Ferroptosis in Traumatic Brain Injury: The Future Direction?

Abstract

Traumatic brain injury (TBI) is a severe acute brain injury caused by external mechanical force, resulting in temporary or permanent impairment of physical, psychological, cognitive functions, and altered consciousness states. Cognitive dysfunction commonly leading to symptoms such as inattention and decline in learning and memory abilities, and may also result in irritability, anxiety or depression. The underlying pathology involves significant neuron loss and limited synaptic remodeling. Abnormal iron metabolism is common in the brains of patients with TBI and cognitive impairment. However, effective intervention measures are still lacking which prompts us to explore new therapeutic targets and develop novel therapies for TBI. As a newly discovered form of regulated cell death, ferroptosis occurs due to excessive iron accumulation in the brain, leading to cellular and neuronal damage. Iron overload and ferroptosis play a significant role in the pathophysiology of secondary brain injury. Therefore, potential therapeutic approaches may involve targeting factors such as iron deposition and ferroptosis inhibition. This chapter provides an overview of ferroptosis’ role in traumatic brain injury (TBI) and advance investigation in related research fields with the aim of enhancing our understanding of TBI treatment through ferroptosis inhibition, ultimately guiding new clinical directions for further TBI research.

1. Introduction

Traumatic brain injury (TBI) is a frequently occurring disease resulting from injuries in the emergency department. It is also a leading cause of disability and paralysis worldwide among trauma-related injuries. According to statistics, approximately 50 million individuals suffer from varying degrees of TBI annually. The primary causes of TBI include traffic accidents, falls from heights, violent attacks, slips and falls. In addition to direct trauma-induced injury, patients typically experience severe neuronal necrosis, brain tissue edema, disruption of the blood-brain barrier (BBB), heightened oxidative stress, and excessive inflammation following TBI. Several factors mechanistically contribute to secondary injury including excitability, mitochondrial dysfunction, oxidative stress, lipid peroxidation neuroinflammation, axonal degeneration, and apoptotic cell death (Figure 1).

Figure 1.

The pathophysiological mechanism of primary and secondary brain injury.

These factors result in consequences such as disruption of the blood-brain barrier (BBB), release of blood metabolites, activation of microglia, thrombin activity, excitatory amino acids, and proinflammatory cytokines (Figure 1). This creates a harmful cycle [1]. Patients with varying degrees of traumatic brain injury (TBI) may experience different levels of secondary damage [2]. Some evidence suggests an increased occurrence of neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease, and chronic traumatic encephalopathy caused by head trauma. Treatment for TBI may involve options such as pharmacotherapy, cognitive therapy, or surgery. There is a growing demand for agents that can potentially alleviate primary brain injury aspects and mitigate the secondary pathological deficits associated with TBI.

In recent years, several studies have indicated that abnormal iron metabolism is prevalent in the brains of patients with traumatic brain injury and cognitive impairment. Furthermore, an increase in brain iron content has been linked to cognitive decline. Iron, as an essential trace element for maintaining normal physiological functions in the human body, plays a crucial role in various aspects such as electron transport, cell respiration, and DNA synthesis. However, like everything else, iron also has its drawbacks and can be a “double-edged sword.” Its balanced metabolism is vital for sustaining overall bodily function; any imbalance can lead to a range of diseases. Studies on chronic traumatic encephalopathy have revealed that tau overdeposition and neurofibrillary tangle formation are typical pathological features observed during autopsies [3, 4]. Excessive deposition of iron has also been found within these neurofibrillary tangles where oxidative stress-induced phosphorylation of tau occurs [5, 6]. Interestingly, earlier reports demonstrated non-heme iron deposition in the deep cortex and hippocampus of postmortem brain tissue from mTBI patients [5]. Consequently, iron has gradually emerged as a potential target for intervention in TBI research. Therefore, it is believed that imbalances in iron homeostasis play a significant role in influencing the pathological processes associated with TBI.

Iron is the most abundant metal trace element in the brain and plays a crucial role in the development of the central nervous system (CNS), contributing to the synthesis of myelin and various neurotransmitters [7, 8]. Insufficient iron levels during lactation can impair neurotransmitter synthesis, leading to developmental disorders in behavioral functions. Conversely, excessive intracellular iron can disrupt neurological function, particularly in brains affected by traumatic brain injury (TBI). TBI-induced damage to brain tissue, disruption of the blood-brain barrier, increased cerebral vascular permeability, and severe local inflammatory reactions result in an influx of iron from the bloodstream into the brain parenchyma. Neuronal death is one of the most serious consequences following TBI.

Ferroptosis is a newly discovered mode of cell death characterized by iron-dependent lipid peroxidation. Specifically, excess iron, in the form of divalent ferrous ion Fe²⁺, can react with hydrogen peroxide (H₂O₂) or organic hydrogen peroxide (ROOH) to produce soluble hydroxyl (HO) or lipid alkoxy (RO•) groups, respectively. These reactions are the main source of reactive oxygen species (ROS), known as the Fenton reaction [9], which ultimately leads to cell death. Neurons, being terminally differentiated cells, experience various cellular stresses throughout their lifespan. Oxidative stress is one of the key ones, which is caused by excessive accumulation of intracellular ROS. Neuronal membranes contain high levels of cholesterol and polyunsaturated fatty acids (PUFAs), making them susceptible to oxidation by ROS and resulting in oxidative damage [10]. Additionally, neurons have limited ability to scavenge ROS autonomously due to relatively low levels of antioxidant factors such as superoxide dismutase (SOD) and glutathione peroxidase (GPX) compared to other tissues in the brain [11, 12]. The aforementioned evidence further supports the notion that neurons are particularly vulnerable to iron overload. These findings have led researchers to speculate that ferroptosis may play a significant role in cognitive dysfunction resulting from traumatic brain injury (TBI). Furthermore, recent studies have indeed demonstrated activation of the ferroptosis pathway after TBI leading to neuronal death.

The aforementioned studies all indicate a strong correlation between disrupted iron homeostasis and unfavorable prognosis in patients with traumatic brain injury (TBI). In this chapter, we aim to delve into the role of iron and ferroptosis in brain injuries, enhance our comprehension of iron metabolism and the significance of ferroptosis in TBI, as well as explore novel effective treatment approaches for managing iron dysregulation and ferroptosis following TBI.

