Following an ischemic stroke, the immune system is critically involved in the neuroinflammatory response. The initial damage from the stroke releases DAMPs (damage-associated molecular patterns), which activate various components of the innate immune system, such as neutrophils, macrophages, and microglia (14, 15). This activation triggers the complement system and toll-like receptors, intensifying the inflammatory response. Concurrently, the adaptive immune system is engaged, involving different lymphocyte populations including T cells, B cells, and regulatory T cells, as well as antigen-specific responses (14, 16, 17). This immune activation can compromise the integrity of the blood-brain barrier (BBB), allowing immune cells and inflammatory molecules to enter the brain, further exacerbating inflammation and stroke injury (14, 18). Importantly, this response presents a wide array of possible therapeutic targets since it involves many signaling pathways over a timescale of days to weeks.

The inflammatory response following a stroke evolves over time, transitioning from an acute phase to a chronic phase. Acute neuroinflammation serves as an initial defense mechanism, with microglial cells, the brain's resident immune cells, clearing out dead cells and debris (19, 20). In intracerebral hemorrhage (ICH), macrophages clear red blood cells, aiding in hematoma resolution, while in ischemic stroke, microglia and macrophages might contribute to ferroptosis, a type of non programed cell death associated with iron and lipid peroxidation (21, 22). Targeting ferroptosis is a promising therapeutic strategy, though the mechanisms are still under investigation (23–25).

Two types of edema characterize the acute and chronic phases of post stroke neuroinflammation. Cytotoxic edema occurs rapidly after an ischemic stroke within minutes to hours and is caused by loss of ATP due to reduced blood flow and glucose supply. This leads to Na+/K+ pump failure and thus cellular swelling. Cytotoxic edema peaks 3–4 days post injury (26, 27). Vasogenic edema is characterized by BBB breakdown that allows water and plasma proteins to leak into the interstitial compartment. This occurs via a transcellular route and a paracellular route through tight junction disruption. Vasogenic edema can begin as early as 6 h after a stroke and peaks 6–7 days. Since vasogenic edema causes direct tissue swelling, it is the primary driver of post stroke elevations of intracranial pressure and subsequent neurologic decline. Severe cerebral edema of either variety (but more commonly vasogenic) can increase mortality rates up to 80% (26, 27).

Current research underscores the importance of platelet adhesion and early activation in mediating thromboinflammatory damage post-stroke, primarily through interactions involving glycoprotein (GP) VI and integrin α₂β₁ with collagen, and GPIbα with von Willebrand factor (VWF) (31, 52). These interactions facilitate platelet adhesion, activation, and aggregation, crucial for thrombus formation. Notably, VWF's role in stroke is evidenced by studies showing that VWF deficiency leads to reduced ischemic damage and improved outcomes (53–55). Therapeutic approaches targeting the VWF-GPIbα axis, such as anti-VWF nanobodies and GPIbα antagonists, show promise in experimental models by reducing both thrombosis and inflammation without increasing bleeding risks (28, 56–58). However, these interventions have not been performed in humans yet.

Platelet activation begins with endothelial damage, which exposes subendothelial collagen and VWF. This exposure results in platelet tethering via the GPIb-V-IX complex. Subsequent firm adhesion is mediated by integrins, notably α₂β₁ binding to collagen and αII_bβ₃ binding to fibrinogen (59). This interaction triggers platelet activation, characterized by shape change, degranulation, and the release of secondary mediators such as ADP and thromboxane A2, which amplify the activation process (60). Activated platelets aggregate to form a thrombus, stabilizing the initial hemostatic plug.

Activated platelets also release polyphosphates, which further activate the intrinsic coagulation pathway through FXII, which leads to thrombin generation and fibrin formation (61). Beyond their hemostatic function, platelets release pro-inflammatory cytokines and chemokines, such as IL-1_β and CD40l, which recruit and activate leukocytes, thereby linking thrombosis with inflammation (62, 63). Additionally, when activated to the inflammatory phenotype, platelets impair normal microvascular function, cause breakdown of microvascular barriers and can affect the extravasation of immune cells through gap junctions (29, 64, 65). These multifaceted roles of platelets highlight their central position in thromboinflammation, making them a prime target for therapeutic intervention.

