Chronic pain states are challenging to control with current drug therapies. Here, we demonstrate that a single dose of psilocybin can produce a sustained anti-nociceptive effect in a mouse model of chronic neuropathic pain. Beyond this, the single dose of psilocybin caused a dramatic increase in the anti-nociceptive potential of gabapentin, a widely used treatment for neuropathic pain, such data are suggestive of establishment of longer lasting changes in network processing.
Article
Psilocybin ameliorates neuropathic pain-like behaviour in mice and facilitates the gabapentin-mediated analgesia
https://doi.org/10.21203/rs.3.rs-5026806/v1
This work is licensed under a CC BY 4.0 License
Version 1
posted
You are reading this latest preprint version
Chronic pain affects millions of people worldwide and presents a huge social and economic burden. Long lasting pain negatively affects the quality of life of patients1 and is associated with significant unmet clinical need. Management of chronic pain is difficult and clinically available drugs are often poorly tolerated and/or potentially addictive2,3. Chronic pain patients often develop affective comorbidities, such as depression and anxiety4 that are frequently associated with worsening clinical outcomes5.
Psilocybin is a classic psychedelic drug that typically causes a profound alteration of perception and mood6. The active metabolite of psilocybin is psilocin, which is known to bind to multiple serotoninergic receptors, of which 5-hydroxytryptamine (5-HT) 2A is necessary for the induction of the psychedelic effect7. There has been a resurgence of interest in the clinical potential of psilocybin for mental health conditions such as major depression disorders8, diseases which are co-morbid with pain. Positive treatment outcomes following a single treatment with psylocibin have been associated with the long-term modulation of intrinsic brain networks and the resetting of aberrant connectivity that reinforced negative patterns of behaviour9.
There are also some early indications that psilocybin may alleviate intractable phantom limb pain10 and migraine11 in humans and reduce mechanical hypersensitivity in an inflammatory pain model in male and female rats12. Investigating neuronal networks that might sustain chronic pain states remains at an early stage13, but maladaptive changes to functional and structural connectivity were demonstrated to precede the onset of chronic subacute lower back pain in human patients14. This led us to consider the potential value of psilocybin as a potential treatment for ‘unlocking’ neuronal networks that support chronic neuropathic pain (see Askey et al, submitted). Moreover, we also examined the hypothesis that psilocybin could positively affect the anti-nociceptive response of analgesics established as treatments for neuropathic pain.
The experimental design is summarised in figure 1a. The spared nerve injury (SNI) mouse model is a preclinical model of neuropathic pain involving partial transection of the peripheral nerves innervating the hind paw.15 A low and higher dose of psilocybin (0.3 and 1mg/kg) were examined across a range of different behavioural tests that measure both reflexive and affective responses to mechanical and thermal stimulation of the hind paw.
Male mice underwent SNI surgery and, when static mechanical hypersensitivity was fully developed (day 12), they received a single intraperitoneal (i.p.) injection of psilocybin (0.3 mg/kg) or saline control (Fig 1 a). The head-twitch response (HTR) is considered a rodent behavioural proxy of the human response to a psychedelic experience16 and an increase in HTR was observed in the psilocybin treatment groups compared to saline control (Fig 1 b) confirming central nervous system exposure to the drug. Mechanical hypersensitivity was reduced (Maximum Possible Effect, MPE=18%) in mice treated with psilocybin with the effect lasting up to day 28 after injection (test day 40) (Fig 1 c). However on test days 56 and 65, there was a maintained reduction in the affective behaviours that characterise dynamic mechanical hypersensitivity to light brush stroke (Fig 1 d) and the licking/biting response to a cold stimulus17 (Fig 1 e). Single dose of psilocybin had no effect on locomotor performance (Fig 1 f), consistent with psilocybin not causing unwanted motor function deficits. Taken together we observed an anti-nociceptive response of psilocybin particularly on affective motivational responses to noxious stimulation.
