Effects of Lithium Ions on tPA-Induced Hemorrhagic Transformation under Stroke

Abstract

Thrombolytic therapy with the tissue plasminogen activator (tPA) is a therapeutic option for acute ischemic stroke. However, this approach is subject to several limitations, particularly the increased risk of hemorrhagic transformation (HT). Lithium salts show neuroprotective effects in stroke, but their effects on HT mechanisms are still unknown. In our study, we use the models of photothrombosis (PT)-induced brain ischemia and oxygen-glucose deprivation (OGD) to investigate the effect of Li⁺ on tPA-induced changes in brain and endothelial cell cultures. We found that tPA did not affect lesion volume or exacerbate neurological deficits but disrupted the blood-brain barrier. We demonstrate that poststroke treatment with Li⁺ improves neurological status and increases blood-brain barrier integrity after thrombolytic therapy. Under conditions of OGD, tPA treatment increased MMP-2/9 levels in endothelial cells, and preincubation with LiCl abolished this MMP activation. Moreover, we observed the effect of Li⁺ on glycolysis in tPA-treated endothelial cells, which we hypothesized to have an effect on MMP expression.

Keywords: hemorrhagic transformation; lithium salts; oxygen-glucose deprivation; photothrombosis; tissue plasminogen activator.

Figure 1

Evaluation of the neuroprotective effect of concurrent thrombolytic therapy with LiCl on the severity of brain damage on the 7th day after PT in rats. (A) Representative brain section obtained by T2-weighed MRI (image covered the center of ischemic area). Hyperintensive regions refer to ischemic areas. (B) Volume of brain lesions caused by PT in the PT evaluated by using MRI with analysis of T2-weighted images. No significant difference between the groups was found (one-way ANOVA test). The data are presented as mean ± SEM.

Figure 2

(A) Neurological status scores estimated in the limb-placing test (3rd and 6th days after the PT). The data are presented as mean ± SEM. *—p ≤ 0.05 in PT vs. PT + tPA + LiCl, **—p ≤ 0.005 in PT + tPA vs. PT + tPA + LiCl group on day 6 (two-way ANOVA test). (B) PT-induced limb asymmetry measured in the cylinder test on the 7th day after the surgery. The data are presented as mean ± SEM.

Figure 3

Representative images of the brain with EBD in the PT area in the right hemisphere (A). (B) The levels of EBD accumulation in brain tissue of damaged and contralateral hemisphere. **—p ≤ 0.005 (two-way ANOVA test).

Figure 4

Survival of EA.hy926 cells exposed to increasing concentrations of tPA. (A) Toxicity of tPA in normoxia. Effect of LiCl pretreatment on tPA-induced cell death in normoxia (B) or after OGD (C). The data are presented as mean ± SEM ***—p ≤ 0.001 (two-way ANOVA test). Viability of control cells was taken as 100%.

Figure 5

MMP levels in EA.hy926 endotheliocytes treated with 1.6 µM tPA and 1 mM LiCl. The content of MMP-2 (A) and MMP-9 (B) measured by Western blotting (One-way ANOVA test). Representative images and densitometry analysis of the signal intensity are presented. Representative zymogram for MMP-2 activity (C) and its densitometry (D) (two-way ANOVA test). The data are presented as mean±SEM. *—p ≤ 0.05, **—p ≤ 0.005.

Figure 6

Effect of tPA on glycolysis and respiration of EA.hy926 cells. Effects of tPA and LiCl on oxygen consumption rate (OCR) curves (A) and respiratory parameters (B): basal respiration (BR), maximal respiration capacity (MRC), and non-mitochondrial respiration (NMR). (C) Effects of tPA and LiCl on extracellular acidification rate (ECAR). OCR and ECAR of EA.hy926 (A,C) were measured after sequential addition of D-glucose (10 mM), ATP synthase inhibitor oligomycin (4.5 μM), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (10 μM), and a mixture of rotenone and antimycin A (2.5 and 4 μM, respectively). (D) Glycolytic parameters: basal glycolysis (BG), glycolytic capacity (GC), and glycolytic reserve (GR). (E,F) GAPDH content in endothelial cells measured by Western blotting (One-way ANOVA tests). The data are presented as mean ± SEM. **—p ≤ 0.005, ***—p ≤ 0.001.