Fundamental Neurochemistry Review: Microglial immunometabolism in traumatic brain injury

Traumatic brain injury (TBI) is a devastating neurological disorder caused by a physical impact to the brain that promotes diffuse damage and chronic neurodegeneration. Key mechanisms believed to support secondary brain injury include mitochondrial dysfunction and chronic neuroinflammation. Microglia and brain‐infiltrating macrophages are responsible for neuroinflammatory cytokine and reactive oxygen species (ROS) production after TBI. Their production is associated with loss of homeostatic microglial functions such as immunosurveillance, phagocytosis, and immune resolution. Beyond providing energy support, mitochondrial metabolic pathways reprogram the pro‐ and anti‐inflammatory machinery in immune cells, providing a critical immunometabolic axis capable of regulating immunologic response to noxious stimuli. In the brain, the capacity to adapt to different environmental stimuli derives, in part, from microglia's ability to recognize and respond to changes in extracellular and intracellular metabolite levels. This capacity is met by an equally plastic metabolism, capable of altering immune function. Microglial pro‐inflammatory activation is associated with decreased mitochondrial respiration, whereas anti‐inflammatory microglial polarization is supported by increased oxidative metabolism. These metabolic adaptations contribute to neuroimmune responses, placing mitochondria as a central regulator of post‐traumatic neuroinflammation. Although it is established that profound neurometabolic changes occur following TBI, key questions related to metabolic shifts in microglia remain unresolved. These include (a) the nature of microglial mitochondrial dysfunction after TBI, (b) the hierarchical positions of different metabolic pathways such as glycolysis, pentose phosphate pathway, glutaminolysis, and lipid oxidation during secondary injury and recovery, and (c) how immunometabolism alters microglial phenotypes, culminating in chronic non‐resolving neuroinflammation. In this basic neurochemistry review article, we describe the contributions of immunometabolism to TBI, detail primary evidence of mitochondrial dysfunction and metabolic impairments in microglia and macrophages, discuss how major metabolic pathways contribute to post‐traumatic neuroinflammation, and set out future directions toward advancing immunometabolic phenotyping in TBI.


| INTRODUC TI ON
The brain is a highly demanding metabolic organ.Despite accounting for only 2% of total body weight, the brain is responsible for >30% of whole-body glucose consumption (Erbsloh et al., 1958;Mergenthaler et al., 2013).Much of the energy demand in neurons is provided by the sequential catabolism of glucose through glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation, enzymatically catalyzed processes that generate up to 36 adenosine triphosphates (ATPs) per glucose molecule, most of which are produced in the mitochondria.Energy demands in the brain can also be sustained by the oxidation of amino acids, ketone bodies, and lipids (Ebert et al., 2003;Jiang et al., 2011;McKenna, 2012;Pan et al., 2002).Importantly, cellular metabolism is not static, but it can adapt to substrate availability or in response to different environmental stimuli.This allows energy substrates to be directed to less "energy efficient" pathways to generate substrates required for specific cell functions, such as glucose shunt to the pentose phosphate pathway (PPP) to make NADPH.Metabolic flexibility in neurons and glial cells is critical for synaptic function, providing a reliable supply of energy substrates during neurotransmission, or allowing for neurotransmitter reuptake (Pellerin & Magistretti, 1994;Zimmer et al., 2017).
As the resident macrophages of the central nervous system (CNS), microglia perform critical functions from pathogen surveillance and tissue repair to synapse pruning and learning-associated neuroplasticity (Jiang et al., 2021;Paolicelli et al., 2022).These diverse and energy-demanding functions require the metabolism of glucose, amino acids, lipids, and ketone bodies, and their intermediate metabolites serve as biosynthetic precursors for reactions that regulate transitions between inflammatory states (Ferreira et al., 2022;Hirschberger et al., 2021;Sumbria et al., 2021;Zhou et al., 2022).The rates at which substrates are used and which pathways are employed for their catabolism are linked to microglial phenotypic states (Button et al., 2014;Guillot-Sestier et al., 2021;McManus et al., 2022).Immunometabolism constitutes the changes in metabolic pathways in cells during immune activation (O'Neill et al., 2016).In fact, impairments in immunometabolic responses can promote detrimental pro-inflammatory microglial phenotypes associated with neurodegeneration (Castillo et al., 2021;Cheng et al., 2021;Yuan et al., 2013).Research into microglial metabolism and current knowledge has mainly relied on transcriptomic data or in vitro cell line studies.However, emerging evidence from in vivo animal models and multi-dimensional proteomic analyses indicate that microglial immunometabolic transitions may be involved in acute neural injury and chronic neurodegenerative disease (Baik et al., 2019;Guillot-Sestier et al., 2021;Xiang et al., 2021).
Traumatic brain injury (TBI) is a leading cause of mortality and disability worldwide accounting for up to 30% of all injury-related deaths (Maas et al., 2017).TBI is caused by a mechanical insult, such as a fall or motor vehicle crash, which impacts and/or rapidly accelerates the head producing focal contusions, hematomas, and/or shear deformation of white matter tracts (Loane & Faden, 2010).TBI can result in the development of complex neurological deficits as a result of both primary and secondary injury mechanisms.Primary injury events encompass the mechanical damage that occurs at the time of trauma to neurons, axons, glia, and blood vessels as a result of shearing, tearing, or stretching (Saatman et al., 2008).In addition, secondary injury evolves over minutes to days and even months after the initial traumatic insult and results from delayed biochemical, metabolic, and cellular changes that are initiated by the primary event.These secondary injury cascades are thought to account for the development of many of the neurological deficits observed after TBI (Loane & Faden, 2010).Secondary injury mechanisms include a wide variety of processes such as depolarization, disturbances of ionic homeostasis, release of neurotransmitters (such as excitatory regulator of post-traumatic neuroinflammation.Although it is established that profound neurometabolic changes occur following TBI, key questions related to metabolic shifts in microglia remain unresolved.These include (a) the nature of microglial mitochondrial dysfunction after TBI, (b) the hierarchical positions of different metabolic pathways such as glycolysis, pentose phosphate pathway, glutaminolysis, and lipid oxidation during secondary injury and recovery, and (c) how immunometabolism alters microglial phenotypes, culminating in chronic non-resolving neuroinflammation.In this basic neurochemistry review article, we describe the contributions of immunometabolism to TBI, detail primary evidence of mitochondrial dysfunction and metabolic impairments in microglia and macrophages, discuss how major metabolic pathways contribute to post-traumatic neuroinflammation, and set out future directions toward advancing immunometabolic phenotyping in TBI.