2. Brain iron metabolism

Iron is the most abundant trace element in the brain and is crucial for brain development and proper functioning. Iron can be found in various components of the brain, including neurons, astrocytes, oligodendrocytes, axons, microglia, and myelin [7, 13, 14]. It plays a vital role in processes such as myelination, neuroectoderm development, oxygen transport, neurotransmitter transmission, and mitochondrial energy production [8, 15].

To reach the CNS, iron must pass through either the blood-brain barrier or the blood-CSF-brain (BCSF) and be taken up by brain tissue cells. The blood-brain barrier is crucial in protecting the brain from fluctuations in systemic iron levels and plays a vital role in brain iron uptake. Iron enters capillary epithelial cells of brain tissue via endocytosis, with Fe²⁺ most likely transported out by Fpn1. Similar cells include BBB endothelial cells, neural tube cells, oligodendrocytes, astrocytes, choroid plexus cells, and ependymal cells.

Iron in the bloodstream enters the brain through the blood-brain barrier in two main forms: the transferrin-bound iron-transferrin receptor complex and non-transferrin-bound iron [16].

Studies have reported that endothelial cells in the choroid plexus also play a role in regulating iron entry into the central nervous system [17]. Astrocytes facilitate the redistribution of iron within the central nervous system with the assistance of ceruloplasmin, which has already crossed the blood-brain barrier to other cells [13]. Ceruloplasmin oxidizes divalent iron to Fe³⁺, which then binds to transferrin and enters the cerebrospinal fluid (CSF) [16].

In addition, in cells that do not express the transferrin receptor, significant amounts of non-transferrin binding iron circulate in the cerebrospinal fluid (CSF) along with ATP, citrate, and ascorbic acid [7, 18]. Oligodendrocytes possess the highest iron content in the brain [19], which may explain their heightened susceptibility to oxidative stress [20]. While oligodendrocytes are primarily responsible for expressing and secreting transferrin, they also uptake non-transferrin-bound iron through Tim-2 receptors via ferritin. Astrocytes acquire non-transferrin-bound iron through DMT1 uptake [17, 21, 22, 23]. Neurons obtain iron from CSF and interstitial fluid by binding it to transferrin.

Lactoferrin [24] and its receptor, lactoferrin receptor (LfR), as well as glycosyl phosphatidylinositol (GPI)-anchored melanotransferrin (MTf) and secreted melanotransferrin, may also contribute to the transportation of iron across the blood-brain barrier [25, 26].

At acidic pH, trivalent iron (Fe³⁺) is released from endosomes and subsequently reduced to divalent iron (Fe²⁺). Divalent metal transporter 1 (DMT1, also known as SLC11A2) facilitates the release of Fe²⁺ from endosomes into unstable iron stores in the cytoplasm [7]. Ceruloplasmin is a GPI-linked membrane protein expressed mainly in astrocytes surrounding brain microvessels within the mammalian central nervous system [27]. Following the discovery of ceruloplasmin’s ferroxidase activity, it was suggested that its role in iron metabolism involves aiding the cellular export of iron by binding to membrane ferroportin [28, 29].

Abnormalities in iron metabolism, including both iron deficiency and iron overload, have a negative impact on overall bodily functions and are associated with various diseases. Iron deficiency specifically reduces cytochrome oxidase activity in the brain, particularly in the hippocampus and prefrontal regions [30], potentially leading to disorders in brain development such as motor skills and cognitive memory [31]. Conversely, excessive levels of iron can accumulate in the brain and contribute to neurodegenerative diseases like Parkinson’s disease (PD) and Alzheimer’s disease (AD) [32, 33]. Additionally, neuroferritinopathies associated with traumatic brain injury (TBI) have also been identified as neurodegenerative conditions characterized by abnormal iron accumulation [34].

3. Ferroptosis and its regulation

3.1 Ferroptosis is a new type of iron-dependent cell death

Ferroptosis is a new mode of cell death discovered by Stockwell’s group at Columbia University in the past decade [9]. When they observed that the small molecule compound Erastin killed RAS mutant tumor cells, they found that the death mode was different from apoptosis, necrosis or autophagy, etc., but it could be effectively inhibited by the iron chelator deferoxamine (DFO), so they named this iron-dependent cell death ferroptosis [35, 36].

3.2 The regulatory mechanism of ferroptosis is complex

With the deepening of research, scientists have found that there is a complex and fine regulatory network of ferroptosis, including inhibition of ferroptosis pathway and activation of ferroptosis pathway (Figure 2).

Figure 2.

Molecular mechanism of ferroptosis.

3.2.1 Inhibition of ferroptosis pathway

The regulatory system of GPX4, along with the GPX4-independent resistance ferroptosis pathway, are included in these mechanisms.

1. The GSH-GPX4 antioxidant system in the cytoplasm and mitochondria is an important pathway for cells to defend against ferroptosis. The cystine/glutamate antiporter (SLC7A11/SLC3A2, also known as System Xc⁻) on the cell membrane transports cystine into the cell and releases glutamate into the extracellular space at a 1:1 ratio [31]. Once inside the cell, cystine can be converted to cysteine, which is further processed by glutamate cysteine ligase (GCL) and glutathione synthetase (GSS). The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) serves as an important reducing agent for synthesizing Glutathione (GSH). By utilizing GSH as a reducing cofactor, lipid peroxidases are ultimately converted to lipid alcohols by cytoplasmic and mitochondrial glutathione peroxidase GPX4 [37].

2. The GPX4-independent pathway of ferroptosis resistance involves the FSP1-CoQ₁₀ pathway, a cell membrane-based inhibitor that prevents lipid peroxide generation by trapping lipophilic free radicals. Myristoylated FSP1 (also known as apoptosis-inducing factor mitochondrial 2, AIFM2) acts as an oxidorereductase of CoQ₁₀ at the plasma membrane and plays a crucial role in this process [38, 39].

3. GPX4-independent ferroptosis resistance pathway, GTP cyclohydrogenase 1-tetrahydrobiopterin (GCH1-BH4) pathway in cytoplasm. GCH1 and its metabolic derivative, tetrahydrobiopterin/dihydrobiopterin (BH4/BH2), protect against ferrodeath by selectively inhibiting the depletion of phospholipids containing two polyunsaturated fatty acyl groups [40, 41].

