The role of the meningeal lymphatics in stroke

Introduction

Stroke is a leading cause of death and acquired disability worldwide and affects approximately 15 million people every year¹. Stroke can be classified into two main categories: ischemic and hemorrhagic. Ischemic stroke, which accounts for over 80% of cases, is caused by a disruption of cerebral blood flow and related parenchymal oxygen deprivation¹. Following an ischemic event, neural cells release damage-associated molecular patterns which, in turn, activate the innate immune system of the brain². At the same time, peripheral immune cells, such as neutrophils, dendritic cells, monocytes, and T cells, traffic to the brain through a disrupted blood brain barrier (BBB) in response to the ischemic injury². Until recently, the mechanism by which the peripheral immune system was activated after stroke was unknown.

In 2015, Louveau et al. discovered the existence of a meningeal lymphatic system, shedding new light on the role of the immune system in the etiology and progression of neuroinflammatory diseases³. Recent studies have now demonstrated the crucial role of the meningeal lymphatic system in the immune response post-stroke^4–6. Meningeal lymphatic vessels (MLVs) act as a physical conduit for antigens and immune cells from the central nervous system to enter the draining cervical lymph nodes (CLNs)⁴. These antigens activate the systemic immune response, allowing immune cells to traffic to the brain through a disrupted BBB⁵. In addition, after ischemic stroke, MLVs invade the injured brain parenchyma, reducing brain edema by creating a pathway for cerebral interstitial fluid to drain, as well as promoting angiogenesis⁶. These multifactorial roles highlight the importance of the meningeal lymphatic system after brain infarction and its potential as a therapeutic target for ischemic stroke.

Structure and Function of the Lymphatic System

The lymphatic vascular system is essential for maintaining tissue fluid homeostasis and for regulating immune cell trafficking and surveillance^7,8. Lymphatic vessels are present in the meninges, which are composed of the dura, arachnoid, and pia mater⁹. In addition, a newly described layer named subarachnoid lymphatic-like membranes has been identified between the arachnoid and pia mater¹⁰. The dura mater, closest to the cranial bones, contains the majority of MLVs, which are concentrated at the base of the skull¹¹. These vessels clear macromolecules, interstitial fluid, and even cerebrospinal fluid from the brain parenchyma^9,11. The collected fluid exits the cranial cavity and eventually drains into deep CLNs^4,12. The meningeal lymphatic system also drains interstitial fluid into the cerebrospinal fluid, creating a bidirectional connection between the cerebrospinal fluid and the systemic circulation¹². Further, MLVs drain antigens and immune cells from the cerebrospinal to the CLNs⁴. They transport antigens and antigen-presenting cells, such as macrophages and dendritic cells, to lymph nodes, allowing for an adaptive immune response¹³.

The Role of Meningeal Lymphatics in Stroke-induced Edema

When the BBB is disrupted after stroke, the increased permeability allows vascular fluid to enter the interstitial space of the brain, leading to the formation of vasogenic edema¹⁴. Ischemic stroke causes cytotoxic edema by depleting ATP reserves, which impairs the functioning of the Na+/K+ pump and results in abnormal levels of fluid entering the cells¹⁵. Brain edema can result in tissue death, higher intracranial pressure, and fatal brain herniation¹⁵. The meningeal lymphatic system acts to reduce edema in the brain in a neuroprotective manner¹⁴.

Lymphatic drainage is essential for removing excess fluid from the skull. The increased diameter of MLVs post-stroke results in a greater outflow rate of drainage under high pressure conditions. This suggests that when edema results in higher intracranial pressure after stroke, meningeal lymphatic drainage is activated, promoting edema clearing^14,16.

When meningeal lymphatics are inhibited, there is a decrease in drainage of brain antigens, waste, and meningeal T cells, which could cause an inflammatory cascade in the brain due to dysregulated meningeal immunity¹⁷. It has also been shown that blocking CLN drainage in rat models of stroke, by removing the bilateral shallow and deep CLNs, results in higher ischemic brain damage due to higher water, sodium, and calcium content in the brain tissue¹⁸. The importance of meningeal lymphatics in stroke is further supported by an increase in severity of ischemic stroke in mouse models of hypoplastic MLVs¹⁶. These mice exhibit higher infarct volume, more severe neurological deficits, and poorer prognosis compared to control mice¹⁶.

Further, enlarged MLVs can actively grow into the injured parenchymal region, ultimately relieving edema⁶. In a photothrombotic mouse model, stroke induced lymphangiogenesis in the infarcted brain region¹⁶. A study in zebrafish has shown that this growth occurs in a vascular endothelial growth factor C (VEGF-C)-dependent manner. In this model, the ingrown lymphatics act as a pathway for the drainage of cerebral interstitial fluid, reducing brain edema⁶. The lymphatic vessels that grow into the parenchyma also promote vascular regeneration by acting as “growing tracks” for nascent blood vessels, most likely via mechanical physical adhesion⁶. Angiogenesis is fundamental to re-nourish the injured brain parenchyma. Once cerebral vascular regeneration is complete, the ingrown lymphatic vessels are cleared through apoptosis, returning the brain to its physiological state⁶.

