As an essential trace element, Zn is required for the function of over 2000 metalloenzymes/proteins. It serves as a crucial component in the regulation of DNA and RNA biosynthesis, in hormone–receptor interactions, and in intracellular signaling, especially for neurotransmission, neurogenesis, or neuronal growth (reviewed in ^[1][2][19,20]). In 1963, the essentiality of Zn in humans was discovered ^[1][19], revealing that Zn deficiency is a worldwide concern related to malnutrition.

Zn content in the body (for a 70 kg adult male) is about 2.5 g; most of the body’s Zn is stored in skeletal muscle, bone, the liver, and the brain, while serum Zn accounts for less than 1% (10–15 µmol/L) ^[2][20].

Zn is absorbed in the small intestine by 2 families of transporters: Zrt/Irt-like proteins (ZIP), a family of 14 proteins, and ZnT, a family of 10 members ^[3][4][5][21,22,23]. ZnT and Zip transporters have opposite roles since Zip transporters increase cytoplasmic Zn concentrations, while ZnTs decrease them, e.g., ZnT1 is located in the small intestine and regulates Zn release from the enterocyte to the general circulation.

Metallothioneins (MTs) are cysteine-rich intracellular proteins that bind metals. They are ubiquitous, and in the intestine’s mucosal cells, they can bind Zn and facilitate its excretion through its loss as cells slough off. In fact, Zn excretion occurs mainly through the intestine, in the pancreatic secretions, while it is lowered through urinary loss and the shedding of epithelial cells.

In serum, Zn²⁺ travels tightly bound to α2-macroglobulin (α2m) and loosely to albumin (Alb) and other proteins, peptides, and amino acids, which serve as a primary source of Zn accessible to all cells. Zn in serum is sharply reduced after severe trauma and inflammation, probably due to the simultaneous increase in the trans-capillary escape rate of α2m and Alb and the increased rate of their catabolism ^[6][24]. The decrease in circulating Zn levels during an acute phase can also be explained by the increased demand for the metal by the liver ^[7][8][9][25,26,27]. In the hepatocyte, Zn facilitates the biosynthesis of acute phase reactants and other essential processes, such as regulation of gluconeogenesis ^[10][11][28,29] in complex processes orchestrated by cytokines, critical modulators of inflammation ^[10][12][13][28,30,31].

Zn plays a central role in brain metabolism. It reaches the brain and crosses the brain capillary endothelial cells (BCECs) that form the Blood–Brain Barrier (BBB), reaching the neurons in the interstitial space. It enters the cerebrospinal fluid (CSF), a biological fluid secreted by the choroid plexus, and surrounds the brain, filling the brain ventricles. Zn is necessary for neurons’ growth and functioning. Transmembrane ZiP proteins facilitate Zn²⁺ ions’ transportation into neurons. Intracellular levels of Zn are regulated by ZnT family members that control Zn²⁺ trafficking and accumulation into vesicles. Zn is enriched into presynaptic vesicles by ZnT3, which loads Zn into glutamate synaptic vesicles and is released into the synaptic cleft together with glutamate. Among the most abundant Zn transporters, ZnT1, -3, and -6 regulate Zn brain levels. Its homeostasis is also controlled by the MT-3 protein that is exclusively expressed in the brain and plays a role in sequestering Zn in synaptic vesicles ^[14][32].

Most all Fe is reduced to Fe²⁺ by reductases before entering the enterocyte. Divalent metal transporter (DMT1) facilitates Fe²⁺ transport within the cell. The Fe²⁺ can be absorbed as heme through the heme carrier protein 1 (HCP1) present on the apical surface of the enterocyte. A heme–oxygenase splits Fe²⁺ from heme and allows it to be stored in ferritin. Ferroportin located in the basolateral enterocyte surface transports Fe²⁺ to the portal plasma. Fe²⁺ is then oxidated to Fe³⁺ by the ferroxidase hephaestin (HP); Fe³⁺ binds to apo-transferrin, forming holo-transferrin (Tf), which transports Fe³⁺ in the blood. Fe levels in the blood are regulated by hepcidin, a hormone that is biosynthesized in the liver. Hepcidin regulates the degradation rate of ferroportin in the enterocyte membrane, and, as a result, the rate of Fe export from the enterocyte to the blood (reviewed in ^[15][33]).

In the liver, Fe is taken up by hepatocytes through endocytosis mediated by the transferrin receptor1 (TfR1). Fe reaches the mitochondria, where it is used for the biosynthesis of heme and Fe–sulfur clusters or for storage in ferritin, the main Fe reserve in the body.

