(2024) 21:135
Mokbel et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03122-7

Journal of Neuroinflammation

Open Access

REVIEW

The contribution of the meningeal immune
interface to neuroinflammation in traumaticbrain injury
Alaa Y. Mokbel1, Mark P. Burns1 and Bevan S. Main1*

Abstract
Traumatic brain injury (TBI) is a major cause of disability and mortality worldwide, particularly among the elderly,
yet our mechanistic understanding of what renders the post-traumatic brain vulnerable to poor outcomes, and susceptible to neurological disease, is incomplete. It is well established that dysregulated and sustained immune
responses elicit negative consequences after TBI; however, our understanding of the neuroimmune interface
that facilitates crosstalk between central and peripheral immune reservoirs is in its infancy. The meninges serveas the interface between the brain and the immune system, facilitating important bi-directional roles in both healthyand disease settings. It has been previously shown that disruption of this system exacerbates neuroinflammationin age-related neurodegenerative disorders such as Alzheimer’s disease; however, we have an incomplete understanding of how the meningeal compartment influences immune responses after TBI. In this manuscript, we will offer
a detailed overview of the holistic nature of neuroinflammatory responses in TBI, including hallmark features observed
across clinical and animal models. We will highlight the structure and function of the meningeal lymphatic system,
including its role in immuno-surveillance and immune responses within the meninges and the brain. We will provide
a comprehensive update on our current knowledge of meningeal-derived responses across the spectrum of TBI,
and identify new avenues for neuroimmune modulation within the neurotrauma field.
Keywords Meninges, Innate immunity, Adaptive immunity, Neuroinflammation, TBI
Background
Traumatic brain injury (TBI) is a leading cause of deathand disability, posing a significant socioeconomic andpublic health burden, with an estimated 64–74 million
people sustaining a TBI each year [1, 2]. While the prevalence of TBI is centered around young adults and theelderly [3, 4], the consequences of TBI are more severein aged populations. Slower recovery, worse functional,*Correspondence:
Bevan S. Main
bsm53@georgetown.edu
1
Department of Neuroscience, Georgetown University Medical Center,
New Research Building‑EG11, 3970 Reservoir Rd, NW, Washington, DC
20057, USA

cognitive, and psychosocial outcomes, all highlight the
influence of age on overall TBI pathogenesis [5–11].
Strikingly, there were 69,473 TBI-related deaths in the
United States alone in 2021 [12], emphasizing the needfor disease modifying interventions. There are no comprehensive pharmacological treatments for TBI, withthe diversity of injury still considered a significant barrier toward the translation of effective therapeutics [13].
Although TBI can be influenced by many variables, it isaccepted that neuroinflammation contributes to negative outcomes after TBI. Evidence suggests that thesynergistic functioning of innate and adaptive immune
cells, crucial for orchestrating and sustaining a healthy
brain microenvironment, becomes dysfunctional after
TBI. This dysfunction results in sustained, uncontrolled© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Mokbel et al. Journal of Neuroinflammation(2024) 21:135

neuroinflammatory responses which detrimentally affectoutcomes and recovery timeframes. Indeed, therapies
targeting inflammatory responses display efficacy in preclinical and single-center trials; however, they fail to show
improvement in larger multicenter clinical trials [14, 15].
This may be due to the fact that the majority of preclinical TBI studies tend to analyze these cellular responses
individually, with interventions focused on targeting a
singular cell modality. It is now evident that neuroinflammatory responses in TBI are not confined to a single cell
type, and it is more likely that a combination of complex
cellular interactions determines the nature of inflammatory cascades. Therefore, in order to develop more effective therapeutics, we require a greater understanding of
the bi-directional crosstalk between the innate and adaptive immune response and how they contribute holistically to TBI-induced neuroinflammation.

Neuroinflammation in TBI
The complexity and challenges in understanding cellular interactions and signaling cascades in TBI is largely
due to the heterogenous nature of injury. Primary injury
causes brain lesions from fractures, intracranial hemorrhage, epidural and subdural hematoma, brain contusionand direct mechanical damage to neural tissue. Secondary injury occurs immediately following head impact,
initiating cascades including neuroinflammation that canpersist for weeks and even years, contributing to neurological impairment. Temporally, primary injury results inblood–brain barrier (BBB) dysfunction, neural damage,and release of endogenous damage-associated molecularpatterns (DAMPs). These DAMPs subsequently engage
pattern recognition receptors (PRR), such as the toll
like receptors (TLRs) on innate (microglia/astrocytes)
or adaptive (myeloid/lymphoid) cells, leading to theirimmune activation. The molecular diversity of DAMPs
binding PRRs is vast (reviewed in [16]), and their significance after trauma is critical, with DAMP level correlating with injury severity and inversely related to clinicaloutcomes [17]. Indeed, clinical insights offer invaluabletranslational knowledge to identify secondary injury triggers and processes, information that may identify avenues for therapeutic intervention.
Clinical hallmarks of neuroinflammation in TBI

Physical trauma to the brain causes BBB disruption, withincreased fibrinogen, immunoglobulin and heightened
cerebrospinal fluid (CSF) to serum albumin quotients
detectable within hours of clinical evaluation, persisting for weeks or even years [18–26]. Increases in serum
and CSF levels of brain specific glial fibrillary acid protein (GFAP), ubiquitin carboxyl-terminal hydrolase
isozyme L1 (UCH-L1) and S100 calcium-binding protein

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B (S100B) are correlated with early barrier permeability[20, 27]. Similarly, increased CSF levels of matrix metalloproteinases (MMPs) [28] and the complement mediators C3, factor B and sC5b-9 are also associated with
BBB dysfunction [23, 29]. The CSF of severe TBI patients
also contains DAMPs, PRRs and mediators downstream
from PRR activation, with elevated levels of high mobility group protein B1 (HMGB1) [30–32], double stranded
DNA (dsDNA), absent in melanoma 2 (AIM2), apoptosis-associated speck-like protein (ASC), NLR Family
Pyrin Domain Containing 1 (NLRP1), and caspase-1 [33,34]. Fluid biomarkers (CSF/serum/blood) show temporal
increases in cytokines and chemokines, including members of respective interferon, interleukin, tumor necrosis
factor, transforming growth factor and C-C motif ligand
families (Table 1).
Indeed, tissue samples from patients with TBI indicate that this inflammatory response is composed ofboth innate and adaptive cellular elements, including monocytes/macrophages, reactive microglia, polymorphonuclear cells, and C­ D4+, ­CD8+ T cells [83–88].
Using positron emission tomography (PET) scans, TBI
patients show elevated translocator protein (TSPO)
expression in microglia, demonstrating microglial activation which could be seen up to 17 years after injury [89,90]. Indeed, in brain tissue, activated microglia (­CD68+,­CD11b+, ­TMEM119+) are highly expressed after injury
across acute and chronic timepoints [83, 85, 91–94],
alongside markers for toll like receptor (TLR) signaling including TLR4 and myeloid differentiation primary
response 88 (Myd88) [95, 96]. Peripheral cell contributions are observed in blood samples after TBI, with acutedecreases of the number ­CD4+ and ­CD8+ and natural killer (NK) cells, followed by transient increases inT regulatory (Treg) cells after injury [35, 97–100]. The
temporal series of these responses may be influenced
by injury severity, with expansion of Th17-type CD4 T
cells alongside IL-17 and IL-22, seen at 5 days post injury[35]. Concurrently NK cells have reduced T-bet expression and lower IFNγ and TNFα, all indicating cell specificresponses after TBI [35].
Hallmarks of neuroinflammation in animal models of TBI

Animal models have proven invaluable for studying TBI
and unraveling the complicated mechanisms underlying both primary and secondary injuries. Similar
to clinical studies, pre-clinical models show BBB permeability within hours, with spontaneous closure at
approximately 7–10 days post-injury [101–105]. Mechanistically, TBI has been shown to affect processes at
the neurovascular unit, with reductions in tight junction proteins (claudins, occludins) and pericyte loss
[104, 106–108]. Across the spectrum of TBI models,Mokbel et al. Journal of Neuroinflammation(2024) 21:135

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Table 1 Cytokines and chemokines modulating neuroinflammation in clinical TBI
Immune signature/trigger

Time

Fluid biomarkers
IFNγ↑ 1–5 days after injury [35, 36], ↑ up to 12 months [37]TNF

↑ 6 h–2 weeks days [24, 35, 38–50], ↑ up to 12 months [37]IL-1β

↑ 1 day–1 month [39, 46, 51–59]

