Oxidative stress results in accumulation of numerous oxidative intermediates, such as reactive oxygen species (ROS) and reactive nitrogen radicals, leading to tissue damage and cell death (Martemucci et al., 2023; Maiese, 2023). Cerebral ischemia and hypoxia in VCI increases ROS levels in cerebrovascular ECs (Zeylan et al., 2024) and can cause disruption of Ca²⁺ homeostasis in the brain, impairing the function of the mitochondrial electron transport chain (ETC) in ECs. Activation of ROS enzymes, such as nicotinamide adenine dinucleotide phosphate oxidase (Nox), xanthine oxidase, and nitric oxide synthase (NOS), causes the production of excess ROS to further promote the occurrence of oxidative stress (Wang et al., 2024; Wang et al., 2023). In rodent models of CCH, Nox2 levels in the brain and in hippocampal neurons are increased and associated with cognitive impairment (Alfieri et al., 2022; Choi et al., 2014). Oxygen generates other ROS, such as hydrogen peroxide (H₂O₂) and hydroxyl free radicals, through a series of disproportionation reactions. In vitro experiments using cells exposed to H₂O₂ to simulate an oxidative stress injury have been performed (Song et al., 2020; Wang et al., 2021). Antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and catalase are decreased in the peripheral blood of VCI patients, whereas the oxidative stress markers malondialdehyde (MDA), reactive ROS, and lactate dehydrogenase are increased (Krishnan and Rani, 2014; Xue et al., 2017). Oxidative stress is the basic pathological process of VCI; however, the mechanism has not been fully elucidated, and further in-depth study is needed.

Neuroinflammation is an immune cascade mediated by microglia and astrocytes in the central nervous system (CNS) (Sanz et al., 2024; Villa-Gonzalez et al., 2024), which is caused by pathological injuries such as infection, ischemia, and hypoxia (Titisari et al., 2024; Thiankhaw et al., 2024). Neuroinflammation triggered by cerebral ischemia and hypoxia in a VCI mouse model causes WMLs, cognitive dysfunction, and learning and memory deficits. In addition, various proinflammatory factors, such as interleukin beta 1 (IL-1β) and IL-6, are significantly increased in brain tissue and serum (Gao et al., 2024; Tang et al., 2024). Neuroinflammation induces neuronal dysfunction and death, leading to cognitive dysfunction (Villa-Gonzalez et al., 2024; Huang et al., 2024). Continuous damage to the vascular system results in the destruction of the BBB, further promoting the inflammatory response and eventually leading to more severe cognitive dysfunction and even dementia (Tang et al., 2024; Nyul-Toth et al., 2024). Additionally, the complement system can also cause neuroinflammation. In the early stages of VCI, complement components (C1q and C3) facilitate phagocytosis and clearance of amyloid fibrils; however, in late stages, C5 activates the membrane attack complex to induce neuroinflammation and neuronal damage (Batista et al., 2024; Wen et al., 2024). In summary, the entire inflammatory process promotes astrogliosis, endothelial dysfunction, BBB disruption, and complement system disorders, leading to neuronal damage.

Mitochondria, known as the “powerhouse” organelles of the cell, provide 95% of cellular energy and play an important role many cell biological processes (Spinelli and Haigis, 2018; Hu et al., 2024). Ischemia and hypoxia reduces the synthesis of ATP in nerve cells, the Na⁺/K⁺ pump on the cell membrane is inactivated, and excitatory amino acid (mainly glutamate) levels increase, which activates the formation of mitochondrial permeability transition pore (mPTP). This leads to Ca^{2 +} overload and disrupts the mitochondrial membrane potential (Wang et al., 2023; Rahi and Kaundal, 2024). The open mPTP also allows other metabolites to enter the mitochondrial matrix space and uncouples the mitochondrial ETC from ATP synthase activity, leading to a reduction in ATP, cessation of oxidative phosphorylation, and finally, production of excessive ROS (Beg et al., 2024; He et al., 2022). ROS destroy the activities of complexes I to IV and impairs mitochondrial respiratory function, resulting in mitochondrial outer membrane damage. Cytochrome C and apoptosis-inducing factor in mitochondria are released into the cytoplasm and promote neuronal apoptosis (Okoye et al., 2023). Additionally, prolonged hypoxia, mitochondrial Ca²⁺ overload, and excessive ROS accumulation may completely inhibit or over-activate mitophagy, leading to mitochondrial homeostasis imbalance and further aggravating cerebral ischemic injury and cognitive impairment (Wang et al., 2024). Therefore, mitochondrial dysfunction is among the fundamental causes of nerve damage and cognitive impairment in VCI.

