N, N-dimethyltryptamine mitigates experimental stroke by stabilizing the blood-brain barrier and reducing neuroinflammation

Abstract

N,N-dimethyltryptamine (DMT) is a psychoactive molecule present in the human brain. DMT is under clinical evaluation as a neuroprotective agent in poststroke recovery. Yet, its mechanism of action remains poorly understood. In a rat transient middle cerebral artery occlusion stroke model, we previously showed that DMT reduces infarct volume. Here, we demonstrate that this effect is accompanied by reduction of cerebral edema, attenuated astrocyte dysfunction, and a shift in serum protein composition toward an anti-inflammatory, neuroprotective state. DMT restored tight junction integrity and blood-brain barrier (BBB) function in vitro and in vivo. DMT suppressed the release of proinflammatory cytokines and chemokines in brain endothelial cells and peripheral immune cells and reduced microglial activation via the sigma-1 receptor. Our findings prove that DMT mitigates a poststroke effect by stabilizing the BBB and reducing neuroinflammation. Such interactions of DMT with the vascular and immune systems can be leveraged to complement current, insufficient, stroke therapy.

Fig. 1.. Effect of DMT on infarct size and barrier integrity in a rat model of stroke.

(A) Representative images of coronal brain sections from a DMT-treated rat, stained with cresyl violet, are shown sequentially from cranial to caudal sections. The infarct area is delineated by a yellow dotted line. (B and C) Comparison of brain infarct volumes and edema between the stroke group (n = 10) and the DMT-treated stroke group (n = 9). DMT treatment reduced both infarct volume (**P = 0.0327) and associated edema (**P = 0.0135); mean ± SD, unpaired t test. (D) Treatment and procedure timeline. (E) Representative magnetic resonance imaging (MRI) images of rats from the stroke group, DMT-treated stroke group, and DMT + BD1063 cotreated group (n = 10 rats per group) are shown in sagittal, axial, and coronal planes. (F) Representative images show green fluorescence, indicating FITC-albumin extravasation into the brain parenchyma. (G) Quantification of FITC-albumin extravasation in the control and injured brain hemispheres in the stroke, DMT-treated stroke, and DMT + BD1063–treated stroke groups (n = 7 rats per group). DMT reduced FITC intensity compared to the stroke group (***P = 0.0093). Cotreatment with DMT + BD1063 also decreased intensity relative to stroke (*P = 0.011) and showed no difference from the DMT treatment group (P = 0.4254); mean ± SD, two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. No change was observed in the noninjured hemispheres. AU, arbitrary units. (H) Quantification of FITC-albumin extravasation to the CSF by fluorescence spectroscopy (n = 5 rats per group). FITC intensity was lower in the DMT-treated group (*P = 0.0454) but not in the DMT + BD1063 cotreatment group compared to the stroke group (P = 0.3821).

Fig. 2.. Effect of OGDR and DMT on a rat primary BBB coculture model.

(A) Experimental setup for impedance measurements. Brain endothelial cells: green, brain pericytes: orange, glial cells: blue. After 6-hour OGD, 24-hour reoxygenation was administered in the presence of DMT or DMT + BD1063. (B) Real-time impedance kinetics of rat primary brain endothelial cell, brain pericyte, and glial cell cultures and area under the curve values of cell index (0 to 30 hours); n = 5 to 13; mean ± SD, one-way ANOVA and Bonferroni’s post hoc test, ****P < 0.0001. (C) Experimental setup for barrier integrity measurements on the coculture model of the BBB in normoxia and OGDR. (D) TEER measurements after 24-hour reoxygenation. Mean ± SEM, n = 8. (E) Permeability for sodium fluorescein (SF) and (F) Evans blue–labeled albumin (EBA) across the BBB model after 24-hour reoxygenation. P_e, endothelial permeability coefficient. Mean ± SD, n = 4; one-way ANOVA with Bonferroni’s post hoc test, #P < 0.05, ##P < 0.01, and ###P < 0.0003 compared to the normoxia group. (A and C) Created with Biorender.com. Vigh, J. (2025) https://BioRender.com/9atsvr6https://BioRender.com/9atsvr6.

Fig. 3.. DMT protects BBB integrity.

