Traumatic brain injury (TBI) and aneurysmal subarachnoid hemorrhage (SAH) are common neurological conditions (1, 2) associated with extensive morbidity and mortality (3, 4). While the two diseases are different entities, they share many common features in their secondary pathophysiology. Furthermore, they provide an experimental paradigm in which multimodality monitoring offers the opportunity to decipher common pathological processes. There are multiple potential mechanisms by which neuronal injury is inflicted following TBI and SAH. The traditional clinical paradigm for management of these conditions has focused on adequate delivery of oxygen and appropriate metabolic substrates to the injured brain, and prevention of secondary injuries caused by hypotension and hypoxia (5). These secondary processes lead to an increasingly inhospitable environment with ongoing excitotoxicity, oxidative stress, blood–brain barrier (BBB) disruption, cortical spreading depression, mitochondrial dysfunction, and subsequent cellular death in the tissue surrounding the initial damage (6, 7). There is an increasing recognition that inflammatory mediators are involved in the mechanistic link that underlies these injurious processes (8–10).

Galen’s original description of inflammation as calor (heat), dolor (pain), rubor (redness), tumor (swelling), and subsequently functio laesa (loss of function) has been refined into a more complex phenomenon that represents the host response to any insult. This complex process involves the release of molecular mediators, the alteration of the cerebral vasculature, the activation and influx of immune cells to eliminate pathogens, damaged cells, and other perceived harmful stimuli. The brain was long thought to be immunoprivileged due to the presence of the BBB, which limits the cross-talk between the blood and brain-resident inflammatory cells, the lack of a classical lymphatic system, and the shielding of neural antigens from peripheral immune surveillance. More recently, all these assumptions have been questioned and revised (11). There is an increasing realization that cerebral inflammation or neuroinflammation occurs within the whole gamut of central nervous system (CNS) pathologies, whether it is an adaptive autoimmune response (e.g., multiple sclerosis) or a response to external stimuli (e.g., accumulation of red blood cells following brain hemorrhage). While microglia are considered the primary immune cells of the CNS, it is becoming increasingly clear that other glial cells (astrocytes and oligodendrocytes) as well as neurons display immune-competent functions. Activated resident cells of the CNS, in combination with migrating inflammatory cells from peripheral blood, form an intricate immune network (12, 13). Although this immune response may be initiated to protect the brain, it is becoming evident that it can result in harmful outcomes for the CNS (14). In fact, an ongoing neuroinflammatory response may not only contribute to increased edema, cellular death, and BBB disruption but also function as a potent scavenger of dead cells and support the regenerative processes in the injured CNS (15, 16). An outline of the pathophysiological mechanisms identified in the literature is illustrated in Figure 1, many of which are described in this review.

Brain hemorrhage triggers a series of pathological processes resulting in neuronal damage and consequent neurological deficit (17–22). Early brain injury (EBI) refers to the damage ensuing in the first 72 h after the bleed caused by transient cerebral ischemia (CI), BBB disruption, edema, cell death, and brain tissue loss. Many of the patients who survive these phenomena deteriorate days later from delayed ischemic neurological deficit (DIND), which is responsible for poor outcomes or death in up to 30% of patients with SAH. DIND was traditionally attributed to narrowing of the large basal cerebral arteries, termed cerebral or angiographic vasospasm, causing a reduction in cerebral blood flow (CBF) and, if critically reduced, progressing to infarction in the relevant vascular territory. However, a growing number of reports shows that angiographic vasospasm does not always correlate with CI, cerebral infarction, or changes in CBF, indicating that DIND is a more complex (and complicated) phenomenon (23–33). The landmark CONSCIOUS-1 trial showed that an endothelin-1 antagonist ameliorates angiographic vasospasm, but fails to improve functional outcomes (34, 35), forever changing the concept of DIND, which is now suspected to arise from the combined effects of angiographic vasospasm, global reduction in blood flow, arteriolar constriction and thrombosis, cortical spreading depressions, and processes triggered by EBI (17–21, 36). With the complex interplay of the underlying pathological substrates, DIND is often (incorrectly) used as a blanket term for vascular spasm, CI, and neurological (clinical) deficit. It remains unclear to what extent these phenomena overlap or represent distinct processes (Figure 2).

It is increasingly recognized that the neuroinflammatory cascade following SAH is the potential mechanistic link between the initial ictus and the progression of neuronal injury in EBI as well as the development of DIND (37–40). Both overlapping phenomena of ischemic injury and vasospasm lead to an unfavorable physiological environment that may inflict neuronal injury (Figure 2), possibly driven in part by neuroinflammatory mediators.

