Effects of psilocybin and related compounds on neuroprotection in human stroke

1. Principal investigators

Ruslan Rust, PhD: RR Google Scholar Link
Patrick D. Lyden, MD: PL Google Scholar Link

Affiliation:

Ruslan Rust, Ph.D.
Assistant Professor
Department of Physiology and Neuroscience
Keck School of Medicine of the University of Southern California
1501 San Pablo Street, Los Angeles, CA 90033
email: rrust@usc.edu
ORCID: 0000-0003-3376-3453

2. Overview

Background

Stroke is a leading cause of disability and death, affecting 1 in 4 people during their lifetime.¹ Although recanalization treatments exist for acute stroke,^2–4 many survivors are left with permanent motor, speech, and/or cognitive deficits that would benefit from new or adjunctive therapies.⁵ Psychedelics including psilocybin and related tryptamines have shown broad therapeutic potential for the treatment of various neuropsychiatric disorders in mice, rabbits, and humans.^6–8 More recently, animal studies now show significant neuroprotection with psilocybin via activation of serotonin (5-HT) receptors. In rat models of stroke, a single post-stroke dose reduced infarct volume, lowered neuroinflammation, and restored locomotor function⁹. Yet, the cellular targets of this protection remain unknown, including which neuronal and glial subtypes respond most strongly. It is also unclear whether the same signaling pathways occur in the ischemic human brain or whether structurally related 5-HT-receptor ligands (e.g., natural compounds baeocystin, norbaeocystin, aeruginascin, and norpsilocin, or next-generation analogues like CYB003, PSIL 001, and PSIL 002), can provide similar or superior benefit.

Overall objective

We will screen and evaluate how psilocybin and related compounds contribute to neuroprotection in the ischemic mouse brain and translate the most promising candidates in a new ischemic living ex vivo human brain slice model (Fig. 1).

Fig:1: Graphical abstract for proposed project: 1) Screen psilocybin and related compounds for neuroprotection in ischemic mouse brain slices, 2) Identify responsive cell types and signaling networks via single-nucleus RNA sequencing, 3) Validate lead compounds in living human brain slices and map conserved neuroprotective pathways.

Own contributions

Our groups have extensive preclinical (Rust, Lyden) and clinical (Lyden) stroke research experience that spans pharmacological, antibody and cell based therapeutics for stroke.^10–15 We recently built an oxygen glucose deprivation brain slice screening platform that maintains metabolic activity for high content screening, which we can combine with established single-cell RNA sequencing technology¹⁴. In collaboration with our neurosurgery department, we now receive resected cortical and hippocampal tissue that can be studied within minutes of removal, offering a new opportunity to study living human brain biology ex vivo.

Specific Aims

1. Screen and evaluate psilocybin and analogs for neuroprotection in living ex vivo mouse brain slices from mouse stroke models and identify activated cell populations and signaling networks using single-cell RNA sequencing.

2. Validate the most effective compounds in living ex vivo human brain slices exposed to ischemia and determine conserved neuroprotective pathways across species through microscopy and single-cell RNA sequencing.

Innovation

This will be the first study to comprehensively assess the molecular and physiological effects of psychedelic compounds directly in living mouse and human brain tissue under stroke-like conditions.

Impact

The project will dissect the cellular and molecular mechanisms by which psilocybin and related tryptamine compounds preserve brain tissue after stroke-like ischemia in humans. These insights could fast-track the application of targeted neuroprotective therapies, directly addressing a critical unmet need for stroke survivors.

3. Methods

All procedures will be performed at the Keck School of Medicine of the University of Southern California (USC), within the Department of Physiology and Neuroscience, which provides access to state-of-the-art facilities for stroke modeling, brain slice preparation, high-resolution imaging, and transcriptomic profiling. All experimental protocols are well established in our laboratories and supported by institutional infrastructure and expertise. We highlight here core experimental procedures.

Stroke induction:

Transient focal ischemia will be induced using the intraluminal filament model of middle cerebral artery occlusion, which is routinely used in our lab. Male and female mice will be anesthetized with 1–2% isoflurane in oxygen and placed in a supine position. Following a ventral neck incision, the common, external, and internal carotid arteries will be exposed. A silicone-coated 6-0 monofilament (Doccol Corp.) will be inserted into the external carotid artery and advanced into the internal carotid to occlude the origin of the MCA. Laser Doppler flowmetry (Moor Instruments MOORLDI2-IR) will be used to confirm a ≥70% reduction in cerebral blood flow (CBF) and guide proper filament placement. MCA occlusion will be maintained for 60 minutes, after which the filament will be withdrawn to allow reperfusion. Mice will be kept on a heated pad during surgery and recovery, and receive postoperative analgesia. The procedures are established in our labs as shown previously.^10–15