IL-6↑ 6 h–2 weeks [24, 36, 46, 47, 49–52, 56, 58–68], ↑ up to 6 months [69]IL-10

↑ 6 h–3 days [24, 39, 46, 51, 68, 70–73]

IL-8 (CXCL8)↑ 2 h–5 days [48, 50, 51, 59, 69, 74], ↑ up to 12 months [37]IL-12p70↑ 1–3 days, peaks at days 3–5 [46, 50, 51]

TGFβ↑ 1–21 days after trauma [75, 76]

CCL2↑ 1–10 days [46, 77, 78], ↑ up to 3 months [58]CCL3

↑ 1–3 days [46, 51]

CXCL8 (IL-8)↑ 1–4.5 days [46, 51, 63, 79]

IL-18↑ up to 10 days [80]sIL-2R↑1–21 days [81]IL-17, IL-22↑ day 5 after injury [35], ↑ up to 12 months [37]IL-2

↑ 24 h post injury [67]IL-9

↑ admission–12 months [37]IL-4

↑ within 48 h post injury [59]iNOS↑24 h post injury [47]NADPH

↑24 h post injury [47]COX-2

↑24 h post injury [47]TissueIFNγ↑ 17 min–5 days injury [70]TNF

↑ 17 min–5 days injury [70]IL-1β

↑ 6 h–5days after injury [70]IL-6

↑ 17 min after injury [56, 70]

CCL2↑ 3 h–15 days post-injury [78]CCL3

↑ 3 h–15 days post-injury [78]IL-8 (CXCL8)↑ 3 h–15 days post-injury [70, 78]

IL-2↑ 17 min–5 days injury [70]NOX2

↑12–24 h post injury [82]NOX4

↑12–48 h post injury [82]various signals and triggers are elevated following
injury, including DAMPS, cytokines, chemokines andsoluble factors similar to those previously identified inclinical studies. Critically, their time and cell dependent function can be influenced by a myriad of context
dependent factors (Fig. 1). Broadly, experimental TBI
increases levels of DAMPs including HMGB1, ATP,heat-shock, GM-CSF, mtDNA and S100 proteins andtheir receptors such as TLRs and receptors of advanced
glycosylation end-products (RAGE) and purinergic
receptors. In addition, alterations in levels of proinflammatory cytokines TNF-α, IL-1β, IL-6, IL-18, IFNγ,
Type-I interferons (IFNs), anti-inflammatory cytokines
Arg-1, IL-4, IL-10, TGF-β and chemokines IL-8, MCP1,
CCL2, CCL5, CXCL2 are all involved within the holistic

nature of inflammation in TBI models (detailed review
in [14, 109, 110]).
Innate CNS responses

Microglia are resident central nervous system (CNS)
immune cells, making about 10–13% of the cell population in the mouse and human brain [111, 112]. Theirprimary functions can be grouped to include surveillance, phagocytosis, and the secretion of soluble factors[113]. To determine functionality, microglia react tochanges and context specific stimuli within CNS, eliciting
a diverse array of tailored responses. Indeed, advancements in technologies (transcriptomics, proteomics
and metabolomics) have highlighted that categorizing
microglia into simplistic dichotomies like "good or bad"

Mokbel et al. Journal of Neuroinflammation(2024) 21:135

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Fig. 1 Hallmarks of neuroinflammation in Traumatic Brain Injury. Summary of hallmark characteristics that encompass the holistic natureof TBI-induced neuroinflammation. Based on decades of clinical research and mechanistic knowledge gained from the diverse range of injury
models, key neuroinflammatory events include: barrier dysfunction, immune triggers and signaling, neural damage or release of soluble factors,
neutrophil infiltration, monocyte recruitment, resident CNS microglial and astrocyte responses and activation of T and B cell responses. Importantly,
all areas of this neuroinflammatory wheel can be influenced by context dependent factors, and should be considered across the spectrumof the neurotrauma fieldor "M1 or M2" does not adequately capture the full spectrum of microglial states across disease settings, and that
updated nomenclatures and terminologies are warranted[113]. We agree with this principle, and to that extent,
in assessing the role of microglia in pre-clinical TBI, we
use the term Trauma Associated Microglia (TAM) to

characterize the unique functionality of microglia in TBI
versus other disease contexts (Fig. 1).
In healthy environments, microglia surveil their environment and can aid in synaptic remodeling, cell survival, and controlled phagocytosis removing CNS
debris and waste products [114–116]. Morphologically,

Mokbel et al. Journal of Neuroinflammation(2024) 21:135

microglia are described as ‘ramified’ in their normal
homeostatic state, and transform to a spectrum of morphologies during disease. In models of TBI, a dynamic
range of TAMs are observed at both acute and chronic
timepoints, initially clearing necrotic debris by adopting
phagocytic states in response to ATP mediated purinergic signaling [117–119]. In the days and weeks following
TBI, microglial cells undertake various functions, including, but not limited to, initiating immune responses, synaptic engulfment and clearance phagocytosis, influencing
neuronal activity through bidirectional synaptic contact
with neurons, and the potential to promote neurogenesis [120–123]. This dynamic functionality is generally
delineated by the expression of Cd11b, CD68, CD86
and MHCII markers and accompanied by the release ofproinflammatory mediators like TNF-α, IL-1β, NOX,
IFNs or alternatively the expression of CD206 and Arg-1
markers and the release of anti-inflammatory factors
including IL-10 and TGFβ [117, 121, 124–129]. Mechanistically, this functionality is determined by immune
triggers that signal microglia to undergo morphologicalalterations resulting in highly activated states where they
release soluble factors [118, 130]. Specifically, microglia
are shown to respond to DAMPs activating PRRs, with
TBI upregulating HMGB1, TLR2, TLR4 and NF-κB [31,131–133]. Moreover, TLR2- and TLR4-deficient micedisplay decreased levels of TNF-α, IL-1, IL-6 and NF-κB
signaling, evidencing their role in the TBI-induced proinflammatory response [134, 135]. In addition to TLRs,
purinergic receptors P2Y6, P2Y12, and P2X4, detect ATP
released from damaged cells and influence microglial
responses after TBI [118, 119, 136]. Alongside the production of cytokines and chemokines, TAM also adopt
functional states that promote oxidative stress throughchronic NOX2 activation [137, 138]. In recent times,
pharmacological depletion of microglia in the subacuteand chronic timepoints after TBI is shown to be neuroprotective and improves outcomes [127, 139], underscoring the key role they play in deleterious TBI-induced
neuroinflammatory responses.
Similar to microglia, astrocytes undergo morphological, molecular, and functional remodeling, with theirclassification now determined by the sum of their multifactorial impact in context specific pathological settings [140]. In healthy contexts, astrocytes contribute to
immune signaling, synaptogenesis regulation, BBB formation and maintenance, neurotransmitter recycling,
ion and water homeostasis, and blood flow control [141–145]. In TBI, astrocytes are best known for the formationof the glial scar as a protective mechanism to limit secondary injury and promote regeneration [104, 146–149].
Astrogliosis is categorized by increased ­GFAP+ andvimentin across TBI models, yet away from the glial scar,

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the morphological significance of this gliosis is temporally and spatially dynamic [14]. They contribute to brain
edema after injury [150–152], and contain decreased
expression of GLT-1, GLAST and EAAT1/2, evidencing
their role in TBI-induced glutamate dysregulation [153–156]. They also contribute directly to immune responses
through DAMP/TLRs signaling as well as proinflammatory cytokine and chronic complement production after
TBI [157–160]. Recent advances in high throughput
sequencing and ‘omic analysis has generated datasets
to begin to unravel the context specific nature of astrocytes in TBI [127, 161–164], yet these studies are stillin early stages, especially when considering the range ofheterogeneous immune responses that underpin the hallmark characteristics of TBI (Fig. 1). Furthermore, these
responses often involve more than just a single cell type,exhibiting bidirectional communication, as evidenced by
HMGB1 release from necrotic neurons, activating microglial TLR4, and subsequently increasing levels of astrocytic aquaporin-4 (AQP4), influencing BBB dynamics
after TBI [31]. Indeed, this communication is now being
explored in disease settings, with microglial release ofC1q, IL1β and TNFα shown to transform astrocytes into
neurotoxic states via their secretion of lipids contained in
APOE and APOJ [165–167]. Blockade of this interaction
is beneficial after stroke [168], suggesting studies of thispathway in TBI may yield promising targets to reduce
inflammation mediated cell death pathways.
Adaptive immune responses

Peripheral interactions and cell specific processes
play important roles in the overall neuroinflammatory response in TBI. Temporally, within minutes aninitial wave of neutrophils crosses the BBB to phagocytose injured tissue, followed by potent chemoattractant
signals that encourage the migration and infiltration of
peripheral macrophages and lymphocytes (Fig. 1). The
temporal series of these cellular events within the brain
parenchyma has been extensively reviewed elsewhere[169, 170]. Within the scope of this review, we would
like to discuss the contribution of the adaptive immune
response in relation to its interaction at meningeal barriers, including the recently discovered immune rich lymphatic vessel interface.