Patients suffering from TBI and SAH are typically treated in specialized NCCUs where extensive intracranial monitoring is implemented in order to detect subsequent secondary insults, prevent further deterioration, and optimize conditions for brain recovery (41, 42). Monitoring neuroinflammatory processes is technically and logistically complex, and this has hampered the development of pharmaceutical compounds that act to modulate the neuroinflammatory cascade (43). As we acquire a greater insight into the interplay between neuroinflammation and secondary brain injury, monitoring the neuroinflammatory events may open an opportunity to both increase our understanding of the molecular basis of the underlying pathology as well as identify novel therapeutic targets.

The aim of this narrative review is to demonstrate how different aspects of the neuroinflammatory response contribute to the pathophysiology of secondary brain injury. In addition, we discuss the methods available to monitor cellular and humoral inflammation in TBI and SAH patients in the clinical setting.

The CNS has several inherent barriers, restricting and controlling the flow of substances. These include the BBB, the blood–cerebrospinal fluid (CSF) barrier, and the CSF–brain barrier. The brain compartment, or specifically the extracellular space of the brain, is accessible by using a microdialysis (MD) catheter. The MD catheter possesses a semipermeable membrane that is able to extract proteins like cytokines directly from the extracellular fluid (ECF) when inserted into the injured brain (13, 45).

The CSF is produced by cells of the choroid plexus located within the cerebral ventricles and provides a medium to embed the brain, maintain a homeostatic environment, and facilitate the transport of nutrients and waste products. Thus, while using the CSF for cytokine measurement may provide a global assessment of cerebral inflammation, it remains less specific in terms of regional monitoring. CSF is more easily accessible through, ideally, external ventricular drainage in sedated patients or via lumbar puncture in conscious patients where several milliliters can be drawn for analyses. While blood is biologically the most remote space from the organ of interest, it represents an easily accessible compartment for repeated sampling. To compare and monitor cytokine concentrations, collection of matched CSF and plasma samples over time allows a composite picture of ongoing immune responses, distinguishing central from systemic cytokine profiles and determining how immune activation in the brain may impact on mechanisms occurring in the periphery and vice versa.

Figure 1 illustrates the intricate relationship between different compartments in the CNS and the potential cross-talk between the different compartments.

Although controversial, some groups have correlated cytokine concentrations measured in blood/plasma in the acute and chronic setting of patients with TBI and SAH to functional outcome (46–48). This suggests that, while these mediators may represent non-brain-specific inflammatory activity, systemic inflammation is triggered by brain injury, potentially having long-term sequelae such as stimulating an acute-phase response in the liver (49). Importantly, the pattern of cytokine production is both qualitatively and quantitatively different in blood compared to CSF following injury (49–52), suggesting the existence of distinct biological processes at play.

Collection of CSF is the oldest and most prevalent method to test for inflammatory mediators following TBI and SAH and, and in contrast to blood sampling, it represents a global measure of cerebral immune activity. Access to ventricular CSF is achieved with surgical insertion of a drain in the ventricles to measure and monitor changes of intracranial pressure. Longitudinal measurement of cytokines in CSF of patients with severe TBI reveals a biphasic immune response being maximal in the first few days after injury followed by a second modest increase by 7–10 days as shown for IL-6 (49).

These data have been confirmed using principal component analysis (PCA) of CSF cytokines and chemokines, resulting in the identification of distinct clusters of cytokine response (53, 54). Interestingly, multivariate analysis has also demonstrated a significant difference in outcome between the two clusters. Thus, assessing inflammatory activity provides a potential window for appropriate pharmaceutical therapies targeting cytokine production (54). The difficulty is in disentangling the roles of individual mediators within these clusters to investigate rationalized treatment targets.

In SAH, CSF samples may not be a true measure of neuroinflammation. The volume of blood present within the subarachnoid space can affect the levels of cytokines present in CSF, and therefore, cytokine concentration in CSF is partly a consequence of the volume of SAH rather than a specific measure of the magnitude of the inflammatory response within the brain.