4. GPX4-independent ferroptosis resistance pathway, known as the dihydroorotate dehydrogenase (DHODH-CoQ₁₀H₂) pathway in mitochondria, plays a crucial role in protecting against ferroptosis. Dihydroorotate dehydrogenase (DHODH), an enzyme dependent on flavin, is located in the inner mitochondrial membrane and primarily functions to catalyze the fourth step of pyrimidine biosynthesis. During this process, dihydroorotate (DHO) is oxidized to orotate (OA), while simultaneously transferring electrons to ubiquinone in the mitochondrial inner membrane. This leads to the reduction of ubiquinone into dihydroubiquinone (CoQ₁₀H₂), which contributes to mitigating ferroptosis within mitochondria. Furthermore, it was found that DHODH inhibits lipid peroxidation and suppresses ferroptosis within mitochondria when GPX4 expression is low. However, under conditions of high GPX4 expression, a combination of ferroptosis inducers such as sulfasalazine and DHODH inhibitors can activate ferroptosis [42].

3.2.2 Activation of ferroptosis pathway

Lipid peroxidation is the main driver of ferroptosis. Intracellular accumulation of free fatty acids is induced by acetyl-CoA carboxylase (ACAC)-mediated fatty acid synthesis or lipophagy-mediated fatty acid release, ultimately leading to ferroptosis [43]. Acyl-coa synthetase long-chain family member 4 (ACSL4) connects polyunsaturated fatty acids (PUFAs), including arachidonic acid and docosatetraenoic acid [44]. These PUFAs are then incorporated into phospholipids by lysophosphatidylcholine acyltransferase 3 (LPCAT3), forming polyunsaturated fatty acid binding phospholipids (PUFA-PLs). PUFA-PLs are susceptible to lipid peroxidation induced by arachidonate lipoxygenase (ALOXs)-mediated free radicals, which ultimately play a crucial role in triggering ferroptosis [45].

Iron ion involvement is a fundamental characteristic of ferroptosis. Iron catalyzes the formation of unstable hydroxyl radicals through the Fenton reaction, which participates in the oxidation of polyunsaturated fatty acids and generates conjugated dienes. This process further leads to the production of lipid peroxidation products such as 4-hydroxynonenoic acid (4-HNE) and malondialdehyde (MDA). It enhances the vulnerability of various cell membranes, impairs cellular function, and ultimately promotes ferroptosis [46, 47]. Wang’s group discovered that key genes involved in iron metabolism pathway, including transferrin and SLC39A14 ferrotransporter [48], ferritin heavy chain (FTH) [35, 49], transferrin receptor (TFR1) [50], FPN pump ferritin [51, 52], iron chaperone binding protein (PCBP1), [53], and nuclear receptor coactivator 4(NCOA4) [54, 55] reported by other research groups have been found to regulate ferroptosis by controlling iron metabolism. It is evident that understanding the biological role of iron and its metabolic pathways in ferroptosis is crucial; therefore, further exploration into underlying mechanisms is necessary.

4. The roles of iron and ferroptosis in TBI

4.1 Imbalance of iron homeostasis in TBI

Studies have confirmed that iron homeostasis in the brain is disrupted following TBI (Figure 3). Several potential causes include abnormal iron deposition due to erythrocyte lysis, mitochondrial and lysosomal dysfunction, and regulatory effects on iron metabolism networks.

Figure 3.

Imbalance of iron homeostasis in TBI.

TBI encompasses both primary and secondary impairments. Primary injury refers to fractures of the head caused by external forces, resulting in cortical or subcortical contusions and lacerations, intracranial hemorrhage (subarachnoid hemorrhage or subdural hematoma), and disruption of the blood-brain barrier (BBB). Diffuse axonal injury (DAI), which is the hallmark injury of TBI, is primarily responsible for long-term complications that may arise from a combination of neuroinflammation and neurodegeneration [56]. Following trauma, axons in the brain’s white matter are particularly vulnerable to damage. The main effects of DAI include mechanical rupture of the axonal cytoskeleton, leading to disrupted axonal transport, resulting in swelling and proteolysis within the axons themselves [57]. Secondary injury refers to subsequent molecular and chemical inflammatory reactions such as release of blood hemoglobin and iron, dysfunction in brain metabolism and cerebral blood flow, further triggering processes related to neuroinflammation, oxidative stress, glutamatergic excitotoxicity, mitochondrial dysfunction, etc., all contributing to additional damage within the brain [58].

Some studies have reported intracerebral iron deposition in TBI patients due to the release of heme iron from intracranial hemorrhage. As brain-specific phagocytes, microglia phagocytose red blood cells and then release degraded iron into the brain interstitium [59]. These mechanisms contribute to an excess accumulation of iron in nerve cells after TBI. The increased levels of iron in both the interstitium and cerebrospinal fluid can be transported to various types of nerve cells through different pathways, ultimately leading to oxidative stress and exacerbating secondary injury following TBI [60].

Iron homeostasis is a dynamic equilibrium regulated by a dynamic network of multiple genes. Following intracerebral hemorrhage, there is an increase in the expression of ferritin, transferrin, and transferrin receptors in the brain, facilitating iron uptake by neurons and oligodendrocytes. Excess Fe can suppress the expression of IRPs in cells and then regulate the increase of ferritin and Fpn1 expression to enhance the capacity of iron pool storage and iron efflux [31, 61]. In patients with TBI, microglia are activated and release toxic substances such as proinflammatory cytokines interleukin (IL-6) and tumor necrosis factor α (TNF-α), complement proteins, and proteases, which contribute to brain injury [62]. With the destruction of the blood-brain barrier and the increase of permeability after TBI, neutrophils and macrophages further infiltrate into the brain tissue [63, 64, 65]. They participate in autophagy, secrete anti-inflammatory factors, enhance the clearance of damaged cells, and reduce the toxic effects caused by cellular degradation. Additionally, they promote cellular repair and restore neural plasticity. However, hepcidin expression also increases during inflammation and iron overload in cells [66]. Studies have indicated that hepcidin has the ability to cross the blood-brain barrier and enter the central nervous system [67]. When the blood-brain barrier is compromised, a significant amount of hepcidin can enter brain tissue. The weakened ability of iron efflux further exacerbates the excessive accumulation of iron, resulting in increased production of toxic free radicals and reactive oxygen species (ROS) such as superoxide free radicals and nitric oxide. This ultimately leads to cognitive dysfunction, brain edema, and other detrimental effects. In addition to ROS generation, it also impairs mitochondrial function, reduces mitochondrial respiration, causes lipid peroxidation, and disrupts protein and enzyme oxidation processes, all contributing to neuronal damage [68]. The increased toxicity of extracellular glutamate and the over-stimulation of glutamate N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors can affect calcium uptake, leading to neuronal damage and cell death [69]. Patients with TBI exhibit elevated levels of extracellular glutamate in the cortex and hippocampus, along with reduced glutamate transporters. These alterations result in decreased gabaergic control and heightened epileptic activity, ultimately causing cell death and affecting cognition in a mouse TBI model [70, 71]. At the same time, it can result in mitochondrial dysfunction and excessive production of free radicals, leading to the activation of the caspase signaling pathway and promoting cell apoptosis. The characteristic features of ferroptosis such as mitochondrial destruction, iron deposition, and accumulation of lipid ROS can be observed [72].