K E Y W O R D S
metabolism, microglia, mitochondria, neuroimmunology, traumatic brain injury amino acids), lipid degradation, mitochondrial dysfunction, and initiation of inflammatory and immune processes, among others.Subsequent biochemical events generate large amounts of toxic and pro-inflammatory molecules such as nitric oxide, prostaglandins, reactive oxygen and nitrogen species, and pro-inflammatory cytokines, which lead to lipid peroxidation, blood-brain barrier (BBB) disruption, and the development of edema (Loane & Faden, 2010).The associated increase in intracranial pressure (ICP) can contribute to local hypoxia and ischemia, secondary hemorrhage and herniation, and additional neuronal cell death via necrosis or apoptosis.These secondary injury mechanisms are chronic and persistent, and have been shown to exist diffusely across brain regions, including brain regions remote to the primary injury site (Giordano et al., 2022;Singh et al., 2006).
In the immediate aftermath of a TBI, there is a major crisis in energy metabolism (Gajavelli et al., 2015;Gilmer et al., 2010;Hermanides et al., 2021;Hill et al., 2017;O'Connell et al., 2005;Pan et al., 2002;Singh et al., 2006).TBI-induced metabolic alterations include increased glycolytic flux, reduced oxidative phosphorylation, and increased glutaminolysis; all of which are associated with injury severity, mortality risk, and development of cognitive impairments in severely injured patients (Kilbaugh et al., 2015;Singh et al., 2006;Stefani et al., 2017).Disruption of metabolic pathways mainly occurs in mitochondria, consistent with the idea that mitochondrial dysfunction plays a central role in secondary injury after TBI.Inefficient mitochondrial calcium handling, excessive ROS production, and reduced energy supply to sustain synaptic activity contribute to neuronal cell death following TBI (Carteri et al., 2019;Lyons et al., 2018;Singh et al., 2006).Changes in the mitochondrial electron transport system (ETS) have been described in TBI patients and pre-clinical models (Carteri et al., 2019;Kilbaugh et al., 2015;Lazzarino et al., 2019;Mkrtchyan et al., 2018;Stovell et al., 2018), indicating that trauma shifts energetic substrate preference in the injured brain.The deleterious consequences of TBI-induced metabolic impairments are reinforced by pre-clinical studies where mitochondriatargeted therapies produce beneficial biochemical, neurobehavioral, and neuroprotective outcomes during the acute and chronic phases of recovery (Carteri et al., 2019;Hubbard et al., 2021;Khellaf et al., 2022;Millet et al., 2016;Pandya et al., 2007;Vekaria et al., 2020;Yonutas et al., 2020;Zhao et al., 2021).
There is limited information on the cell-type specificity of mitochondrial dysfunction following TBI.Studies using whole-brain homogenates, isolated mitochondria, and mitochondria-containing dissociated pre-synaptic terminals ("synaptosomes") identified severe mitochondrial impairments after TBI (Carteri et al., 2019;Kilbaugh et al., 2015;Lyons et al., 2018;Singh et al., 2006), which are predicted to disrupt critical neuronal and astrocyte signaling and other cell-cell interactions, including those regulated by microglia.Because the environmental milieu may be a driver of immune cell polarization (Loane & Kumar, 2016;Simon et al., 2017), altered metabolism may sustain pro-inflammatory microglial activation during secondary injury.Several key enzymes that are chronically impaired in microglia following TBI rely on intermediates of energy metabolism pathways, such as the TCA cycle, glycolysis, and glutaminolysis.These pathways feed mechanisms associated with microglial pro-inflammatory activation, including ROS production (Bordt & Polster, 2014), reinforcing the important role that metabolic remodeling could play in supporting chronic microglial activation and neuroinflammation.
Here, we review emerging literature linking altered metabolic function in microglia with secondary injury responses following TBI.We also discuss limitations in experimental approaches used to investigate microglial metabolism and review cutting-edge technologies that may identify new mechanisms of immunometabolic regulation of microglial phenotypes after TBI.

| G LUCOS E UP TAK E AND CELLUL AR ME TABOLIS M IN MI CROG LIA
Glucose is the major energy substrate for brain cells and glycolysis is the initial pathway through which it is oxidized (Mergenthaler et al., 2013).Glycolysis consists of 10 stepwise enzymatic reactions that convert one molecule of glucose into two molecules of pyruvate to store biochemical energy in the form of two ATP molecules.Other products of glycolysis include cytosolic lactate and NADH.Key enzymes within this pathway, hexokinase (HK), phosphofructokinase-1 (PFK-1), pyruvate kinase (PK), and pyruvate dehydrogenase (PDH), regulate the rate of carbohydrate used to supply both energy and substrates for other critical cellular functions.Adaptations in glycolysis are linked with immune cell function and suggest increased glycolytic metabolism is a key modulator of pro-inflammatory immune response (Dionisio et al., 2023;Loftus & Finlay, 2016).