Meningeal structures and barriers
The meninges, essential components of the CNS’s protective and functional architecture, are composed ofthree distinct layers: the innermost pia mater, the intermediate arachnoid, and the outer dura mater. Structurally, the dura predominantly comprises collagen fibersthat anchor it to the skull, as well as thin layer of fibroblasts demarcating the dura from the arachnoid [171]. InMokbel et al. Journal of Neuroinflammation(2024) 21:135

addition, the dura contains fenestrated blood vessels andlymphatics that form a connection with the periphery[171–176]. Beneath the dura lies the arachnoid barrier
layer, composed of an outer layer of epithelial-like cells
interconnected by tight junctions [177, 178]. It is thisbarrier that serves as a key component of the blood-CSF
barrier, restricting the movement of molecules from thedura to the subarachnoid space [171, 175, 179]. The subarachnoid space plays host to vasculature networks andimmune cells, with arachnoid trabeculae consisting of
flattened fibroblasts-like cells connecting the arachnoid
to the pia [180, 181]. CSF flows within this space and provides brain nourishment and buoyancy, as well as waste
removal via venous flow and resorptive transport in thechoreoid plexus [182, 183]. Tightly adhered to the brain,
the pia mater is composed of a thin layer of fibroblasts,
followed by a basement membrane that separates it fromthe underlying glia limitans. Collectively the arachnoid
and pia are commonly referred to as the leptomeninges [184], within which the non-fenestrated vasculature
contains tight junctions to form the leptomeningeal barrier [184–187]. Lastly, the glial limitans is constituted byastrocytic end feet processes to form a boundary between
the brain parenchyma and the pia.

Meningeal lymphatic vessels (mLVs)
The presence of lymphatic vasculature within the meninges, though hinted at and often misconstrued in various
anatomical accounts in the seventeenth century [188],
was first visually depicted in the late eighteenth century by the anatomist Paolo Mascagni [189]. However,subsequent to this, knowledge of these lymphatic vessels lapsed into relative obscurity until the mid-twentieth century [190]. Its ’rediscovery’ in modern times inboth mice [173, 175] and humans [174, 176] had led toa resurgent wave of interest within the neurosciences,challenging the long-lasting dogma of the “immuneprivileged” brain. Anatomically, healthy meningeal lymphatic vessels (mLVs) are found alongside dural sinuses,
arteries and veins, including the superior sagittal sinus,transverse sinus, sigmoid sinus, retroglenoid vein, rostralrhinal vein, middle meningeal artery, and pterygopalatine artery [173, 175, 191–194]. Insights into the mechanistic development of lymphatic vessels is largely drawn
from studies of networks in peripheral tissues. In mice,lymphatic vasculature (LV) formation is predominantly
venous-derived, originating from the cardinal vein during embryogenesis. At embryonic day 9.5 (E9.5), a subpopulation of venous endothelial cells express Sox18 (aSRY-related HMG-box transcription factor), which activates prospero homeobox 1 (PROX1) [195–198]. This
PROX1 activation induces specific lymphatic endothelial
cell (LEC) gene expression, and inhibits blood endothelial

Page 6 of 22cell-specific genes by binding to the nuclear receptors
COUP-TFII [199–202]. Indeed, ­Prox1−/− endothelial
cells fail to express LEC markers, instead retaining blood
vascular endothelial properties [195], evidencing theirrole as a master regulator of lymphatic identity. Ultimately Sox18-PROX1 activation initiates the acquisitionof LEC properties for the subsequent creation of lymph
sacs and lymphatic vessel networks [203, 204]. Additional lymphangiogenic mediators include lymphatic
vessel endothelial hyaluronan receptor 1 (LYVE-1),
which increases the expression of the platelet aggregating protein podoplanin (PDPN), in LECs at E12.5. Vascular endothelial growth factor C (VEGF-C) activation ofvascular endothelial growth factor receptor-3 (VEGFR-3)
is also essential for the sprouting of LVs from the embryonic veins [205, 206], allowing for the ability of LECs tomigrate and form the lymphatic sacs, visible at E12.5[207]. Buds and sprouts progress until E14.5, by whichtime the lymphatic development stage is completed [208,209].
In contrast to the embryonic formation of peripheral
LV, the development of the intracranial mLV occurs postnatally, in a VEGF-C dependent manner [210]. Starting at
the base of the skull LEC sprouting begins at birth, postnatal P0. Temporally, lymphangiogenesis continues in acharacteristic pattern alongside veins, arteries, and cranial nerves during the first weeks. The mLVs appear at thecribriform plate at P2, middle meningeal artery after P4,
and grow into the transverse sinus at P8 [210]. Between
P13 and P20, lymphatic vessels expand along the transverse sinus appearing at the confluence of sinuses at P16[191, 210], before extending the length of the superior
sagittal sinus toward the olfactory bulb by P20 [191]. At
this time, the mLVs become functional, draining content
from the CNS into the peripheral lymphatic system at thebase of the skull, with the mLV network fully developed
at approximately P28 [175, 191, 192, 210–212].
In terms of functionality, mLVs can be categorized intothin-walled “initial” lymphatic vessels, and the larger“collecting” vessels. In mice, initial lymphatic vessels arehighly permeable and are located dorsally near both thetransverse and superior sagittal sinus. Initial lymphatics lack smooth muscle cell (SMC) coverage, and arecomposed of discontinuous button-like LEC junctions[173, 175, 213]. These button-shaped junctions consist of tight junction- and adherens associated proteins,that attach adjoining LECs at the base of interdigitating
flaps between cells [214–216]. This flap-like overlap creates mini-valves that permit the entry of interstitial fluid,
macromolecules, soluble antigens and immune cells
including antigen presenting cells (APCs) [215–221].
In contrast, collecting lymphatic vessels at the base of
the skull are surrounded by SMCs to help propel fluidMokbel et al. Journal of Neuroinflammation(2024) 21:135

movement and contain tight continuous zipper-like connections and secondary intraluminal valves [217, 220].
This makes them virtually impermeable, facilitating unidirectional drainage, whilst simultaneously preventing
lymphatic backflow [222, 223]. In both mice and humans,collecting meningeal lymphatic vessels extend along thejugular vein, exit the skull through various foramina,
before merging with peripheral collecting lymphatics that
predominantly drain into the deep cervical lymph nodes(dCLNs) [173, 174, 192, 210, 224–226]. On the journey
between initial to collecting lymphatics, fluid transitions
into specialized pre-collecting lymphatic vessels. Locatedat the basal foramina, they exhibit features of both initial and collecting vessels, as they lack SMC, but containone-way valves and a mix of both button- and zipperlike junctions [192, 227]. These findings emphasize thedistinct morphological characteristics of various mLV
subsets, providing insight into their specialized roles in
immune cell and CSF waste clearance cascades from the
CNS to the periphery.

The (previously missing) link from the brain
to the periphery
The 2015 multi-group discovery of mLVs in the duramater, evidenced a role for these vessels as an initial collection site for CSF before its drainage to the dCLNs.
Physically, the arachnoid barrier delineates the CSF
contained in the subarachnoid space from the duramater; however, the mechanism by which the CSF carrying waste products and immune signals from the brain
reaches these mLVs in the dura was unclear. It has previously been suggested that arachnoid granulations,which are protrusions of the arachnoid mater into thevenous sinuses of the dura mater, enabled drainage of
CSF directly to the bloodstream [228], but tracers administrated into the CNS or CSF can drain directly to thedCLNs, demonstrating that there must be a direct routefrom the CSF to the dura that bypasses the bloodstream[194]. In 2024, a pioneering study identified arachnoid
mater cuffs around the bridging veins that connect thesubarachnoid space to the dura [229]. This finding was
made by experiments that involve injecting tracers into
the intracisternal magna (i.c.m) and observing theirtransport. The experiments revealed that the i.c.m. tracers were being transported into the dura before they
had reached either the blood or the dCLNs, indicatingthat the drainage occurred directly from the CSF into
the dura before being drained out by lymphatic vessels.
This connection between the dura mater and the subarachnoid space consists of bridging veins that create discontinuities in the arachnoid barrier, forming structurestermed arachnoid cuff exit (ACE) points. ACE points
are present in humans and can work bi-directionally,

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enabling molecules and immune cells to enter from thedura mater into the brain [229]. This may explain how
cytokine responses from meningeal immune cells influence brain function, a discovery that will shape the way
we think about neuroinflammation in future TBI studies.

