Hemoglobin, the primary component released by erythrocytes, is a potent mediator of OS-induced injury. Katsu et al. found that intracerebral injection of hemoglobin into rat striatum induced hydroethidine signals, which was found colocalized within vessel walls by assessing situ gelatinolytic activity (27). Yan et al. reported that injection of hemoglobin into striatum dramatically boosted the level of carbonylation and formation of malondialdehyde compared with that in the contralateral striatum at 72 hours (33). Prominently, hemoglobin oxidative injury is caused by the formation of free radicals (mainly through Fenton-type mechanism) (7), while deoxyhemoglobin spontaneously and nonenzymatically oxidizes to methemoglobin (34).

Hemin, the degradation substance of hemoglobin, is highly cytotoxic due to the large amount of ROS it can produce (35). In vivo or in vitro, ROS production can be significantly elevated by treatment with hemin in rat brain or in cultures of primary cortical neuron (36) and cortical astrocytes (35). The mechanism of hemin-related oxidative damage is thought to be due to its breakdown to iron by heme oxygenase (13). Indeed, hemin can directly induce the production of free radicals and damage intracellular structures (37). Hemin is a lipophilic substance that promotes lipid peroxidation by inserting into lipid membranes (38).

Iron is a product of the degradation of hemoglobin/hemin. In a collagenase ICH model, iron was found to deposit around the hematoma 1 day after ICH, peaked at 7 days, and remained at a higher level at 14 days (39). OS induced by ferrous iron leads to DNA injury and brain damage after ICH. The mechanism of iron overload leading to ROS production after ICH mainly includes neuronal toxicity of iron via transferrin receptor transport system (40), and ferroptosis, an iron-dependent lipid peroxidation injury (41). Recently, ferroptosis was documented to occur in a mouse model of ICH (42). Ferrostatin-1, a specific inhibitor of ferroptosis, exerts neuroprotective effects by inhibiting lipid peroxidation, prostaglandin-endoperoxide synthase-2 and cyclooxygenase-2 (42). The iron chelator, deferiprone and clioquinol, decreased iron levels and ROS production, improved neurological outcomes and attenuated brain edema (43). Consistent with these results, Takehiko et al. reported neurological dysfunction and brain edema, and the high expression of 8-Hydroxy-2-deoxyguanosine in ICH was significantly alleviated after deferoxamine treatment (44). These observations indicate that deferoxamine and other iron chelators may be neuroprotective through reduction of OS triggered by iron.

Bilirubin is the end product of hemin catabolism. Low level of bilirubin induces OS, neuroinflammation, and cell death (45). Besides hemoglobin and hemin, bilirubin oxidation products are also considered as a source of ROS after ICH. OS damage caused by bilirubin oxidation products via hemoglobin and Fenton reaction following ICH was first reported in 2008. Bilirubin oxidation products and malondialdehyde were significantly increased in brain tissues of the porcine ICH model, suggesting a strong oxidative stress environment (46). However, there have been no thorough information regarding bilirubin oxidation products in ICH since then.

Apoptosis is caspase dependent programmed cell death (71). Han and colleagues reported that hydroxyl radicals and malondialdehyde dramatically increased in an ICH animal model; terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells appeared at 6 hours, peaked at 3 days, and decreased at 7 days after ICH. These authors also reported that apoptosis was positively correlated with malondialdehyde and free radical content in brain tissue, suggesting that increased oxidative stress injury after ICH may lead to brain cell apoptosis (72). Moreover, intracellular ROS and microglia apoptosis were significantly attenuated by the ROS inhibitor N-acetyl-l-cysteine, probably via a thioredoxin interacting protein pathway (65). Thus, inhibition of ROS/thioredoxin interacting protein pathway may ameliorate microglial apoptosis.

OS triggers apoptosis through a number of mechanisms. For example, ROS and oxidation of mitochondrial proteins cause mitochondrial permeability transition pore to open, and cytochrome C is then released to activate cell death intrinsic pathways (70, 73). As well, mitochondrial membranes are destroyed by peroxidated lipids, resulting in the outflow of Ca²⁺ and loss of cell membrane function leading to mitochondrial permeability transition pore formation (70). Moreover, cellular viability is decreased by the 4-hydroxy-trans-2-nonenal-derived OS, which is mainly related to caspase independent apoptosis (74).

Necrosis can be induced by a variety of chemical or biological insult. Necrosis is characterized by swelling of the organelles, rupturing of membranes, and release of cell contents.