Substrate uptake is the initial step in cellular energy metabolism and is mediated by membrane-bound transporters.For glucose, the glucose transporter protein (GLUT) superfamily is the main gateway to cell metabolism, while α-ketoacids such as lactate and pyruvate are shuttled through monocarboxylate transporters (MCT).Most glucose uptake in immune cells, including microglia, is provided by insulin-independent GLUT-1, GLUT-5, and GLUT-6 (Freemerman et al., 2014;Maedera et al., 2019;Payne et al., 1997;Wang et al., 2019).Following uptake, glucose is committed to cellular metabolism upon phosphorylation to glucose-6-phosphate (G6P) by HK (Sokoloff et al., 1977).G6P is at the crossroads of carbohydrate metabolism, feeding glycolysis/gluconeogenesis, the PPP, and glycogen metabolism.Thus, HK activity is a critical regulator of these three important metabolic pathways.
Furthermore, there is increased PKM2 nuclear translocation following LPS stimulation, such that inhibition of PKM2 nuclear transport attenuates pro-inflammatory microglial activation by disrupting the interaction between PKM2 and activated transcription factor 2 (ATF2) (Li et al., 2021).

| G LYCOLYS IS AND S ECONDARY NEURO INF L AM MAT I ON FOLLOWING TB I
[18]F-fluorodeoxyglucose (FDG) positron emission tomography (PET) neuroimaging in humans and animal models has identified significant TBI-induced changes in brain glucose metabolism (Brabazon et al., 2017;Byrnes et al., 2014;Jaiswal et al., 2019;Okonkwo et al., 2019;Yasmin et al., 2022).In pre-clinical models, TBI produces divergent effects in FDG uptake according to injury severity and time post-injury.Diffuse mild TBI in mice results in increased FDG uptake through 5 days post-injury, whereas there is an acute reduction in FDG uptake following severe contusion TBI (Israel et al., 2016).Also, reduced glucose uptake acutely after severe TBI increases N,Ndiethyl-2-[4-(2-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo [1,5-a] pyrimidine-3-acetamide ([18F]DPA) binding, an established PET marker of neuroinflammation (Israel et al., 2016).Other pre-clinical studies demonstrate post-traumatic glucose hypometabolism is associated with increased microglial reactivity in the corpus callosum acutely after moderate TBI in rats (Brabazon et al., 2017).This association is transitory because during the chronic stages of recovery, there is increased glucose uptake correlated with microglial activation in the injured cortex and hippocampus (Brabazon et al., 2017).In a co-culture model of microglia and needle-scratched injured neurons, there are increased glycolysis-derived levels of 3-phosphoglycerate, phosphoenolpyruvate, pyruvate, and lactate (Liu et al., 2023), which results in increased NLRP3 inflammasome activation and IL-1β production.Inhibition of glycolysis in this co-culture system using the PFKB3 inhibitor, 3PO, results in reduced glycolysis intermediary levels and reduced expression of pro-inflammatory mediators (Liu et al., 2023) (Figure 1).

| PENTOS E PHOS PHATE PATHWAY (PPP) ME TABOLIS M AND MI CROG LIA
Following phosphorylation by HK, G6P may also be diverted from the glycolytic pathway to the PPP.This pathway produces reduced coenzymes that fuel energy-dependent processes such as lipid synthesis and ROS production during oxidative burst.The PPP consists of two distinct sequences of reactions.The first one, known as the oxidative phase, promotes the conversion of G6P into ribose-5-phosphate, and is the major contributor to the intracellular pool of NADPH (Curi et al., 2020).Under certain conditions, a second, non-oxidative phase takes place to allow ribose-5-phosphate to be redirected toward the glycolytic pathway in the form of fructose-5-phosphate and glyceraldehyde-3-phosphate.The contribution of PPP during oxidative burst in immune cells is attributed to the enzymatic activity of NADPH oxidase 2 (NOX2), which catalyzes the oxidation of NADPH to produce the superoxide anion (O 2 − ).NOX2 is highly enriched in phagocytes, including brain-resident microglia, where it contributes to microglial pro-inflammatory polarization (Choi et al., 2012), particularly in the context of TBI (Kumar, Barrett, et al., 2016;Wang et al., 2017).Because of the stoichiometry of NADPH, increased NOX2 activation must be accompanied by NADPH turnover in the cytosol.Key non-oxidative PPP enzymes are necessary for oxidative burst, indicating a rate-limiting role for the PPP in immune cell ROS production (Lajqi et al., 2021).In fact, knockout of transketolase, an indispensable enzyme from the nonoxidative phase of PPP, leads to a fatal autoimmune disease in mice, potentially linked to reduced glycolysis and increased oxidative phosphorylation (Liu et al., 2022).Transketolase content is also reduced in Treg cells in autoimmune disease patients, suggesting a role for non-oxidative PPP in regulating immune function (Liu et al., 2022).
Furthermore, neutrophil non-oxidative PPP is remodeled to adopt a cyclic metabolism that recycles G6P from PPP products (mainly fructose-6-phosphate) to increase NADPH yield per glucose molecule at the cost of reduced ATP production (Britt et al., 2022).This suggests that in immune cells, increased glucose uptake can be used to match NADPH demand through the PPP to fuel pro-inflammatory responses (Figure 1).
The mechanisms linking glucose uptake, the PPP, and proinflammatory immune activation in microglia are beginning to be explored.In LPS + IFNγ-stimulated microglia, iNOS and nitric oxide (NO) are attenuated under low-glucose conditions (Castillo et al., 2021).NO levels are preserved when NADPH, or malate, a precursor to NADPH, is reintroduced into the culture system (Castillo et al., 2021), which suggests that increased glucose uptake in pro-inflammatory microglial activation may be primarily driven by PPP-mediated NADPH production, and not energy supply via glycolysis (Castillo et al., 2021).