The immunological environment
within the meninges
It is now recognized that the meningeal layers act as vital
immunological reservoirs, hosting an array of adaptive
immune cells that first arrive in development via bloodvessels or calvarium bone marrow that connects the dura
via specialized vascular channels [211, 230–235]. Collectively, the meningeal layers contain lymphoid cells,
macrophages, mast cells, eosinophils, dendritic cells(type 1 and type 2 classical dendritic cells, plasmacytoid
dendritic cells and migratory dendritic cells), neutrophils, innate lymphoid cells (ILCs), natural killer cells,
plasma cells, B cells (immature and mature) and T cells(CD4, CD8 and T cell receptor gamma/delta (TCRγδ))[186, 211, 231, 236–240]. Proportionally, the distributionof these cells varies across layers, with the dura largely
thought to contain a higher diversity and frequency of
immune cells versus the leptomeningeal layer [186]. Thismay be attributed to the need for tissue specific support,with the niche environment around dural sinuses containing innate lymphoid cells, macrophages, T cells, B
cells and plasma cells [234, 241]. In leptomeningeal layers, macrophages, CD4, and CD8 cells, are found in largepart around blood vessels in the CSF containing subarachnoid space [242–245].
Meningeal macrophages
Macrophages are heterogeneous groups of cells that carryout distinct functions depending on their location andphenotype [246]. Meningeal macrophages belong to a
specialized group called border associated macrophages(BAMs), originating from yolk sac ­CD206+ myeloid progenitor cells [230]. In the adult, leptomeningeal BAMs
can be transcriptionally delineated by CD206, Lyve1,P2rx7, and Egfl7 expression [186, 236]. In contrast, adultdural BAMs differ to their leptomeningeal counterpartsas they lack Lyve-1 expression, and can be transcriptionally divided into subgroups based on major histocompatibility complex II (MHCII) expression [247, 248].
This may represent divergent roles under homeostaticor disease conditions, with M­ HCIIHi BAMs displaying
enhanced levels of CCR2, whereas ­MHCIIlow BAMs contain gene signatures that include Clec4n, Clec10a andFolr2 expression [186, 211, 246, 249]. Of note, a determining factor that drives postnatal MHCII high or low
differentiation, is the fms intronic regulatory element(FIRE), a highly conserved super enhancer of the colonyMokbel et al. Journal of Neuroinflammation(2024) 21:135

stimulating factor 1 receptor (CSF1R) and its downstream signaling [250, 251]. This may be pertinent toconsider in studies examining microglial driven inflammation in CSF1R deletion models (or via Pexidartinib(PLX) inhibition), as the effect of CSF1R in modulating
facets of meningeal immunity has yet to be fully investigated. In healthy settings the functional role of BAMs islargely unknown, however evidence suggests they can act
as sentinels in anti-viral defense. Following lymphocytic
choriomeningitis virus infection (LCMV), BAMs become
rapidly activated, acquire the viral antigen and trigger
engagement by infiltrating cytotoxic T lymphocytes thatare essential to resolve the infection [252]. Further, thetype-1 interferon-Stat1 anti-viral pathway is induced in
BAMs, blocking further LCMV infection [253]. Transcranial delivery of PLX3397 or PLX5622 to specificallydeplete BAMs resulted in the death of LCMV-infected
mice [253], emphasizing the importance of further investigation into BAMs in CNS disease models that containinflammatory components within their etiology.

Border associated dendritic cells
Dendritic cells (DCs) are professional APCs that play
key roles in immune surveillance in both peripheral andmeningeal layers [254]. DCs in the meninges and choroid plexus arise from circulating and skull bone marrowprecursor-DCs, which transform into conventional dendritic cells (cDCs) at these sites [233, 255, 256]. Investigations into their trafficking ability, revealed that peripheral
DCs can migrate into the CNS across barrier sites via
the C–C motif chemokine receptor 2, chemokine ligand
2 (CCR2-CCL2) signaling pathway [257]. This migration can be bi-directional, with DCs observed in thecervical and auxiliary lymph nodes 3–7 days after theirinjection into the CSF or brain parenchyma [258–260].
Meningeal mediated migratory drainage of DC occurs
via initial mLVs in a CCR7 dependent manner. CCR7 isa potent receptor of DC migration toward the ligands
CCL19 and CCL21, which are highly expressed by lymphatic vessels (Fig. 2). In pathological states, DCs recognize and capture antigens, upregulate CCR7 and migrate
to initial mLVs before draining into the dCLNs where
they can activate antigen-specific T-cell inflammatory
responses [173, 225, 239, 261]. Indeed, dural and cribriform plate mLVs express CCL21, facilitating a gradientfor DC migration and drainage into peripheral lymph
nodes [175, 211, 225, 262]. Additionally, dural fibroblastlike cells express CCL19, providing further stimulus for
CCR7 mediated signaling within the meninges; however,
the role of these fibroblasts in relation to CNS lymphaticdrainage is yet to be elucidated [211]. Collectively, DC
populations within the meninges and CSF may containboth parenchymal infiltrating DCs as well as migratory

Page 8 of 22

draining DCs in transit to lymph nodes, suggesting thatthe holistic neuroinflammatory environment unique tospecific disease states influences DC capacity, drainage
and antigen presentation.

Border associated T cells
The interplay between the innate CNS and adaptive
immune response contributes to neuroinflammation
within disease contexts. T cells are found in the meningeal space, with populations identified at dural and cribriform lymphatic sites [173, 192, 262]. Initial evidence of T
cell migration into dCLNs was noted 12–48 h after their
CNS injection into healthy or lesioned mice [263], withsubsequent lymphatic ablation studies offering extensive
characterization of T cell migration pathways via dural
mLVs [225]. Mechanistically, drainage of T cells into thedCLNs via mLVs occurs in a CCR7 dependent manner[225], a process similar to that seen in inflammatoryinduced peripheral T cell migration [264, 265]. Indeed,
CCR7 expressing T cells are found in human CSF, mostof which are thought to be central memory T cells conducting immune surveillance within the healthy subarachnoid space [266, 267]. While trafficking to dCLNs
may reduce meningeal T cell pools, they are replenishedby blood circulating T cells that enter the brain via thedural sinuses, and adopt tissue-resident phenotypes [211,231]. Indeed, evidence shows elevated densities of APCs
and T cells at dural sinuses, serving as an active site for
APCs to acquire blood- or brain-borne pathological antigens, presenting them to local T cells [211]. Functionally,
the role of a well-regulated adaptive immune response
is paramount, as ­CD4+ and TCRγδ cells can influence
CNS neuronal, glial, and homeostatic activities throughcytokine secretions including IL-4, IL-17 and IFNγ [231,
268–272]. Furthermore, mice lacking functional T cells,
and consequently deficient T cell drainage to dCLNs,
have notable deficits in exploratory, social, and cognitive
behaviors. [268, 273, 274]. These deficits are reversed following adoptive transfer of T cells or secondary lymphoidorgan lymphocytes, further evidencing their importantrole in the regulation of CNS behaviors [268, 274, 275].
Border associated B cells
B cells play versatile roles within the adaptive immune
response. Known predominantly for their antibody production and as precursors to antibody-producing plasma
cells, they also function as APCs, and producers of proinflammatory cytokines and chemokines (reviewed in[276]). In humans, the expression of B cells and plasma
cells is sparse within the CNS parenchyma [277, 278],
however they are abundantly located in the meninges[277], specifically the dural layer [279]. In mice, the identification of meningeal B­ 220+ ­CD11c− B cells was firstMokbel et al. Journal of Neuroinflammation(2024) 21:135