The kallikrein or contact-kinin pathway is, initiated by factor XII (FXII) activation, and it significantly contributes to thromboinflammation by promoting both coagulation and inflammation (66, 67). This complex of serine proteases both triggers thrombus formation and helps regulate the subsequent local response via kinin mediated signaling cascade. FXII activation leads to a cascade involving plasma kallikrein and bradykinin release, which exacerbates BBB permeability and inflammation, worsening the risk of subsequent hemorrhage (68, 69). Inhibition of this pathway, either genetically or pharmacologically, has been shown to reduce stroke severity in animal MCA occlusion mouse models, highlighting its potential as a therapeutic target. For instance, blocking plasma kallikrein or bradykinin receptors mitigates BBB disruption, inflammation, and thrombus formation, which suggests these interventions may provide neuroprotective effects without impairing hemostasis.

Upon vascular injury, FXII binds to exposed endothelial surfaces and becomes activated to FXIIa. This initiates the kallikrein-kinin system (KKS), where FXIIa converts plasma prekallikrein to kallikrein. Kallikrein then cleaves high-molecular-weight kininogen (HMWK) to release bradykinin, a potent vasodilator and inflammatory mediator. Bradykinin binds to its receptors, B1 and B2, on endothelial cells, which increases vascular permeability and promotes the recruitment of immune cells.

This pathway also interacts with the complement system, which is another critical component of the inflammatory response. FXIIa can activate the complement cascade, which leads to the production of anaphylatoxins such as C3a and C5a that further recruit and activate leukocytes. This crosstalk between the coagulation and complement systems amplifies the inflammatory response and contributes to tissue damage and edema in the ischemic brain.

Therapeutic interventions targeting the kallikrein-kinin system (KKS), such as FXII inhibitors, kallikrein inhibitors, and bradykinin receptor antagonists, have demonstrated substantial promise in preclinical MCA occlusion mouse models. For instance, FXII inhibitors like garadacimab have been shown to reduce thrombus formation and limit inflammation, resulting in decreased infarct sizes. Kallikrein inhibitors, such as lanadelumab, effectively block the conversion of prekallikrein to kallikrein, thereby diminishing the production of bradykinin and subsequent inflammatory responses. Bradykinin receptor antagonists, including icatibant, prevent bradykinin from binding to its receptors B1 and B2, which helps to stabilize the BBB and reduce cerebral edema. A phase 1 clinical trial ReMEDy1 (NCT03290560), testing DM199 a recombinant form of human tissue kallikrein-1 (KLK1) showed promising initial results in reducing the rate of secondary strokes. A follow up phase 2/3 trial ReMEDy2 (NCT05065216) is ongoing, though it was paused for nearly 2 years due to reports of three adverse events regarding hypotension on drug administration (70–72).

These treatments collectively reduce inflammation and stabilize the BBB, which lead to smaller infarct sizes and better overall outcomes in animal models. Importantly, these therapeutic agents have not been associated with a significant increase in bleeding risk, and they are attractive candidates for further clinical development. Their efficacy in managing thromboinflammatory processes in ischemic stroke presents a promising avenue for improving patient care and reducing the long-term impacts of stroke.

The adaptive immune system, particularly T cells, plays a critical role in stroke-induced thromboinflammation. Studies have demonstrated that T cell infiltration into the ischemic brain exacerbates neuronal damage through interactions with platelets and endothelial cells (35). Regulatory T cells (Tregs) and other subsets of T cells modulate this inflammatory response, with evidence suggesting that their absence can either worsen or ameliorate stroke outcomes depending on the context (73, 74). Current research is exploring the modulation of T cell activity as a therapeutic strategy, with promising results from interventions targeting T cell adhesion molecules and cytokine signaling pathways (75).

After a stroke, the damaged BBB allows immune cells to infiltrate the brain parenchyma (76). CD4+ and CD8+ T cells, which are normally involved in immune surveillance, become pathogenic in the context of ischemic injury. CD4+ T cells contribute to inflammation by releasing pro-inflammatory cytokines such as IFN-γ and TNF-α, which exacerbate neuronal damage and promote further immune cell recruitment (77). CD8+ T cells, on the other hand, directly kill neurons through perforin and granzyme B, compounds typically used to eliminate virus-infected cells (45, 77).