Ex vivo mouse / human brain-slice screen

Acute 300 µm cortical slices are generated on a vibratome in chilled, carbogenated NMDG-aCSF following rapid perfusion (mouse stroke) or surgical resection (human). Slices recover 10 min in warmed NMDG-aCSF, then enter a controlled oxygen-glucose deprivation (OGD) chamber where perfusate is switched to OGD-aCSF (95 % N₂/5 % CO₂) to mimic ischemia. Lead psychedelic compounds, prepared at working dilutions immediately before use, are applied during the OGD or early reperfusion phase. After defined OGD (e.g., 60 min) slices are reperfused with normoxic aCSF under carbogen and incubated for 2 h. Viability is quantified by Propidium Iodide versus Calcein-AM (or LDH release). Surviving tissue is fixed (4% PFA), cryoprotected (30% sucrose), sectioned, and immunostained for neuron, astrocyte, microglia, and endothelial markers to map cell-type-specific protection and analysed with established automated pipelines. To identify and classify cell death, we will stain slices additionally with cleaved-Caspase-3 and TUNEL for apoptotic cells, and pMLKL or Fluoro-Jade C to detect necroptotic or degenerating neurons. All buffers are pH-titrated (7.30–7.40), gas flow and temperature are logged, and slice yield targets ≈ 20 high-quality sections per mouse brain, with identical workflows for human tissue.

Single-nucleus RNA sequencing

To study molecular changes, cell-cell interactions and identify receptor-ligand interactions, brain tissue will be snap-frozen, nuclei isolated, and processed for single-nucleus RNA-seq using 10x Genomics protocols. Cell hashing will preserve sample identity. Sequencing data will be processed with Cell Ranger v7, and downstream analysis including differential expression, pathway enrichment, and receptor-ligand mapping will be performed in Seurat v5.0. Ligand-receptor interaction networks will be reconstructed using CellChat and other established pipelines to identify signaling pathways that drive neuroprotection. The procedures and all downstream analysis are established in our labs as previously described.^13,14

Sample size, randomization and blinding

We will use adult (3–6-month-old) male and female mice. We will use in total 12 stroke animals, yielding ~12-20 high-quality slices per brain, randomized to vehicle, psilocybin, and selected analogues. For human tissue, we aim to collect slices from ~10 neurosurgical donors, with 5–10 viable slices per donor. Sample size is based on prior studies using similar slice-based designs, allowing detection of moderate-high effects. Slice allocation will be randomized within each donor or animal and balanced across treatment conditions. The researchers performing the experiments and analysis will remain blinded to group assignment until quality control and analysis scripts are finalized.

Statistics

All data will be analyzed using linear mixed-effects models (with treatment as a fixed effect and animal or donor as a random intercept) to account for within-subject correlation across slices. Group differences in the Neuroprotection Index and individual histology readouts will be assessed using two-sided tests, with Holm correction applied for multiple comparisons, where appropriate. All exploratory analyses, including pathway enrichment and ligand–receptor mapping from single-nucleus RNA-seq data, will be performed in R with validated specialized packages including Seurat v5 or CellChat v3.

Planned Analyses

1. Neuroprotective Screen of Psilocybin and related compounds in ischemic stroke mouse brain slices (Aim 1)

Neuroprotection screen of psilocybin and related compounds in ex vivo mouse stroke

We will calculate a composite Neuroprotection Index that we infer from Propidium Iodide/Calcein-AM ratios and cleaved-Caspase-3, TUNEL, and pMLKL staining to rank the ability of each tested psychedelic compound to reduce ischemia-induced cell death.
We will also analyse standard brain tissue responses including inflammatory responses (Iba1), glial scarring (GFAP) and neuronal nuclei count (NeuN).

Identify cell populations affected by psilocybin and related compounds and dissect underlying signaling networks using single nucleus RNAseq

snRNA-seq data will be processed with Cell Ranger v7 and analyzed in Seurat for differential expression, pathway enrichment of individual brain cells
CellChat ligand-receptor mapping to reveal cell-type-specific signaling cascades that correlate with protection.

2. Validation of lead compounds in human ischemic brain slices (Aim 2)

Validation of neuroprotection of lead compounds from Aim 1 in human ischemic ex vivo brain tissue

The same Neuroprotection Index (see Aim 1) will confirm efficacy of top candidates in resected human cortical and hippocampal slices subjected to OGD.