The Role of Meningeal Lymphatics in Post-Stroke Immune Response

While the brain, under normal conditions, does not contain immune lineage cells other than microglia, the meninges are home to many diverse immune cells¹⁹. Conversely, in ischemic stroke, BBB disruption results in the accumulation of leukocytes, particularly neutrophils and macrophages within the brain parenchyma as well as in the MLVs²⁰. The lymphatic system’s critical role in the immune response after stroke is underscored by the higher levels of brain-derived antigens associated with lymph node macrophages, including MAP2 and myelin basic protein, found in tissue samples collected from ischemic stroke patients compared to samples from controls (Table 1)²¹. Brain-derived antigens in the meningeal lymphatic system activate dendritic cells, which act as antigen presenting cells for peripheral B and T cells⁵. Cerebrospinal fluid drains antigens, T cells, and other immune cells to the CLNs through the meningeal lymphatic system⁵. Dendritic cells migrate to the CLNs through upregulation of the chemokine receptor CCR7, binding to the ligand CCL21 expressed by MLVs¹³. Dendritic cells move along the lymphatic endothelium with adhesive interactions, then detach and are passively transported to the CLNs¹³. The significance of CCR7 in this process is illustrated by studies in CCR7-deficient mice, where dendritic cells and T cells fail to drain into the CLNs²⁴. In the CLNs, dendritic cells activate and trigger the proliferation of antigen-specific T cells, including CD4+ helper T cells, CD8+ cytotoxic T cells, and regulatory T cells^13,25. Effector B cells are also activated, either by soluble antigens binding to B-cell receptors or protein antigens internalized by dendritic cells and presented to CD4⁺ T cells¹³. The activated effector T and B cells and humoral factors including antibodies are transported into the blood circulation through efferent lymphatic vessels and enter the brain through the disrupted BBB, enacting proinflammatory or anti-inflammatory effects^4,5 (Figure 1).

Table 1.

After ischemic stroke, both human and mouse models have shown an increase in immune cell levels in the brain,^22,23.

Cell Type	Model	Levels Post Stroke	Onset Post Stroke	Duration
MAP2-immunopositive cells	Human	↑	varies	Varies
MBP-immunopositive cells	Human	↑	varies	Varies
CD69+ T cells	Human	↑	varies	Varies
Regulatory CD4+ T cells	Mouse	↑	Several days	30 days
CD8+ T cells	Mouse	↑	3–24 hours	30 days
Neutrophils	Mouse	↑	3 hours	7 days
Monocytes	Mouse	↑	24 hours	14 days
Lymphocytes	Mouse	↑	12 hours	30 days
Microglial cells	Mouse	↑	12 hours	Varies
Dendritic cells	Mouse	↑	24 hours	Varies

Figure 1.

In the lymphatic drainage system, immune cells are transported to the cervical lymph nodes where they are activated, and then return to the brain through the bloodstream to exert their effects.

An area of debate is whether meningeal lymphatics may have deleterious effects on stroke recovery by facilitating neuroinflammation, despite their important role in clearance. First, the activated lymphatic endothelium can stimulate macrophages to secrete proinflammatory factors like tumor necrosis factor-α and interleukin-1β, which leads to breakdown of the BBB, aiding entrance of pro-inflammatory cells into the brain parenchyma⁴. Next, pre-clinical mouse models of stroke exhibit dilated MLVs, which can increase neuroinflammation by increasing immune response to stroke^4,16. A potential mechanism for this dilation is through an increase in paracrine and endocrine VEGF-C in the meninges, which can bind to vascular endothelial growth factor receptor 3 (VEGFR3)²⁶. Injection of VEGFR3-specific recombinant VEGF-C into the cisterna magna of mice has been shown to result in increased diameter of MLVs³. In addition, brain ischemia induces proliferation of lymphatic endothelial cells in the CLNs by secreting VEGF-C⁴. VEGF-C/VEGFR3 signaling increases lymphatic growth and draining of brain antigens to the CLNs⁵. When this signaling in inhibited, the immune response is less inflammatory and there is a reduction in brain infarct volume²⁷. Similarly, removal of the CLNs has been shown to diminish the stroke-induced immune response in the brain, resulting in a decrease in infarct size^4,27. In addition, after middle cerebral artery occlusion in rat models, meningeal lymphatic dysfunction worsened neuroinflammation, neurological deficits, and increased ischemic injury²⁸.

Interestingly, in a preclinical model of hemorrhagic stroke, ablation of MLVs or ligation of the deep CLNs facilitated hematoma clearance via VEGF-C resulting in reduced neuronal loss and improved outcome. This suggests an important role of the MLVs in both ischemic and hemorrhagic stroke⁸.

Conclusion

Recent studies on the role of meningeal lymphatics and CLNs in stroke indicate that restoring the function of lymphatic drainage systems could both aid in resolution of cerebral edema and improve the anti-inflammatory response, providing neuroprotection after infarction. This highlights the importance of the MLVs in brain injury after stroke. More research is needed to better understand the complex biology of this system, which will likely be an important therapeutic target for stroke treatment in the future.

Non-standard Abbreviations and Acronyms

BBB

blood brain barrier

CLNs

cervical lymph nodes

MLVs

meningeal lymphatic vessels

VEGF-C

vascular endothelial growth factor C

VEGFR3

vascular endothelial growth factor receptor 3