Excitotoxicity is one of the earliest discovered and widely recognized molecular mechanisms of injury after ischemic stroke (Burch et al., 2024). Due to disordered metabolism in the ischemic area in VCI, excessive release of the excitatory neurotransmitter glutamate in the synaptic cleft increases activation of the N-methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and kainic acid receptors, causing increased Ca²⁺ influx. The dramatic increase in intracellular Ca²⁺ activates neutral proteases, endonucleases, and phospholipases, resulting in the destruction of DNA and the neuronal cytoskeleton, simultaneously producing various free radicals and apoptotic bodies and eventually causing neuronal apoptosis (Rajeev et al., 2023; Deng et al., 2019). Moreover excitability toxicity and excessive NMDA receptor activation triggers the JNK/c-Jun/AP-1 signal transduction pathway, further expanding ischemic cell death (Nuzzo et al., 2019). Furthermore, NMDA activation stimulates NOS to produce a high concentration of NO, which further promotes neuronal damage (Wang et al., 2024; de Sousa Maciel et al., 2022). In summary, excitotoxicity is an important neurobiological phenomenon that plays a key role in various neurological diseases and injuries.

Glial activation, particularly microglial activation, results in polarization towards proinflammatory M1 and anti-inflammatory M2 phenotypes (Ye et al., 2024; Lokesh et al., 2024). M1 microglia secrete a variety of proinflammatory cytokines, whereas M2 microglia secrete a series of anti-inflammatory cytokines that promote endocytosis and eventually reduce neuronal damage (Mo et al., 2022; Wang et al., 2024). Microglial cells in the brain of VCI patients multiply, especially in the white matter, and quickly activate, causing morphological changes (Zhao et al., 2024) that mainly promote the polarization of microglia cells to the proinflammatory M1 phenotype. Secreted proinflammatory factors aggravate brain tissue damage after ischemia (Pang et al., 2023). In vitro experiments showed that 2,3,5,6-tetramethylpyrazine had an anti-inflammatory effect, restraining the inactivation of the NF-κB signaling pathway to inhibit the polarization of M1 microglia and reducing the expression of inducible NO synthase and CD86 M1 markers and proinflammatory cytokines TNF-α and IL-6, thereby reducing lipopolysaccharide (LPS)-induced neuroinflammation (Chen et al., 2023). Another study showed that paeoniflorin inhibited activation of the NF-κB signaling pathway, thereby blocking the polarization of M1 microglia in the hippocampal CA1 region of VCI rats and reducing the expression of inflammatory mediators IL-1β, IL-6, TNF-α, and NO. Furthermore, paeoniflorin can upregulate the expression of anti-inflammatory factors IL-10 and TGF-β1 in M2 microglia, thereby reducing neuroinflammation in the hippocampal CA1 region (Luo et al., 2018). In conclusion, microglia can promote neuroinflammation in chronic cerebral ischemia by activating the NF-κB signaling pathway (Chen et al., 2024).

Programmed cell death is a regulatory mechanism mediated by signal transduction pathways. Abnormal regulation of programmed cell death is associated with neurodegenerative diseases and cancer (Yang et al., 2022). In CCH animal models, as ischemic time was prolonged, apoptosis of hippocampal neurons and Bax expression increased, while expression of the anti-apoptotic factor Bcl-2 decreased (Niu et al., 2021; Zhang et al., 2021). Expression of the autophagy markers P62 and LC3-II/LC3-I increased (Xu et al., 2023; Wang et al., 2023), whereas ferroptosis-related proteins such as solute carrier family 7 member 11 and GPX4 were downregulated (Fan et al., 2024; Lou et al., 2024). Additionally, expression of pyroptotic proteins caspase-3, nod-like receptor pyrin domain-containing protein 3 (NLRP3), GSDMD-N, caspase-1 p20 (its active form), and ASC were increased (Zhu et al., 2024; Zhang et al., 2023). Necroptosis plays an important role in hippocampal neuron loss and white matter damage after chronic cerebral ischemia (Nikseresht et al., 2019; Chen et al., 2018). Various cell death modes mediate VCI cell loss; however, necroptosis plays a particularly prominent role and is a potential target for regulating VCI neuron loss.