We next tested whether repeated injection of psilocybin (0.3mg/kg) could amplify the anti-nociceptive effects observed. Psilocybin was injected every 7 days for 3 weeks in male mice that had undergone SNI or sham surgery. A reduction of mechanical hypersensitivity was observed in SNI mice. Repeated injections of psilocybin (0.3 mg/kg) substantially prolonged and amplified the antinociceptive effect of psilocybin for several weeks (Fig 1 g) in comparison to a single dose (Fig 1 c) (%MPE= 62.6 in the same group of mice (SNI) before and after psilocybin injection). No changes in mechanical threshold were observed in sham mice.
In a second series of experiments, a single higher dose of psilocybin (1 mg/kg i.p.) (Fig 2 a) induced a robust HTR (Fig 2 b). At this dose, psilocybin-induced reduction in mechanical hypersensitivity (MPE=25.5%) (Fig 2 c) was comparable to the lower dose (MPE=18% 0.3mg/kg) and persisted until day 34 after injury. Psilocybin was able to reduce hypersensitivity to light brush that develops after peripheral nerve injury (Fig 2 d) at day 17 and 29 after injury. Here, we used the thermal place preference test (TPP)18 to assess cold sensitivity and recorded the total amount of time mice spent on the cold plate before (Bs) and after nerve injury (day 1 to day 30). Mice treated with psilocybin spent substantially more time on the cold plate compared to saline-injected mice by day 30 (Fig 2 e) (psilocybin, 65.10 s ± 23.4; saline, 18.9 s ±3.6 s). Next, we analysed faecal output as a measure of stress in mice19 after SNI for psilocybin (1mg/kg) vs saline controls. Psilocybin treatment reduced faecal boli output in mice after SNI surgery (Fig 2 d), this was also associated with increased body weight (Fig S1). These data are consistent with the hypothesis that psilocybin may reduce the stress that follows injury.
Overall, we have shown here, for the first time, that a multiple low dose treatment with psilocybin can attenuate pain-like behaviour for over 30d following peripheral nerve injury and that this treatment regimen was as effective as a single large dose of drug.
The mechanisms underlying the effects of psilocybin are not fully understood but are thought to involve the modulation of normal patterns of communication between different areas of the brain. Altered brain functional connectivity in chronic pain patients and micehave been observed20,21 and the analgesic effect of psychedelic drugs could be due to their capacity to drive neuroplasticity and reset aberrant connections that support chronic pain22. Given that the effects of a single dose of psilocybin can last for many months in both people with depression23 and control groups, we hypothesised that psilocybin might be able to influence pain processing networks in mice beyond the period when alteration in pain behaviours were seen. We therefore investigated the effect of psilocybin on response to gabapentin. Gabapentin is widely used in clinical practice to treat neuropathic pain, but not all the people with neuropathic pain achieve adequate pain relief with gabapentin24; moreover, gabapentin use is also associated with side effects24 and a risk of addiction25. Mice received a single i.p. injection of gabapentin (50mg/kg) at day 45 after surgery when the anti-nociceptive effect of psilocybin was no longer measurable (Fig 2 g). In mice treated with psilocybin (1mg/kg), a dramatic prolonged anti-nociceptive effect of gabapentin from 2h to 96h was observed compared to mice treated with saline vehicle (Fig 2 g).
Developing safer, effective treatments for chronic pain has proven challenging. Here, we have demonstrated that psilocybin can reduce neuropathic pain-like behaviour in male mice for up to 30 days, particularly the affective component, that this reduction in pain behaviour can be amplified by repeated treatment with low dose psilocybin and finally that at later time points psilocybin can potentiate the effect of gabapentin, a standard treatment for neuropathic pain in humans.
