Microglia are centrally involved in neuroinflammation. They belong to the mononuclear phagocyte lineage and are CNS-resident cells. Microglia usually present a ramified morphology in the normal brain, regulate brain development, maintain neuronal networks, and repair damage (30, 31). Inflammation, ischemia, and alterations in brain homeostasis can cause dramatic changes in the morphology, gene expression, and functional behavior of microglia, which is termed “microglial activation (32).” Microglial activation can be classified as classically or alternatively activated type (M1 or M2 type, respectively). The M1 type is characterized by the production of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and C-C chemokine ligand 2, which exacerbate the inflammatory response. Contrastingly, the M2 type is characterized by the production of cytokines, such as IL-10, transforming growth factor-β, IL-4, IL-13, and insulin-like growth factor-1, which suppress inflammation and promote tissue repair (33).

Moreover, the microglia-mediated inflammatory cascade is rapidly activated after the onset of severe CVT. Specifically, immediately after CVT onset, endoplasmic reticulum (ER) oxidative stress can be detected as IRE1α by activated microglia, which promotes programmed neuronal death. This indicates that microglia are involved in the early pathophysiology of severe CVT (19). Rashad et al. (18) observed extensive microglial infiltration in the superior sagittal sinus as early as 1 day after severe CVT. On the third day of cerebral venous infarction, microglia infiltration peaked and extended to the subarachnoid space and infarcted cortex. Moreover, microglia showed proinflammatory morphology, with an amoeboid shape rather than the usual ramifications, which is suggestive of M1 microglia.

Following CVT-induced disruption of cerebral blood flow, the adhesion of circulating neutrophils (22) and the release of inflammatory media, including cytokines and chemokines, ultimately induce a series of brain injuries, including BBB disruption, brain edema, and venous infarction (18–20). Activated microglia produce IL, TNF, chemokines, and other inflammatory mediators involved in inflammation regulation; further, they produce cytotoxic substances, including reactive oxygen species, to aggravate brain tissue injury (34, 35). Taken together, microglial activation may be significantly involved in the pathophysiology of severe CVT; however, its specific role and the phenotypic evolution of M1 and M2 remain unclear.

Studies on innate inflammatory responses in the CNS have mainly focused on microglia, which are the CNS macrophages. The contribution of other cell types to inflammatory responses has been overlooked for a long time. However, there is emerging evidence indicating that astrocytes are actively involved in the brain's inflammatory response, with complex biphasic roles in the local regulation of inflammatory responses. Astrocytes, which are the most abundant glial cells of the CNS, originate from the neuroectoderm and are essential BBB components. They are crucially involved in maintaining brain homeostasis and neuronal function (36).

Similar to microglia, reactive astrocytes have two polarization states (the proinflammatory phenotype [A1] and anti-inflammatory phenotype [A2]), which both interact with microglia. M1 microglia activate A1 astrocytes by secreting factors such as IL-1 and TNF-α to set off an inflammatory response amplifying cascade (36). Upon activation, A1 astrocytes increase the expression of numerous genes of the classic complement cascade and inflammatory factors that communicate with M1 microglia and damage neural cells and the BBB (36, 37). Additionally, M2 microglia and A2 astrocytes work together to produce anti-inflammatory cytokines, including IL-6, IL-10, and cardiotrophin-like cytokine factor 1. A2 astrocytes promote BBB remodeling through the aforementioned beneficial cytokines. Specifically, interactions between M2-type microglia and A2-type astrocytes promote neuronal survival and repair (36).

On the third day after the onset of severe CVT in rats, there is dense staining of glial fibrillary acidic protein, which is specifically expressed by astrocytes, in the outer area of the venous infarction. This suggests that astrocytes are strongly activated and recruited in the cerebral venous infarct area, which is consistent with the region of microglia expression. This indicates that astrocytes are also involved in the initial stage of severe CVT (18). This is further indicated by reports of astrocyte infiltration in brain tissues with infarction or severe cerebral edema (19, 38). Astrocyte expression peaks on day 7 after severe CVT, with accompanying glial scars, which suggests that astrocytes may be involved in repair after cerebral venous infarction (18, 20). Furthermore, a clinical case report described that a patient who had isolated cortical venous thrombosis with severe seizures presented parenchymal destruction accompanied by reactive astrocyte proliferation in the excised lobar lesions (21). Taken together, besides microglia, astrocytes may be also actively involved in the inflammatory processes of severe CVT and mediate subsequent neural repair.