The BBB is an important physiological barrier between the CNS and peripheral blood circulation. It selectively transports nutrients, expels toxic substances and metabolites (Zhang et al., 2024; Zhou et al., 2024), and maintains the dynamic stability of the internal environment of the CNS (Shang et al., 2024; Bai and Ge, 2024).

The mechanism of BBB dysfunction in VCI is not fully understood; however, oxidative stress, excitotoxicity, and neuroinflammation promote dysregulation of EC apical tight junction proteins, leading to increased BBB permeability. This promotes the entry of cytokines, immunoglobulins, and self-secreted serum factors into the brain, resulting in collagen deposition around the corpus callosum. Subsequently, WMLs are formed, resulting in cognitive decline (Rajeev et al., 2022; Rajeev et al., 2022). Additionally, increased endothelial transendocytosis after CCH leads to BBB dysfunction (Shang et al., 2024). In cerebral ischemia, excitatory toxicity from increased Ca²⁺ concentration in the EC cytoplasm induces metabolic disorders, mitochondrial dysfunction, protease activity, and the activation of phospholipase and ROS generation. These common factors cause EC membrane injury and vascular cell death, damaging the integrity of the BBB (Shah et al., 2024; Zhao et al., 2022). Animal experiments have shown that CCH-induced cognitive impairment can be improved by alleviating neuroinflammation and protecting the BBB (Wang et al., 2023; Li et al., 2022).

WMLs are predominantly involved in brain structure changes, and the main pathological features are demyelination, gliosis, loss of nerve fibers and oligodendrocytes, and microglial activation (Lin et al., 2024; Xiao et al., 2023). WMLs are associated with a 73% increased risk of VCI (Hu et al., 2021). CCH induces oxidative stress, inflammation, oligodendrocyte apoptosis, and microglial activation, promotes demyelination, decreases nerve fiber microtubule-associated protein 2 (MAP2) levels, and increases myelin basic protein levels, leading to impaired cognitive function and further aggravating WMLs (Jiang et al., 2024; Guo et al., 2022). Therefore, extensive cerebral ischemia-induced WMLs are now considered the key drivers and most important pathological features of VCI and dementia and are an important cause of cognitive deficits.

Pathophysiological studies on cerebral ischemia suggest that glial cell activation, cell death pathways, BBB function, and therapeutic interventions for WMLs may inhibit the pathological progression of VCI.

In the classical necroptotic pathway, TNF-α binds to its receptor TNFR1 and then combines with TNF receptor-associated death domain protein (TRADD), RIPK1, TNF receptor-associated factor family proteins (TRAFs), and inhibitor of apoptosis protein cIAP1/2 and linear ubiquitin chain assembly complex (LUBAC) to form complex I (Wu et al., 2024; Wendlocha et al., 2024). In response to deubiquitinating enzymes such as cylindromatosis (CYLD), RIPK1 dissociates from complex I and recruits Fas-associated death domain protein (FADD), which then binds to pro-caspase-8 to generate complex IIa. When caspase-8 is inhibited, RIPK1 is autophosphorylated at Ser166 and binds to RIPK3 via the RIPK homotypic interaction motif to form complex IIb, and RIP3 is phosphorylated at Ser227 to promote MLKL phosphorylation. Phosphorylated MLKL (p-MLKL) oligomerizes and translocates to the cell membrane, leading to the disruption of cell and organelle membranes and eventually to necrosis (Ranjan and Pathak, 2024; Tran et al., 2024) (Figure 2).