Page 9 of 22

Fig. 2 The meningeal neuroimmune interface influencing inflammatory responses in TBI. Simplified schematic of the meningeal interface
in healthy and injured settings. In homeostatic conditions (left), border-associated immune cells may synergistically support brain environments
through cytokine secretion (IL-4, IL-17), which can directly influence neurons. A network of initial and collecting mLVs expressing Lyve1, drain
CSF solutes/molecules to the dCLNs. Immune cell drainage of dendritic and T cells into the dCLNs occurs via chemokine gradients (E.g. CCR7
expressing cells migrating towards its ligands CCL19 and CCL21). TBI (right) causes BBB breakdown and mLV dysfunction, resulting in reduced L­ yve+mLVs, and impaired drainage of solutes to the dCLNs. This TBI-induced lymphatic dysfunction and immune activation at the meningeal interface,
exacerbates neuroinflammation by resident CNS cells within the brain parenchyma

reported within the dural mLVs [173], with subsequent
single-cell sequencing studies identifying a diverse rangeof progenitor, early and resident B cell populations [234,279–281]. Constitutively, B cells represent ~ 15–30% ofall ­CD45hi cells within the dural meninges, encompassing multiple stages of B cell development from pro-B tomature B cells [234, 279]. Meningeal B cells derive locallyfrom the calvaria bone marrow niche before migrating
to the meninges through specialized skull vascular channels [234]. Following this calvarial–meningeal migration,

B cells complete their development locally, with factors
such as CXCL12 and CXCR4 critical for their survival
and differentiation [234]. IL-7 has also been implicatedin supporting B cell development, alongside other nichefactors in the meninges including the CNS specific antigen myelin oligodendrocyte glycoprotein [281]. For themost part, the majority of mature B cells in the CNS arenaïve ­IgM+ cells, with small numbers of I­ gA+ B cells [234,279]. In terms of migration, B cells have the capacity todrain to the dCLNs via mLV in a similar fashion to their

Mokbel et al. Journal of Neuroinflammation(2024) 21:135

T cell counterparts. Plasma cells ­(IgA+) are also found in
the meninges, with a portion of this population derived
from gut plasma cells [282]. This meningeal plasma cell
expression is age dependent, with young mice displaying
IgA+ cells, switching to an I­gG+ and I­gM+ plasma cell
phenotype in aged mice [234].

Neuroimaging of meningeal disruption in clinical
TBI
Neuroimaging provides valuable data to assist in patient
evaluation and diagnosis following TBI. Common imaging modalities include conventional non-contrast computed tomography (CT) and magnetic resonance imaging(MRI), often used in the clinical work-up for injury indications in patients with GCS < 13 [283]. CT scans provide initial class I assessment of injury extent (lesion,
fracture), extradural, subdural or intracranial hemorrhage, traumatic subarachnoid hemorrhage and ventricular abnormalities [284, 285]. This data is beneficial for
diagnosing the mechanically induced primary trauma,
yet sophisticated imaging techniques are necessary todetect subtle injuries to the brain structure and secondary injury processes. Enhanced imaging of dysregulated
secondary injury cascades including glucose metabolism(functional MRI), diffuse axonal injury (diffusion tensor
imaging, DTI), protein accumulation and neuroinflammation (radiotracer positron emission tomography, PET)
could lead to the discovery of new imaging biomarkers
and therapeutics [286].
Advancements in MRI using contrast agents (such as
gadolinium) have improved the specificity of diagnostic
images, allowing for identification of novel biomarkers
and insights into disease progression [287, 288]. Ordinarily gadolinium is unable to cross the blood–brain
barrier, and therefore acts as a surrogate for leakage ofproteins and other macromolecules when detected outside of the vasculature [289]. Gadolinium post-contrast
fluid-attenuated inversion recovery (FLAIR)-MRI merges
a high intensity of T2 weighting with the attenuationof cerebrospinal fluid (CSF) signal. This allows for thedetection of leakage of the gadolinium contrast agent
across damaged barriers into the CSF space, and highlights hyperintense regions at CSF adjacent borders.
This is relevant in the context of TBI, as vascular injury
can occur in the parenchyma and vessels traversing themeninges [290]. Meningeal arteries and veins in the duraand subarachnoid space are particularly vulnerable to theprimary impact of TBI. Indeed, recent findings suggestthat Traumatic Meningeal Enhancement (TME), associated with meningeal injury and inflammation, is a novel
biomarker observed by FLAIR-MRI in patients following
TBI (Table 2).

Page 10 of 22

Although meningeal enhancement has been observedin neuroinflammatory contexts and in neurological disease [299–301], its identification in TBI is a relatively
recent development (Table 2). In 2014, Roth and colleagues first reported focal enhancement of the meninges
in approximately 50% of patients following a mild TBI(mTBI) [119]. A temporal follow up study identified similar TBI induced TME positive patients (50%) [293], of
which 76% displayed resolution by approximately 22 days
post-injury; however, 17% had persistent TME for several
months (72 to 103 days post injury) [293]. This TME timecourse may be associated with, and influenced by, injury
severity, with TME detected in moderate to severe TBI
patients at 1-year post injury [296]. Collectively, theselikely represent chronic inflammatory or other secondary injury cascades occurring within this compartment in
the weeks, months, and years post-injury. Anatomically,
TME positive mTBI patients display thick linear meningeal enhancement of the dura, including diffuse andlocalized convexity patterns, as well as enhancement of
the falx cerebri [119, 289, 291, 292]. As the volume of dataand studies expand, the diagnostic value of positive TME
findings has become more apparent. TME signatures
have been shown to be associated with loss of consciousness, implying clinically significant head injury [291],
and FLAIR imaging protocol demonstrate superiority indetecting trauma-related abnormalities not visible on CT
scans, distinguishing between acute trauma from nonspecific conditions [292]. Specifically, FLAIR protocols
outperform T1W1 post-contrast sequence in identifying the presence or absence of TME, where T1W1 failed
to show it in 38% of patients where it was readily shownusing FLAIR [295]. To gain insights into biological mechanisms associated with TBI-induced meningeal enhancement, transcriptomic analysis from patients with TME
identified 76 differentially expressed genes, including
increases in IgA, FCαR, MCTP2, GPR2, and decreases in
CD79A [294]. Furthermore, innate lymphoid cells (ILCs)
are found in the dura and CSF of TBI patients, suggesting meningeal immune interactions [298]. Increases in
VEGF2 expression in meningeal vasculature [297], suggests regenerative capacity of dural vessels; however, thisfinding should be repeated in subjects with clinical classification data, for meaningful translational interpretation.

Meningeal lymphatic dysfunction after TBI
Neuroinflammation, characterized by the intricate interplay between innate and adaptive immune
responses, contributes to the pathophysiology of TBI(Fig. 1). A fine balance exists whereby acute activationof neuroimmune cells may be a beneficial physiological response to promote injury resolution, in contrast
to aberrant neuroimmune activation that may manifest

Mokbel et al. Journal of Neuroinflammation(2024) 21:135

Page 11 of 22

Table 2 Imaging of meningeal-brain border disruption in clinical TBI
TBI
Severity

Age
(IQR)

MRI

Image time (h/d)

ResultN (%)

Meningeal enhancement Refs.
observationsmTBI
GCS = 15

THINC (Age undisclosed)

FLAIR

Within 48 hME

69/142 (48.6%)

Focal enhancement

[119]
2014

mTBI
GCS ≥ 1359.8
(26–84)

FLAIR

3.2 d
(0.2–14 d)

TME

32/54
(59%)

Diffuse (12/32), Localized(8/32), Falx (12/32)

[291]
2014

mTBI
GCS = 1548
(41–55)

FLAIR

5.4 h
(3.6–5.3)

TME

10/22 (45%)

Diffuse (4/10), Localized(3/10), Falx (3/10)

[292]
2017

mTBI
GCS > 1242
(28–55)
THINC

FLAIR

11.6 h
(4.9–20.2 h),
Temporal follow-ups

TME

104/209 (50%)

ME resolved in 79
patients (76%) at 22d(7–37 d). Chronic ME
in 17% of patients at 87 d
(72–103 d)

[293]
2018

mTBI
GCS ≥ 1339.8
(22.7–57.8)
THINC

FLAIR

21 h
(14–26 h)

TME

17 TME+
13 TME-76 DEGs in TME + vs TME↑ IgA ↑FCαR, ↑MCTP2,↑ GPR27, ↓CD79A

[294]
2017

mTBI
GCS ≥ 1349
(35–62)
THINC

FLAIR

6.07 h
(4.3–19.6 h)