Microdialysis is inevitably a spatially restricted approach that can be used to target pericontusional/infarcted tissue at risk for secondary injury and thus allow us to monitor ongoing or worsening focal brain damage. Notably, cytokine analysis revealed large variations in their concentrations when comparing CSF with brain-ECF (Table 1). However, the difference in cytokine levels between CSF and brain-ECF is smaller compared to the concentration gradients between these compartments and serum (55). Measurement of cytokines using MD have the additional complication of relative recovery. Relative recovery within the microdialysate fluid refers to the recovered fraction of the true concentration in the ECF, which is governed by a range of physicochemical factors including the molecular weight of the cytokine, its isoelectric point (which in turn determines the charge the protein carries at neutral pH and its solubility in water), and its native oligomerization characteristics (56). For some species, the relative recovery can be as low as 5% (exemplified in Table 1) making it technically difficult to reliably monitor.

There is an intricate interplay between the various cellular elements (both resident and recruited) within the brain, which are both the source and target of these mediators (13, 57, 58), further complicating the interpretation.

Using either a qualitative approach or an unbiased quantitative analysis (such as PCA), cytokine production appears as an intrinsic and ordered component among the multiple biochemical pathways contributing to delayed brain damage (57, 59). This fact would argue against the inflammatory response being a secondary form of injury per se, and rather a sequential cascade of molecular events [in the words of Julius Caesar: alea iacta est (the die is cast)], with a predetermined sequence following the initial injury.

MD is commonly used in the NCCU for metabolic monitoring of the injured brain, recovering small metabolites such as lactate, pyruvate, glucose and glycerol from the ECF (45). For these metabolites, the relative recovery is circa 70% from catheters inserted into the brain, using a flow rate of 0.3 µl/min (60). Importantly, the pore size of MD membranes used for metabolite analysis is significantly smaller (20 kDa) compared to the size used for larger proteins such as cytokines (100 kDa) (61, 62). By using 100 kDa cutoff catheters with the standard crystalloid solution, the relative recovery for cytokines has been shown to be typically 30%, but as low as 5% for cytokines that oligomerize (e.g., TNF) (56). In order to improve poor recovery rates, colloids such as human albumin solution (HAS) or dextran may be added to the perfusion fluid (63). The addition of colloids does not always improve the recovery of all cytokines; however, the relative recovery for TNF in 3.5% HAS has been shown to increase from 4.4 to 31.2%, a sevenfold increase (56). A 3% Dextran-500 solution shows equally encouraging results compared to crystalloid solutions (64), and has the capacity to improve extraction of even larger inflammatory mediators (65, 66) and has been empirically demonstrated to remain within a 100 kDa membrane catheter (67). In summary, the optimal recovery of cytokines within the MD samples is dependent upon perfusate supplemented with albumin or dextran, favoring an improved relative recovery and no net loss of fluid from the catheter. In our opinion, this is an essential adjunct for protein microdialysis in general and specifically in recovering inflammatory mediators.

While the neuroinflammatory cascade following TBI is complex and characterized by substantial collinearity and functional overlap among mediators, many studies have focused on single cytokines utilizing univariate comparative statistics without adequately assessing the interrelationships with other factors (68–70). A more appropriate statistical approach would be to incorporate several cytokines into multivariate analysis methods, such as PCA (71). By using PCA, it is possible to detect the greatest sources of variance in the dataset and thus highlight patterns in the cytokine response (59). A regression extension of PCA such as partial least square discriminant analysis (PLS-DA) can be used to identify variations in a particular domain such as time (58, 72). PLS-DA has the disadvantage of introducing bias, but can be used to test prespecified hypotheses.

A systematic review has identified 32 studies describing the analysis of CSF cytokines in 1,363 severe TBI patients, of which 19 studies reported the analysis of cytokines via MD in 267 severe TBI patients (73). The majority of the MD studies have been published from a few centers of excellence in TBI research, known for their work in the field of cerebral MD. From these studies, it is evident that MD catheter positioning plays a crucial role in the amount of cytokines recovered, with peri-lesional or lesional locations displaying greatly different cytokine profiles compared to “healthy” non-lesioned tissue. There is also a stereotyped response to injury whereby the peaks of MD derived cytokines such as interleukin-1 beta (IL-1β), IL-6, and IL-8 occur within the first 48–72 h post-TBI, with IL-10 remaining elevated throughout the acute period up to 5 days of monitoring. Therapeutic modulation of the neuroinflammatory response to TBI has been demonstrated clinically in patients using subcutaneous administration of recombinant human IL-1 receptor antagonist, with the aim of attenuating the experimentally proven IL-1-mediated cellular damage in the injured brain (57, 58). Several studies have attempted to correlate CSF cytokine concentrations [IL-1β, interleukin-1 receptor antagonist (IL-1ra), IL-6, IL-8, IL-10 and TNF] with patient outcome, demonstrating a number of conflicting data [summarized in a review article (74)]. We would argue that this approach is flawed as it ascribes a unitary role for a given cytokine (as “good” or “bad”) as well as ignoring the high degree of collinearity between all these mediators (75). It is important to recognize the delicate interplay between mediators such that the ultimate effect of a given cytokine is determined by the specific cellular, chemical, and temporal milieu in which the cytokine is acting. Finally, the clinical complication profile with brain-ECF and CSF-based cytokine sampling appears to be quite low, attesting to the safety of both procedures in critically ill TBI patients. All these findings are necessarily limited by the small number of studies identified in the accompanying systematic review.