At this point we have only looked at male mice and it will be important to determine whether sex differences occur26 , although recent research has reported no differences between males and females in response to psilocybin in a rat model of inflammatory pain12. Further studies will also be required to determine the mechanism by which psilocybin mediates the anti-nociceptive effects demonstrated here. In this regard, there is growing evidence that there are changes in frontal lobe connectivity associated with chronic pain27 and that psilocybin and other psychedelic drugs can drive neural plasticity and to reorganise neural networks associated with the frontal cortex28. Taken together it seems likely that gabapentin and psilocybin synergise to produce a powerful long-term effect on neuronal networks that generate neuropathic pain. It will therefore be important to determine if this unique observation can be extended to the actions of other drugs used to target chronic neuropathic pain such as amitriptyline and duloxetine29. Together, these data provide the first preclinical demonstration that psilocybin could be an effective tool for the management of chronic pain due to nerve injury and also to provide a new therapeutic adjunct for the control of chronic pain.
Experimental design
This study was designed to evaluate the effect of psilocybin on pain sensitivity. In all experiments, mice were randomly assigned into treatment groups. The experimenter was always blind to treatment. The number of mice in each group are designated in the individual figures.
Animals
Animal use for pain studies comply with the ARRIVE guidelines30. All efforts were made to minimize animal suffering (UK Animal Act, 1986) and to reduce the number of mice used (3Rs). Experiments used the minimum number of animals to provide sufficient statistical power calculated, based on our previous experience with behavioural assays.
Adult male C57BL/6J mice (8 to 12 weeks old) were purchased from Charles River (UK) and housed to acclimatize for 1 week prior to experiments.
All mice were kept in their home cage in a temperature-controlled (20° ± 1°C) environment, with a light-dark cycle of 12 hours (lights on at 7:30 a.m.). Food and water were provided ad libitum.
This study was conducted under Project Licence PPL PP9720547.
Mouse model of neuropathic pain: spared nerve injury (SNI)
The SNI surgery was performed as previously described15. Briefly, under isoflurane anaesthesia, the skin on the lateral surface of the thigh was incised, and a section made directly though the biceps femoris muscle, exposing the sciatic nerve and its three terminal branches: the sural, the common peroneal, and the tibial nerves. The common peroneal and the tibial nerves were tightly ligated with 5 − 0 silk suture and sectioned distal to the ligation. Great care was taken to avoid any contact with the spared sural nerve. Complete haemostasis was confirmed, and the wound was sutured.
Drugs
Psilocybin (COMP360, a proprietary formulation of synthetic psilocybin) was provided by Compass Pathfinder, (a subsidiary of Compass Pathways) and was dissolved in saline. Control mice received the same volume of saline.
Gabapentin was purchased from Sigma and was dissolved in saline.
Behavioural testing
The experimenters were always blind to treatment group for all behavioural tests.
Von Frey filament test for static mechanical sensitivity
For the assessment of mechanical sensitivity, the von Frey filament test was used as previously described17. Mice were placed in Plexiglas chambers, located on an elevated grid, and allowed to habituate for at least 1h. After this time, the plantar surface of the paw was stimulated with a series of calibrated von Frey monofilaments, mechanical sensitivity threshold was determined using the up-down-method31. The data were expressed as log of the mean of 50% pain threshold ± SEM.
Brush test for dynamic mechanical sensitivity
Dynamic allodynia was tested by light stroking (velocity ~ 2cm/s) of the external lateral side of the injured hind paw in the direction from heel to toe using a paintbrush. The protocol was adapted from Duan and colleagues32. Mice were placed in Plexiglas chambers, located on an elevated grid, and allowed to habituate for at least 1h. Observed responses were scored: 0, no response or moving the stimulated paw; 1, single withdrawal, flick or stamp of the stimulated paw; 2, multiple withdrawals of the stimulated paw in rapid succession; 3, licking of the plantar surface or continued elevation/withdrawal of the stimulated paw. The stimulation was repeated three times at intervals of at least 3min and the average scores was obtained for each mouse.
Acetone test for cold sensitivity
For assessment of cold sensitivity, the acetone test was used as previously described17. Mice were placed in Plexiglas chamber located on an elevated grid for 1h and then a drop (~ 50µl) of acetone was applied to the external lateral side of the injured hind paw. Total time licking/biting of the hid paw was recorded for 20 s.