Fe³⁺ moves into the brain, transported by transferrin via the brain capillaries. By binding to the TfR1, it crosses the BCEC that forms the BBB, reaching the neurons in the interstitial space.

The TfR1 on the neuron surface mediates Fe endocytosis. Presynaptic vesicles bear ferroportin, suggesting that Fe²⁺ can travel via the synaptic vesicles to the synaptic cleft, where Fe²⁺ is released from the vesicles.

Glycosylphosphatidylinositol (GPI)-anchored ceruloplasmin (Cp-GPI) on the outside of the tip of astrocytes facilitates the oxidation of Fe²⁺ to Fe³⁺, allowing Fe uploading into apo-transferrin.

The role of Cu in human biology is essential: it serves as a protein cofactor in basic redox reactions in energy production involving cellular respiration, as well as free radical defense, collagen structure, neurotransmitter function, and Fe metabolism. Cu is absorbed in the small intestine into the enterocyte, and a pool of low-molecular-weight, soluble Cu²⁺ complexes is reduced by reductases to Cu¹⁺ that is imported by CTR1. Within the enterocyte, the Cu-transporting P-type ATPase (ATPase7A) pumps Cu²⁺ out of the basolateral membrane via the vesicular compartment. Cu²⁺ then travels in serum to the liver through the portal vein, mostly bound to amino acids, peptides, micronutrients, and Alb as a pool of low molecular-weight Cu, known as non-ceruloplasmin (non-Cp) Cu.

It is then absorbed by the hepatocyte, through the CTR1. In the hepatocyte, ATPase7B, the homolog of enterocytes’ ATPase7A, incorporates Cu into ceruloplasmin that tightly binds 75–95% of Cu, while the residual 5–15% loosely binds to and is exchanged among Alb, α2m, amino acids, peptides, and several micronutrients (non-Cp Cu). Hepatocytes regulate non-Cp Cu levels in the blood to 0.008–1.6 µmol/L (after an overnight fast) ^[16][35]. Cu excess prompts ATPase7B to move from the trans-Golgi network to the canalicular membrane (via a vesicular compartment), where it mediates the release of the metal into bile.

Cu travels to the brain capillaries, mainly as non-Cp Cu, where it crosses the BBB ^[17][36], reaching CSF, where it has values in the range of 0.5–2.5 µmol/L. In the choroid plexus, Cu is taken up, mainly as non-Cp Cu from the blood, and is then released into the brain by processes mediated by CTR1, ATPase7A, and ATPase7AB ^[17][36].

ARAS is an extensive network of more than 20 nuclei in each cerebral hemisphere and of interconnecting fibers, including the ‘diffuse modulatory systems’. They include noradrenergic neurons of the locus coeruleus, serotonergic neurons of the raphe nuclei, cholinergic neurons of the brainstem and basal forebrain, and the dopaminergic neurons of the substantia nigra and of the ventral tegmental area. The neurons of the diffuse modulatory systems have extensive and divergent axon projections, and a single neuron can make contact with 100,000 post-synaptic neurons, releasing neurotransmitters into the extracellular fluid that diffuses to numerous neurons. The diffuse modulatory systems of the ARAS control the rhythms of the thalamus.

Dopamine, noradrenalin (catecholamines), and serotonin play a central role in the cortical–subcortical circuitry of ARAS that takes part, alongside the role in consciousness, either in the regulation of mood, emotions, and sexuality or in cognitive function, primarily in executive functions, as well as in the regulation of sleep and appetite ^[18][19][37,38].

Cu (II) and serotonin have a potential risk of toxicity via oxidation of the serotonin and formation of compounds that are assumed to be unfavorable for neuronal survival ^[20][39].

In cells, including neurons, DihydrOxyPhenylAlanine (DOPA) is synthesized from tyrosine, and it is then used as a substrate by tyrosinase, a Cu-bearing enzyme, to catalyze the synthesis of melanin. The isomer L-DOPA is produced in neurons by tyrosine hydroxylase, a Fe-containing enzyme, and is converted to dopamine by the enzyme L-dopa-decarboxylase. The balance of the catecholamine is regulated by the enzyme beta-hydroxylase, which facilitates the synthesis of norepinephrine from dopamine ^[21][40]. Finally, monoamine oxidase (MAO) controls catecholamine hydrolysis.