Necroptosis involves several non-classical pathways. Although the activation pathways are not identical, RIPK and MLKL are activated. For example, Z-DNA binding protein 1(ZBP1) activates RIPK3-dependent cell death upon binding to Z-form nucleic acids, including Z-RNA produced by certain viral or endogenous retroviral elements (Yang et al., 2023; Liu et al., 2023). Toll-like receptor 3 (TLR3) recognizes and binds to double-stranded RNA molecules in the cytoplasm and recruits and phosphorylates RIPK1 and RIPK3 via the bound toll-like receptor linker molecule 1 (TRIF). TLR4 is a membrane receptor that combines with LPS signaling molecules and relies on TRIF recruitment of RIPK1 and RIPK3 (Muendlein et al., 2022; Chen et al., 2022). Type I/II interferon can activate double-stranded RNA-dependent kinase, which binds RIPK1 and RIPK3 (Rosenberg and Sibley, 2021). TRAIL can convert apoptosis into an RIPK1-dependent necrosis pathway in an acidic environment (Park et al., 2020). TNFR lacking the death domain can initiate necrosis via the RIPK1-FADD-caspase-8 complex (Abd El-Aal et al., 2022; Pham et al., 2019). In some cases, MLKL can be directly activated independently of RIPK3 activation.

RIPK1 has a unique hydrophobic pocket that modulates its kinase activity through structural changes. All RIPK1 inhibitors reported to date bind to this pocket (Xie et al., 2013). Nec-1 was first discovered by screening necroptotic inhibitors (Chevin et al., 2022). Nec-1 significantly reduces ischemia-induced RIPK1 and increases the number of neurons in a rat MCAO cerebral ischemia model, thus showing a protective effect on brain injury (Deng et al., 2019). In an LPS-induced neuritis mouse model, Necs can inhibit microglia activation by inhibiting RIPK1 phosphorylation, thereby inhibiting neuroinflammation (Kim et al., 2023). GSK481, GSK772, GSK963, and GSK547 are also RIPK1 inhibitors (Mifflin et al., 2020). RIPK1 inhibitors such as SAR443820, SIR2446M, GFH312 have been tested in phase I clinical trials in healthy subjects, It has a good safety, pharmacokinetic and pharmacodynamic profile (Hincelin-Mery et al., 2024; Sun et al., 2024; Lickliter et al., 2023). RIPK1 inhibitors showed various neural inflammatory disease treatment effects but did not specifically inhibit necroptosis because they can also inhibit apoptosis.

Unlike RIPK1, RIPK3 does not affect apoptosis, and targeted inhibition of RIPK3 more specifically controls necroptosis (Prasad Panda et al., 2023). Therefore, specific RIPK3 inhibitors are important for necroptosis-related drug research and development. Classical RIPK3 small-molecule inhibitors include GSK840, GSK84, GSK872, and GW39B (Sun et al., 2024; Xu et al., 2023). GSK872 administration decreased RIPK3, p-JNK, and IL-6 expression and neuronal death and improved neurobehavior in rats (Hu et al., 2020). Moreover, GSK872 inhibited neuronal necroptosis in intracerebral hemorrhage mice through the death domain-associated protein signaling pathway (Bai et al., 2024). Many RIPK3 inhibitors, such as AZD5423, Compound-42, and Zharp-99, have been developed to inhibit necrotizing apoptosis in mouse inflammatory models and acute kidney injury, thereby alleviating kidney injury and systemic inflammation (He et al., 2023; Xu et al., 2022). Therefore, RIPK3 inhibitors could also have a neuroprotective effect in VCI.

MLKL, which comprises a four-helix bundle (4HB) and pseudokinase (psK) domains, acts as an executor of necroptosis and is an important drug target, and many inhibitors have been tested in previous studies. These include covalent inhibitors, ATP competitive inhibitors, and noncovalent inhibitors (Tang and Zhuang, 2024; Martinez-Osorio et al., 2023). As irreversible covalent inhibitors, necrosulfonamide and compound TC13172 both bind to Cys86 of human MLKL to protect cells from necroptosis; however, they do not affect necroptosis in mouse cells (Ji et al., 2021). GW806742X was the first molecule discovered that protects the cell from necroptosis by competing with ATP binding at the psK domain of MLKL (Pierotti et al., 2020). Another noncovalent inhibitor binds to the 4HB domain of MLKL, thereby inhibiting its action (Cui et al., 2022). However, the current molecular regulators of MLKL are in the early drug development stage and still have some limitations, so they are mainly applied in research.