TME

9/25 (36%)

Conspicuity of TME
is higher on FLAIR MRI
than on post-contrast
T1WI

[295]
2020

Mild 7,
Mod 19, Severe 4

46.4(SD 16.5)

FLAIR

within 48h

TME

16/30 (53%)

Of 16 with TME, 10 (63%)
resolved 1-year follow-up,
6 TME persisted

[296]
2020

mTBI
GCS = 1539.8
(22.7–57.8)
THINC

FLAIR

19 h
(5.9–44 h)

TME

12/36 (33%)

Regions- 11 Falx, 6 Vertex,
5 Frontal, 4 Temporal, 3Occipital, 1 Cerebellar

[289]
2020

mTBI
GCS = 14–15

57(41–67)
THINC

FLAIR

4.3 h
SD ± 0.87

TME, ECSAS

44/75(59%),­2ndscan 23/32(72%)

18/32 (56%) positive
for ECSAS

[289]
2020

Fatal vertical falls

Data not provided

Dural
Tissue24 h for Histology+ IHC

4 samples↑VEGFR2 in blood vessels
in injured dura mater

[297]
2020

Moderate-to-severe

39.2(28–56)

Dural
Tissue (1 ­m2)

Resected from decompressive craniectomy

Flow, ex vivo assays

5 males,
1 female

ILC1:
[298]
­CD45+Lin−CD127+CD161+ 2021
­NKp44+, produce IFNγ;
ILC2: ­CD45+Lin− ­CD127+­GATA3+ ­CRTH2+, produce
IL-5/IL-13;
ILC3: ­CD45+ ­Lin− ­CD127+
RORγt+AhR+, produce IL-17

mTBI: mild traumatic brain injury; GCS: Glasgow Coma Scale; MRI: magnetic resonance imaging; IQR: interquartile range; FLAIR: fluid-attenuated inversion recovery;
ME: Meningeal enhancement; TME: traumatic meningeal enhancement; THINC: Traumatic Head Injury Neuroimaging Classification study (NCT01132937); DEGs:
differentially expressed genes; ECSAS: extravasation of contrast into the subarachnoid space; HARM: hyperintense acute reperfusion marker; ILCs: innate lymphoidcells

toward deleterious chronic outcomes. Identification of
the immune rich lymphatic system at the meningeal
barrier interface, has triggered a new area of previously
unexplored research, investigating the contribution ofthis neuroimmune crosstalk to TBI-induced neuroinflammatory responses (Table 3) In 2020, Bolte et al.
reported seminal findings evidencing the temporalseries (across seven timepoints) of meningeal lymphatic disruptions in a closed head impact model ofTBI. Here, they detail mLV drainage of CSF molecules
to the dCLNs is impaired as early as 2 h, and persists for at least 1-month, following injury [302]. This
impaired drainage was associated with changes to lymphatic vasculature, with a decrease in Lyve-1 expression observed within the first 2 to 24 h post-injury[302]. Interestingly, mLVs demonstrated a capacity
for regenerative lymphangiogenesis, evidenced by the
spontaneous increase of Lyve-1 coverage and complexity in meningeal whole mount preparations 1–2 weeks
post injury [302]. Photothrombotic vascular damage
to the dura mater and pia mater causes immediate vascular degeneration followed by revascularization at
7d post-injury [297], suggesting that changes in mLVs
may occur in parallel with regenerative endothelial
angiogenesis at dural sites (Fig. 2). However, across the
spectrum of TBI, the extent of this naturally occurring vascular and lymphangiogenic responses varies
upon injury type and severity. Lyve-1+ mLV coverage/
expression is decreased at 3d (mice, impact 1.9atm)[303] and 7d (rats, impact 2.6 atm) [304] after lateralMokbel et al. Journal of Neuroinflammation(2024) 21:135

Page 12 of 22

Table 3 Meningeal insights in pre-clinical models of traumatic brain injury
TBI model

Animal/Age/Sex

Interval time

Tissue/Cell type

Injury-induced meningeal
signature

Refs

Thinned skull, manual
blade compression mTBI

Male, Female
C57Bl/6J mice
(8–12 wks)

6 h, 1 d, 4 d

Whole mount meninges,
QPCR analysis

At 6 h, 1 d ↑Cxcl1 (neutrophils), ↑Ccl2, Ccl12­(CCR2himonocyte) ↑Cxcl10,
Il1β
At 1 d, 4 d↑ vascular leakage
At 4 d ↑wound-healing­CD206+ Lyve-1+ macrophages

[293]
2018

Photo-thrombotic injury

C57Bl/6J mice
(8–10 wks)

1 d, 7 d,Whole mount dura and pia, Dura mater revascularization
Dura–RNA & protein
by 7 d post injury

[297]
2020

Adapted CCI hit and run
Male, Female(closed head, 2 mm,
C57Bl/6J mice
5.2 m/s, dwell time 100 ms) (8–10 wks)

2 h, 1 d, 4 d, 1  w,
2 w, 1 m, 2 m

Whole mount meninges& dCLNAt 2 h, 1 d, 4 d, 1 w, 2 w, 1 m↓ MLVs drainage to dCLN. At
2 m drainage restored
At 1 w, 2 w↑Lyve1 coverage,at 1 m, 2 m ↑loops/complexity

[309]
2020

Thinned skull, manual
blade compression mTBI

Male, Female
C57Bl/6J mice
(8–12 wks)

1 d, 3 d, 4 d,5 d, 7 dWhole mount meninges

At 1 d ↓endothelial vascular
integrity. Revascularization
at 3 d, 5 d,7 d. Inhibitedby infection
At 1 d, 3 d, 4 d and 7 d ↑­CD11b+, ­CD206+ myeloid
cells

[321]
2021

CCI (3 mm, 3 m/s, dwell
time 85 ms)

Male
C57Bl/6J mice
(9–10 wks)

1 d, 7 d, 1 year

Meninges,
Meningeal ILCs
(Innate lymphoid cells)

At 1,7d ↑ILC1-3. At 1y ↑ILC2/3 [298]Metabolic dysregulation
2021
with ↓AMPKα1 at 1d*CCI + IL-33 (1μg) ↑pAMPKαCHIMERA (single × 1 &repeat × 4)

Male, Female C57Bl/6
mice (6–7 wks)

1 d, 7 dWhole brain MRI

At 1d & 7d ↑ meningeal
enhancement in both single
and repeat impacts

[305]
2022

CCI (0.5 mm or 1 mm,
5 m/s, d well undisclosed)

Male
C57Bl/6J mice
(9–10 wks)

3 dpi, 6 wks

Dura mater–RNAseq,
Whole mount meninges

3dpi - ↑CD45+ myeloid (Bcell) & CD11b+ cells
6 wks - ↑Ccl8, Il1β, Ccl2
Ccl7 ↑immune pathways,
including interferon gamma
response

[316]
2022

CCI (2 mm, velocity undisclosed)

Male
C57Bl/6 mice
(10 wks)

3d

Meningeal lymphatic
endothelial cells (LECs)

Flow cytometry ↓ Lyve1 LECs
Microarray ↑ DEGs involvedin FCERI signaling, antibodymediated complement,
Inflammatory response

[307]
2022

Adapted CCI hit and run
Male(closed head, 2 mm,
C57Bl/6J mice
5.2 m/s, dwell time 100 ms) (8–12 wks & 20 months)

7 d, 1.5 m

Whole mount meninges
Meninges for bulk &scRNA-seq

At 7d, ↑DEG s related
to macrophages, fibroblasts,
and adaptive immune cells.
↑IFNβ, IRF5, IFNAR1
At 1.5m ↑ collagen, fibroblasts ↑T/B cell DEG. Aging
amplified

[302]
2023

FPI (11°, 2.6 ± 0.16 atm)

Male, Sprague–Dawley
rats

7d

Whole mount meninges& dCLN*FPI ↓MLVs drainage to dCLN
↓Lyve1, Prox1, Foxc2, VEGFR3*FPI + VEGF-C/Ketoprofen/
RA improves Lyve1 + dCLN
drainage

[304]
2023

CCI (1.5 mm, 3m/s, d welltime 120 ms)(8–10 wks)

4 d, 7 d,
14 d

Whole mount meninges& dCLN*CCI ↓MLVs drainage to dCLN [306]
4 d, 7 d,14 d. ↑Lyve1 7 d
2023*CCI + VEGF-C 156S&EVs@Gel
restores dCLN drainage