Similar to the findings identified in the review on TBI, the studies reporting on the analysis of cytokines via MD and CSF in aneurysmal SAH cytokine can be found in the parent systematic review, included in this Research Topic in Frontiers in Neurology (Zeiler et al., 2017, II submitted). Overall, there are 9 studies describing the analysis of cytokines via MD in 246 aneurysmal SAH patients. Another 20 studies reported the analysis of CSF cytokines in 630 patients. As with the TBI cytokine literature, the MD and CSF studies identified within the review originated from a small number of centers of excellence in NCCU and SAH based research. This body of literature is smaller than that identified within the TBI population. Thus, the conclusions that can be made within the SAH systematic review are somewhat limited. However, as with the TBI literature, some interesting trends should be highlighted. Again, MD catheter location plays a role in the cytokine levels obtained in SAH patients, with lesioned tissue (i.e., ICH, ischemic brain) and peri-lesional tissue displaying higher cytokine levels than the “healthy” non-lesioned tissue. Second, SAH patients displayed peaks in MD IL-1β, IL-6, and IL-8 within the first 12 h’ post-hemorrhage, while (as in TBI) IL-10 appears to remain elevated within the acute period. Third, CSF-based cytokines in SAH appear to correlate with patient outcome, but as with the TBI data, our interpretation is that this type of solitary comparative analysis for each given cytokine may be misleading as there will be a potent collinearity between all these mediators. The association between cytokine profiles and the incidence of clinical vasospasm post-SAH has been documented in a previous systematic review and meta-analysis, with IL-6 and TNF linked to the development of vasospasm and delayed ischemic neurological deficit (DIND) (76). In addition, chronic hydrocephalus and shunt dependency may be associated with TGF-β levels within the CSF for SAH patients.

Positron emission tomography relies on radioligands, which bind to a specific receptor or mimic a biological molecule of interest, and can be used in in vivo studies of neuroinflammation (80). Translocator protein (TSPO) is a 18 kDa mitochondrial membrane protein involved in steroid biosynthesis and is by far the most studied target to quantify microglial activation following TBI (81, 82). The first-generation TSPO ligand, [¹¹C]-PK11195 is the most commonly employed; however, there are now several second-generation agents with improved signal to noise ratio and reduced non-specific binding. The second-generation agents all demonstrate variable binding between individuals relating to a specific genetic polymorphism resulting in different TSPO affinity for the radiolabeled ligands (83). PET studies have revealed that the binding of TSPO ligand PK-11195 remains elevated chronically, many years following TBI (81, 82), and that the degree of binding, and therefore chronic microglial activation, correlates with the degree of cognitive impairment (81). The second-generation TSPO ligand [¹¹C]DPA-713 has been used to demonstrate cellular neuroinflammation in retired American football players (84, 85).

Translocator protein ligands are limited by their overexpression in reactive astrocytes, complicating the interpretation of the resulting binding (86, 87). Moreover, expense and the ability to generate radioligands on-site in the acute phase make the logistics of scanning complex. Nevertheless, currently, TSPO PET remains the only method for human in vivo imaging of microglial activation.

Other putative PET ligands to assess neuroinflammation include cannabinoid-2-receptor (88), cyclo-oxygenase (COX) (89), and matrix metalloproteinases (90) but these have not been adequately studied in acute brain injury. Furthermore, it is also possible that (¹⁸F)-FDG-PET may be applicable to imaging neuroinflammation, because of the apparent linkage between metabolic programing and inflammation (see below).