(A) CLDN5 immunofluorescent staining in brain sections. (B) Quantification of CLDN5 immunostaining. Mean ± SD, one-way ANOVA with Bonferroni’s post hoc test, ####P < 0.0001 compared to the control, uninjured side group, ****P < 0.0001 compared to the stroke group; n = 5 to 15. (C) CLDN5 immunofluorescent staining in cell culture. (D) Quantification of CLDN5 immunostaining. Mean ± SD, one-way ANOVA with Bonferroni’s post hoc test, #P = 0.0399 and ##P = 0.0058; compared to the normoxia group, n = 13 to 17. (E) AQP4 and GFAP immunofluorescent double staining in brain sections. (F) Quantification of AQP4 immunostaining. Mean ± SD, one-way ANOVA with Bonferroni’s post hoc test, ##P < 0.005 compared to the control side group, n = 5 to 15. (G) Quantification of GFAP immunostaining. Serum ELISA of (H) CLDN5, (I) MMP9, and (J) GFAP proteins. n = 9 rats per group, mean ± SD, one-way ANOVA with Bonferroni’s post hoc test, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared to the sham group, *P < 0.05, **P = 0.0076, and ****P < 0.0001 compared to the stroke group.

Fig. 4.. The anti-inflammatory effects of DMT in cultured brain endothelial cells after OGDR.

(A) Principal components analysis (PCA) of normoxia and OGDR groups. (B) Volcano plot of the OGDR versus normoxia comparison. Log₂FC, log₂(fold change); FDR, false discovery rate. (C) Pathway analysis showing down-regulated and (D) up-regulated Gene Ontology (GO) terms and transcriptions factor (TF) binding sites (TF) in the OGDR group compared to normoxia. HIF-1α, hypoxia-inducible factor-1α; BP, biological process; MF, molecular function; CC, cellular component. (E) Volcano plot showing the effect of DMT and (F) the addition of BD1063 in OGDR on the brain endothelial transcriptome. The schematic figure above (G) shows the BBB coculture model and the luminal and abluminal fluid compartments. (G to N) Levels of TNFα, IL-1β, IL-6, and IL-10 cytokines in supernatant samples from the luminal and abluminal compartments of the rat triple coculture BBB model. n/d, not detected; n.s., not significant. n = 3 to 6, mean ± SD, one-way ANOVA with Bonferroni’s post hoc test, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared to the normoxia group; ***P < 0.001 and ****P < 0.0001 compared between the OGDR groups.

Fig. 5.. The anti-inflammatory effects of DMT in a rat unilateral stroke model.

(A to G) Serum levels of cytokines TNFα, IL-1β, IL-6, IL-10, CXCL-1, CXCL-10, and BDNF measured by ELISA. (H to M) Expression of genes of cytokines TNFα, IL-1β, IL-6, IL-10, and chemokines CXCL-1 and CXCL-10 in PBMCs measured by quantitative polymerase chain reaction (qPCR). n = 9 rats per group, mean ± SD, one-way ANOVA with Bonferroni’s post hoc test. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared to the sham group; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared between stroke groups.

Fig. 6.. DMT helps to preserve microglia morphology in a rat unilateral stroke model.

(A) IBA1 immunofluorescent microglia cells in brain sections in all experimental groups. (B) Quantification of IBA1 immunofluorescence intensity. n = 5 to 15 images per group, mean ± SD, one-way ANOVA with Bonferroni’s post hoc test, ##P < 0.01 and ####P < 0.0001 compared to the control side, **P < 0.01 compared to the stroke + DMT group. (C) Morphology of IBA1 immunolabeled microglia cells in brain sections in all experimental groups. (D) Ratio of extensively ramified, homeostatic (cluster 0, gray), moderately ramified, intermediate (cluster 1, green), and less ramified, highly responsive (cluster 2, black) microglia detected in brain sections in all experimental groups. n = 5 animals per group; n = 10 images per side per animal; n = 553 cells per group.

Fig. 7.. BBB changes in stroke and the protective effects of DMT.

Schematic representation of the neurovascular unit (NVU) in physiological condition and in stroke. Arrows and inhibition signs (cyan) show the effect of the DMT in pathological condition. In stroke, tight junctions (red line between brain endothelial cells) are disrupted, and CLDN5 tight junction protein is decreased; in the blood soluble neurovascular unit, proteins appear, and the level of the inflammatory cytokines increases and that of anti-inflammatory cytokines decreases. In the brain, the morphology of microglia changes, the ratio of less ramified microglia cells increases, the AQP4 distribution changes, and the level of inflammatory cytokines increases. DMT decreases the level of soluble CLDN5, MMP9, and GFAP proteins and inhibits inflammatory cytokines in the blood, while it increases the level of BDNF and the anti-inflammatory cytokine IL-10. In the brain, DMT inhibits the morphological changes in microglia and the redistribution of AQP4 in astroglia and decreases proinflammatory cytokine production. Created with Biorender.com. Vigh, J. (2025) https://BioRender.com/o95e437.