Evidence suggest that excessive superoxide generated by nicotinamide adenine dinucleotide phosphate oxidase in cells leads to the switch from apoptosis to necrosis (75). Conversely, administration of nicotinamide adenine dinucleotide decreases OS induced by H₂O₂ and protects cells from necrosis (76). Recent data show that inhibiting mitochondrial dysfunction by AdipoRon increased ATP levels, lowered ROS, and reduced neuronal necrosis and the apoptotic rate at 72 hours after ICH in mice (77). These results suggest that changes in intracellular redox status influence the process of cell necrosis. Mechanisms of cell necrosis induced by OS after ICH includes: 1) Mechanical compression of the hematoma on the adjacent tissue and activation of NMDAR by excessive glutamate. Both can result in an influx of calcium, which then causes the depolarization of mitochondrial membrane and the activation of the mitochondrial permeability transition pore, resulting in release of ROS and necrosis (78); and 2) Activated microglia/macrophages and infiltrating neutrophils following ICH can release proteases and oxidants. Moveover, they can also directly induce cell necrosis by attacking complement-bound cells (79).

Necroptosis is a receptor-interacting protein and mixed lineage kinase domain-like protein mediated programmed necrosis that can be induced by ROS. The ROS scavenger, butylated hydroxyanisole, lowered the number of necroptotic cells; corresponding, necrostatin-1 significantly decreased ROS accumulation induced by hemin via inhibiting receptor-interacting protein 1 in HT-22 cells (80). These results suggest that antioxidants ameliorate heme-induced necroptosis (76). There is considerable evidence that ROS can drive necroptosis through caspase independent pathways (81). However, the mechanisms of OS in inducing necroptosis still need be further explored.

Autophagy is a lysosome-mediated degradation mechanism of cellular contents. Moderate autophagy promotes cell survival, while excessive autophagy induces cell death (82). A recent study revealed that ICH involves autophagy and iron plays an important role in this process (83). Consistent with this result, another study reported that autophagy appeared at 6 hours after ICH, the autophagic flux was damaged at 3 days, and returned to normal at 7 days (84). Interestingly, OS is a key mediator to induce autophagy after ICH (71). The mechanisms of OS-mediated autophagy after ICH primarily include three aspects. First, OS promotes autophagy up-regulation to clear dysfunctional mitochondria, destroyed intracellular proteins and other damaged organelles, maintaining intracellular homeostasis (85). Second, OS activates IKKα/NF-κB/I-κB kinase β-mediated pro-inflammatory signaling pathways through autophagy. At the same time, the accumulation of large amounts of ROS in mitochondria and cytoplasm can cause OS damage and induce autophagy. Third, the molecular network of ROS damage, including toll-like receptor-4, PI3K-Akt-mTOR, and AMPK, is closely related to the activation of autophagy-related genes (85).

Ferroptosis is an iron-dependent lipid peroxidation injury of cell. It is characterized by excessive involvement of antioxidant enzymes and the lethal peroxidation of lipid bilayer membrane. Ferroptosis is elicited in ICH and its inhibition is beneficial in vivo and in vitro ICH conditions (40). The antioxidant enzyme glutathione peroxidase 4 may alleviate secondary brain injury following ICH by inhibiting ferroptosis (86). OS is involved in the process of ferroptosis. Moreover, increased lipoxygenase activity leads to sustained oxidation of phosphatidyl ethanol and arachidonic acid, which is an indispensable part of ferroptosis (81). Administration of ferrostatin-1 decreased lipid oxidation products, and inhibited gene expression of cyclooxygenase-2 and prostaglandin-endoperoxide synthase-2 (42). Therefore, the imbalance of OS due to intracellular iron overload and depletion of antioxidant enzyme may be an important factor of ferroptosis after ICH.

Iron released by hemoglobin, the degradation product of erythrocytes within hematoma, has been implicated in apoptosis, OS, inflammation, and autophagy, which contribute to secondary brain injury after ICH (99). Thus, targeting iron is a promising strategy against OS in ICH.

Deferoxamine is an iron chelating agent that crosses the BBB to reach high therapeutic concentrations in brain tissue (100). It can prevent iron from entering the Haber–Weiss reaction to produce hydroxyl radicals by forming stable compounds with chelated ferric iron and hemosiderin (91). Moreover, deferoxamine regulates the expression of iron metabolism regulation genes, the activity of binding proteins, and it promotes the gene expression of antioxidant enzyme that inhibit apoptosis. In addition, deferoxamine down-regulates the expression of prolyl 4-hydroxylase and c-Jun N-terminus kinase genes, while it elevates heme oxygenase-1 gene expression after ICH (101). More importantly, deferoxamine has both anti-phagocytic and anti-inflammatory effects (102). Therefore, deferoxamine may be a promising therapy for ICH. Systemic administration of deferoxamine (200 mg/kg, intraperitoneal) lowered ROS, reduced iron deposition, attenuated the activity of microglia and infiltrated neutrophils, and decreased neuronal death in a collagenase-induced ICH model (91). More recently, a clinical trial has confirmed the safety of deferoxamine in ICH patients (103). A post hoc analysis of the intracerebral hemorrhage deferoxamine trial (iDEF Ttrial) reported that deferoxamine treated patients with moderate hematoma volume (10-30 mL) achieved favorable outcomes (104).