4.2 Iron-related brain injury after TBI

4.2.1 Brain edema after trauma

Brain edema after traumatic brain injury (TBI) is the most common and important pathophysiological reaction, which can lead to increased intracranial pressure (ICP) and even lead to brain tissue displacement and herniation. It is a major cause of clinical death and severe disability, with approximately 50% of TBI patients dying from cerebral edema and its associated lesions [73, 74, 75, 76]. Typically, brain edema peaks within 2 to 3 days after TBI. Eliminating brain edema has become a key target for the treatment of TBI, which is directly related to the prognosis of patients with traumatic brain injury.

There are two main types of brain edema after TBI, including vasogenic edema and cytotoxic edema. Vasogenic edema is caused by fluid and protein leaking into the brain tissue space due to increased capillary permeability after damage to the blood-brain barrier (BBB), resulting in edema around the brain tissue, which can be seen as a widening of the intercellular space. Cytotoxic edema is caused by an increase in intracellular osmoactive substances, usually due to ion pump dysfunction or subsequent entry of extracellular hypotonic fluid into cells, leading to cell edema. Therefore, cytotoxic edema does not lead to an increase in ICP because it refers only to an increase in intracellular water without a change in overall brain water content [77]. This type of edema occurs in all cell types but mainly results in astrocyte cell edema during the acute phase of TBI. The mechanism of traumatic brain edema is still not fully understood. So far, it is believed that the occurrence and development of traumatic brain edema involve a variety of mechanisms, including blood-brain barrier destruction, Ca²⁺ overload, oxygen-free radical toxicity, cerebral microcirculation disorders, energy metabolism disorders, neurotransmitter toxicity, cytokine release, and autonomic nervous dysfunction.

Recent studies have shown that ferroptosis is also involved in TBI, and ferroptosis inhibitor Ferrostatin-1 can mitigate the levels of IL-1b and TNF-a levels as well as the destruction of BBB, thereby reducing brain edema [78]. At the same time, TBI can disrupt the balance between oxidative and antioxidant systems, potentially leading to brain edema [79]. A decrease in GPX4 is a characteristic marker of ferroptosis occurrence, which is caused by the increase of ROS. In animal models of TBI, overexpression of GPX4 reduced brain edema and blood-brain barrier disruption [78]. PUFAs such as arachidonic acid increase the amount of ROS, leading to ferroptosis. An increase in AA and polyunsaturated fatty acids in the brain leads to an elevation in water and sodium levels, while causing a reduction in potassium and ATP-dependent NaC/KC pumps; consequently, this leads to the development of brain edema [80].

4.2.2 Posttraumatic cognitive dysfunction

Posttraumatic cognitive dysfunction primarily presents as disturbance of consciousness, memory, attention, and learning and processing abilities [81, 82], significantly impacting the patients’ quality of life. Individuals with mild TBI may experience temporary disruptions in consciousness, mild confusion, lack of focus, and amnesia immediately following the injury; however, most recover within 6 months [83], with a favorable long-term prognosis. Only a small percentage of individuals with mild TBI will suffer from persistent cognitive impairments [84]. Patients with moderate or severe TBI endure the loss of consciousness for more than 30 minutes or posttraumatic amnesia lasting over 24 hours [85].

Currently, the etiology of cognitive dysfunction remains inconclusive; however, research has postulated the involvement of ferroptosis, diffuse axonal injury (DAI), neuronal abnormalities, and blood-brain barrier (BBB) disruption in this process. Studies have demonstrated reduced levels of glutathione in patients with traumatic brain injury (TBI) [86], as well as iron deposition, which may be associated with cognitive impairment [87]. Intraventricular administration of ferrostatin-1 in animal models of TBI has shown a significant reduction in iron deposition and neuronal degeneration along with improved cognitive function, suggesting a potential role for ferroptosis in the pathogenesis of cognitive impairment [88]. Many studies have demonstrated that head injury is a risk factor for Alzheimer’s disease (AD) [89]. The primary characteristic of AD patients is cognitive impairment. Neuronal deficits in AD patients, particularly hippocampal neuronal damage accompanied by increased ferroptosis, suggest the activation of the Nrf2/GPX4 signaling pathway, which may also contribute to cognitive impairment in traumatic brain injury (TBI) patients [90, 91]. Animal models of AD have indicated that blood-brain barrier (BBB) damage occurs early in the course of the disease [92]. Therefore, BBB impairment in TBI patients may also play a role in cognitive dysfunction.

DAI often occurs after TBI and is characterized by loss of consciousness and extensive axonal damage in the cerebral hemispheres, cerebellum, and brain stem. White matter axons are particularly susceptible to injury following TBI. The shear stress caused by head trauma on white matter axons can result in their rupture, leading to disrupted transport and subsequent physiological changes such as swelling. Almost all patients with severe TBI present with DAI [93]. Studies have demonstrated that axonal abnormalities persist in the brains of TBI patients for years after the initial injury, and DAI may trigger long-term progressive neurodegenerative processes [94].