In PD models induced by either LPS or 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine (MPTP) injections in mice, the rate-limiting enzyme of PPP, glucose-6-phosphate dehydrogenase (G6PD) is upregulated (Tu et al., 2019).Specifically, G6PD expression and activity are elevated in LPS-stimulated midbrain neuron-glia cultures (i.e., an in vitro PD model) and in the substantia nigra of PD mice (Tu et al., 2019).G6PD physiologically operates far below maximum catalytic capacity as a result of NADP + levels in the cytosol being below the respective K m to G6PD (Britt et al., 2022).This provides G6PD with significant flexibility to increase NADPH production on demand.Therefore, the increase in G6PD activity in LPS-stimulated microglia may reflect a very significant change in cytosolic NADPH turnover.Interestingly, elevated G6PD activity in microglia produces excessive NADPH, providing abundant substrate to drive NOX2 activity, which leads to excessive ROS production (Tu et al., 2019).
Notably, flux through PPP and NOX2 activity is reduced when G6PD inhibitors (6-aminonicotinamide and dehydroepiandrosterone) or G6PD siRNA are administered to LPS-stimulated microglia (Tu et al., 2019), which indicates that glucose uptake is driven by NADPH-dependent NOX2 activity.There is also reduced carbohydrate kinase-like (CARKL) expression, a negative regulator of the non-oxidative branch of PPP, which promotes the interconversion of carbohydrates to increase NADPH production (Haschemi et al., 2012;Lajqi et al., 2021).This provides further evidence that PPP may be rewired during pro-inflammatory microglial activation and ROS production (Figure 2).
The rate-limiting capacity of NADPH supply and PPP activity on NOX2-dependent ROS production is established, but flux through microglial PPP following TBI has not been investigated.Therefore, important questions remain unanswered, including (1) to what extent the increased glucose uptake by microglia following TBI is serving as substrate to PPP-derived NADPH production, and is this a response to increased NOX2 activity?(2) Do chronically activated microglia possess metabolic flexibility with regard to glucose metabolism and PPP, or is flux through the PPP chronically elevated after TBI? (3) Could therapies that target NADPH production by PPP in microglia attenuate NOX2-driven chronic neuroinflammation and improve outcomes following TBI?

| MI CROG LIAL AC TIVATI ON IS SUPP ORTED BY RE WIRING THE TC A C YCLE
Following glucose entry into the glycolytic pathway, pyruvate can be further oxidized inside the mitochondria via the TCA cycle and oxidative phosphorylation.These pathways allow for the complete oxidation of glucose and an optimum yield of 32-36 moles of ATP per one mole of glucose.Pyruvate is incorporated into mitochondrial metabolism as acetyl-CoA, produced by pyruvate dehydrogenase (PDH).In the mitochondria, acetyl-CoA enters the TCA cycle, a metabolic pathway that is composed of nine enzymatic steps that promote the interconversion of tricyclic α-ketoacids.This pathway produces energy-rich coenzymes NADH and FADH 2 , which can be used to produce ATP through oxidative phosphorylation by the FoF1-ATP Synthase.Many other metabolic pathways intersect the TCA cycle, including those involved in amino acid and lipid metabolism.
Further, influx of substrates into the TCA cycle may occur through acetyl-CoA-producing enzymes such as PDH, as well as through other anaplerotic routes.TCA intermediates can be diverted to provide substrates for the synthesis of other molecules, such as amino acids, lipids, and cytokines (Hooftman et al., 2020;Jha et al., 2015;Tannahill et al., 2013).Therefore, the capacity to regulate flow direction of substrates from the TCA cycle allows for the rapid adaptation of energy supply to match the trophic demands of the cell.
In macrophages, PDH, an enzyme that feeds acetyl-CoA, and aconitase-2 (ACO2), a TCA cycle enzyme, are inhibited following LPS + IFNγ stimulation, suggesting that during pro-inflammatory activation, macrophages reduce the incorporation of pyruvate into the TCA cycle and increase their dependence on glycolysis to meet The destination of glucose across the spectrum of microglial functional phenotypes.Microglial plasticity and functional responses are accompanied by changes in metabolism.Glucose flux through either glycolysis or pentose phosphate pathway is tailored to support metabolic requirements of microglia.This metabolic flexibility allows microglial metabolism to be skewed from the full conversion of glucose into pyruvate, supporting aerobic metabolism in phagocytic/surveillant microglia, to a cyclic pentose phosphate pathway that provides increased NADPH yield in preference of pyruvate production to support NADPH oxidase activity in inflammatory microglia.In chronic TBI or aging, chronically activated microglia display features such as increased levels of NOX2 and lipid accumulation, which suggest that glucose metabolism in microglia under these conditions may be irreversibly shifted toward NADPH production.DAMPs, Damageassociated molecular patterns; DHAP, Dihydroxyacetone phosphate; F6P, Fructose-6-phosphate; G6P, Glucose-6-phosphate; GA3P, Glyceraldehyde-3-phosphate; Glu, Glucose; R5P, Ribulose-5-phosphate; ROS, Reactive oxygen species.Created with BioRe nder.com.energy demands (Jha et al., 2015;Palmieri et al., 2020).Inhibition of ACO 2 during pro-inflammatory stimulation disrupts the TCA cycle and the accumulation of intermediates, such as citrate and cis-aconitate (Palmieri et al., 2020).Inhibition or down-regulation of the expression of TCA cycle enzymes can lead to the shunting of TCA cycle intermediates into the cytosol.In immune cells, this causes the TCA cycle to be "broken," becoming fragmented into independent sequences of reactions (O'Neill, 2015).During proinflammatory stimulation, TCA cycle intermediates can be transported to the cytosol and serve as biosynthetic precursors of immunomodulatory molecules (Hooftman et al., 2020;Tannahill et al., 2013).In contrast, when macrophages are stimulated with anti-inflammatory IL-4, there is increased influx of substrates into the TCA cycle, particularly lactate (Jha et al., 2015).The rescue of oxidative metabolism by lactate reflects an increase in mitochondrial respiration that is mediated by cytosolic acetyl-CoA lyase and histone acetylation (Noe et al., 2021).Interestingly, this mechanism not only provides substrates for epigenetic modifications necessary for anti-inflammatory transcriptional changes but also provides energy support for tissue healing mechanisms (Noe et al., 2021).In microglia, transcriptional down-regulation of TCA cycle genes (e.g.,

Idh2
) occurs following LPS simulation, but this does not occur when microglia are repolarized toward an anti-inflammatory phenotype by IL-4 (Hu et al., 2020).