Regulatory T cells (Tregs) play a dual role in stroke. In the acute phase, Tregs can limit excessive inflammation and tissue damage through the release of anti-inflammatory cytokines like IL-10 and TGF-β (78). However, in some contexts, Tregs may also inhibit beneficial immune responses necessary for tissue repair and clearance of dead cells (79). The timing and context of Treg activity are therefore critical in determining their overall impact on stroke outcomes.

The role of B cells in stroke-related thromboinflammation has been less extensively studied compared to T cells, but emerging evidence suggests they play important roles. Recent research indicates that B cells may have both protective and pathogenic roles in stroke. Regulatory B cells producing IL-10 have been shown to limit CNS inflammation and reduce infarct volume in experimental stroke models (80). However, B cells can also contribute to thromboinflammation by producing autoantibodies against brain antigens exposed after BBB disruption (81). The net effect of B cells likely depends on the specific subsets involved and the timing relative to stroke onset. More research is needed to fully elucidate the diverse functions of B cells in stroke pathophysiology.

Therapies aimed at modulating T cell responses include monoclonal antibodies targeting adhesion molecules (such as ICAM-1 and VCAM-1) that facilitate T cell entry into the brain, as well as cytokine inhibitors that block the action of pro-inflammatory cytokines (82). Early clinical trials with these approaches have shown potential in reducing inflammation and improving neurological outcomes, although more research is needed to refine these strategies (83–85).

Thromboinflammation post-reperfusion in ischemic stroke involves complex interactions between cellular and humoral immune mediators. Reperfusion triggers an influx of neutrophils, monocytes/macrophages, and T cells, which peak at different time points and contribute to both protective and damaging effects (86, 87). This process in the CNS differs from peripheral thromboinflammation due to the presence of the BBB and resident microglia (88). The repercussions of reperfusion on a leaky BBB include increased permeability to both small and large molecules, facilitating the entry of inflammatory mediators and potentially exacerbating tissue damage.

The von Willebrand factor (VWF) A1 domain plays a crucial role in mediating the inflammatory response during cerebral ischemia/reperfusion by recruiting inflammatory monocytes, neutrophils, and T cells (56). This recruitment process contributes to the thromboinflammatory cascade, which involves highly interconnected thrombotic and neuroinflammatory signatures. The central-peripheral immune communication after ischemic stroke is facilitated by various mechanisms, including the release of damage-associated molecular patterns (DAMPs) into the periphery and the transmission of inflammatory signals from the brain to the spleen via the sympathetic nervous system and hypothalamic-pituitary-adrenal axis (89, 90).

BBB disruption, a major hallmark of stroke, is initiated by ischemia and continues to deteriorate with sustained hypoperfusion. This breakdown occurs partially through the overexpression of matrix metalloproteinases (MMPs), which can degrade the endothelial glycocalyx, a known mechanotransducer (8). The sustained increase in BBB permeability is likely due to a neuroinflammatory response, which, combined with other consequences such as brain edema, contributes to longer-term permanent loss of neurological function (91).

Antiplatelet agents such as clopidogrel or ticagrelor are already in widespread clinical use and target this pathway (92, 93). Other agents remain under investigation. For example, GPVI antagonists are being tested for their efficacy in reducing stroke severity and improving outcomes (94). Additionally, the potential repurposing of drugs used in other inflammatory conditions, such as multiple sclerosis, for stroke treatment is being investigated (95, 96). Overall, understanding the intricate interplay between thrombosis and inflammation in stroke is paving the way for novel therapeutic approaches that could significantly enhance patient outcomes by addressing the underlying thromboinflammatory mechanisms.

Targeting the Kallikren-Kinin pathway is another promising approach. FXII inhibitors, such as garadacimab, have shown efficacy in reducing thrombus formation and inflammation in animal models (97). A clinical trial of this agent reduced the angioedema attacks in patients with hereditary angioedema with dysfunction of factor XII (98, 99). This would be an interesting agent to test in the stroke population. Similarly, bradykinin receptor antagonists, such as icatibant, have demonstrated protective effects by reducing BBB permeability and edema (100).

Anti-inflammatory strategies are also being explored, including the use of Toll-like receptor 4 (TLR4) antagonists and interleukin-1β (IL-1β) inhibitors (47, 101). Early clinical trials have shown promising results, with agents like ApTOLL and Canakinumab demonstrating potential in reducing inflammation and improving recovery (102, 103). Fingolimod, a sphingosine-1-phosphate receptor modulator used in multiple sclerosis, has also shown potential in reducing infarct size and improving outcomes in stroke patients (95, 96, 104).