4.2.3 Posttraumatic epilepsy (PTE)

Posttraumatic epilepsy (PTE) is a common symptom following traumatic brain injury (TBI), with approximately 30% of severe TBI patients developing PTE [95]. About 80% of PTE patients experience seizures within 2 years after the initial injury [96]. Risk factors for PTE include the severity of brain injury, acute cerebral or subdural hematoma, diffuse hydrocephalus, and penetrating brain injury [97]. Posttraumatic seizures (PTS) occur once after head trauma, while PTE is characterized by recurrent epileptiform symptoms in temporal or frontal lobes that can worsen memory and cognitive impairment, sleep disorders, and depression caused by TBI. Antiepileptic drugs such as phenytoin, phenobarbital, levetiracetam, and carbamazepine are commonly used to prevent acute attacks after TBI.

The occurrence of PTE may be associated with damage to the blood-brain barrier (BBB) and intracranial hemorrhage caused by TBI. Infiltration of red blood cells and blood components leads to hemolysis and breakdown of hemoglobin, resulting in the accumulation of free iron and iron-rich compounds such as heme. Studies have indicated that metals can trigger seizures [98]. Iron and ferrous ions initiate an inflammatory response that enhances the generation of free radicals and fragmentation of mitochondria. Oxidative stress leads to lipid peroxidation, causing damage to lipids and promoting the development of epilepsy. This theory was supported by evidence showing that a model of epilepsy could be created by injecting FeCl₃ into mice [99]. In TBI epilepsy models, Nrf2, a transcription factor involved in ferroptosis, is significantly reduced, which promotes the expression of multiple antioxidant, anti-inflammatory, and neuroprotective proteins [100]. NMDA receptor-mediated glutamate excitotoxicity, formation of reactive oxygen species (ROS), subsequent membrane lipid peroxidation, and neuronal cell death can exacerbate cerebral ischemia in TBI patients. Further depolarization results in a lower seizure threshold, leading to seizures [101].

4.2.4 Posttraumatic hydrocephalus

Posttraumatic epilepsy, which is a specific type of hydrocephalus occurring after TBI, was first identified in children by Dandy in 1914 [102]. It is more commonly observed in the pediatric population [103]. The cause of PTH may be associated with intracranial hemorrhage and the adhesion of inflammatory mediators following TBI, affecting the outflow and absorption of cerebrospinal fluid (CSF) [104, 105]. Additionally, iron excess in the brain might also contribute to its occurrence. A study demonstrated that injecting red blood cells into the cisterna magna of dogs resulted in increased iron levels and hydrocephalus formation [106]. Furthermore, another study revealed significantly elevated levels of iron and ferritin in the CSF of SAH patients.

Patients with ubarachnoid hemorrhage (SAH) after TBI are more likely to develop PTH, with a significantly higher risk than patients with other types of TBI [107]. The elevated ferritin may be attributed to the robust inflammatory response in the subarachnoid space, leading to an influx of inflammatory cells and stimulation of ferritin synthesis by inflammatory cytokines. Animal studies have also demonstrated that hemoglobin injection enhances heme oxygenase (HO) activity, accelerates heme metabolism [108, 109], induces erythrocyte lysis, and results in iron overload in the brain. Iron accumulation can contribute to ventricular dilatation. Deferoxamine (DFO), an iron chelator, can upregulate HO-1 expression and reduce acute hydrocephalus caused by ventricular dilatation following TBI [110]. DFO has a concentration-dependent ability to bind and remove iron from ferritin and eliminate approximately 10–15% of iron from saturated transferrin but not from hemoglobin [110]. Additionally, increased lipid peroxidation leads to vascular changes that can also contribute to hydrocephalus development [111].

5. Agents targeting iron and ferroptosis for TBI treatment

Traumatic brain injury (TBI) can result in neuronal damage and even cell death while frequently causing various neurological dysfunction encompassing motor deficits, cognitive impairment, dementia, as well as psychiatric disorders like anxiety and depression. These studies indicate a correlation between iron overload, lipid peroxidation along with GSH depletion with neurodegeneration, and subsequent neurological impairments post-TBI. Here, we summarized an overview of the current research on the treatment of TBI in cases involving ferroptosis (Figure 4 and Table 1).

Figure 4.

Scheme of several pathways implicated in ferroptosis in neurons.