Microglial plasticity (i.e., the capacity to shift from cytotoxic to regenerative phenotypes or vice versa) can be mediated by TCA cycle intermediaries.Metabolites, such as itaconate, provide signaling not only to sustain responses but also to shift from one phenotype to another (Hooftman et al., 2020).Itaconate derivatives, when cultured with microglia stimulated with LPS + ATP, inhibit the NLRP3 inflammasome and IL-1β release.(Yang et al., 2021).
In vivo administration of itaconate to experimental autoimmune encephalomyelitis (EAE) mice, an experimental model of multiple sclerosis, reduces disease severity and suppresses microglial activation (Kuo et al., 2020).Other cells also alter TCA cycle metabolism during immune responses.Anaplerotic supply of fumarate by glutaminolysis is essential for trained immunity induction in monocytes (Arts et al., 2016).In human immunopathologies, extreme pro-inflammatory signaling during sepsis is associated with suppressed mitochondrial metabolism in circulating immune cells, which feed on TCA cycle substrates (Japiassu et al., 2011;Martinez-Garcia et al., 2019).Moreover, decreased mitochondrial function of mononuclear cells in critically ill patients is associated with increased mortality rates (Nedel et al., 2021).In critically ill patients, mitochondrial Complexes I and II functions were negatively correlated with increased pro-inflammatory cytokines IL-1β and IL-6 (Nedel et al., 2022).The mechanisms that support metabolic adaptations in microglia/macrophages are still under investigation.There is evidence to support TCA cycle "breakage" as a result of pro-inflammatory NO production and release (Palmieri et al., 2020); however, metabolic adaptations, including increases in lactate-transporting MCTs, are necessary for anti-inflammatory polarization (Colegio et al., 2014).As such, additional studies are needed to define metabolic adaptations more clearly in microglia/ macrophages under various pathological conditions, including TBI.

| G LUTAMINOLYS IS FUEL S ANTI -INFL AMMATORY RE S P ONS E S
Among major anaplerotic routes, amino acid metabolism also plays an important role in feeding the TCA cycle with intermediaries.Glutamine is the most abundant amino acid in the blood and CSF and can be used by cells as an energy substrate.Glutaminolysis is a result of biochemical reactions that facilitate the deamination of glutamine to glutamate and transamination of glutamate to α-ketoglutarate (αKG).This process occurs within the mitochondrial matrix and directly provides integration of glutamine to oxidative metabolism through αKG and the TCA cycle.Secondary metabolic routes also allow glutamine to generate other TCA cycle intermediaries, such as succinate (Sonnewald et al., 2006).
There is a high demand for glutamine in both macrophages and microglia.TBI-induced changes in glutamine metabolism were first described back in the 1980s (Ardawi & Newsholme, 1982;Newsholme et al., 1987;Parry-Billings et al., 1990), but mechanisms by which glutamine may regulate immune functions have only recently been discovered.Glutamine is a major fuel for anti-inflammatory immune cells, whereby glutaminolysis in bone marrow-derived macrophages (BMDM) promotes the expression of anti-inflammatory genes such as Arg1, Ym1, and Retnla, while decreasing pro-inflammatory gene expression (e.g., Il1b, Tnf, and Il6; Liu et al., 2017).There is reduced expression of genes associated with glutaminolysis in LPS-simulated BMDM, including glutaminase Gls1, glutamate transaminases, Got1, Got2, and Gpt2 (Liu et al., 2017).Genes for other TCA cycle enzymes that feed the cellular αKG pool, such as isocitrate dehydrogenase Idh2, are also decreased in LPS-stimulated BMDM (Liu et al., 2017).In the absence of glucose, glutaminolysis can step in to sustain microglial functional responses elicited by a lesion in brain slices (Bernier et al., 2020).Furthermore, glutamine metabolism in microglia is regulated by NLRP3, such that loss of NLRP3 increases glutamine/ glutamate-related metabolism, which results in enhanced mitochondrial and metabolic activity (McManus et al., 2022).
Although glutamine degradation is associated with antiinflammatory immune function, glutaminolysis may also sustain pro-inflammatory signaling by breaking the TCA cycle.During proinflammatory activation, alternative pathways such as the GABA shunt contribute to the conversion of glutamine/glutamate to succinate in the TCA cycle (Kieler et al., 2021;Tannahill et al., 2013).
Succinate accumulation is a common metabolic feature of proinflammatory macrophages, which stabilizes the transcription factor HIF1α boosting glycolytic flux by up-regulating key enzymes and transporters (Tannahill et al., 2013).This allows glutamine oxidation to stimulate glycolysis and provides a mechanism by which impaired glutamine/glutamate metabolism could drive pro-inflammatory activation of macrophages.

| MITOCHONDRIAL ELEC TRON TR AN S FER SYS TEM AND OXIDATIVE PHOS PHORYL ATI ON IN MI CROG LIA
Changes in TCA cycle flux culminate in altered oxygen consumption and ATP production through the ETS.Transcriptional regulators, such as peroxisome proliferator-activated receptorγ (PPARγ), can elevate oxygen consumption levels, in part by increasing the net flow of substrates to the ETS (Bernardo et al., 2013;Coletta et al., 2009;Ghosh et al., 2007;Wilson-Fritch et al., 2004).This is followed by an increase in anti-inflammatory gene expression (e.g., Il4, Il10, and Arg1) (Miao et al., 2022;Peng et al., 2019;Ramkalawan et al., 2012).Pioglitazone, a PPARγ agonist, robustly attenuates proinflammatory iNOS expression in microglia that typically precedes dopaminergic neuron loss in a PD model (Breidert et al., 2002).