Rotarod test
Locomotor activity was analysed as previously described34. Briefly, in this study we used an accelerating rotarod apparatus with a 3cm diameter rod starting at an initial rotation of 4rpm and slowly accelerating to 40rpm over 100s. Mice were expected to walk at the speed of rod rotation to keep from falling. Mice were not tested at baseline to minimize the number of tests on the apparatus. Time taken to fall from the rod was recorded.
Fecal pellet output
Mice were placed in Plexiglas chamber located on an elevated grid for 1h, after this time the number of fecal pellets were counted for each mouse.
Data and statistical analysis
All statistical tests were performed using the IBM SPSS Statistic Programme (version 27), and P < 0.05 was considered statistically significant. Data are means ± s.e.m. and independent experiment unit (n) is animals. As previously described17, data of von Frey filaments test was log transformed to ensure a normal distribution34.
Difference in sensitivity was assessed using repeated measure mixed model ANOVA. In all cases, “time” was treated as within-subject factors and “treatment” was treated as between-subject factor.
The MPE (Maximum Possible Effect) was calculated as previously described and according to the formula:
where log(0.6) is our maximum von Frey’s force applied.
The protocols used in this study have been reviewed and approved by the University of Reading Animal Welfare and Ethical Review Body (AWERB) and the study was carried out under the authority of Maria Maiarú’s Home Office PPL number PP9720547
Contributions
TA, MA, SPH, GJS and MM conceived and designed the experiments; TA, DAR and MM performed the experiments; TA and MM analysed the data; TA, MA, SPH, GJS and MM drafted the paper and MM wrote the manuscript. All authors revised and edited the manuscript.
Competing interests
The authors declare no competing interests.
Funding
This work was supported by Compass Pathfinder Limited (a subsidiary of Compass Pathways) and the University of Reading Strategic PGR Studentship (to support TA) and by the Academy of Medical Sciences Springboard SBF008\1092 awarded to MM
- Mills, S. E. E., Nicolson, K. P. & Smith, B. H. Chronic pain: a review of its epidemiology and associated factors in population-based studies. Br J Anaesth 123, e273–e283 (2019).
- Finnerup, N. B. et al. Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. The Lancet Neurology 14, 162–173 (2015).
- Volkow, N. D. & McLellan, A. T. Opioid Abuse in Chronic Pain — Misconceptions and Mitigation Strategies. N Engl J Med 374, 1253–1263 (2016).
- Robinson, E. S. J. Translational new approaches for investigating mood disorders in rodents and what they may reveal about the underlying neurobiology of major depressive disorder. Phil. Trans. R. Soc. B 373, 20170036 (2018).
- Bair, M. J., Robinson, R. L., Katon, W. & Kroenke, K. Depression and Pain Comorbidity: A Literature Review. Arch Intern Med 163, 2433 (2003).
- Hanks, J. B. & González-Maeso, J. Animal models of serotonergic psychedelics. ACS Chem Neurosci 4, 33–42 (2013).
- Madsen, M. K. et al. Psychedelic effects of psilocybin correlate with serotonin 2A receptor occupancy and plasma psilocin levels. Neuropsychopharmacol. 44, 1328–1334 (2019).
- Carhart-Harris, R. et al. Trial of Psilocybin versus Escitalopram for Depression. N Engl J Med 384, 1402–1411 (2021).
- Daws, R. E. et al. Increased global integration in the brain after psilocybin therapy for depression. Nat Med 28, 844–851 (2022).
- Ramachandran, V., Chunharas, C., Marcus, Z., Furnish, T. & Lin, A. Relief from intractable phantom pain by combining psilocybin and mirror visual-feedback (MVF). Neurocase 24, 105–110 (2018).
- Schindler, E. A. D. et al. Exploratory Controlled Study of the Migraine-Suppressing Effects of Psilocybin. Neurotherapeutics 18, 534–543 (2021).
- Kolbman, N. et al. Intravenous psilocybin attenuates mechanical hypersensitivity in a rat model of chronic pain. Current Biology 33, R1282–R1283 (2023).