The complex interplay between thrombotic and inflammatory processes in stroke highlights the need for multifaceted therapeutic approaches, but also offers promise because of the many potential therapeutic targets. Combination therapies that target both thrombosis and inflammation may offer the best outcomes. Ongoing research and clinical trials will be critical in identifying the most effective strategies and translating these findings into clinical practice.

Ischemic stroke disrupts the BBB and triggers a robust inflammatory response. The primary ischemic injury sets off a chain reaction involving excitotoxicity, oxidative stress, and the release of damage-associated molecular patterns (DAMPs), which further exacerbate the injury (Figure 2). Excessive release of glutamate leads to overactivation of NMDA receptors, which in turn results in calcium overload and neuronal injury. Reactive oxygen species (ROS) generated during ischemia/reperfusion damage cellular components, leading to lipid peroxidation, protein oxidation, and DNA damage. The ischemic insult elevates levels of DAMPs, activating microglia and astrocytes and attracting peripheral immune cells to the injury site (105). This immune response, while initially protective, often becomes dysregulated, leading to further neuronal damage.

Neutrophils are among the first peripheral immune cells to infiltrate the ischemic brain, which typically peaks within the first 3 days post-stroke. These cells exacerbate tissue damage through the release of proteolytic enzymes, ROS, and pro-inflammatory cytokines (106). Elevated levels of matrix metalloproteinases (MMPs), particularly MMP-9, are associated with BBB breakdown and hemorrhagic transformation (107). Neutrophil depletion or inhibition of MMP activity has been shown to reduce BBB permeability and infarct size, which suggests that neutrophil inhibition may be an attractive therapy for acuter ischemic stroke (108). Interestingly, in animal models there is evidence that neutrophils from the bone marrow of the skull can utilize microvascular channels to migrate to the neuroinflammatory site, potentially indicating that there may be a role for blockading this local bone marrow effect (109).

Monocytes are recruited to the ischemic brain shortly after neutrophils, where they differentiate into macrophages. These cells play a dual role in stroke pathology. In the early stages, macrophages contribute to inflammation and tissue damage by producing pro-inflammatory cytokines such as TNF-α and IL-1β (90). However, during the later stages, macrophages can adopt an anti-inflammatory phenotype, promoting tissue repair and remodeling by secreting growth factors and anti-inflammatory cytokines like IL-10 (90, 110).

Microglia, the resident immune cells of the central nervous system (CNS), play a crucial role in the immediate and long-term response to ischemic stroke. Following the ischemic insult, microglia are rapidly activated, migrating to the site of injury. This activation is essential for debris clearance and initial neuroprotection; however, prolonged or excessive activation can lead to exacerbated neuroinflammation and secondary cell death (20, 111).

Microglia exhibit a range of phenotypes post-stroke, including both proinflammatory (M1) and anti-inflammatory (M2) subtypes (112). This categorization has fallen out of favor as it does not capture the complex range of responses, however it is still a useful structural framework to conceptualize the response. The M1 phenotype is typically associated with the production of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, and is linked to neurotoxicity and the perpetuation of inflammation (113, 114). On the other hand, the M2 phenotype is associated with the release of anti-inflammatory cytokines, such as IL-10 and TGF-β, and is involved in tissue repair and neuroprotection (114).

The differentiation between these subtypes is influenced by various signals in the microenvironment (115). For example, interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) can drive microglia towards the M1 phenotype, while IL-4 and IL-13 promote the M2 phenotype (20, 114). Single cell RNA studies have indicated that there can be many microglial cell subsets within an area of ischemia neural tissue (111, 113, 116, 117).

The interplay between microglia and adaptive immune cells is critical in determining the extent of secondary cell death, and this relationship is poorly understood in stroke patients. For instance, microglia can present antigens to T cells, influencing their activation and differentiation (111, 114). This interaction can either amplify the inflammatory response or contribute to its resolution, depending on the context and the signals involved.

This plasticity of microglial responses underscores their potential as therapeutic targets in managing neuroinflammation and secondary cell death post-stroke.