Mitochondrial complexes respond specifically to different inflammatory signals.While reduced mitochondrial respiration following LPS + IFNγ stimulation in BV2 microglia is linked to inhibition of mitochondrial respiration, stimulation with LPS alone inhibits Complex II activity (Chausse et al., 2020).In contrast, inhibition of Complex II prior to LPS + ATP stimulation attenuates pro-inflammatory caspase 1 cleavage, NLRP3 inflammasome activation, and IL-1β release in BMDMs (Billingham et al., 2022).Furthermore, treatment of microglia with Complex I inhibitor, rotenone, can increase proinflammatory expression of iNOS, TNFα, and IL-1β (Gao et al., 2002(Gao et al., , 2013;;Yuan et al., 2013;Zhou et al., 2007Zhou et al., , 2008)).Complex I, II, III, and IV inhibitors also produce an inhibitor-type and dose-dependent increase in pro-inflammatory marker expression (e.g., TNFα, IL-1β, IL-6), with the greatest up-regulation with Complex I and III inhibitors, and reduced levels with Complex II and IV inhibitors (Ye et al., 2016).
Altogether, this suggests that ETS complexes respond selectively to inflammatory signals in microglia.
Other inflammatory mechanisms in microglia are influenced by mitochondrial metabolism.Mitochondria are a major source of ROS, but during pro-inflammatory microglial activation, phagocytic NOX2 also generates O 2 − and promotes neuroinflammation (Choi et al., 2012;Qin et al., 2004Qin et al., , 2005)).There remains controversy regarding the overlap between mitochondrial-and NOX-derived ROS in regulating immune function, including in microglia (Bordt & Polster, 2014).One known pro-inflammatory mechanism thought to be triggered by ROS is the activation of the NLRP3 inflammasome, which initiates pyroptosis, an inflammatory form of cell death (Dostert et al., 2008;Tschopp & Schroder, 2010;Xia et al., 2021;Zhou et al., 2010).The regulation of NLRP3 has been attributed to ROS of either mitochondrial or NOX enzyme origins (Bordt & Polster, 2014).Nonetheless, even though NLRP3 inflammasome activation may not depend on mitochondrial ROS production, it does require mitochondrial Complexes I, II, and III activity (Billingham et al., 2022).This indicates that while mitochondrial ROS may not be necessary for NLRP3 inflammasome activation, mitochondria may provide metabolic support to the effector function of O 2 − derived from NOX2.

| OXIDATIVE PHOS PHORYL ATION AND K REBS C YCLE DYS FUN C TI ON FOLLOWING TB I
Electron microscopy analysis of human tissue reveals morphologically altered mitochondria in synapses following TBI (Balan et al., 2013), consistent with data showing reduced Complex I activity and whole-brain mitochondrial respiration in clinical and preclinical TBI studies (Carteri et al., 2019;Fischer et al., 2016;Hubbard et al., 2023;Kilbaugh et al., 2015).The specific contribution of microglia to the overall reduction in mitochondrial function following TBI is unknown because metabolic and neuroimmune mechanisms coexist during secondary injury.
There is reduced serine and pyruvate metabolism after TBI that is associated with increased expression of pro-inflammatory cytokines (e.g., IL-1β and TNFα) in experimental models (Arora et al., 2022).
Evidence for a mitochondrial ROS-mediated mechanism of posttraumatic microglial/macrophage activation is currently lacking.
Other mechanisms of mitochondrial dysfunction described in human TBI samples, such as excess calcium influx, reduced membrane potential and mitochondrial respiration (Carteri et al., 2019;Kilbaugh et al., 2015;Singh et al., 2006), are phenomena also observed in LPS-stimulated BV2 microglia (Pereira Jr. et al., 2021).In addition, metabolic analysis of microglia co-cultured with needle scratchinjured neurons demonstrates an increase in glucose conversion to citrate, aconitate, and αKG, but not malate (Liu et al., 2023), indicating a disruption in the TCA cycle following injury.
Post-traumatic metabolic dysfunction and neuroinflammation are not static injury events; they continue to evolve with secondary injury progression and during functional recovery (Pandya et al., 2021).Therefore, it is critical to understand how neuroimmune and metabolic alterations are paired over time, how one may be contributing to the other, and any hierarchy within their relationship (i.e., who drives who?).There may also be significant spatiotemporal differences within brain regions ipsilateral to the primary lesion (e.g.,

| LIPID OXIDATI ON REG UL ATE S MI CROG LIAL FUN C TI ON AND N E U R O I N F L A M M AT O R Y R E S P O N S E S
Fatty acids (FAs) can be used as energy substrates in the brain.FA oxidation within the mitochondria generates ATP and NADH, which feed the TCA cycle and ETS, respectively.To be properly transported to the mitochondrial matrix for oxidation, FA must either be permeable to the mitochondrial inner membrane or transported by carrier proteins.Mitochondrial membranes are impermeable to FA with very long acyl chains.However, peroxisomal oxidation allows very long FAs to be partially metabolized, without the generation of ATP, to mitochondria permeable FAs.This process also produces large amounts of H 2 O 2 , which may be used for oxidative bursts (Chausse et al., 2021;Di Cara et al., 2017).
