The Role of Glutamate in Pathogenesis of Brain Edema in Intracerebral Hemorrhage

Abstract

This chapter is dedicated to the impressing molecule of glutamamte—both an amino acid and a major excitatory neurotransmitter in the brain. The chapter focuses scientific on review of our work in the past decade, stressing the role of glutamate excitotoxicity as significant and sensitive biomarker for quantification of the volume of brain edema in intracerebral hemorrhage, which is important in the trajectory of clinical deterioration. We explain several developed mathematical models based on multiple regression analysis for the purposes of prognostication and potential clinical implications. These mathematical models can contribute to clinical decision making and resolving the dilemma between conservative and operative treatment in patients with hemorrhagic stroke, especially in the first 4–5 days.

1. Introduction

Almost all living organisms on the planet use glutamic acid in protein biosynthesis. This incredible molecule is an α-amino acid and is a non-essential nutrient for humans due to the ability of our body to synthesize enormous quantities of glutamic acid needed for building protein blocks—the main building blocks of our cells. The molecular formula of glutamic acid is C₅H₉NO (symbol Glu or E), and its systematic IUPAC name is 2-aminopentanedioic acid. Nevertheless, glutamic acid is more known for its anionic form—glutamate (⁻OOC − CH(NH₃⁺) − (CH₂)₂ − COO⁻), which naturally occurs in the body under physiological pH values.

Glutamate is the major constituent of a wide variety of proteins and is considered as one of the most abundant amino acids in the human body [1]. Nevertheless, besides its involvement in protein synthesis, glutamate plays a crucial role as the main “master” excitatory neurotransmitter in both the central and peripheral nervous systems [2]. Glutamatergic transmission is the major excitatory transmission, accounting for over 90% of the synaptic connections in the human brain, and its pathways highly interconnect with numerous other neurotransmitter pathways [3]. Glutamate is produced within the central nervous system through the conversion of glutamine in the glutamate-glutamine cycle, facilitated by the enzyme glutaminase [4]. This conversion takes place either within the presynaptic neuron or in nearby glial cells. Glutamate receptors are also distributed extensively broadly across neurons and glial cells throughout the brain and spinal cord. Moreover, glutamate also serves as the precursor for the synthesis of gamma-aminobutyric acid (GABA), the chief inhibitory neurotransmitter in the brain, catalyzed by the enzyme L-glutamic acid decarboxylase [5] in the GABAergic neurons.

Glutamate influences biological processes by attaching to and stimulating receptors on the post-synaptic cell surface. Mammals possess four categorized families of these receptors [6]: amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AMPA receptors (GluA1–GluA4), kainate receptors (GluK1–GluK5), N-methyl-D-aspartate NMDA receptors (GluN1, GluN2A–GluN2D, GluN3A, and GluN3B), and metabotropic glutamate receptors. The first three families are ionotropic glutamate receptors (ligand-gated ion channels), that is, integral membrane proteins composed of four large subunits that form a central ion channel pore [6]; glutamate binding activates these channels by opening them, enabling ion passage, and thus stimulating fast excitatory neurotransmission. In contrast, the metabotropic family consists of G protein-coupled receptors (mGluR), which exert their effects through second messengers such as diacylglycerol and cAMP [1].

Given its dual role as an amino acid and neurotransmitter, glutamate serves a diverse range of vital physiological functions. Studies strongly suggest that glutamate is critical for sustaining optimal energy levels essential for numerous CNS functions, especially neuroplasticity, which is vital for adapting to environmental changes [2]. Consequently, disruptions in glutamate function can have significant repercussions in both disease and injury contexts.

2. The role of glutamate in pathological conditions: a focus on intracerebral hemorrhage (ICH)

Many studies point out also to the crucial roles of glutamate in several pathological conditions. The harmful effect of glutamate on the CNS was first observed in 1954 by Dr. Takashi Hayashi (Keio University School of Medicine, Tokyo), a Japanese scientist who detected the occurrence of motor deprivation as a result of the direct effect of glutamate on the CNS. This report went unnoticed for several years until 1957, when the toxicity of glutamate was highlighted again by D. R. Lucas and J. P. Newhouse. They proved this by applying a subcutaneous injection of monosodium glutamate in newborn mice, and subsequently determined the destruction of neurons in the inner layers of the retina [7].

Later, in 1969, John Olney discovered that the damage was not limited to the retina, but also to other parts of the CNS, and he introduced the term excitotoxicity. He also postulated that cell death is limited to postsynaptic neurons that have receptors for activation by glutamate agonists and that damage can be prevented by blocking these agonists [8].