- Castellanos, J. P. et al. Chronic pain and psychedelics: a review and proposed mechanism of action. Reg Anesth Pain Med 45, 486–494 (2020).
- Hashmi, J. A. et al. Shape shifting pain: chronification of back pain shifts brain representation from nociceptive to emotional circuits. Brain 136, 2751–2768 (2013).
- Decosterd, I. & Woolf, C. J. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 87, 149–158 (2000).
- Halberstadt, A. L., Chatha, M., Klein, A. K., Wallach, J. & Brandt, S. D. Correlation between the potency of hallucinogens in the mouse head-twitch response assay and their behavioral and subjective effects in other species. Neuropharmacology 167, 107933 (2020).
- Maiarù, M. et al. Substance P-Botulinum Mediates Long-term Silencing of Pain Pathways that can be Re-instated with a Second Injection of the Construct in Mice. The Journal of Pain S1526590024003407 (2024) doi:10.1016/j.jpain.2024.01.331.
- Caporoso, J. et al. A Thermal Place Preference Test for Discovery of Neuropathic Pain Drugs. ACS Chem. Neurosci. 11, 1006–1012 (2020).
- Matsumoto, K. et al. Juvenile social defeat stress exposure favors in later onset of irritable bowel syndrome-like symptoms in male mice. Sci Rep 11, 16276 (2021).
- Drake, R. A., Steel, K. A., Apps, R., Lumb, B. M. & Pickering, A. E. Loss of cortical control over the descending pain modulatory system determines the development of the neuropathic pain state in rats. eLife 10, e65156 (2021).
- Baliki, M. N., Mansour, A. R., Baria, A. T. & Apkarian, A. V. Functional Reorganization of the Default Mode Network across Chronic Pain Conditions. PLoS ONE 9, e106133 (2014).
- Corder, G. et al. An amygdalar neural ensemble that encodes the unpleasantness of pain. Science 363, 276–281 (2019).
- Goodwin, G. M. et al. Single-Dose Psilocybin for a Treatment-Resistant Episode of Major Depression. N Engl J Med 387, 1637–1648 (2022).
- Wiffen, P. J. et al. Gabapentin for chronic neuropathic pain in adults. Cochrane Database of Systematic Reviews 2020, (2017).
- Bonnet, U. & Scherbaum, N. How addictive are gabapentin and pregabalin? A systematic review. European Neuropsychopharmacology 27, 1185–1215 (2017).
- Ghazisaeidi, S., Muley, M. M. & Salter, M. W. Neuropathic Pain: Mechanisms, Sex Differences, and Potential Therapies for a Global Problem. Annu. Rev. Pharmacol. Toxicol. 63, 565–583 (2023).
- Huang, J. et al. A neuronal circuit for activating descending modulation of neuropathic pain. Nat Neurosci 22, 1659–1668 (2019).
- Siegel, J. S. et al. Psilocybin desynchronizes the human brain. Nature 632, 131–138 (2024).
- Finnerup, N. B., Kuner, R. & Jensen, T. S. Neuropathic Pain: From Mechanisms to Treatment. Physiological Reviews 101, 259–301 (2021).
- Percie Du Sert, N. et al. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. British J Pharmacology 177, 3617–3624 (2020).
- Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M. & Yaksh, T. L. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55–63 (1994).
- Duan, B. et al. Identification of Spinal Circuits Transmitting and Gating Mechanical Pain. Cell 159, 1417–1432 (2014).
- Decosterd, I. & Woolf, C. J. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 87, 149–158 (2000).
- Mills, C. et al. Estimating Efficacy and Drug ED50’s Using von Frey Thresholds: Impact of Weber’s Law and Log Transformation. The Journal of Pain 13, 519–523 (2012).
- Maiarù, M. et al. Selective neuronal silencing using synthetic botulinum molecules alleviates chronic pain in mice. Sci Transl Med 10, eaar7384 (2018).
There is NO Competing Interest.