The chemical structure of FAs varies in chain length, number, and position of double covalent bonds, and even ramifications in the carbon chain.These chemical properties determine the metabolic routes and products obtained from each FA.Some intermediates of FA metabolism used for both anabolic reactions and immune responses can only be produced from exogenous lipids, particularly polyunsaturated fatty acids (PUFA; Hamilton et al., 2022;Kapralov et al., 2020;Lowry et al., 2020;Madore et al., 2020).When compared to lipid content in whole brain, there is differential lipid composition in microglia with higher levels of PUFAs, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA; Cisbani et al., 2021).
PUFA metabolism is intrinsically linked to immune function and is modulated by master regulators, such as STING (Vila et al., 2022).
In dendritic cells, activation of lipid-regulated PPARγ leads to an increase in genes involved in pro-inflammatory activation (e.g., Tlr4, Trim22, Il21r) and lipid catabolism (e.g., Acsl1, Apoc1, Pltp).This results in reduced cellular FA content and increased differentiation of blood-borne monocytes toward dendritic cell lineages (Szatmari et al., 2007).Lipids also have significant signaling properties.In Microglia are capable of oxidizing FA's, and this alternative energy supply may support critical neuroimmune functions.Under pathological conditions associated with reduced brain glucose uptake, such as in AD models, lipid oxidation compensates for ATP requirements (Leng et al., 2022).Selective HK inhibition in microglia promotes increased expression of lipoprotein lipase (LPL), a key regulator of lipid oxidation, and reduces NADPH levels (Leng et al., 2022).This positive regulation of lipid oxidation is attenuated by the supplementation of PPP (G6P and F6P), but not glycolytic (F16BP), substrates.
This indicates that increased microglial lipid oxidation following inhibition of glycolysis is not only a consequence of reduced ATP production but may also contribute to critical cellular immune function (e.g., phagocytosis) (Leng et al., 2022).
Although microglial catabolism of saturated (16:0) and monounsaturated (18:1) lipids provide equivalent support for mitochondrial respiration, they diverge significantly when initiating metabolic and immunomodulatory effects.Cellular changes associated with saturated FA catabolism include the accumulation of PUFA that serves as substrates for pro-inflammatory lipid peroxidation (Chausse et al., 2019).In contrast, monounsaturated FA catabolism results in increased accumulation of triacylglycerol lipid droplets and reduced redox balance (Chausse et al., 2019;Figure 4).Furthermore, intravenous ϖ-3 PUFA administration reduces NLRP3 inflammasome activation, reactive microgliosis, and on-going demyelination in an experimental model of spinal cord injury, thereby demonstrating potent anti-inflammatory actions of ϖ-3 PUFA (Baazm et al., 2021).
Despite these studies not discriminating between key metabolic and immune signaling effects of ϖ-3 PUFA, they suggest an integral relationship between lipids and neuroimmunity following CNS injury.
LPS-stimulated microglia transform their lipidomic profiles by accumulating saturated FA (SFA) and reducing monounsaturated FA (MUFA) levels (Button et al., 2014).The reduction in MUFA is caused by reduced stearoyl-CoA desaturase expression, an enzyme involved in MUFA oxidation (Button et al., 2014).This indicates that lipidomic alterations in LPS-stimulated microglia cannot be explained simply by the selective increase of MUFA oxidation but may involve active diversion of lipid metabolites into secondary metabolic pathways.
Furthermore, incubation of microglia with either MUFA or SFAs in the absence of LPS increases IL-6 secretion (Button et al., 2014), which indicates that changes in lipid oxidation are part of the metabolic adaptations of microglia.It also indicates that lipids may possess other signaling properties that could drive pro-inflammatory immune responses (Figure 4).
Cholesterol is a critical regulator of microglia.Cholesterol and oxysterol derivatives that do not contribute to ATP production are mainly catabolized in the liver.Apolipoprotein E (APOE) contributes to cholesterol uptake and transport by way of its high-affinity interaction with lipoprotein receptors, including the low-density lipoprotein (LDL) receptor.APOE is the major lipoprotein in the CNS, and one of its alleles, APOE4, is a major genetic risk factor for developing sporadic AD (Corder et al., 1993;Strittmatter et al., 1993).In humanized APOE models, APOE4 mice that are predisposed to poor cholesterol efflux have increased numbers of microglia with an activated morphology in the hippocampus and increased IL-1β expression when compared to APOE3 mice that have normal brain cholesterol clearance (Seaks et al., 2022).In CRISPR-edited iPSC-derived microglia, APOE4 induces pro-inflammatory priming and promotes adaptations to neuronal-microglial communications that drive lipid accumulation (Victor et al., 2022).Genes encoding rate-limiting enzymes of lipid metabolism, such as fatty acid synthase (FASN) and Acyl-CoA synthase (ACSL1), and transcriptional regulator of sterol biosynthesis, sterol responding element-binding protein 1 (SREBP1), are increased in APOE4 microglia, and provide the metabolic mechanisms necessary to promote lipid accumulation (Victor et al., 2022).
The pro-inflammatory phenotype of APOE4 microglia is further aggravated by the loss of triggering receptor expressed in myeloid cells 2 (TREM2) (Fitz et al., 2020).Loss of TREM2 function results in increased cholesterol accumulation in microglia and is considered a major risk factor for AD (Guerreiro et al., 2013;Nugent et al., 2020; of lipid metabolism (Nugent et al., 2020).Interestingly, functional APOE3 myelin-phagocyting microglia consume the cholesterol biosynthetic precursor desmosterol, which initiates anti-inflammatory signaling through LXR receptors to resolve inflammation and promote tissue repair by increasing cholesterol trafficking to oligodendrocytes (Berghoff et al., 2021).

| K E TONE BOD IE S INHIB IT MICROG LIAL AC TIVATION
In another example of their remarkable metabolic flexibility, microglia oxidize ketone bodies such as 2(b)-hydroxybutyrate (BHB) and acetoacetate (AcAc).The oxidation of BHB and AcAc depends on a supply of TCA cycle anaplerotic substrates, and this involves glutaminolysis, whereby glutamine and AcAc synergically increase mitochondrial respiration in microglia (Nagy et al., 2018).