Drugs		Targets	Protective effects	Potential mechanisms	References
Deferoxamine; (DFO)	Iron chelator	Iron chelator	↓ Brain lesion volume, improve cognitive dysfunction; ↑ Cerebral blood flow, improve cerebrovascular function; Inhibit ferroptosis and neuroinflammation,; Improve neurological dysfunction	↓ FTH、FTL、iron deposition, ROS; ↑ GPx activity	[112, 113, 114]
Fer-1	First-generation ferroptosis inhibitor	Inhibitors of lipid peroxidation	↓ Iron deposition, Volume of injury; Improve cognitive dysfunction	↓ xCT, FTH, TFR1, FPN, 4HNE, iron deposition, LPO; ↑ GSH	[88, 115, 116]
Melatonin	hormone	Activate MT2 and inhibit FTH	↓ Brain lesion volume, cytoplasmic shrinkage or nuclear pyknosis, neurodegeneration; Improve cognitive dysfunction, alleviate anxiety-like behavior	↓ Iron deposition, xCT, COX2, TfR1, Fpn, Nox2, FTH, FTL, 4HNE, MDA, Slc7a11, Ptgs2, ERS; ↑ GSH, MT2; regulate circPtpn14/miR-351-5p/5-LOX signaling	[116, 117]
Baicalein	lipoxygenase inhibitor	Inhibit lipid peroxidation (12/15-LOX inhibitor)	↓ Oxidation of phosphatidylethanolamine,; ↑ Neurological function,; Improve spatial memory acquisition	↓ Hippocampal neuronal apoptosis, AA/AdA-PE, 15-LOX, ACSL4; ↑ GSH	[118]
Polydatin	lipoxygenase inhibitor	Activate GPX4 pathway and inhibit TFR, inhibit lipid peroxidation	↓ Acute neuronal damage; Improve motor deficits and memory dysfunctions	↓ Iron deposition, MDA, Slc7a11, Ptgs2, Atp5g3; ↑ GPX4 activity, Gpx4,	[119]
miR-212-5p	Agonist	Micro-RNA Inhibit Ptgs2	Improve learning and spatial memory	↑ miR-212-5p, Ptgs2; ↓ ferroptosis	[120]
Prokineticin-2; (Prok-2)	Ferroptosis inhibitor	Activate GPX4 pathway Inhibit lipid peroxidation	Inhibits lipid peroxidation to prevent neuronal cell death	↓ biosynthesis of arachidonic acid-phospholipid (a lipid peroxidation substrate, accelerates FBXO10-driven ubiquitination, Ptgs2, ACSL4, ferroptosis; ↑ GPX4	[121]
Ruxolitinib	Tyrosine kinase inhibitor	Activate GPX4 pathway and inhibit TfR	↓ Neurodegeneration, brain edema, brain lesion volume, the shrinkage and hyperchromatic morphology; Improve motor deficits and memory dysfunctions, and anxiety-like behaviors	↓ Iron deposition, COX2, TFR1, MDA, 4-HNE↓; ↑ GPX4, GSH, LC3II/I, SCL7A11, FTH	[49]
Anacardic acid	Histone acetyltransferases Inhibitor	Activate GPX4 pathway	Improve neurological function	↓ Ferroptosis	[122]
Orexin-A	G protein-coupled receptor OX1R agonist	Activate Nrf2/HO-1 pathway	↓Brain edema; Improve neurological function	↓ Iron overload, ROS; ↑ Nrf2, HO-1, NQO1	[123]
Tetrandrine	Potassium channels activator	Regulate autophagy	Improve neurological function,	↓ MDA, FTH, p62↓; ↑ GSH, GPX4, SCL7A11, BECN1, LC3II/I,	[124]
Hinokitiol	Natural monoterpene small molecule compounds	Activate GPX4	↓ cerebral edema, mNSS, brain contusion lesions; Protect neurons, Improve neurological function	↓ ROS, LPO, Fe^2+, MDA, p62; ↑ HO-1, Nrf2, GSH, GPX4, SCL7A11,FTH, BECN1	[125]
Pioglitazone	PPAγ agonist	Activate PPARγ	↓ NSS, injured area, neuronal loss; Improve neurological function	↓COX2, DNA; ↑PPARγ	[126]
Netrin-1; (NTN1)	Neurite guidance factors	Activate UNC5B/Nrf2/GPX4 pathway	Protect neurons, improve neurological function	↓MDA, ROS, ACSL4, LOX; ↑Nrf2, GPX4	[127]
Ferristatin II	Iron uptake inhibitors		Protect neurons	↓TfR1, Fe(III), iron overload, MDA, ROS,	[128]
Annexin A5	immune checkpoint inhibitor	Activate NF-kB/HMGB1andNrf2/HO-1pathways	Protect neurons, Ameliorates neuroinflammation, oxidative stress and anti-ferroptosis	↓TfR1, Fe(III), iron overload, MDA, ROS,; ↑Nrf2, HO-1, SOD activity, ratio of M2/M1,	[129]
N-acetyl serotonin	TrkB agonist	Activate TrkB/PI3K/Akt/Nrf2 pathway	↓ Iron accumulation in the ipsilateral cortex,; Alleviate TBI-induced neurobehavioral deficits, lesion volume and neurodegeneration. Rescue mitochondrial shrinkage, improve neurological function	↓xCT, FTH, TfR1; ↑Nrf2, p-TrkB, p-PI3K, p-AKT, GPX4	[130]
HucMSC-derived exosome	Exosome derived from pluripotent stem cells	Suppress PINK1/Parkin-mediated mitophagy	↓brain edema, neuron death, inhibit neuron apoptosis, pyroptosis and ferroptosis; improve neurological function	↓xCT, FTH, TfR1, MDA, 8-OHdG; ↑ Fpn, GPX4	[131]

Table 1.

Agents targeting iron and ferroptosis for TBI treatment.

Given the evidence of iron overload in the brain tissue of TBI patients, drug removal of excessive deposited iron might be an effective therapeutic approach for improving the prognosis of TBI patients. Desferriamine (DFO), as an iron-chelating agent, can bind to excess deposited iron in tissues and cells under pathological conditions. It undergoes oxidative deamination and excreted through the kidney into urine. DFO is commonly employed in clinical treatment for iron overload diseases. Clinical trials have demonstrated that DFO can facilitate hematoma and edema absorption following TBI, while also reducing neuronal degeneration, myelin injury, and inflammation after experimental cerebral hemorrhage. DFO has been shown to alleviate long-term neurotoxicity induced by iron as well as acute cerebral edema caused by TBI in the mouse model, thus alleviating brain damage and cognitive dysfunction in mouse models of TBI [113, 132].

Liproxatin-1 (Lip-1) is a potent antioxidant and ferroptosis inhibitor, comprising amides and sulfonamide subunits that directly inhibit ferroptosis by trapping ROS through the inhibition of xCT or inactivation of GPX4 [94]. It effectively protects cells from ferroptosis. Lip-1 has demonstrated superior efficacy compared to other ferroptosis inhibitors such as DFO and Fer-1. Moreover, Lip-1 exhibits the ability to preserve the integrity of the blood-brain barrier (BBB) in animal models of subarachnoid hemorrhage by inhibiting ferroptosis, suggesting its potential therapeutic value in central nervous system repair by effectively suppressing GPX4-induced oligodendrocyte ferroptosis. In mouse models of TBI, Lip-1 reduces brain injury volume, mitigates neurodegeneration, and improves cognitive dysfunction [116]. These findings provide a promising avenue for clinical treatment of TBI.

Fer-1 is a specific inhibitor of ferroptosis [88, 115, 116], and it has been demonstrated its ability to inhibit cell death in various disease models, including HD, acute brain injury, periabdominal leukomalacia, and kidney failure. It has also shown promising results in stroke and PD models by effectively inhibiting glutamate-induced ferroptosis. In TBI models, intraventricular injection of Fer-1 has been proven to reduce iron accumulation, alleviate neuronal degeneration, and improve cognitive and motor impairment caused by TBI. However, further studies are still needed to validate other clinically applicable methods of administration such as intraperitoneal or intravenous administration.

Melatonin, a hormone secreted by the pineal gland, possesses anti-inflammatory and antioxidant properties. Its lipophilicity enables easy diffusion across cell membranes and subcellular components, effectively blocking a wide range of reactions triggered by TBI, including the ferroptosis pathway. Recent studies have demonstrated that melatonin improves neurological dysfunction following TBI by reducing iron deposition and neurodegeneration [116]. Furthermore, it has been shown that melatonin can effectively enhance nerve function and alleviate motor dysfunction after TBI through inhibition of ferritin heavy chain-mediated ferroptosis [117].