Neutrophils are the earliest peripheral leukocytes recruited to the CNS after brain injury (34, 39). Additionally, they can respond to DAMPs and upregulate adhesion receptors after activation by inflammatory mediators such as TNF-α and interferon-gamma (INF-γ), which promotes their adhesion to endothelial cells and migration into inflammatory tissues. Neutrophil recruitment can enhance CNS damage by releasing lysozymes and secreting inflammatory mediators (39), which further activate microglia (26). Additionally, neutrophils can produce neutrophil extracellular traps, extracellular DNA fibers containing histones, and neutrophil antibacterial proteins, which promote the formation of venous thrombosis through various effects (40).

Eosinophilic necrotic foci and scattered neutrophils are commonly observed in the brains of rats with acute severe CVT (41). Additionally, brain water content and BBB permeability after severe CVT could be dependent on leukocyte-endothelial cell adhesion, with both showing a temporal increase during the acute phase. Although the mechanism underlying the relationship between leukocyte-endothelial cell adhesion and brain edema remains unclear, post-CVT brain edema and BBB dysfunction can be prevented by inducing neutrophil reduction using a CD-18 antibody (22).

A retrospective study on inflammatory markers and temporal changes in CVT suggested that compared with patients with chronic CVT, those with acute and subacute CVT showed a higher rate of adverse clinical outcomes and higher absolute neutrophil counts in the peripheral blood (42). Therefore, neutrophil regulation in early-stage CVT could be a potential therapeutic target.

The interleukin-1 cytokine family is comprised of 11 proteins. They bind to their respective ligands and initiate inflammatory signaling and regulate immune response; among them, IL-1β and IL-18 act as the proinflammatory family members (44). IL-1β and IL-18 are closely associated with venous thrombosis (45, 46). Specifically, IL-1β is a key accelerator of venous thrombo-inflammation, which promotes venous thrombosis through several mechanisms, including leukocyte recruitment, remote signaling through thrombogenic microparticles, and platelet integrin activation (45). IL-1β and IL-18 are indicators of microglial and inflammasome activation, which intensifies the CVT severity (18). There are increased IL-1β (18, 19, 47) and IL-18 (18, 19) levels in the area of cerebral venous infarction in rats with acute/subacute severe CVT. IL-1β begins modestly increasing as early as 6 h after CVT, peaking on day 3 (18, 19) and remaining at a high level until day 7, followed by a return to baseline levels by day 14 (19). The trends of IL-18 and IL-1β expression are similar (18). After severe CT, immunofluorescence staining reveals significant microglial IL-1β levels (18), which further illustrate the inflammatory role of microglia in severe CVT.

Interleukin-6 can be produced by various cells, including monocytes, macrophages, endothelial cells, adipocytes, and the Th-2 subset of T-helper cells. IL-6 can transmit signals to endothelial cells, leukocytes, and hepatocytes; promote the synthesis of the coagulation factors (e.g., fibrinogen, tissue factor, and factor VIII); and stimulate platelet production. Specifically, IL-6 can mediate the coagulation cascade through various actions, which are closely associated with arteriovenous thromboses (48). An exploratory study on the acute CVT animal model reported significantly increased levels of proinflammatory cytokines, including IL-6 and IL-1β, compared with the sham group. Upon reduction of the levels of the aforementioned proinflammatory factors, there was a significant reduction in the cerebral venous infarct volume, which suggests that inhibiting inflammation may improve outcomes in severe CVT (47).

Notably, IL-6 can promote C-reactive protein (CRP) production by hepatocytes. IL-6 and plasma CRP levels increase during acute inflammation, which synergistically acts during the acute inflammatory phase. They are among the most commonly tested biomarkers for acute inflammation in clinical practice (49). Several studies have reported significantly higher serum IL-6 (17, 50) and Hs-CRP (17, 51)/CRP (52) levels in patients with severe CVT than in healthy controls, with IL-6 levels peaking on the first day of CVT diagnosis (50). This suggests that IL-6 and Hs-CRP levels are strikingly increased during the acute and subacute stages of CVT, followed by a decrease in the chronic phase (17, 51).