Excitotoxicity is a pathological process through which nerve cells are damaged and destroyed due to excessive stimulation by foreign neurotransmitters, such as glutamate and similar substances. Pathophysiologically, excessive activation of glutamate receptors in the CNS (NMDA and AMPA receptors) occurs as a result of increased glutamate levels, and consequently activation of the mechanism of excessive uptake of calcium ions (Ca²⁺) in the cell [9]. The latter, in turn, activates several intracellular enzymes, such as phospholipase (PLC), endonuclease, protease, which secondarily damages the cellular structure (components of the cytoskeleton, cellular membrane, and DNA).

Many studies stress the role of glutamate as an excitatory neurotransmitter in the pathogenesis of cerebral ischemia [10]. Confirmation for this association are many experimental studies in which glutamate antagonist drugs were applied, where a reduction in the volume of the infarcted region occurs. The usefulness of the glutamate antagonist is based on the hypothesis that excitotoxicity persists at least for several hours after the occurrence of a stroke [11].

Increased levels of glutamate and consequent excitotoxic effect on the CNS, except in ischemic cerebrovascular insults, have also been detected in other pathological conditions, such as traumatic brain and spinal cord injuries, hearing loss due to exposure to excessive noise or ototoxicity, and neurodegenerative diseases of CNS (multiple sclerosis, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Huntington’s and Parkinson’s disease) [12, 13]. Other conditions that were shown to cause increased levels of CNS glutamate are alcoholism, hypoglycemia, and status epilepticus [14, 15].

It is particularly important to emphasize that, unlike cerebral ischemia and traumatic brain injury where glutamate-induced excitotoxicity role in triggering cell death, its contribution to the development of brain injury was well documented, and its role in intracerebral hemorrhage (ICH) was not clearly defined in patients before 2015 [16]. The first findings of elevated extracellular glutamate levels after ICH were obtained using in vivo brain microdialysis techniques in experimental animals with induced ICH. Namely, a fourfold increase in glutamate was detected ipsilateral to the hematoma at 30 min after ICH, and these levels also remained elevated for 5 h [17]. Similarly, only a few studies have examined the role of blocking glutamate accumulation on brain damage after ICH. Mendelow [18] suggested that the NMDA receptor antagonist D-CPP reduces edema in rats. Also, in animal models of ICH with collagenase, it was shown that the non-competitive antagonist of NMDA receptors —memantine, causes a decrease in hematoma expansion, a decrease in cell death, and a decrease in infiltration of immune cells [19]. These results indicate that excitotoxicity may be an important mechanism for cell death after ICH. Later, Sharp et al. [20], using a genomic approach, identified a 20-fold increase in the expression of a member of the Src family—Lyn in the brain after ICH, which deals with the regulation of NMDA receptors through phosphorylation. Based on this, they suggested that ICH induces thrombin production after hemorrhage, which results in Src activation, which contributes to NMDA receptor phosphorylation, leading to neuronal damage. Nevertheless, although some authors suggested that certain mechanisms through which glutamate contributes to ICH-induced brain damage were specific [21], and differed from those in ischemic damage, and there were no data in human patients with ICH. It seemed that glutamate-triggered excitotoxicity contributes to secondary brain damage in ICH, but the interaction with the other mechanisms of brain damage, especially inflammatory processes, was not well investigated.

There is considerable evidence to support the theory of TNF-α involvement in the development of perifocal edema. In the brain, TNF-α is synthesized by microglia and astrocytes, while TNF-α receptors are found on glial cells and neurons. Although TNF-α has neuromodulatory abilities in the healthy brain, its function in the post-ICH brain is clearly neurotoxic and detrimental [22]. One of the factors leading to an increase in the brain level of TNF-α after ICH is the production of thrombin during hematoma formation [23, 24], and thrombin directly stimulates the production of TNF-α. Elevation of TNF-α levels causes activation of microglial cells and astrocytes after the injury, regulation of blood-brain barrier permeability, glutaminergic transmission, and synaptic plasticity [22]. After brain injury in experimental animals, elevated levels of TNF-α were detected in the tissue adjacent to the injury site, which contributes to the development of edema through the increased permeability of the blood-brain barrier. In addition, TNF-α increases excitatory synaptic transmission via elevated AMPA receptor expression and reduces inhibitory transmission, thus causing excitotoxicity [25]. In summary, there is rational preclinical evidence that increased TNF-α concentrations have detrimental effects on the brain after ICH, involving glutamate excitotoxicity. Based on the proposed model, TNF-α causes additional release of glutamate from synaptosomes, as well as inhibition of glutamate reuptake from the synaptic cleft back to the presynaptic neuron, causing excitotoxicity. Therefore, there is a possibility of an interaction effect between excitotoxicity and inflammatory mechanisms in the development of brain secondary injury, but before 2018, there were no studies examining this interaction effect (whether it is additive, synergistic, antagonistic, etc.).

Studies on peripheral glutamate levels in patients with ICH are very rare. In this context, for example, Castillo et al. showed significant differences in glutamate levels between patients with good and poor neurological outcomes [26], but this study did not include a control group for comparison of their values.