Statins, which are classical cholesterol-lowering agents, have pleotropic effects including anti-inflammatory, antioxidative, and neuroprotective outcomes (105). Statin-mediated neuroprotection may stem from several mechanisms, including reduction of ROS production and down-regulation of mevalonate and its derivatives; statins also target the main signaling pathways of cell proliferation, adhesion, migration and cytokine secretion (106). In rats with cecal ligation and puncture, treatment with simvastatin reduced nitric oxide level, increased catalase activity, and normalized reduced/oxidized glutathione (GSH/GSSG) ratio in the brain (107). Atorvastatin decreased TUNEL-positive cells, inducible nitric oxide synthase expression, and myeloperoxidase-positive or OX42-positive cells in the perihematomal regions in ICH in a dose-dependent manner (92). However, another group observed that prior use of statins before ICH did not impact neurological outcomes (108). A clinical trial found that application of statins before onset of ICH reduced the volume of brain edema around hematoma (109) and 30-day mortality (110). These contrary results may be due to species differences or the mode of administration of statin. A phase III trial (Statins In Intracerebral Hemorrhage [SATURN]) is now underway to determine the benefits of statin by continual or discontinuous administration; and whether the individual’s apolipoprotein-E genotype may affect the decision to continue/discontinue statins. This study should provide new insights and guidelines to the use of statin in ICH patients.

PPAR-γ is a ligand-activated nuclear transcription factor which plays an important role in augmenting the phagocytosis activity of microglia/macrophage, and regulating oxidative stress and inflammation. Rosiglitazone, a PPAR-γ agonist, upregulated the CD36 phagocytic receptor in phagocytes, enhanced the ability of phagocytes to engulf erythrocytes, promoted hematoma absorption and reduced ICH-induced neurological defects in a mouse model of ICH (111). Consistent with this, another study revealed that PPAR-γ agonists remarkablely enhanced the expression of PPAR-γ targeted genes (CD36 and catalase); they inhibit the transcription of pro-inflammatory factors, reduce extracellular H₂O₂ level, and promote neurological function recovery (93). Based on these encouraging preclinical research results, a clinical trial was conducted to test the safety of pioglitazone in the absorption of hematoma after ICH. However, although the study is completed (ClinicalTrials.gov Identifier: NCT00827892), the results have not appeared in the published literature.

Minocycline, a tetracycline derivative, is FDA-approved for bacterial infection and acne vulgaris. It can cross the BBB, and significantly reduce the infiltration of neutrophils and macrophages; it decreases the activation of microglia and ameliorates irreversible damage of cells (112, 113). Minocycline is also a potent matrix metalloproteinase inhibitor; it is an iron chelator and has anti-inflammatory properties (114). In cortical cell cultures treated with ferrous sulfate, minocycline prevented neuronal loss and decreased the level of malondialdehyde (94), which corroborated the anti-OS property of minocycline. Recently, it was shown to significantly improve neurobehavioral outcomes in ICH (115). Moreover, a small clinical trial showed that minocycline at 400 mg per day achieved reasonable serum concentrations in ICH patients (116). A review of the literature has proposed that a high dose twice daily IV minocycline, initiated promptly after ICH with hematomal evacuation, is a reasonable strategy to pursue in a treatment trial in ICH to improve its prognosis (117).

NXY-059 is a free radical scavenger developed for use in acute ischemic stroke. In a collagenase induced ICH model, NXY-059 treatment significantly inhibited the infiltration of neutrophils, reduced TUNEL-positive cells around the hematoma, and improved the neurological dysfunction of rats 48 hours after ICH (95). A clinical trial was conducted in 2004, and confirmed that NXY-059 was safe to begin within 6 hours of ICH and wass well tolerated with no serious adverse effects; however, there were no obviously differences in neurological deficit scores and disability rates at 3 months after ICH compared with the placebo-treated patients (118). Therefore, the translation of NXY-059 from bench to clinic needs to be further investigated.

Edaravone is a ROS scavenger widely used in ischemic stroke within 24 hours of onset. In ICH animal model, edaravone significantly alleviated brain edema, inhibited the activation of caspase, and decreased the levels of interleukin 1β and NF-κB (96). Moreover, edaravone improved behavioral recovery scores, ameliorated cerebral edema and BBB permeability, decreased interleukin 1β and tumor necrosis factor levels, and ameliorated neuronal apoptosis (119). However, there is no clinical evidence of the effectiveness of edaravone in ICH patients.