Baicalin (BAI) is a natural flavonoid compound found in the scutellaria plant. It acts as a polyphenol antioxidant and 12/15-LOX inhibitor, possessing anti-inflammatory and antioxidant effects that are widely utilized in clinical practice. Numerous studies have demonstrated the diverse physiological and pharmacological effects of BAI. BAI has been shown to be effective in treating cerebral ischemia by reducing cerebral infarction size and improving neurological deficits in rats with focal cerebral ischemia. Furthermore, it significantly enhances motor function scores and improves motor coordination in animal models after cerebral ischemia, potentially through its involvement in regulating synaptic plasticity and axon growth. Additionally, when injected into the brain tissue of TBI mice, BAI improves memory dysfunction following TBI [120].

Polydatin is a plant extract known for its potent antioxidant and neuroprotective effects. Studies conducted on mouse models of traumatic brain injury (TBI) have demonstrated that polydatin can safeguard neurons, enhance motor deficits in these models, reverse the accumulation of free iron, increase MDA levels, and reduce GPX4 activity in affected brain regions. In vitro studies have also revealed that polydatin can effectively counteract the damage caused by chlormethemoglobin-induced oxidative stress on Neuro2A cells [118].

miR-212-5p, found in various tissues, plays a crucial role in synaptic plasticity, memory formation, and maintaining the integrity of the blood-brain barrier in the brain. In HT-22 and neuro2A cell lines, overexpression of miR-212-5p has been shown to mitigate iron-induced ferroptosis, while downregulation of miR-212-5p promotes ferroptosis through targeting prostaglandin intracperoxidase synthase 2 (PTGS2) [119]. Furthermore, it has been demonstrated that miR-212-5p can significantly enhance spatial learning and memory abilities in TBI mice [119], suggesting its potential to alleviate neuronal iron-induced ferroptosis by targeting PTGS2.

Prokineticin-2 (Prok2) is an important secreted protein likely involved in the pathogenesis of several acute and chronic neurological diseases through currently unidentified regulatory mechanisms. Following traumatic brain injury, neurons undergo mechanical damage which triggers multiple secondary responses including cell death programs such as ferroptosis. It was reported that Prok2 prevents neuronal cell death by inhibiting the biosynthesis of lipid peroxidation substrates, arachidonic acid-phospholipids, via accelerated F-box only protein 10 (Fbxo10)-driven ubiquitination and degradation of long-chain-fatty-acid-CoA ligase 4 (Acsl4), thereby suppressing lipid peroxidation. Mice injected with adeno-associated virus-Prok2 before controlled cortical impact injury exhibit reduced neuronal degeneration and improved motor and cognitive functions [121].

Ruxolitinib, a JAK1/2 inhibitor, is utilized for treating myeloid fibroma. Research has demonstrated that rusotinib exerts neuroprotective effects by inhibiting the JAK-STAT pathway in experimental TBI models [49]. Additionally, it can reduce iron deposition, neurodegeneration, brain edema, and brain injury volume in TBI model mice. This is evidenced by improved motor deficits, memory dysfunction, and anxiety-like behaviors.

Sumac acid (AA), the natural component extracted from cashew nut shells, has been found to have neuroprotective effects in cases of traumatic brain injury. Studies conducted on mouse models with TBI have shown that AA has a strong inhibitory effect on ferroptosis, significantly reducing neural function damage and cognitive impairment caused by TBI [122].

Orexin A (OXA) has been shown to have a protective effect against neuronal damage in various neurological diseases. Studies indicate that OXA effectively inhibits brain tissue injury and nerve function deficits in rats with traumatic brain injury (TBI), while also reducing oxidative stress and iron deposition in the cerebral cortex of these rats. Additionally, OXA may reduce brain damage after TBI by activating the Nrf2/HO-1 signaling pathway, which helps prevent iron-induced ferroptosis [123].

Tetrandrine (Tet) is a natural bibenzyl isoquinoline alkaloid that exhibits promising anticancer, anti-inflammatory, and analgesic properties [124]. Tet effectively mitigates neurotoxicity in 5XFAD mice with cognitive dysfunction by suppressing microglial inflammatory activation. Furthermore, it can alleviate subacute neuronal damage caused by ischemia/reperfusion (I/R-) through the reduction of oxidative stress, apoptosis, and autophagy. Additionally, Tet treatment improves modified NSS (mNSS) scores following traumatic brain injury (TBI), thereby alleviating cerebral contusions and edema in TBI mice [124]. Tet has the potential to ameliorate TBI by attenuating iron-induced cell death via autophagy activation. These findings suggest that Tet may hold promise as a clinical drug for treating TBI.

Hinokitiol (β-thujaplicin, HIK) is a natural monoterpene small molecule that possesses various biological effects, including antibacterial, anti-inflammatory, and anti-tumor activities. Studies have demonstrated that cypress oleophenol can mitigate glutamate-induced intracellular reactive oxygen species (ROS), lipid peroxidation, and Fe²⁺ accumulation in HT-22 cells. Furthermore, it upregulates the expression of heme oxygenase-1 (HO-1), promotes the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), inhibits microglia and astrocyte activation after traumatic brain injury (TBI). Additionally, cypress oleophenol significantly reduces brain tissue damage and neuronal loss while alleviating iron deposition in an in vivo TBI model constructed by controlled cortical impact (CCI), thereby mitigating nerve damage. These findings suggest that cypress oleophenol exhibits neuroprotective effects by rescuing cells from TBI-induced neuronal iron enrichment. Moreover, Hyacinol effectively reduces neuronal damage through activation of the Nrf2/Keap1/HO-1 signaling pathway, highlighting its potential as a therapeutic candidate for TBI [125].

Pioglitazone is a specific agonist for peroxisome proliferator-activated receptor-γ (PPARγ), which has been shown to have a protective effect against damage in the central nervous system [126]. PPARγ levels are reduced in neurons that have undergone iron-induced cell death after traumatic brain injury (TBI), but pioglitazone can reverse this reduction [126]. Further studies have demonstrated that PPARγ activation can prevent iron-induced neuronal death. Administration of pioglitazone effectively reduces oxidative damage, as well as nerve severity scores (NSS), damaged areas, and neuronal loss in TBI mice. These findings suggest that pioglitazone may have potential therapeutic effects against iron-induced cell death in the treatment of TBI. Additionally, rosiglitazone (RSG), another PPARγ agonist, exhibits similar neuroprotective effects to pioglitazone by reducing inflammation and neuronal loss after TBI, inhibiting autophagy and apoptosis in cortical neurons. This suggests that RSG could also be used to improve clinical treatment outcomes for TBI patients.

