2026 ECNP Congress

PS01-1001

Blood-brain barrier and neuroimmune alterations induced by methamphetamine: a preclinical study

Ana Paula Silva — R. Valls, D. Simões, J. Almeida, V. Alves · Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine- University of Coimbra, Coimbra, Portugal

2026 ECNP Congress

Introduction

Methamphetamine (METH) is a widely abused psychostimulant with high addictive potential. Beyond its impact on neuronal circuits, METH can also trigger neuroinflammation and disrupt the blood–brain barrier (BBB) [1-3]. Interestingly, blockade of C–C chemokine receptor type 5 (CCR5) was shown to reduce the reinforcing effects of cocaine, providing the first evidence linking CCR5 to drug abuse. Nevertheless, little is known about the cellular mechanisms triggered by CCR5 or the role of its ligands, such as CCL5, in brain function. Thus, the present study aimed to investigate the impact of METH on the brain CCR5/CCL5 axis.

Methods

Male C57BL/6 mice (2–3 months) were subjected to a conditioned place preference (CPP) paradigm. Control animals received daily intraperitoneal injections, whereas the METH group received vehicle (morning) and METH (2 mg/kg; afternoon) for 4 consecutive days. Afterwards, blood and brain (prefrontal cortex) were collected for analysis. Immune cell populations were characterized by flow cytometry, and CCR5/CCL5 mRNA and protein levels by RT-PCR and ELISA, respectively. Additionally, human cerebral microvascular endothelial cell line (hCMEC/D3) was used to assess BBB function by transendothelial electrical resistance (TEER) and FITC-dextran (4kDa) permeability. Monocyte transmigration (THP-1 cells) across brain endothelial cells was also evaluated. Statistical analyses were assessed using unpaired t-test (GraphPad Prism 8). All procedures were approved by the Portuguese Ethical Committee (DGAV, Ref: 34500/25-S).

Results

METH induced drug-seeking behavior in 71% of mice. Blood cell population of these animals, when compared to controls, showed a clear increase of neutrophils (CD45+CD11b+Ly6G+; CTR 6.8±2.1% of CD45+ cells; METH 28.6±6.1%; p=0.01) and inflammatory monocytes (CD45highLy6G-CD11b+Ly6Chigh;CTR 3.0±0.5% of CD45+ cells; METH 4.6±0.4%; p=0.03), as well as an upregulation of CCR5 expression on both cell types (neutrophils: CTR 114±10.33 a.u.; METH 154.8±9.8; p=0.02; monocytes: CTR 199.5±5.8 a.u.; METH 225.4±6.9; p=0.02). Within the frontal cortex, METH increased the number of macrophage-derived monocytes (CD45highLy6G-CD11b+Ly6Clow; CTR 57.9±4.4% of CD11b+ cells; METH 72.1±2.2%; p=0.01) and the number of CCR5+ infiltrated monocytes (CTR 89.8±3.1% of monocytes; METH 96.6±0.8%; p=0.04). Additionally, blood total CCL5 protein levels were decreased (CTR 109.7±1.9 pg/mL; METH 51.4 ± 12.7 pg/mL; p=0.04), whereas brain levels tended to increase. At the BBB level, METH induced downregulation of mRNA levels of tight (claudin-5, occludin, ZO-1) and adherens junctions (VE-cadherin) molecules, and upregulation of adhesion molecule ICAM-1. In vitro, METH also induced functional impairment of brain endothelial cells, characterized by decreased TEER values (43.4±6% of control; p<0.0001) and increased paracellular permeability (1.2 ± 0.07 fold change; p=0.01). In accordance with METH results, CCL5 by itself also impaired barrier function. Moreover, METH promoted a significant migration of monocytes across endothelial cell monolayer (151.1±15.1% of control; p=0.0002), together with a downregulation of claudin-5, ZO-1 and VE-cadherin, as well as an upregulation of ICAM-1.

Conclusions

METH induced drug-seeking behavior with a concomitant BBB dysfunction, monocyte transmigration, and upregulation of CCR5 in these immune cells.

References

  1. Coelho-Santos, V., Leitão, R. A., Cardoso, F. L., Palmela, I., Rito, M., Barbosa, M., Brito, M. A., Fontes-Ribeiro, C. A., & Silva, A. P. (2015). The TNF-α/NF-κB signaling pathway has a key role in methamphetamine-induced blood-brain barrier dysfunction. Journal of cerebral blood flow and metabolism, 35(8), 1260–1271.
  2. Gonçalves, J., Leitão, R. A., Higuera-Matas, A., Assis, M. A., Coria, S. M., Fontes-Ribeiro, C., Ambrosio, E., & Silva, A. P. (2017). Extended-access methamphetamine self-administration elicits neuroinflammatory response along with blood-brain barrier breakdown. Brain, behavior, and immunity, 62, 306–317.
  3. Leitão, R. A., Fontes-Ribeiro, C. A., & Silva, A. P. (2022). The effect of parthenolide on methamphetamine-induced blood-brain barrier and astrocyte alterations. European journal of clinical investigation, 52(4), e13694.