Ferroptosis Contributes to Neuronal Death and Functional Outcome After Traumatic Brain Injury

Abstract

Objectives: Traumatic brain injury triggers multiple cell death pathways, possibly including ferroptosis-a recently described cell death pathway that results from accumulation of 15-lipoxygenase-mediated lipid oxidation products, specifically oxidized phosphatidylethanolamine containing arachidonic or adrenic acid. This study aimed to investigate whether ferroptosis contributed to the pathogenesis of in vitro and in vivo traumatic brain injury, and whether inhibition of 15-lipoxygenase provided neuroprotection.

Design: Cell culture study and randomized controlled animal study.

Setting: University research laboratory.

Subjects: HT22 neuronal cell line and adult male C57BL/6 mice.

Interventions: HT22 cells were subjected to pharmacologic induction of ferroptosis or mechanical stretch injury with and without administration of inhibitors of ferroptosis. Mice were subjected to sham or controlled cortical impact injury. Injured mice were randomized to receive vehicle or baicalein (12/15-lipoxygenase inhibitor) at 10-15 minutes postinjury.

Measurements and main results: Pharmacologic inducers of ferroptosis and mechanical stretch injury resulted in cell death that was rescued by prototypical antiferroptotic agents including baicalein. Liquid chromatography tandem-mass spectrometry revealed the abundance of arachidonic/adrenic-phosphatidylethanolamine compared with other arachidonic/adrenic acid-containing phospholipids in the brain. Controlled cortical impact resulted in accumulation of oxidized phosphatidylethanolamine, increased expression of 15-lipoxygenase and acyl-CoA synthetase long-chain family member 4 (enzyme that generates substrate for the esterification of arachidonic/adrenic acid into phosphatidylethanolamine), and depletion of glutathione in the ipsilateral cortex. Postinjury administration of baicalein attenuated oxidation of arachidonic/adrenic acid-containing-phosphatidylethanolamine, decreased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells in the hippocampus, and improved spatial memory acquisition versus vehicle.

Conclusions: Biomarkers of ferroptotic death were increased after traumatic brain injury. Baicalein decreased ferroptotic phosphatidylethanolamine oxidation and improved outcome after controlled cortical impact, suggesting that 15-lipoxygenase pathway might be a valuable therapeutic target after traumatic brain injury.

Figure 1.. Inhibition of ferroptosis rescues HT22 neurons from pharmacologically-induced cell death and in vitro traumatic brain injury (TBI).

(A) Ferroptosis was induced using RSL3 (50 nM; inhibitor of GPX4) resulting in a significant increase in cell death (lactate dehydrogenase release). Administration of Fer-1 (0.2-1 μM; lipid radical-trapping antioxidant), triacsin C (9.6 μM; ACSL4 inhibitor), or baicalein (5 μM; 12/15-LOX inhibitor) resulted in a significant reduction in RSL3-induced cell death. (B) HT22 neurons were subjected to severe mechanical stretch injury (3-4 psi), resulting in a significant increase in cell death that was rescued with Fer-1 (0.4 μM), triacsin C (10 μM), baicalein (5 μM), or liproxstatin-1 (40 nM; lipid radical-trapping antioxidant) administration. (Data are Mean ± SD, N=3-4/group, *p<0.05 vs control, #p<0.05 vs vehicle). Abbreviations: arachidonic acid (AA), adrenic acid (AdA), hydroperoxyl-phosphatidylethanolamine (PE-OOH), hydroxy-phosphatidylethanolamine (PE-OH), acyl-CoA synthetase long-chain family member 4 (ACSL4), lipoxygenase (LOX), glutathione peroxidase 4 (GPX4), glutathione (GSH), glutathione disulfide (GSSG), L-buthionine sulfoximine (BSO), RAS-selective lethal compound 3 (RSL3), deferoxamine (DFO), liproxstatin-1 (Lip-1), ferrostatin-1(Fer-1).

Figure 2.. Baicalein administration reduces phosphatidylethanolamine (PE) oxidation in the cortex after controlled cortical impact (CCI).

(A) CCI resulted in a significant increase in total PE oxidation at 4 h after injury. The increase in PE oxidation was attenuated with baicalein administration. (B) Contour map demonstrating the speciation of PE oxidation in the cortex of vehicle-treated animals compared to naive (top) and baicalein-treated animals compared to vehicle-treated animals (bottom) at 4 h post-CCI. (Data are Mean ± SD, N=4-5/group, *p<0.05 vs naive, #p<0.05 vs vehicle).

Figure 3.. Controlled cortical impact (CCI) results in protein and glutathione (GSH) changes consistent with ferroptosis activation in the cortex.

Cortical expression of 15-lipoxygenase 2 (15-LOX2), acyl-CoA synthetase long-chain family member 4 (ACSL4), and glutathione peroxidase (GPX4) were measured in naive and CCI groups. (A, B) CCI resulted in a significant increase in 15-LOX2 (60-fold and 45-fold) and ACSL4 (6-fold and 4-fold) expression in cortex vs naive at 4 and 24 h after injury, respectively. GPX4 expression did not significantly differ between naive and CCI groups. (Data are Mean ± SD, N=3-4/group, *p<0.05 vs naive). (C) GSH was significantly depleted in cortex at 4 h post-CCI with respect to naive. (Data are Mean ± SD, N=4-5/group, *p<0.05 vs naive).

Figure 4.. Baicalein administration results in decreased TUNEL positivity and improved Morris water maze (MWM) performance after controlled cortical impact (CCI).

(A) White boxes indicate areas of interest, shown in higher magnification in panel B. (B) Representative fluorescent images of DAPI and TUNEL staining in ipsilateral dentate gyrus (DG), CA1, and CA3 at 24 h after sham or CCI with baicalein or vehicle treatment. . (C) Quantitativape assessment of TUNEL-positive cells in the hippocampus showed a significant decrease in TUNEL positivity with baicalein compared to vehicle treatment following CCI. (Data are Mean ± SD, N=4-5/group, *p<0.05 vs sham, #p<0.05 vs vehicle). (D) Line graph showing the swim latency to reach the hidden platform on days 10-14 post-CCI and visible (v) platform on day 15 post-CCI in MWM testing. There was no significant difference in time to reach the visible platform between sham, CCI+vehicle, and CCI+baicalein groups, indicating a lack of motor of visual deficit. (Data are Mean ± SE, N=9-10/group, *p<0.05 vs. sham and baicalein-CCI).