The clinically used MRI sequences may be used to explore specific features of neuroinflammation (91), most commonly by assessing BBB integrity through the addition of the contrast agent gadolinium (92). In preclinical experimental settings, there are more advanced MRI applications, employing microparticles iron oxide, endothelial vascular cell adhesion molecule-1 (VCAM-1) (93), and macrophage-specific epitopes (94). However, in clinical studies in humans, MRI has not been employed to directly assess neuroinflammation, rather indirectly provide a measure of BBB dysfunction and endothelial cell activation.

The prevalent form of in vivo spectroscopy is ¹H magnetic resonance spectroscopy (¹H-MRS, also called proton MRS). This is employed to assess relative levels of cerebral metabolites. MRS is also known as NMR spectroscopy, but usually the term MRS refers to in vivo measurements and NMR to ex vivo or in vitro measurements. The most abundant signal in the brain ¹H spectrum is usually N-acetylaspartate (NAA). The peptide N-acetylaspartylglutamate (NAAG) is a product of NAA and the two are interconvertible. NAAG is a small peak in the brain ¹H-MRS that is difficult to distinguish from NAA. Often both are considered together as “total NAA” that is interpreted as a marker for neuronal health, viability, and/or number of neurons, particularly their mitochondria. Reduction in NAA is regarded as indicating dysfunction (permanent or temporary) of neuronal tissue. Other ¹H signals include creatine (combined signal from creatine and phosphocreatine), glutamate and glutamine (often considered together as Glx as the two species’ signals are incompletely resolved), gamma-aminobutyric acid, and lactate. ¹H MRS also allows detection of elevated levels of myo-inositol (osmolyte present in glial cells) and choline containing compounds. Commonly, MRS is used to measure metabolic activity in TBI (95), but these substances have also been shown to act as a surrogate of neuroinflammation (91, 96).

Another form of MRS less commonly used is ¹³C-MRS, which necessitates administration (usually intravenously, or sometimes orally) of substrates such as glucose, acetate, lactate etc. that have been manufactured to be artificially enriched in ¹³C (e.g., 99%), in contrast to the natural abundance of ¹³C that is only 1.1% of all carbons. ¹³C-MRS methodology is a powerful means of quantifying metabolic fluxes in vivo, e.g., the tricarboxylic acid cycle and glutamate–glutamine cycling (97). Also, in recent years, ¹³C hyperpolarization has evolved, a technique that boosts the ¹³C-MRS signal albeit for a very brief duration. Clinically, it utilizes hyperpolarized 1-¹³C pyruvate, given intravenously, to measure glycolysis vs TCA cycle, by means of signals for the respective products 1-¹³C lactate and ¹³C HCO₃⁻ (98). To date, it has mostly been used to image tumors (99), although results have emerged recently on ¹³C hyperpolarization in preclinical TBI (100, 101). ¹³C-MRS modalities may be useful in future in studies of neuroinflammation. A recent development (preclinically) is the use of hyperpolarized [6-¹³C] arginine to detect inflammatory cell function in cancer (102), and this could presumably be used in brain injury. A final MRS modality is ³¹P-MRS. No artificial enrichment is needed as ³¹P is virtually 100% of all naturally occurring phosphorus atoms. ³¹P-MRS is not commonly used, despite being able to measure energy-related phosphorus species non-invasively in vivo, including phosphocreatine, ATP, and inorganic phosphate. Notably, in a ³¹P-MRS study of TBI patients, Garnett et al. (103) suggested that the changes (vs healthy controls) might be due to reactive gliosis in the injured brain. The ³¹P-MRS modality is being actively investigated in acute brain injury.

The cellular components of the adaptive immune system can be measured in blood by identifying specific subpopulations of peripheral lymphocytes using flow cytometry. In severe TBI, this technology has revealed that the number of T-cells, specifically CD8⁺ cytotoxic cells, decrease significantly following TBI, while the number of B-cells remain constant (105).

The humoral adaptive immune system is also activated following TBI as a result of presentation of previously novel neural antigens to the peripheral immune system. Acute brain injury triggers the production of autoantibodies toward brain-specific proteins, including the brain-enriched proteins glial fibrillary acidic protein (106) and S100B (107). Moreover, Cox and coworkers showed that peripheral blood mononuclear cells isolated from patients with TBI have the capacity to proliferate in vitro when stimulated by myelin basic protein, the most abundant protein of the myelin sheath expressed in the brain by oligodendrocytes (108).

These adaptive responses are long lasting and are a candidate mechanism in the later stages of the pathophysiology of brain injury that may underlie chronic neurodegeneration (109); however, the mechanism by which empirically identified autoantibodies inflict neuronal injury is yet to be proven.