Mokbel et al. Journal of Neuroinflammation(2024) 21:135

Page 13 of 22

Table 3 (continued)
TBI model

Animal/Age/Sex

Interval time

Tissue/Cell type

Injury-induced meningeal
signature

Refs

FPI (1.9 ± 0.2 atm)

Male, Female C57Bl/6
mice (8–10 wks)

3d

Whole mount meninges& dCLN*FPI ↓MLVs drainage to dCLN [303]↓Lyve1, ↓VEGFC/VEGFR3
2023*FPI + IL-33 (i.c.m, 20ng/μl,
5 μl), restores dCLN drainage,
Lyve1, VEGFC/VEGFR3

CCI (2 mm, 3.5 m/s, dwell
time 500 ms)

Male
28 d
C57Bl/6 mice (3 months)

Regrown cavity leptomeninges (pia, arachnoid)

Brain Fibroblasts (BFB 2–5)
↑Fmod (BFB2/3), ↑Dpp4(BFB4), ↑Slc47a1 (BFB5) (drugtransporter)

[184]
2023

CCI (depth,
velocity, undisclosed)

Male
C57Bl/6 mice (6–10 wks)

28 dWhole mount meninges*CCI ↓Lyve-1 area at 28d*CCI+ pVEGFC (i.v 50µg
@1 d, 3 d, 5 d) restores Lyve1(lymphangiogenesis)

[308]
2023

CCI (1.5 mm, 5.25 m/s,
dwell time 100 ms)

Male
C57Bl/6J mice
(8–12 wks & 18–19 m)

7 d, 1 m

Meningeal bulk RNA-seq

At 7d ↑laminin, collagen[317]
and T-cell DEGs. ↑ TGFβ, IFNα. 2023
At 1 m ↑immunoglobulin
production and B cell DEGs.
↑Ccr2 (young), IL-33 (aged)
upstream regulators. ↑IFNα/β

CCI: controlled cortical impact; FPI: fluid percussion injury; MLVs: meningeal lymphatic vessels; dCLN: deep cervical lymph nodes; meningeal lymphatic endothelial
cells (LECs); CHIMERA: closed-head impact model of engineered rotational acceleration; ILCs: innate lymphoid cells

fluid percussion injury (FPI). MRI of a closed-head
impact model of engineered rotational acceleration(CHIMERA) shows meningeal enhancement 7d after
single and repeat injury, reflecting clinical observations[305]. Controlled cortical impact (CCI) in mice presents mixed findings: Lyve-1+ morphology is increased
at 7 d (impact 1.5 mm) [306], with contrasting studies
showing decreases at 3 d (impact 2 mm) [307], and 1
month (impact undisclosed) [308], highlighting thenuanced and variable nature of spontaneous lymphangiogenic responses across varied mechanisms of impact
and injury parameters. Regardless, if plasticity of thelymphatic network can be induced, it may be beneficial
to improve outcomes (Fig. 2). Indeed, across models of
TBI, overexpression of VEGF-C drives lymphangiogenesis, enhances functional drainage and improves injury
induced neurological deficits, including those seen inaged animals [303, 304, 306, 308–310].

The meningeal interface influencing TBI induced
immunological responses
TBI‑induced meningeal damage

The meninges host a diverse array of immune cells, as
previously outlined, yet the precise details of their locations (whether in the leptomeninges or dura) and trafficking patterns in TBI still remain largely unknown.
Initial studies at the meningeal interface classified thenature of damage to the barrier itself, followed by the
temporal response to direct meningeal injury. Acutely,closed head meningeal compression injury (achieved bya unique skull thinning model) is characterized by rapid
meningeal macrophage cell death attributed to vascularleakage and the release of reactive oxygen species [119],and also causes secondary damage to the glial limitans
and brain parenchyma within the first few hours [119,293]. The initial injury is followed by neutrophil swarming into the meninges (within 1 h), that is essential forregeneration of the initially damaged glial limitans [119].
Elevated proinflammatory cytokines IL1α/IL1β are seenin the meninges at 6 h to 1 d, as well as the chemoattractants Cxcl1 (neutrophils) and Ccl2/Ccl12 (monocytes)[293]. Over the course of a week after injury, infiltrating
myeloid cells ­(CX3CR1lo–negCCR2hi monocytes) scavenge
dead cells at the meningeal lesion core, while woundhealing macrophages (­CX3CR1hiCCR2lo–negCD206+)
proliferate along the lesion perimeter to promote angiogenesis through the clearance of fibrin and production of
MMP-2 [293]. These studies at the level of the meninges
evidenced the importance of the previously unexploredbarrier immunity, setting the stage to address bi-directional interactions containing both extra-axial meningeal
cascades and intra-axial brain parenchyma responses.
Modulating meningeal lymphatics in TBI

Impairment studies shed more light on how the meningeal compartments influence CNS responses, with ablation of mLVs exacerbating resident glial cell (­GFAP+,­IBA1+) immunoreactivity, increasing complement,
reducing neuronal health markers and which collectively

Mokbel et al. Journal of Neuroinflammation(2024) 21:135

influenced cognitive outcomes [309]. Furthermore,administration of the pro-lymphangiogenic VEGF-C toaged mice reduced brain gliosis and improved cognition.
Integrating adaptive immunity, TBI induces the accumulation of Granzyme ­B+ ­CD8+ cytotoxic T cells, a response
preceded by increases in IL-17-producing ­CD4+ T cells
and IFNγ-producing C­ D4+ T cells [311]. This sequence issignificant, given Th17 cell responses through IL-17 andIL-21, can enhance the cytotoxic capability of C
­ D8+ T
cells [311]. In K14-VEGFR3-Ig (TG) mice that have defective growth and lack mLVs alongside sclerotic dCLNs,a reduction in the perilesional infiltration of T-cells isobserved [312]. Specifically, TBI-K14-VEGFR3-Ig miceshow reduced levels of infiltrating C­ D4+ T cells, suggesting that trauma induced brain-derived antigens may bepartially drained through the mLVs to the dCLNs to elicit
Th-2 mediated responses [312]. Indeed, lymphatic vessels
and LECs themselves play a direct chemoattractant rolein the maturation of T cells, turning naive T cells into amemory-like subset of quiescent yet antigen-experienced­CD8+ cells that can rapidly differentiate into an effector
upon inflammatory antigenic challenge [313]. Dendritic
antigen presenting cells can also survey the inflammatory CNS milieu, traverse the mLVs in a CCR7, CCL19
and CCL21 manner, and interact with T cells in thedCLNs. During trauma, DCs are elevated and influence
post-injury immune responses [129, 259, 314, 315], yet
this meningeal migration of DCs downstream activationof adaptive immune responses hasn’t been studied in thecontext of TBI.
Insights from transcriptomic approaches

In order to gain a greater understanding of the cell specific responses that occur in the meninges after TBI, we
and others have taken a transcriptomic approach [184,302, 316, 317]. scRNA-seq shows increased meningeal
macrophages, ­CD8+T cells, T helper cells Th2 and Th17,
immature-mature B cells, dendritic cells, and fibroblasts
in meningeal compartments at 1-week post injury [302].
Similarly, we identified time dependent alterations in themeningeal transcriptome. T-cells respond acutely, followed by chronic B-cell and immunoglobulin production
in an age dependent manner, which may be regulated
by upstream Type-1 IFNs, Ccr2 and IL-33 interactions[317]. Supporting this, meningeal macrophages display strong type-1 IFN signatures (elevated IFNβ andIRF5) and sub cluster into “inflammatory” and “resolution” classes [302]. Resolution macrophages express ofantigen presentation-related genes (H2-Eb1, H2-Ab1,
H2-Aa, Cd74) and anti-inflammatory genes Stab1, Nrros,and Dab2 which are involved in suppression of type I
IFN responses. Conversely, meningeal inflammatory

Page 14 of 22

macrophages are defined by their expression of Ccr2 andchemotaxis genes Ccr7, Ccl22, and Ccl5.
Future avenues for neuroimmune investigation