Tanphaichitr et al. [27] reported reference values of peripheral glutamate levels of 22.4 ± 3.2 nmol/mL glutamate in the blood plasma of healthy individuals, while according to Tsai and Huang [28], the values were estimated as 33.2 ± 15, 4 μmol/L. In the same study, the authors point out that the concentration of blood plasma glutamate is one of the lowest compared to other amino acids, but also one of the most constant, that is, with the least degree of variability in terms of the diet [28].

The study by Rendevski et al. [29] of our research group aimed to evaluate the role of peripheral plasma glutamate and TNF-α levels as biomarkers for ICH. Furthermore, to examine the prognostic role and possible interactions of these variables in the development of edema volume 5 days after ICH. The study was based on the hypothesis that since an increased variability of the blood-brain barrier (BBB) is a typical result after ICH [30], the excitotoxic and pro-inflammatory mediators can transfer from the brain in the blood and be detected peripherally. In this study, significantly higher blood plasma glutamate and TNF-a levels were detected in ICH patients at admission, when compared to healthy controls, which stresses the importance of these mediators in the pathology of the ICH. The glutamate values were more than threefold increase in patients (median value of 107.75 μmol/L) when compared to the median of the healthy population analyzed in the study (31.13 μmol/L)—a value very similar to those reported by Tsai and Huang [28]. Since the difference in glutamate levels between healthy individuals and patients with ICH was striking, it was concluded that glutamate is an important molecular marker of excitotoxicity, the concentration of which increases significantly immediately after the onset of intracerebral hemorrhage. In North Macedonia, no study results of reference values of glutamate in human plasma have been published so far; namely, these were also the first results for reference values for glutamate in the healthy human population.

The study also showed that the anatomic localization of ICH (lobar/deep; left/right hemisphere) did not influence the influx of glutamate and TNF-α and equally induced excitotoxicity and inflammation, regardless of ICH position. Glutamate levels within the patient group did not differ significantly between males and females, or between the different age groups. Moreover, it was demonstrated that the symptom severity and the initial volume of ICH were the major drivers for the variability of the glutamate levels in patients with ICH.

Peripheral glutamate levels were shown as significant predictors for the formation of brain perifocal edema 5 days after ICH [29]. Namely, it was demonstrated that glutamate and TNF-α independently contribute (without any effect of interaction) to the development of the edema, regardless of the localization of the ICH. This was a very important discovery since the worst deterioration in ICH patients occurs due to the formation of brain perifocal edema, a well-proven factor of mortality and poor outcomes after ICH. Hence, aiming at the prevention of formation of large edema volumes in patients after ICH, the ability to predict its formation is crucial benefit.

The later study by Rendevski et al. [31] has also separated glutamate as an independent and significant predictor of development of the brain perifocal edema. This study focused on advanced 3D modeling for prediction and quantification of the perihematomal brain edema formation after ICH. Several 3D models and interactive plots were constructed, which could accurately predict 77% of the variability in the volumes of the edema within patients of ICH. The model was focused on glutamate and TNF-α, suggesting the primary role of excitotoxic and inflammatory mechanisms in the secondary brain damage and the pathogenesis of the edema. The model was also characterized by very high significance (one-way ANOVA resume: F = 33.7592, *p = 7.4 ∙10⁻¹⁹), and with good overall characteristics and fit.

The proposed equation for mathematical quantification of the edema was given as:

$$\begin{array}{l}{V}_{\text{edema}}=0.3292·c_{TNF-\alpha }+0.2484·c_{\text{glutamate}}+0.3162·V_{ICH}\\ +1.6299·CSS+0.9283·c_{\text{glucose}}–49.4949+a\end{array}$$E1

where a—summarizes the effects of anatomic localization and the presence of diabetes (Figure 1).

Figure 1.

3D model for prediction and quantification of the brain edema volume after ICH, based on glutamate and TNF-α values. The model summarizes the differences in prognostication between deep and lobar ICH, within ICH diabetic patients.

3. Conclusions

The necessity for urgent care of patients presenting in the emergency department with intracerebral hemorrhage is undeniable. The worst neurological deterioration in these parents was associated with the formation of perifocal brain edema, a proven predictor for poor outcome, and peripheral glutamate levels resulting from excitotoxicity-induced ICH were shown as a promising marker for edema prediction. The constructed glutamate-based models and the developed interactive plots for prediction of the formation of the brain edema could be beneficial for clinical decision-making between conservative treatment and surgical intervention, especially in the group of threatened ICH patients where high volumes of the edema are expected to occur during the parent’s hospitalization trajectory. In summary, since glutamate-mediated excitotoxicity was one of the proven mechanisms operating during ICH, monitoring of the peripheral glutamate plasma levels, originating from the brain via the disrupted blood-brain barrier, can tell a lot about the severity of the insult and its possible progression, mainly in the “evolution” of edema development.