N,N-Dimethyltryptamine attenuates spreading depolarization and restrains neurodegeneration by sigma-1 receptor activation in the ischemic rat brain

N,N-Dimethyltryptamine (DMT) is an endogenous indole alkaloid with recognised activity at multiple receptor systems, including the sigma-1 receptor (Sig-1R). Earlier work has suggested that DMT concentrations rise in mammalian brain under severe stress, and that exogenous DMT can protect cultured human cortical neurons from hypoxia and reduce infarct size and inflammation in rodent focal ischemia models. Sig-1Rs are intracellular chaperone-like proteins at the mitochondria-associated endoplasmic reticulum membrane that regulate calcium trafficking, reactive oxygen species, and cell survival pathways; prior studies indicate Sig-1R activation can be neuroprotective in ischemia. Szabó and colleagues designed an in vivo study to test whether intravenous DMT given during acute, transient global forebrain ischemia can limit pathological events that promote lesion progression and thereby reduce early neurodegeneration, and whether such effects are mediated by Sig-1R activation. To increase metabolic stress and create a model relevant to penumbral injury, the investigators superimposed three induced spreading depolarizations (SDs) and a brief episode of hypoxia onto two-vessel occlusion (2VO). They compared DMT with a selective Sig-1R agonist and antagonist, and probed potential contributions of serotonin receptors using a broad-spectrum 5-HT receptor antagonist; outcomes included electrophysiological indices of SD, cerebral blood flow (CBF), histological markers of cell death and glial integrity, Sig-1R binding, tissue DMT levels, and cellular localisation of Sig-1R.

Adult male Sprague-Dawley rats (initially n = 69; experimental interventions reported for n = 56) were used. Anaesthesia, temperature control and physiological monitoring followed established protocols; femoral artery and vein cannulation allowed blood sampling, mean arterial blood pressure (MABP) monitoring and continuous intravenous drug infusion. Incomplete global forebrain ischemia was produced by bilateral common carotid artery occlusion (2VO). Two small craniotomies over the right parietal cortex permitted electrophysiological recording and the induction of SDs by topical 1 M KCl. Three SDs were evoked at ≥15 min intervals starting 10 min after 2VO, followed by a brief controlled hypoxic episode (maximum ~1 min) and then reperfusion for 1 h. Drugs (all dissolved in physiological saline) were infused intravenously at 1 mg/kg/h throughout the protocol. Treatment groups were: DMT alone (n = 7), PRE-084 (selective Sig-1R agonist, n = 8), NE-100 (Sig-1R antagonist, n = 6), NE-100 combined with DMT (n = 6), asenapine (broad-spectrum 5-HT receptor antagonist, n = 6), asenapine combined with DMT (n = 6), and vehicle (saline control, n = 7). NE-100 or asenapine infusions began 10 min before ischemia when co-applied with DMT. Electrophysiology used a saline-filled glass capillary microelectrode inserted 700 µm into somatosensory cortex to record direct current (DC) potential and electrocorticogram (ECoG). Local CBF was monitored by laser-Doppler flowmetry (LDF) placed near the recording site. Blood gases, MABP and heart rate were measured serially. SD metrics extracted from downsampled DC traces included peak amplitude, duration at half amplitude, rates of depolarization and repolarization, and area under the curve (AUC); CBF responses to SD were characterised principally by the hyperemic phase. Histological analyses were performed after perfusion fixation at 2 h after ischemia onset and 1 h after reperfusion. Coronal 20 µm sections were immunolabelled for cleaved caspase-3 (CC3, apoptosis), 4-hydroxynonenal (4-HNE, marker of lipid peroxidation/ferroptosis), NeuN (neurons), GFAP (astrocytes) and Iba-1 (microglia). CC3 and 4-HNE-positive cells were counted per mm2 by blinded observers; 4-HNE optical density in perinuclear cytoplasm was quantified. Astrocytic and neuronal area coverage were estimated from masked images; microglial activation was assessed using a ramification index. For cellular localisation, Sig-1R was co-labelled with NeuN, GFAP and Iba-1 in naïve rats and imaged by confocal microscopy. An in vitro competitive radioligand binding assay used rat brain membrane homogenates and [3H]-(+)-pentazocine to assess Sig-1R affinity of DMT and reference ligands; Ki values were derived using nonlinear fitting and the Cheng-Prusoff equation. Plasma and brain DMT concentrations were measured by two-dimensional liquid chromatography coupled to high-resolution tandem mass spectrometry (2D-LC-HRMS/MS) in a subset of animals (n = 4 per group for vehicle and DMT). Data were analysed with SPSS and GraphPad Prism; normality testing and appropriate parametric or non-parametric tests were applied, significance set at p < 0.05.