Identify affected cell populations using single-nucleus RNAseq and cross species comparison

Human snRNA-seq profiles will be integrated with the mouse dataset via ortholog mapping and label transfer to pinpoint conserved cell populations and pathways, highlighting the most translatable neuroprotective mechanisms.

Ethics and Data Management

We have active institutional licenses and ethical approvals for both human tissue collection and proposed animal experimentation. Human brain tissue is obtained in collaboration with our neurosurgery department under IRB-approved protocols, ensuring timely and ethical access to resected cortical and hippocampal samples. All animal procedures comply with IACUC regulations and follow standard stroke model protocols established in our laboratories.

All generated raw data will be deposited in permanent open-access repositories such as zenodo.org with assigned DOIs to ensure transparency and long-term availability. RNA sequencing data, including raw FASTQ files, count matrices, and metadata, will be deposited in specialized public repositories such as NCBI GEO.

We commit to provide regular updates on ResearchHub and to uploading a preprint version of these findings to both bioRxiv and ResearchHub as soon as results are available. We will also report and share any negative results, which we believe are equally important for advancing science and improving reproducibility in the field.

4. Budget

We request 15’000 USD for animal stroke studies, supplies for living mouse and human brain slices assay, neuroprotection assays, imaging reagents (e.g., antibodies), Psilocybin & analogs, RNAseq library prep kits, sequencing and bioinformatics, imaging and data sharing. All aims will be completed within 12-18 months. We have active funding to cover all other project-related expenses, including the salaries of staff and trainees involved in the study, as well as costs related to equipment usage and routine laboratory operations.

5. References

1. Kazi, D. S. et al. Forecasting the Economic Burden of Cardiovascular Disease and Stroke in the United States Through 2050: A Presidential Advisory From the American Heart Association. Circulation 150, e89–e101 (2024).

2. Goyal, M. et al. Endovascular thrombectomy after large-vessel ischaemic stroke: a meta-analysis of individual patient data from five randomised trials. Lancet 387, 1723–1731 (2016).

3. Nogueira, R. G. et al. Thrombectomy 6 to 24 Hours after Stroke with a Mismatch between Deficit and Infarct. New England Journal of Medicine 378, 11–21 (2018).

4. Kwiatkowski, T. G. et al. Effects of tissue plasminogen activator for acute ischemic stroke at one year. National Institute of Neurological Disorders and Stroke Recombinant Tissue Plasminogen Activator Stroke Study Group. N. Engl. J. Med. 340, 1781–1787 (1999).

5. Feigin, V. L. et al. Global, regional, and national burden of stroke and its risk factors, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet Neurology 20, 795–820 (2021).

6. Goodwin, G. M. et al. Single-Dose Psilocybin for a Treatment-Resistant Episode of Major Depression. New England Journal of Medicine 387, 1637–1648 (2022).

7. Warren, A. L. et al. Structural pharmacology and therapeutic potential of 5-methoxytryptamines. Nature 630, 237–246 (2024).

8. Armstrong, S. B. et al. Prospective associations of psychedelic treatment for co-occurring alcohol misuse and posttraumatic stress symptoms among United States Special Operations Forces Veterans. Military Psychology 36, 184–191 (2024).

9. Yu, S.-J. et al. Neuroprotective effects of psilocybin in a rat model of stroke. BMC Neuroscience 25, 49 (2024).

10. Lyden, P. et al. Randomized, controlled, dose escalation trial of a protease-activated receptor-1 agonist in acute ischemic stroke: Final Results of the RHAPSODY trial. Ann Neurol 85, 125–136 (2019).

11. Lyden, P. D. et al. The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results. Stroke 53, 1802–1812 (2022).

12. Morais, A. et al. Biological and Procedural Predictors of Outcome in the Stroke Preclinical Assessment Network (SPAN) Trial. Circulation Research 135, 575–592 (2024).

13. Weber, R. Z. et al. Neural xenografts contribute to long-term recovery in stroke via molecular graft-host crosstalk. 2024.04.03.588020 Preprint at https://doi.org/10.1101/2024.04.03.588020 (2025).

14. Weber, R. Z. et al. A molecular brain atlas reveals cellular shifts during the repair phase of stroke. J Neuroinflammation 22, 112 (2025).

15. Rust, R. et al. Nogo-A targeted therapy promotes vascular repair and functional recovery following stroke. Proc Natl Acad Sci USA 201905309 (2019) doi:10.1073/pnas.1905309116.

6. Conflict of interest

R.R. is an editor at ResearchHub. The authors declare no other competing interests.

Funding

Funding

Effects of psilocybin and related compounds on neuroprotection in human stroke

Funding Progress

Nonprofit Recipient

Funders

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