The adaptive immune system also plays a critical role in the progression of secondary cell death following stroke. The activation of the adaptive immune response involves T cells, B cells, and regulatory T cells (Tregs), each contributing to the inflammatory milieu post-stroke (86, 87, 118). CD4+ and CD8+ T cells infiltrate the CNS following BBB disruption. CD4+ T cells can differentiate into various subsets, including Th1 and Th17 cells, which produce pro-inflammatory cytokines that exacerbate neuroinflammation and tissue damage (35). CD8+ T cells can directly induce neuronal apoptosis through cytotoxic mechanisms. Regulatory T cells (Tregs), on the other hand, have a neuroprotective role by secreting anti-inflammatory cytokines such as IL-10 and TGF-β, which can suppress the activation of harmful immune responses and promote tissue repair (34, 119).

B cells and the antibodies they produce may also contribute to post-stroke inflammation, though evidence suggest they are less important than T cells (38, 120). Autoantibodies targeting CNS antigens can exacerbate neuronal damage and inflammation. However, B cells can also have regulatory roles, producing IL-10 and other factors that help modulate the immune response and promote resolution of inflammation (38).

Chronic inflammation following stroke leads to sustained injury and impaired recovery. Persistent activation of microglia and astrocytes, along with continuous recruitment of peripheral immune cells, creates a hostile environment for neuronal survival and regeneration (48). The prolonged inflammatory response damages the BBB and allows further infiltration of immune cells and perpetuates a cycle of injury. Chronic inflammation is associated with ongoing neuronal loss and white matter damage, which contributes to cognitive decline and functional deficits (121, 122). Secondary neurodegeneration following stroke is characterized by progressive neuronal death and gliosis in regions connected to the primary lesion site, such as the thalamus, hippocampus, and corpus callosum (123). Biomarkers such as neurofilament light chain (Nf-L), tau protein, and S100b reflect the extent of brain injury and are associated with secondary neurodegeneration and correlate with worse functional outcomes post stroke (124, 125).

It is essential to highlight how the complex interplay between different immune cell types plays a crucial role in shaping the neuroinflammatory response after ischemic stroke. While more challenging to study than single cell type effects, this network of interactions better represents the physiologic environment and involves both resident and infiltrating immune cells, each contributing to the progression and resolution of inflammation in unique ways.

Given the significant role of the adaptive immune system in secondary cell death, targeting these immune responses presents a promising therapeutic strategy. Modulating microglial activation, promoting Treg function, and inhibiting pro-inflammatory T cell responses are potential approaches to reduce secondary damage and improve outcomes following stroke. Pharmacological agents that can skew microglia towards a neuroprotective phenotype, such as minocycline or laquinimod, show promise in reducing neuroinflammation (132, 133). Additionally, therapies aimed at enhancing Treg function, such as low-dose IL-2 or Treg adoptive transfer, could provide neuroprotection by modulating the adaptive immune response (134, 135). The use of stem cell therapy has also been explored to modulate the immune response and promote recovery. Mesenchymal stem cells (MSCs), for example, can secrete factors that promote the M2 phenotype in microglia and enhance Treg function, providing a multifaceted approach to reducing inflammation and promoting tissue repair (136–138).

Secondary cell death in stroke is a complex process driven by both the initial ischemic insult and the subsequent immune response. The adaptive immune system, through its various cellular components and interactions with microglia, plays a pivotal role in this process. Understanding these mechanisms provides valuable insights into potential therapeutic strategies aimed at reducing secondary damage and promoting neurological recovery following stroke. Further research and clinical trials are necessary to translate these findings into effective treatments for stroke patients.

The BBB is a highly selective structural and functional barrier that regulates the exchange of substances between the blood and the central nervous system (CNS). It is composed of endothelial cells, pericytes, astrocytes, neurons, microglia, and perivascular macrophages, collectively known as the neurovascular unit. This unit maintains CNS homeostasis and protects the brain from harmful substances and pathogens. However, ischemic stroke induces significant BBB disruption, leading to increased permeability and the infiltration of peripheral immune cells (45).

Three key processes combine to disrupt the BBB: oxidative stress, Matrix Metalloproteinases (MMPs) and Inflammatory Cytokines. Following ischemic stroke, excessive production of reactive oxygen species (ROS) damages endothelial cells and other components of the neurovascular unit (Figure 1). This oxidative stress compromises the structural integrity of the BBB, contributing to its breakdown. Antioxidant mechanisms are impaired, further exacerbating BBB damage. MMPs, particularly MMP-2 and MMP-9, are rapidly upregulated in response to ischemic injury (139–142). These enzymes degrade extracellular matrix components and tight junction proteins, leading to increased BBB permeability. Elevated MMP activity is associated with vasogenic edema and hemorrhagic transformation, highlighting the detrimental role of these proteases in BBB disruption (141, 143).

Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) are released by activated microglia and infiltrating immune cells (22, 142). These cytokines induce the expression of adhesion molecules on endothelial cells, promoting leukocyte adhesion and transmigration across the BBB. The resulting infiltration of immune cells further amplifies inflammation and BBB breakdown (144).

The integrity of the BBB is crucial for determining the extent of brain damage and the clinical outcome of stroke patients. Imaging techniques such as dynamic contrast-enhanced MRI and CT scans are used to assess BBB permeability in vivo and are discussed separately. Increased BBB permeability is associated with worse neurological outcomes and a higher risk of hemorrhagic transformation following thrombolytic therapy (145).

The spleen, a crucial lymphatic organ, mounts an immune response post-stroke that worsens the pathology. It acts as a repository for immune cells, deploying monocytes to the injury site (90). Studies have shown that splenectomy (removal of the spleen) prior to stroke significantly reduces cerebral infarction and the presence of monocytes in the brain, highlighting the spleen's role in stroke-induced inflammation.

The spleen serves as a reservoir for immune cells, including monocytes, which are rapidly deployed to sites of injury. After a stroke, monocytes from the spleen migrate to the brain, where they differentiate into macrophages and exacerbate inflammation. Post-stroke, the spleen undergoes contraction, releasing stored monocytes into the bloodstream which differentiate into macrophages and dendritic cells. This rapid deployment of immune cells to the brain can worsen the inflammatory response and contribute to secondary cell death.

Research involving splenectomy in animal models has demonstrated mixed results. Splenectomy prior to permanent middle cerebral artery occlusion (MCAO) significantly decreases cerebral infarction (149). However, in cases of transient MCAO, splenectomy reduces monocyte infiltration in the brain but does not significantly alter cerebral infarction, suggesting the involvement of different monocyte subsets in stroke pathology (90).

The spleen contains heterogeneous populations of monocytes, particularly the pro-inflammatory Ly6C^hi and anti-inflammatory Ly6C^lo subsets (150–153). Stroke induces the deployment of both subsets from the spleen to the brain, where they contribute to the inflammatory response. Ly6C^hi monocytes express high levels of the chemokine receptor CCR2 and are recruited to the injury site by monocyte chemoattractant protein-1 (MCP-1) (150). Their rapid deployment to the brain post-stroke exacerbates inflammation. Ly6C^lo monocytes are predominately anti-inflammatory monocytes express high levels of the receptor CX3CR1 and are recruited to normal tissues for tissue repair and maintenance (154). Their deployment to the brain post-stroke is more sustained and contributes to the resolution of inflammation.

Another aspect of the peripheral immune response to stroke involves cervical lymph node hypertrophy, likely from CNS drainage coupled with the large-scale release of vascular endothelial growth factor C (VEGF-C) (156, 157). Blocking this receptor reduces endothelial inflammation in the cervical lymphatic system, decreases macrophage activation and has been shown in mice studies (male C57BL6 mice) to lessen brain tissue infarction (156). Brain to cervical lymph node signaling has also been shown to modulate CD4 and CD11b T cell neuronal infiltration and subsequent secondary brain damage after injury in animal models (158). In animal experiments with a cervical lymphadenectomy can partial ameliorate some of the peripheral immune system's negative affects post stroke (156, 157).

The disruption of the BBB is a hallmark of neuroinflammation, and it plays a critical role in the pathophysiology of various neurological conditions, including stroke. Imaging the permeability of the BBB provides insights into the extent and dynamics of this disruption. BBB permeability can be assessed using the extravasation of contrast agents or radioactive tracers, which result in image contrast enhancement (45, 168). This enhancement allows for the visualization of areas where the BBB is compromised.

Dynamic contrast-enhanced MRI (DCE-MRI) and CT (DCE-CT) scans are commonly employed techniques to quantitatively measure BBB permeability, though non-contrast techniques have also been described (169, 170). These imaging modalities facilitate the derivation of key parameters such as the extraction fraction, blood-to-brain transfer constant (K_trans), and the permeability-surface area product (PS) (171–173). These quantitative measures provide detailed information about the degree and spatial distribution of BBB disruption, offering a more precise assessment of neuroinflammatory processes.

