus, while levels of Mucispirillum and Ruminococcaceae were decreased by 26.3 and 2.3 fold, respectively, compared to saline control group.

Discussion

Chronic low dose ketamine altered gut microbial ecology, with Lactobacillus and Turicibacter being increased, whereas Mucispirillum and Ruminococcus were reduced. These changes may be related to ketamine’s antidepressant properties.

Ketamine has been shown to reduce the levels of microorganisms associated with inflammatory processes, including Mucispirillum and Ruminococcus. This may be an additional novel mechanism for ketamine’s anti-inflammatory effects.

Changes in the gut ecosystem can be caused by changes in low-abundance groups such as Lactobacillus, Sarcina and Turicibacter, which can degrade complex polysaccharides to short chain fatty acids such as butyrate, which can be used as a source of energy by the host. Mucispirillum is an anaerobic gram-negative bacterium that can constantly generate lipopolysaccharide (LPS), which can lead to inflammation and dysbiosis. Exercise-induced stress may have similar effects on microbiome.

Ketamine’s reduction of Ruminococcaceae and reversal of the behavioral deficits induced by social defeat suggest that Ruminococcaceae may play a role in stress-induced depressive behavior. Ketamine’s elevation of gut genera Lactobacillus, Sarcina and Turicibacter might be a contributory factor to its anti-inflammatory effects.

The gut bacteria Mucispirillum can influence the colonic mucus layer, leading to leaky gut, which is associated with depression and intestinal disorders. Ketamine might also have anti-inflammatory and antidepressant effects through reduction of Mucispirillum and gut permeability.

Elucidating the direct microbiome influence on neurobiological substrates of mood and on peripheral and central mediators of inflammatory processes may provide novel therapeutic targets in these disorders. Furthermore, manipulation of gut microbiota may also be a novel approach in combatting inflammatory disorders including colitis.

Conclusion

Ketamine administration results in increased levels of low-abundance bacteria genera and decreased levels of opportunistic pathogens in male Wistar rats. This may contribute to the sustained antidepressant and anti-inflammatory effects of ketamine.