Fer-1 alleviates traumatic brain injury-induced cognitive impairment by inhibiting hippocampal ferroptosis

Abstract

The hippocampus plays a pivotal role in the development of cognitive disorders induced by traumatic brain injury (TBI). Recent studies have elucidated the involvement of ferroptosis in the pathophysiological cascade of TBI. However, the specific role of ferroptosis and the therapeutic potential of ferrostatin-1 in mitigating hippocampal damage post-TBI remain unclear. In this study, we established a TBI model and conducted a series of behavioral and histomorphological experiments in mice to assess outcomes. Our assessments included evaluating ferroptosis levels using Perl's staining, assessing neuronal cell death through PI staining, examining mitochondrial shrinkage with transmission electron microscopy, and analyzing the expression levels of key proteins such as glutathione peroxidase 4 (GPX4), transferrin receptor, and SLC40A1 via Western blotting. Our results revealed that TBI led to iron disrupted iron metabolism, mitochondrial atrophy, neuronal cell death, and downregulation of GPX4 expression in the hippocampus. Importantly, administration of Ferrostatin-1 into the lateral ventricle significantly mitigated iron accumulation and neuronal cell death in the hippocampus, thereby attenuating injury and long-term cognitive impairment following TBI. This study highlights the therapeutic potential of targeting hippocampal ferroptosis as a novel treatment approach for TBI.

Keywords: Ferroptosis; Ferrostatin-1(Fer-1); Hippocampus; Traumatic brain injury (TBI).

Fig. 1

Neuronal Cell Degeneration and Iron Accumulation after TBI (A) Field of interest and representative images of Perl's staining in the CA1 region at indicated times post-TBI. Scale bar: 100 μm. (B) Field of interest and representative images of PI staining in the CA1 region at indicated times post-TBI. Scale bar: 100 μm. (C) Quantification of iron-positive cells in the CA1 region. Data presented as mean ± SD (n = 6). **P < 0.01, ***P < 0.001 vs Sham group. (D) Quantification of mean fluorescence density in the CA1 region. Data expressed as mean ± SD (n = 6). *P < 0.05, **P < 0.01 vs Control group.

Fig. 2

Dysfunction of Iron Metabolism, in Hippocampus after TBI (A) Western blotting results of GPX4, FPN, and TfR in the hippocampus at various time points post-TBI. (B, C, D) Histograms showing quantitative analysis of GPX4, FPN, and TfR after TBI. Data expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs Sham group.

Fig. 3

Ultrastructural and biochemical evidence of ferroptosis after TBI. (A) Representative TEM image of Hippocampus CA1 Region. Arrows indicate damaged mitochondria exhibiting ferroptotic features, including remarkable shrinkage and high electron density. Scale bar: 500 nm. (B) Quantification of damaged mitochondria. Data expressed as mean ± SD (n = 3). ***P < 0.001 vs Sham group. ##P < 0.01 vs TBI or TBI + DMSO group. (C) Quantification of glutathione (GSH) levels in the hippocampus. Data expressed as mean ± SD (n = 6). ***P < 0.001 vs Sham group. ##P < 0.01 vs TBI or TBI + DMSO group. (D) Quantification of malondialdehyde (MDA) levels in the hippocampus. Data expressed as mean ± SD (n = 6). ***P < 0.001 vs Sham group. #P < 0.05 vs TBI or TBI + DMSO group. (E) Hippocampus ROS fluorescence intensity. Data expressed as mean ± SD (n = 6). ***P < 0.001 vs Sham group. ###P < 0.001 vs TBI or TBI + DMSO group.

Fig. 4

Ferrostatin-1 Treatment Attenuated Neuronal Degeneration and Iron Accumulation after TBI. Mice were sacrificed 3 days post-TBI. (A) Field of interest and representative images of Perl's staining in CA1 region. Scale bar: 100 μm. (C) Quantification of iron-positive cells in CA1 region respectively. Data expressed as mean ± SD (n = 6). ***P < 0.001 vs Sham group, ## P < 0.01 vs TBI or TBI + DMSO group. (B) Field of interest and representative images of PI staining in CA1 region. Scale bar: 100 μm. (D) Quantification of mean fluorescence density. Data expressed as mean ± SD (n = 6). **P < 0.01 vs Sham group, # P < 0.05 vs TBI or TBI + DMSO group. (E) Representative western blotting of GPX4. (F) Quantitative evaluation of GPX4. Data expressed as mean ± SD (n = 3). ***P < 0.001 vs Sham group, ## P < 0.01 vs TBI or TBI + DMSO group.

Fig. 5

Treatment with Fer-1 Attenuated TBI-Induced Cognitive Impairment (A) Representative track plot of OFT in each group. (B) Representative track plot of NOR in each group. (C) Quantification analysis of total movement distance. (D) Quantification analysis of distance traveled in the center area. (E) Quantification analysis of time spent in the center area. (F) Quantification analysis of discrimination index. (G) Representative track plot of memory phases of MWM test on 21 days after TBI. (H) Quantification analysis of escape latency. (I) Quantification analysis of numbers of crossing the platform. (J) Quantification analysis of time in target quadrant. Data expressed as mean ± SD (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001 vs Sham group. #P < 0.05, ##P < 0.01 vs TBI group and TBI+DMSO group.