Cu is a component of dopamine β-hydroxylase and MAO, and Fe of tyrosine hydroxylase, all involved in the catecholamine balance ^[21][40] that is altered in disorders of Cu metabolism, e.g., in Wilson disease, the paradigm of non-Cp Cu disbalance ^[16][22][35,41], and in a specific subtype of Alzheimer’s disease (AD), the main form of dementia in the elderly, namely, the ‘CuAD’ ^[23][42], typified by non-Cp Cu values higher than normal reference values (>1.6 µmol/L) ^[16][24][35,43].

In the brain, Zn is mainly present in the hippocampus, neocortex, amygdala olfactory bulbs, and hypothalamus ^[25][44], in structural, mostly bound to proteins, and labile, 15–30% forms placed in the synaptic vesicles of glutamatergic glycine- and ϒ-aminobutyric acid-A (GABA)-ergic neurons ^{[26][27][28][29][30]}[45,46,47,48,49].

Zn ions are enriched into presynaptic vesicles by the ZnT3 transporter and released into the synaptic cleft upon neuronal activity ^[31][50]. Upon release, free Zn in the synaptic cleft can bind and modulate pre- and postsynaptic receptors and channels, including the glutamate receptors N-methyl-d-aspartate (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors, glycine, GABAA receptors, and voltage-gated calcium (Ca) channels ^[32][33][34][51,52,53]. In turn, Zn bound to proteins mainly acts as a reservoir of Zn in the synapse; proteins, such as metallothioneins (e.g., brain MT-3) and Zn-finger proteins, can bind to Zn and regulate its availability in the synapse and in different regions of the brain. Zinc bound to proteins also regulates the activity of enzymes, transcription factors, and signaling molecules, affecting synaptic transmission and plasticity ^[14][25][35][32,44,54].

Early work revealed toxic effects of Zn in TBI, mainly related to oxidative stress burst, excitotoxicity, and mitochondrial dysfunction, eventually leading to neuronal apoptosis and/or necrosis (reviewed in ^[36][55]). As Ca, Zn can partake in NMDAR-mediated excitotoxicity, eventually leading to cell death ^[30][49]. Intraneuronal release of Zn²⁺ may also impair mitochondrial functioning ^[37][56]: the metal can prompt the permeabilization of the mitochondrial membrane through the activation of the mitochondrial permeability transition pore that facilitates the release/production of proapoptotic factors, such as cytochrome c, and the apoptosis-inducing factor ^[38][57].

Seminal studies revealed ^[32][33][34][51,52,53] that within the first 6 h after injury, Zn²⁺ is released from presynaptic buttons and is associated with glutamate excitotoxicity processes that lead to postsynaptic neuronal death ^[32][39][51,58]. Zn displacement in these first few hours after injury is the result of rapid transfers from synaptic vesicles, from binding metallothioneins MT-3 in the brain, and from mitochondrial Zn stores, leading to increased cytoplasmic Zn²⁺, which is toxic ^{[32][33][34][36][39]}[51,52,53,55,58]. Upon presynaptic buttons release, Zn appears in the cell bodies of injured postsynaptic neurons; free Zn appears in damaged somata neurons and penetrates the somata through both voltage and ligand-gated ionic channels ^[32][51]. A subsequent study revealed the appearance of Zn²⁺ in injured postsynaptic neurons also at 24 h, then again at 7 days ^[39][58]. Chelating agents, such as CaEDTA, reduced the number of damaged neurons after TBI in the CA3 region of the hippocampus, but only if the treatment occurred within the first 6 h after injury ^[40][41][59,60]. Chelating treatments at later times had deleterious effects; chemical blocking of vesicular Zn ions worsened the effects on the aftermath of TBI by increasing the number of necrotic and apoptotic cells within the first 24 h after TBI ^[42][61], suggesting the beneficial effect of Zn in the reactive processes that follow the acute phase, as will be discussed later.

Compared with Zn, the number of studies on the involvement of Fe and Cu is small, although the clinical significance of these two transition metals in both primary and secondary TBI reactive processes is unquestionable, as demonstrated primarily in studies targeting neurodegeneration. Experimental models of TBI reveal that Fe increases in the acute phase (6 h after injury) and has a major increase in the region closest to the lesion ^[39][58].

Subsequent increases have been revealed at 72 h, then at 7 and 14 days, as well as a maximum increase at 28 days with a concomitant increase in ferritin ^[39][58].