Animal studies utilizing advanced imaging in a mouse model (Claudin5-GFP) have also offered new insights into the mechanisms of BBB disruption in ischemic stroke. Fredriksson and colleagues established a protocol for longitudinal two-photon imaging in mice, enabling the observation of BBB breach at single-vessel resolution as early as 30 min after stroke induction (174). They reveals that the disruption of BBB, along with vascular leakage, can be tracked in real time, contributing to a deeper understanding of the hemodynamic changes and vascular remodeling, such as intussusception and angiogenic sprouting, over both acute and chronic phases. Importantly, this method allows the study of vessel-specific vulnerabilities along the arteriovenous axis, which could guide the development of targeted therapeutic strategies to mitigate the long-term impacts of stroke (Figure 4).

Advanced MRI and PET imaging techniques have been developed to detect and quantify leukocyte infiltration and microglial activation (175, 176). For instance, the intravenous injection of ultrasmall particles of iron oxide can highlight areas of inflammation due to their uptake by circulating monocytes or phagocytosed by macrophages or microglia (45, 177, 178). PET imaging with radiolabeled ligands targeting specific inflammatory markers, such as the 18-kDa translocator protein (TSPO), provides high sensitivity and specificity for detecting microglial activation (179, 180).

PET imaging using radiotracers targeting specific inflammatory markers, such as the somatostatin receptor (SSTR) and chemokine receptors like CXCR4 and CCR2, have shown promise in detecting and monitoring neuroinflammatory processes with good specificity (181, 182).

The expression of cell adhesion molecules like VCAM-1 and ICAM-1 can be visualized using targeted molecular imaging probes. For example, MRI enhanced with iron oxide particles functionalized with antibodies against VCAM-1 has been used to detect upregulation in and around stroke lesions (183, 184). Similarly, dual-tracer PET studies have demonstrated differential time courses for markers like MMPs and TSPO, highlighting their roles in the neuroinflammatory cascade after stroke (45, 180).

Recent studies have demonstrated the utility of advanced imaging techniques in both preclinical and clinical settings. For example, multimodal imaging combining PET and MRI has enabled detailed mapping of neuroinflammatory processes, such as the time-course of microglial activation and BBB permeability changes following stroke (178, 184). These imaging approaches not only enhance our understanding of stroke pathology but also aid in the development and monitoring of therapeutic interventions.

While many imaging modalities have shown promise in preclinical studies, their routine clinical application remains limited. Future research should focus on developing standardized protocols for image acquisition and analysis, as well as translating these techniques into clinical practice. The development of biocompatible and biodegradable contrast agents with high specificity for human cells and epitopes will be crucial for effective clinical implementation. Additionally, multimodal and multifunctional theranostic nanoprobes that combine diagnostic and therapeutic capabilities could revolutionize the management of neuroinflammation in stroke.

Recent studies have illuminated significant sex differences in the immune response to stroke, revealing intricate interactions between biological sex and the immune system's behavior post-stroke. Factors such as host genetics and chromosomal sex play a critical role in shaping the host immune system and neuroimmune response to brain injury (185–187). Epidemiological data indicate that older women have poorer functional outcomes compared with men, partly due to the older age at which they experience their first stroke and the increased comorbidities seen with aging (188). This disparity is also attributed to differences in the immune response between men and women, which leads to altered inflammatory events contributing to sex differences in post-stroke recovery.

Further, evidence from preclinical studies suggests that males and females exhibit distinct activation states of both the innate and adaptive immune compartments post-stroke (188, 189). For instance, females show differentially expressed genes acutely following cardioembolic stroke, which is not observed in males (190, 191). Experimental stroke MCA occlusion mouse C57BL/6N models indicate that aged males have greater brain infiltration of neutrophils compared with age-matched females, corroborated by higher levels of neutrophil-specific cytokines such as MCP-1 and G-CSF in the circulation of aged males (192). This neutrophil infiltration may contribute to an increased incidence of hemorrhagic transformation in males (193). The study also found that females exhibit higher expression levels of Toll-like receptors (TLRs) on dendritic cells, leading to more effective antigen sensing, processing, and presentation, potentially mediated by X chromosome-encoded genes (194).