Mouse models demonstrated an upregulation of Netrin-1 (NTN1) mRNA and protein levels following TBI. The administration of NTN1 shRNA resulted in an increase in the number of FJB-positive cells, as well as elevated levels of malondialdehyde (MDA) and reactive oxygen species (ROS). Conversely, the application of NTN1 recombinants had the opposite effect. Furthermore, knockdowns or inhibition of GPX4, Nrf2, and UNC5B counteracted the effects caused by NTN1 recombinants. Intravenous administration of NTN1 recombination reduced neuronal loss after CCI and improved motor and cognitive function [127]. Therefore, it can be concluded that NTN1 exhibits a neuroprotective effect after TBI while inhibiting iron enrichment through activation of the UNC5B/Nrf2 pathway. These findings may offer potential treatment strategies for TBI.

Ferristatin II (Fer-II) is a substance that inhibits iron uptake and TfR1, leading to the down-regulation of TfR1 through receptor degradation. When administered, Fer-II can reduce the expression of TfR1 in neurons, which helps protect them from damage and neurodegeneration caused by TBI [98]. The molecular mechanism behind Fer-II involves reducing Fe3+ content and iron deposits; reversing the expression of proteins related to iron homeostasis such as R1 and lipid peroxidation genes; and lowering high levels of MDA after TBI [128].

When recombinant Annexin A5 (A5) was administered via the tail vein in TBI mice, it exhibited ameliorative effects on neurological deficits, weight loss, cerebral hypoperfusion, cerebral edema, blood-brain barrier disruption, neuronal apoptosis, and iron-induced cell death. Moreover, A5 effectively attenuated neuronal iron accumulation, p53-mediated cell death, and oxidative stress damage. Additionally, A5 downregulated the HMGB1 and NF-kB signaling pathways while upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) pathway. Collectively, these findings demonstrate that A5 exerts neuroprotective effects in traumatic brain injury by modulating the NF-kB/HMGB1 pathway and Nrf2/HO-1 antioxidant system to mitigate neuroinflammation, oxidative stress damage, and ferroptosis [129].

The N-acetyl-5-hydroxytryptamine (NAS), acting as a TrkB agonist and direct melatonin precursor, exhibits multiple advantageous effects on traumatic brain injury (TBI) [130]. Administration of NAS reduces TBI-induced neurobehavioral deficits, lesion volume, and neurodegeneration. Moreover, NAS effectively mitigates TBI-induced mitochondrial contraction, alterations in iron enrichment-related molecular expression, and iron accumulation in the ipsilateral cortex. Additionally, NAS activates the TrkB/PI3K/Akt/Nrf2 pathway in mouse models of TBI. Inhibition of PI3K and Nrf2 weakens the protective effect of NAS on iron enrichment both in vitro and in vivo. These findings suggest that the TrkB agonist therapy with NAS improves brain function following TBI by inhibiting iron enrichment through activation of the PI3K/Akt/Nrf2/Fth pathway. Consequently, these results provide evidence supporting the potential use of NAS as a promising agent for combating iron enrichment during further treatment of TBI.

Researchers established mouse and neuronal models of traumatic brain injury (TBI). Following treatment with exosomes derived from human umbilical cord mesenchymal stem cells (HUCMSCs), it was observed that exosome therapy can enhance neural function post-TBI, mitigate brain edema, and alleviate brain damage. Administration of exosomes suppressed TBI-induced cellular death, apoptosis, pyroptosis, and ferroptosis. Mitochondrial autophagy mediated by the PINK1/Parkin pathway plays a pivotal role in neuroprotection following TBI [131].

6. Conclusions and prospects

The excessive accumulation of iron and ferroptosis, along with disturbances in iron metabolism, is closely associated with traumatic brain injury (TBI). Iron overload can lead to cell death through processes like ferroptosis and autophagy. It also contributes to the phosphorylation of tau protein in brain tissue, leading to the formation of neurofibrillary tangles. Furthermore, it increases the risk of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease among TBI patients. The potential for neural network damage is also a concern. However, our current understanding of these processes in TBI remains in its early stages, underscoring the importance of delving deeper into their intrinsic relationship.

There are many aspects worth considering. Firstly, in terms of translational medicine, the majority of existing studies primarily focus on a singular factor, and the strategies that can be effectively translated into treatments remain immature. What is the specific regulatory mechanism of ferroptosis on the clinical manifestations of TBI? Sole treatment with antioxidants and anti-excitatory amino acids cannot alleviate cell damage induced by iron overload. Correspondingly, there are certain limitations to improving cognitive function. This suggests that comprehensive consideration of multiple factors is more suitable for selecting clinical treatments. Although iron chelators have been utilized in treating diseases related to iron overload, they have not yet been formally employed in clinical treatment for TBI patients. Rigorous clinical experiments are required to verify their efficacy, which constitutes a long-term project necessitating multi-party cooperation. Secondly, regarding mechanism research, most experimental animal models are used to simulate the pathophysiological characteristics of TBI in humans; while some substances with protective effects on nerve function have been discovered, these animal models fail to fully reflect the complete pathological characteristics observed in humans [112]. The role of iron in the production of ROS and neurotoxicity requires further investigation. Iron chelators are currently only utilized experimentally due to their nephrotoxic and cardiotoxic effects. The clinical application and long-term prognosis of iron chelation therapy in TBI patients also deserve further study. Most likely, the investigation of drug research targeting ferroptosis holds the potential to usher in a new era in the treatment of traumatic brain injury.

Acknowledgments

This work was supported by grants from Medicine and Health Science and Technology Plan Projects of Zhejiang Province (YS, 2020PY029, 2023KY1227), Science and Technology Innovation Special Project of Jiaxing Science and Technology Bureau (YS, 2020AY30003), Key Research Projects of the Affiliated Hospital of Zhejiang Chinese Medical University (YS, 2022FSYYZZ22) and Jiaxing Key Laboratory of Diabetic Angiopathy Research; Jiaxing Key Laboratory of Integrative Rehabilitation of Cerebrovascular Disease.

Conflict of interest

The authors declare no conflict of interest.