The amount of red blood cell residual from a cerebral hemorrhage in TBI is the greatest source of Fe deposition in the tissue. It causes brain injury: heme oxygenase 1 (HO-1) catalyzes heme oxidation and Fe release from red blood cells, then free Fe²⁺ can trigger oxidative stress ^[39][43][44][58,62,63] via the Fenton reaction. Free Fe²⁺, heme, hemoglobulin, and other blood-derived products are potent cytotoxic agents that can boost oxidative stress, inflammation, and cell signaling disruption, eventually leading to cell death (reviewed in ^[45][64]). Further free Fe²⁺ is released by microglia that engulf red blood cells and release free Fe²⁺ into the interstitial space of the brain ^[46][65]. Dysfunction in mitochondria and lysosomes occurs in TBI and can account for severe oxidative injury within the cell. A large amount of Fe and Ca ions move in, disrupting the normal function of the mitochondria, primarily via ROS generated by Fe through the Fenton reaction ^[47][66]. An important contribution to TBI damage is also provided by the release of Fe²⁺ contained in lysosomes: free Fe²⁺ pooling in the cytoplasm is the most powerful producer of reactive oxygen species (ROS) in cells ^[48][67]. Fe accumulation and overload in the site of injury can also change the size and number of lysosomes, affect autophagic flux, and cause autophagic cell death ^[49][68]. Increased free Fe²⁺ and glutamate overload as a result of excitotoxicity phenomena activation in the synaptic cleft may inhibit glutathione (GSH) synthesis and then facilitate ferroptosis, a Fe-dependent cell death characterized by GSH depletion and a build-up of lipid peroxides that results in cell death (reviewed in ^[50][13]). The increase in Fe at 7, 14, and 28 days, as has been revealed in experimental animal models ^[39][58], is associated with secondary injury processes related to head trauma that include the release of blood metabolites, microglial activation, thrombin activity, and proinflammatory factors, contributing to the final severity and recovery of nerve injury after TBI ^[51][69]. In fact, the accumulation of Fe and ferritin, the Fe handling protein, has been observed greatly distal to the cortical injury site in the brain after injury ^[39][43][58,62]. In humans, once the acute phase is over, the series of secondary injury cascades of TBI can still lead to a poor prognosis. Metal-altered metabolism, and in particular Fe accumulation in tissues, is also part of mild traumatic brain injury (mTBI) that is often ignored because its initial symptoms do not seem serious. By using magnetic field correlation MR imaging in humans that is sensitive to the presence of non-heme Fe, Fe accumulation has been demonstrated at sub-thalamic regions greatly distal to the cortical site of injury ^[44][63].

Studies in humans revealed that decreased serum transferrin and Fe and increased ferritin were associated with severe cerebral edema volume and with a poor prognosis ^[52][70].

Experimental models of TBI ^[39][58] show that Cu concentrations increased in the ipsilateral cortex adjacent, but not closest, to the impact zone only 28 days after the injury. If, on the one hand, this elevation might be related to Cu-dependent processes of demyelination or remyelination, on the other hand, it may be cause for concern in relation to the potential chronic oxidative stress toxicity based on Cu abnormal metabolism, as it has been observed in neurodegenerative disorders and more specifically for AD.

Elevated intracranial pressure (ICP) is a major secondary pathology after TBI and a major contributor to morbidity and mortality. Elevated ICP is defined as a measurement of 25 mmHg for at least 5 min that is verified twice in a 24 h period or on 2 consecutive days. Lower serum ceruloplasmin levels within the first 24 h after trauma are prognostic of elevated ICP in patients with TBI. A cut-off of 14 mg/dL had a sensitivity of 87% and specificity of 73% for identifying patients who developed ICP.

Similarly, a low serum total Cu level (less than 20.76 µmol/L) was also predictive of high ICP (sensitivity 86%, specificity 73%). Three days after the injury, ceruloplasmin levels increased.

This dynamic resembles the Cu dynamics after Myocardial Infarction (MI) ^[53][54][71,72], demonstrating an 80% release of Cu from the damaged myocardial tissue caused by myocardial infarction and a subsequent uptake of Cu by the liver; the increase in serum ceruloplasmin at 3 days in response to myocardial infarction was the result of the increased Cu uptake of serum non-Cp Cu released from the damaged myocardium, rather than resulting from an inflammatory response.

Cu is a particularly potent pro-oxidant and can form hydrogen peroxide and subsequent hydroxyl radicals at a high rate, significantly higher than that of Fe ^[39][58].