These seminal pre-clinical studies of the meningeal
response after TBI prompt numerous inquiries for future
investigation within the field of neurotrauma. Not only
do the meninges harbor immune cells that contributeto a vast array of responses, the mLVs play a crucial roleas the waste-removal system in draining CSF, ISF, and
CNS-derived molecules from the brain parenchyma to
the dCLNs. The effect of their damage caused by primary injury and how that influences removal of proteinssuch as Aβ and tau remain to be characterized. Additionally, stratification of the level of damage to the LVs,
subsequent lymphangiogenic recovery, and the effect on
the flow of CSF throughout the brain is still to be characterized. Transcriptomic studies suggest inflammatory
molecules may regulate meningeal derived inflammatory responses, particularly chemokines. Indeed, modulation of the CCR2-CCL2 signaling axis in TBI has beenshown to be beneficial [77, 318–320]. ­CCL2−/− miceshow delayed reductions in lesion size beginning at 28
days post injury. It might be postulated that this delayed
response may be due to deficiency in meningeal mLV
acutely, prior to lymphangiogenic recovery, in combination with altered CCL2 chronic immune responses.
Indeed, ­CCR2−/− mice show altered monocyte/macrophage influx and reduced crosstalk with innate cells,
inhibiting the generation of type-1 IFN microglial
responses after TBI, with pharmacological blockade of
CCR2 improving cognitive outcomes at 28-day timepoints [318, 319]. Populations of macrophages after TBI
were ­Mrc1+ ­Lyve1+ [319] suggesting microglial crosscommunication between meningeal cells, and as of now
investigations into TBI mediated CCR2-CCL2 axis at themLV immune interface are yet to be conducted, potentially warranting future investigation.
Alongside CCL2, CCL7 has been observed in themeninges single TBI [316]. CCR7 is expressed in a variety of peripheral immune cells including DCs and T-cells.
Given ­CCR7+ immune cells migrate to the dCLNs via a
CCL19 and CCL21 chemokine gradient (Brandum et al.,2021), suggests the potential DCs (that have phagocytosed TBI-induced antigens) and naïve T-cells migrate
into LVs and dCLNs and present antigens to naïve C­ D8++
T-cells and ­CD4 T-cells and activate them in response
to TBI. Future studies investigating this potential using
agonists/antagonists or conditional CCR7Ko models
may be of interest to examine pathway influences neuroimmune outcomes and cognition in TBI, similar tothat observed in 5xFAD models [239]. Most importantly,
these questions may be influenced by factors like age,

Mokbel et al. Journal of Neuroinflammation(2024) 21:135

Page 15 of 22sex, and injury severity. Future studies should incorporate these variables to stratify the immune response at
the meningeal interface following traumatic brain injury(TBI).

Conclusions
TBI introduces a complex interplay between neuroinflammatory responses and immune dysregulation. We
now understand that the meninges play a role in modulating this neuroinflammatory response, serving as theinterface between the brain and the immune system;
however, we still do not fully understand the mechanisms underlying prolonged neuroinflammation or how
the temporal progression of damage to the meningeal
lymphatic vessels influences cell trafficking and resolution. After TBI, immune cells (monocytes, macrophages,
neutrophils) are available at the meningeal interface toimmediately influence the brain, and infiltrating cells
such as T and B cells can still be found in the brain andmeningeal interface months following the injury. Does
the presence of meningeal immune cells and the soluble factors released in response to trauma signal to, andamplify, the innate microglial responses? Are these cells
being constantly trafficked into the brain, attracted bycontinuous damage signals? Do invading immune cells
enter the CNS to mediate damage, but are unable to exit
due to trauma-induced mLV dysfunction? Targeting theability of immune cells to enter the CNS in response to
injury, enhancing their ability to drain from the CNS, andgaining a better understanding of the crosstalk between
the brain, meninges, and dCLN will allow us to harness
the power of the immune system to enhance recovery
from TBI.
Abbreviations
ACE	Arachnoid cuff exit
APC	Antigen-presenting cells
AQP4	Aquaporin-4
Arg	ArginaseASC	Protein apoptosis associated speck-like proteinb2M	Beta-2-microglobuli
BAMs	Border associated macrophages
BBB	Blood brain barrier
BDNF	Brain-derived neurotrophic factor
C3	Complement component 3
CCI	Controlled cortical impact
CCL	C-C motif ligand
CCR​	C-C chemokine receptor.
CCR2-CCL2	Chemokine receptor 2, chemokine ligand 2
CD	Cluster of differentiation
cDCs	Conventional dendritic cells
CHIMERA	Closed-head impact model of engineered
acceleration
COX	Cyclooxygenase
CR	Complement receptor
CRP	C-reactive protein
CSF	Cerebrospinal fluid
CSF1R	Colony stimulating factor 1 receptor
CT	Computed tomography
CXCL8	C-X-C motif chemokine ligand

rotational

DAMPS	Damage-associated molecular patterns
DC	Dendritic cells
dCLNs	Deep cervical lymph nodes
DEGs	Differentially expressed genes
DNAM	DNAX accessory molecule
ECSAS	Extravasation of contrast into the subarachnoid space
FBG	Fibrinogen
FIRE	Fms intronic regulatory element
FITC	Fluorescein isothiocyanate
FLAIR	Fluid-attenuated inversion recovery
FPI	Fluid percussion injury
GCS	Glasgow Coma Scale
GFAP	Glial fibrillary acidic protein
HARM	Hyperintense acute reperfusion marker
HLA	Human leukocyte antigen
HMGB1	High mobility group box 1
HSP	Heat-shock protein
i.c.m.	Intracisternal magna
i.v.	Intravenous
ICAM	Intracellular adhesion molecule
IFN	Interferon
IgG	Immunoglobulin GIL	Interleukin
ILCs	Innate lymphoid cells
iNOS	Inducible nitric oxide synthase
IQR	Interquartile range
KIR	Killer-cell immunoglobulin-like receptors
LCMV	Lymphocytic choriomeningitis virus infection
LEC	Lymphatic endothelial cell
LV	Lymphatic vasculature
LYVE-1	Lymphatic vessel endothelial hyaluronan receptor 1
MCP	Monocyte chemoattractant proteinME	Meningeal enhancement
MHCII	Major histocompatibility complex II
mLVs	Meningeal lymphatic vessels
MMP	Matrix metalloproteinases
MPO	Myeloperoxidase
MRI	Magnetic resonance imaging
MRP	Macrophage-related protein
mTBI	Mild traumatic brain injury
Myd88	Myeloid differentiation primary response 88
NADPH	Nicotinamide adenine dinucleotide phosphate
NLRP1	NLR Family Pyrin Domain Containing 1
NF-κB	Nuclear factor kappa B
NK	Natural killer
NOX	NADPH oxidase
NT	Neurotrophin
PDPN	Platelet aggregating protein podoplanin
PK	Ligand [11C](R)PK11195
PLX	Pexidartinib
PROX1	Prospero homeobox 1
PRR	Pattern recognition receptors
RAGE	Receptor for advanced glycation end-products
S100B	S100 calcium-binding protein B
sIL	Soluble interleukin receptor
SMC	Smooth muscle cell
TAM	Trauma Associated Microglia
TBI	Traumatic Brain Injury
TCRγδ	T cell receptor gamma/delta
TGF	Transforming growth factor
THINC	Traumatic Head Injury Neuroimaging Classification study(NCT01132937)
TLRs	Toll like receptors
TME	Traumatic Meningeal Enhancement
TMEM	Transmembrane protein
TNF	Tumor necrosis factor
T-reg	Regulatory T cells
UCH-L1	Ubiquitin carboxyl-terminal hydrolase isozyme L1
VEGF-C	Vascular endothelial growth factor C
VEGFR-3	Vascular endothelial growth factor receptor-3
ZO	Zonula Occludens

Mokbel et al. Journal of Neuroinflammation(2024) 21:135

Acknowledgements
The authors would like to thank past and present members of the Burns lab
for valuable discussions.
Author contributions
BM conceptualized the manuscript. AM and BM wrote the manuscript. AM,
MB and BM reviewed, edited and revised the manuscript. The authors declarethat outside of the funding agencies outlined above, they have no conflict ofinterests to declare. All authors read and approved the final manuscript.
Funding
This work was supported by the National Institutes of Health (NIH) / National
Institute of Neurological Disorders and Stroke (NINDS) (R01NS107370 &R01NS121316) to MB.
Availability of data and materials
All data generated or analyzed during this manuscript are included in thisarticle.

Declarations
Ethics approval and consent to participate
Human subjects’ consent to participate is not applicable. Ethical approval foranimals also not applicable.
Consent for publication
All contributing authors have given their consent for the publication of thisstudy.
Competing interests
The authors declare that they have no competing interests.
Received: 17 February 2024 Accepted: 3 May 2024

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