Contrastingly, another study reported no significant increase in the levels of pro-inflammatory factors, including IL-6, in patients with a history of CVT (53). This further indicates that inflammation occurs soon after the onset of severe CVT and is mainly involved in the acute/subacute phase. Additionally, IL-6 levels are positively associated with the risk of adverse prognostic events (17). This demonstrates that inflammatory factors are associated with severe clinical manifestations of CVT; moreover, they may be used as diagnostic biomarkers with predictive utility in the clinical treatment of severe CVT.

Tumor necrosis factor-α is a common proinflammatory factor involved in coagulation. TNF-α can regulate thrombogenic proteins, activate complements, and stimulate endothelial cells and macrophages to produce tissue factors, which ultimately promote coagulation (54). TNF-α is mainly produced by microglia in the CNS. In animal models of ischemic stroke, TNF-α-induced apoptosis of endothelial cells resulted in BBB disruption (55). Similarly, there was increased TNF-α expression in the area of cerebral venous infarction in rats with early-stage CVT. Notably, TNF-α expression is substantially reduced by recombinant human soluble thrombomodulin (rhs-TM), which inhibits the pro-inflammatory factors to prevent further brain damage in rats, and therefore, improves adverse outcomes (47).

The high mobility groupbox-1 is a ubiquitous nuclear protein that maintains nucleosome integrity and promotes gene transcription; moreover, it is a DAMP protein. It can be released by granulocytes or necrotic cells into the extracellular matrix to activate macrophages and neutrophils, which initiate an acute inflammatory response via proinflammatory cytokines such as TNF-α (56).

The high mobility groupbox-1 levels are significantly increased in infarcted brain segments (47, 57) and peripheral blood (57) in rats with early-stage CVT. There is a simultaneous increase in the proinflammatory cytokines TNF-α, IL-1β, and IL-6 (47). Pharmacological inhibition of the HMGB1 inflammatory pathway using rhs-TM (47) or glycyrrhizin, which is a natural anti-inflammatory drug (57), was found to alleviate neurological deficits and reduce the volumes of cerebral venous infarction in rats with CVT. Additionally, mechanical thrombectomy combined with glycyrrhizin significantly reduced brain injury in rats with severe CVT (57). This suggests that intravascular therapy combined with anti-inflammatory drugs may have additive or synergistic neuroprotective effects and may be a potential treatment option for severe CVT.

Matrix metalloproteinases are a class of zinc-dependent endopeptidases extensively involved in CNS diseases, including neuroinflammation, stroke, epilepsy, and multiple sclerosis (58). Activated microglia, neutrophils, and endothelial cells can produce MMPs, which damage cerebral vessels and the BBB (26). Their gene expressions are activated by various inflammatory mediators, including TNF-α, IL-1β, IL-8, IL-17, and IL-18 (59). MMPs contribute to the neuroinflammatory pathway by acting as signaling molecules, activating the pathway, shedding death molecules, and directly hydrolyzing cerebrovascular basement membrane and tight junction proteins, which further impairs vascular integrity and ultimately causes BBB disruption and leakage (57).

Matrix metalloproteinases-9, which is among the most studied MMPs in stroke, was significantly increased in animal models of CVT (18). A recent study reported higher baseline levels of MMP-9 in patients with CVT with parenchymal brain injury than those in healthy controls (60). MMP-9 levels are associated with persistent venous occlusion, which suggests that MMP-9 is involved in brain injury resulting from severe CVT (60). However, we recently observed no significant differences in serum MMP-9 levels between patients with CVT with or without venous infarction. Additionally, none of the patients showed MMP-9 present in the cerebrospinal fluid (51). Although hypoxia increases the MMP-9 expression (61), hypoxia in venous infarction may not be as significant as that in arterial infarction, which explains the inconsistent findings. Further research is warranted to clarify the role of MMP-9 in severe CVT.