Emerging evidence suggests that there are innate sex difference in the microglia population even in the healthy adult brain. It has been observed that microglia in males are more prone to an inflammatory phenotype whereas in females the anti-inflammatory phenotype predominates (185, 195, 196). Male microglia display higher expression of interferon-stimulated genes, whereas female microglia express genes associated with development and morphogenesis, which are linked to the inhibition of the inflammatory response (196, 197). These sex-specific gene expression patterns suggest that female and male microglia inherently differ even in the healthy brain and contribute to their differential responses to ischemic stroke.

Clinical studies identified distinct biomarkers and immune responses between males and females. For example, CCL20, ICAM1, and PTGS2 were identified as sex-specific targets, with males exhibiting a stronger CD8+ T-cell response and females showing a more pronounced monocytic response (198). Additionally, sex differences in inflammatory markers such as IL-6, CRP, and S100B were observed, with these biomarkers being more elevated in female stroke patients (199). The studies also reported that females tend to have a higher risk of adverse outcomes, such as poorer recovery and increased inflammation, compared to males (185).

Collectively, these studies underscore the importance of considering sex as a biological variable in stroke research. They highlight the necessity for more sex-specific research to better understand the mechanisms underlying these differences, which could lead to more effective, tailored therapeutic strategies for both men and women post-stroke.

While the acute phase of neuroinflammation following a stroke is well-studied, there is a lack of comprehensive research on the long-term impact of chronic neuroinflammation on stroke recovery and rehabilitation. Understanding how persistent inflammation affects neuroplasticity, functional recovery, and the risk of recurrent strokes is crucial. Investigating the temporal dynamics of neuroinflammation and its long-term effects could lead to better strategies for managing stroke recovery and preventing chronic neurological deficits and provide more potential therapeutic targets.

Although several imaging techniques have been developed to visualize and quantify neuroinflammation, there is a need for more sensitive and specific biomarkers that can accurately reflect the extent and nature of neuroinflammation in stroke patients. Current imaging modalities, such as MRI and PET, have limitations in detecting subtle inflammatory changes and differentiating between various types of immune cell activation. Advancing imaging technology to include more precise and reliable biomarkers would improve the diagnosis, monitoring, and treatment of neuroinflammation in stroke patients.

Enhanced Sensitivity and Specificity: Developing new imaging agents and techniques that can more precisely target and visualize specific inflammatory cells and molecules is crucial. For instance, using novel PET tracers that bind to unique markers of microglial activation or specific cytokines could improve sensitivity and specificity.

The quest for a reliable biomarker of stroke has been challenging due to several factors inherent to the condition's complexity and the biological processes involved. Despite extensive research, a universally accepted, highly specific, and sensitive biomarker for stroke remains elusive. There are several currently under investigation, several of which could also provide information about the post stroke inflammatory cascade.

Glial fibrillary acidic protein (GFAP), an astrocyte-specific protein, shows promise in distinguishing between ischemic and hemorrhagic strokes (202). Elevated blood levels of GFAP are typically associated with hemorrhagic events, aiding in critical treatment decisions. Matrix metalloproteinase-9 (MMP-9) is being studied as a marker of BBB disruption and hemorrhagic transformation risk, particularly in patients treated with tissue plasminogen activator (tPA) (203, 204).

Neurofilament light chain (NfL), a structural protein found in neurons, serves as an indicator of axonal damage. Its levels in blood or cerebrospinal fluid correlate with infarct volumes and functional outcomes, making it valuable for prognostication (205). MicroRNAs, particularly miR-124 and miR-223, have also shown potential in stroke diagnosis and prognosis due to their rapid expression changes in response to cerebral ischemia (206).

While these biomarkers are promising, stroke's complexity means that no single marker is likely to provide all necessary information. Current research focuses on developing panels of multiple biomarkers and integrating this data with clinical information and advanced imaging techniques. This comprehensive approach aims to improve stroke diagnosis, prognosis, and treatment strategies, ultimately enhancing patient care and outcomes (207, 208).

Due to multiple technical and logistical challenges in studying the immune response from stroke patients, much of what is known about the immune response comes from animal studies. There is a paucity of human data which may explain why several promising therapies in animal models have not shown successful results in early clinical trials.

The Table 1 summarizes the current ongoing clinical trials that are designed to test various hypothesis about the role of the immune system in acute stroke and that have been registered on ClinicalTrials.Gov.