In transient limb ischemia, remote ischemic conditioning confers neuroprotection in part by countering injury-induced ROS generation in a Nrf2-dependent fashion (120, 121). In animals with ICH, remote ischemic conditioning can attenuate brain edema by upregulating heme oxygenase-1, transferrin, and transferrin receptor (122), and promoting hematoma resolution via increasing anti-inflammatory macrophage activation (123). In a multicenter, randomized, controlled clinical trial of 40 patients with ICH, treatment with remote ischemic conditioning for seven consecutive days was safe and well tolerated, promoted the rate of hematoma resolution, and reduced perihematomal edema (124). However, functional outcomes at 90 days were not significantly different from those in the control group possibly due to the small sample (124). A phase III randomized clinical trial with 452 participants is currently underway to evaluate the effect of remote ischemic conditioning on the clinical outcomes of patients with ICH (ClinicalTrials.gov Identifier: NCT04657133). The results of this study are eagerly awaited.

Heme oxygenase degrades heme into biliverdin, carbon monoxide, and iron. Besides, the breakdown of hemoglobin can be partially catalyzed by heme oxygenase (125). As mentioned above, hemoglobin and its degradation products are key mediators of OS after ICH. Thus, targeting heme oxygenase may be of utility for neuroprotection after ICH.

Heme oxygenase-1, a core enzyme in heme and iron-catalyzed metabolism, is highly expressed in microglia and astrocytes, but has low levels in neurons (32). Hemoglobin can induce heme oxygenase-1 upregulation to reduce ROS accumulation and apoptosis (35). Conversely, downregulating the expression of heme oxygenase-1 by siRNA-mediated knockdown of Nrf2 enhanced the toxicity of hemin on astrocytes (126).

Heme oxygenase-2 is mainly expressed by neurons. Deletion of heme oxygenase-2 attenuated OS injury in the whole-blood injected ICH model in mice (125). Moreover, heme oxygenase-2 gene deletion alleviated the level of carbonylation and malondialdehyde induced by hemoglobin injection (33). In addition, heme oxygenase-2 knockout markedly reduced ROS production and cell damage caused by heme or hemoglobin (97).

Nrf2 is the master regulatory component of antioxidant stress injury. It initiates the expression of various antioxidant enzymes such as superoxide dismutase, glutathione peroxidase-1, heme oxygenase-1, glutathione transferases, and NAD(P)H quinone oxidoreductase-1 (127). Nrf2 knockout mice showed greater neurological injury, increased hematoma volume, cytochrome C release, and DNA oxidative injury than wild-type ICH mice (98). Similarly, exacerbated neurological injury in Nrf2 knockouts was also reported in the blood injection ICH model (128).

miRNAs are a group of small single-stranded RNAs without coding function; they negatively modulate the expression of target genes and proteins (16, 127, 129). OS regulates miRNAs biogenesis by acting on major molecules that influence miRNAs maturation, and modifies miRNAs to alter their integrity, stability and binding activity. Most importantly, multiple miRNAs are involved in antioxidant stress response of cells (127). Accumulating research show that miRNAs are expressed abnormally in perihematomal tissue and blood of patients with ICH (129, 130). However, the effect of different miRNAs on ICH is quite complex and should be discussed within a specific context.

The expression of miR-146a was dramatically reduced in ICH rats (131) and was negatively correlated with OS status in the brain (132). Overexpression of miR-146a inhibited OS injury and inflammation partly through inhibiting the TNF receptor-associated factor 6/NF-κB signaling axis (131). Similarly, overexpressed miR-183-5p protected brain cells and improved neurological deficit function score following ICH via blocking heme oxygenase-1 production, indicating heme oxygenase-1 mediated close relationship between Nrf2 and miRNA (129). Moreover, overexpression of microRNA-139 increased Nrf2 expression and decreased NF-κB expression (133). Taken together, miRNAs may exert a protective role by reducing inflammation and OS injury after ICH.

However, the role of miRNA could also be negative. The inhibition of miR-27b, an OS-responsive microRNA, alleviated brain injury and upregulated the level of heme oxygenase-1, superoxide dismutase and Nrf2 after ICH via the Nrf2/ARE pathway; dual-luciferase reporter analysis demonstrated that miR-27b acts directly on Nrf2 mRNA (127). Moreover, miR-155 in the parietal cortex and hippocampus was reported to be engaged in neural damage in ICH, and down-regulation of miR-155 expression was beneficial for neurological recovery by reducing the products of OS and enhancing vascular endothelial growth factor (134). In addition, increased miR-130a in serum is closely associated with severe brain edema and poor prognosis after ICH (16).