Physiological variables remained broadly within expected ranges. During reperfusion there was a statistically significant rise in arterial pCO2 and a fall in pO2, with a trend to lower pH; MABP and heart rate were stable overall and DMT infusion produced no significant alteration in MABP or baseline CBF. After 2VO, residual CBF stabilised around 40% of baseline; DMT did not change baseline perfusion or the overall CBF trajectory during ischemia, hypoxia and reperfusion. Electrophysiological assessment showed that intravenous DMT attenuated key features of SD. Compared with vehicle, DMT reduced SD peak amplitude (mean −16.5 ± 4.1 mV vs −20.1 ± 1.3 mV), slowed the depolarization rate (−2.62 ± 1.28 vs −3.48 ± 0.94 mV/s) and shortened cumulative SD duration (140 ± 38 s vs 191 ± 42 s). Administration of the selective Sig-1R agonist PRE-084 produced similar changes (amplitude −16.2 ± 5.6 mV; depolarization rate −2.26 ± 1.06 mV/s; cumulative duration tended shorter at 149 ± 31 s), while NE-100 alone had no notable effect. Co-application of NE-100 with DMT abolished the SD-suppressing effects of DMT (metrics similar to vehicle). The broad-spectrum 5-HT receptor antagonist asenapine increased SD amplitude versus vehicle (−22.6 ± 2.6 vs −20.1 ± 1.3 mV); when DMT was co-applied with asenapine SD amplitude returned to control levels (asenapine + DMT −19.1 ± 1.3 mV). None of the pharmacological manipulations, including DMT and PRE-084, produced meaningful changes in the SD-associated hyperemic CBF response (AUC and peak hyperemia similar across groups). Histological endpoints revealed selective neuroprotective signals. NeuN immunopositivity did not differ substantially between naïve, vehicle and DMT groups at the early time point studied. In contrast, apoptotic cell counts (CC3+) increased markedly after ischemia/hypoxia/reperfusion in vehicle-treated animals, and DMT reduced CC3+ cell counts in several regions: somatosensory cortex (66 ± 33 vs 105 ± 56 CC3+ cells per mm2, DMT vs vehicle), hippocampal CA1 (532 ± 268 vs 893 ± 249) and dentate gyrus (1367 ± 311 vs 1649 ± 278). Ferroptosis marker 4-HNE was evident in hippocampal neurons; while counts were not significantly different, the optical density of perinuclear 4-HNE staining was lower after DMT (0.025 ± 0.002 vs 0.048 ± 0.012, DMT vs vehicle), consistent with reduced lipid peroxidation. GFAP-labelled astrocytic area decreased after ischemia in vehicle animals; DMT partially rescued GFAP coverage in cortex and striatum (cortex DMT 3.2 ± 1.9% vs vehicle 1.5 ± 0.3% and naïve 4.7 ± 0.9%), but not consistently in hippocampus. Microglial activation, assessed by ramification index, was greater ipsilateral to SD induction but was not altered by DMT. Competitive binding assays showed that DMT has micromolar affinity for Sig-1R, with an inhibitory constant (Ki) reported as 15.1 μM in rat brain membranes, consistent with earlier measurements in other tissues. Asenapine did not displace the Sig-1R radioligand and did not change the Sig-1R binding affinity of DMT when co-applied in vitro. Measurement of exogenous DMT after infusion confirmed its presence: plasma levels at ~50 min averaged 90.5 ± 37.9 ng/mL (approximately 0.5 μM) and brain tissue concentrations at the end of the protocol were 4.6 ± 1.9 ng/g (reported as ~0.02 μmol/kg); vehicle samples were below detection. Fluorescent co-labelling demonstrated Sig-1R expression in neurons, astrocytes and resting microglia.

Szabó and colleagues interpret their findings as showing that intravenous DMT given during the acute phase of experimentally induced global forebrain ischemia limits the evolution of recurrent spreading depolarizations and reduces early markers of cell death, and that these effects are mediated predominantly via Sig-1R activation. The reduction in SD amplitude, depolarization rate and cumulative duration by both DMT and the selective Sig-1R agonist PRE-084, together with the loss of DMT efficacy when the Sig-1R antagonist NE-100 was co-administered, supports a Sig-1R-dependent mechanism. The authors suggest that Sig-1R activation may modulate intracellular calcium handling and thus blunt SD, drawing on prior literature showing Sig-1R effects on NMDA receptor responses and voltage-gated calcium currents. DMT did not alter the SD-associated hyperemic CBF response in this ischemic setting, nor did it produce overt cardiovascular or cerebrovascular changes; the investigators attribute the lack of haemodynamic effects to the slow continuous infusion and controlled anaesthesia. Histologically, DMT preserved astrocytic GFAP signal in cortex and striatum and decreased apoptotic CC3+ cells in cortex and hippocampus; it also reduced perinuclear 4-HNE staining, indicating an anti-ferroptotic effect consistent with Sig-1R roles in limiting reactive oxygen species. By contrast, DMT did not modify microglial activation induced by SD and trepanation in this acute preparation. The authors note that the measured tissue DMT concentrations and the micromolar Sig-1R affinity of DMT are compatible with biological activity given possible receptor reserve, although they acknowledge rapid peripheral and tissue metabolism of DMT which may lead to underestimation of peak concentrations. Key limitations acknowledged include the early histological time point (2 h post-ischemia, 1 h reperfusion), which may be insufficient to detect mature infarct or fully capture SD-driven cell death, and technical constraints that hinder precise cell counting in densely packed hippocampal layers. The authors also recognise that microglial responses provoked by ischemia and SD in vivo may be less sensitive to Sig-1R modulation than inflammatory stimuli used in cell culture. Finally, they emphasise that DMT measurement may underestimate true exposure because of rapid enzymatic degradation during tissue handling. On the basis of these data, the investigators conclude that timely DMT administration could be considered as an adjuvant pharmacological approach in acute cerebral ischemia, pending further work to confirm efficacy and safety.