Notably, a ketogenic diet (i.e., high-fat, low-carbohydrate content diet, 3.1:1 lipid-to-carbohydrate and protein ratio, respectively) administered to spinal cord injury rats transforms microglia from pro-to anti-inflammatory phenotypes following injury, resulting in improved long-term functional recovery (Kong et al., 2021).The  et al., 2021).Ketone oxidation therefore occurs concomitantly with other metabolic pathways known to be associated with immune response.This overlap limits the evaluation of its individual contribution to immune response.Nonetheless, even under conditions that limit ketone body oxidation, pretreatment of BV2 microglia with BHB prior to LPS+ATP stimulation results in a reduction of pro-(NLRP3, cleaved caspase-1, IL-1β release) and an increase in antiinflammatory markers (Arg1) (Kong et al., 2021;Youm et al., 2015).
Ketone bodies have membrane receptors, such as GPR109a, which are enriched in immune cells, including macrophages and neutrophils (Maciejewski-Lenoir et al., 2006).GPR109a is also expressed in microglia and is up-regulated in in vivo models of PD and hyperalgesia (Fu et al., 2015;Viatchenko-Karpinski et al., 2022;Wakade et al., 2014).Upon binding, GPR109a activates antiinflammatory signaling pathways, including Nrf2/HO-1, and reduces p38 MAPK and NF-κB signaling (Viatchenko-Karpinski et al., 2022;Zhang et al., 2022).This is supported by in vivo data, demonstrating a reduction in microglial p38 MAPK/NF-κB signaling following BHB supplementation in a heat-stress model of neuroinflammation (Huang et al., 2022).The ketogenic diet is used clinically as non-pharmacological adjuvant therapy for neurological disorders, such as multiple drug-resistant epileptic syndromes where it significantly reduces the number and frequency of epileptic seizures (Caraballo, Flesler, et al., 2014;Caraballo, Fortini, et al., 2014;Sondhi et al., 2020;Wang et al., 2020).The neuroprotective mechanisms of the ketogenic diet remain elusive, but its application results in increased synaptic plasticity (Trotta et al., 2022), which is thought to be structurally regulated by microglial phagocytic pruning.BHB supplementation also decreases microglial activation and increases neurotrophins production (e.g., BDNF) following demyelinating injury induced by cuprizone (Sun et al., 2022) and following retinal lesion (Trotta et al., 2022).Thus, ketone bodies have pleiotropic beneficial effects on metabolism and immune signaling in microglia.

| TB I D IS RUP TS MI CROG LIAL LIPID ME TABOLIS M AND IN CRE A S E S K E TONE BODY UP TAKE
In humans, TBI can alter the composition of plasma lipids, which are associated with neuropsychiatric comorbidities (Huguenard et al., 2020).This may indicate that post-traumatic lipid changes are an organized metabolic response that can contribute, along with other factors, to diverse clinical outcomes (Huguenard et al., 2020).
In pre-clinical TBI models, plasma lipid composition is altered with repetitive impacts in rats, which could be a consequence of active modulation of lipid content to match their roles in the dynamic processes of neurological recovery after repetitive brain trauma (Anyaegbu et al., 2022).For example, within 4 h of experimental TBI, there is an increase in both pro-and anti-inflammatory oxidized free  et al., 2021).Furthermore, nutritional supplementation of ϖ-3 PUFA in injured wild-type mice reduced microglia/macrophage activation and improved long-term neurological recovery to similar levels observed in injured fat1 + transgenic mice (Desai et al., 2021).Thus, the anti-inflammatory and neuroprotective effects of PUFA may be mediated directly by lipid signaling, or alternatively via microbiomederived lipid metabolites that influence brain immunity and behavior through the gut-brain axis (Brown et al., 2023).
Experimental TBI also results in the accumulation of lipid-laden microglia (Zambusi et al., 2022).In aged brain tissue in humans and mice, lipid droplet accumulating microglia have a pro-inflammatory activation phenotype that is associated with increased ROS production and reduced phagocytic activity (Marschallinger et al., 2020).
In TBI patients, there are reduced levels of ApoA, a lipoprotein responsible for transporting cholesterol, and an elevated presence of ApoB lipoproteins (Santacruz et al., 2022).This could imply that TBI triggers specific responses that alter cholesterol metabolism.
Mass spectrometry imaging in rats subjected to moderate-level CCI revealed an increase in cholesterol ester content in the perilesional cortex at day 3 post-injury (Roux et al., 2016).Although the connection between cholesterol metabolism and post-traumatic neuroimmune responses is not fully understood, in vitro studies indicate that reducing cholesterol trafficking in microglia, achieved through the knockout of ATP-binding cassette (ABC) proteins ABCA1, intensifies pro-inflammatory microglial activation (Karasinska et al., 2013).
Furthermore, Abcg1 knockout mice subjected to moderate-level CCI had increased IL-1β production as well as increased levels of gasdermin D, ASC, and cleaved caspase 1 in the injured cortex when compared to levels in wild-type CCI mice (Xu et al., 2023).Additionally, the deficiency in cholesterol metabolism caused by Abcg1 knockout resulted in a larger lesion volume, underscoring the importance of cholesterol metabolism in resolving post-traumatic neuroinflammatory responses (Xu et al., 2023).In another study, lipidomic analyses revealed that microglial cholesterol esters increase acutely after TBI and persist for 1 week post-injury in both the damaged cortex and hippocampus (Agrawal et al., 2023).In a diffuse injury model in rats (fluid percussion injury), ABCA1 was increased at 10 days post-injury, but brain cholesterol levels did not correlate with liver pro-inflammatory markers IL-1α, IFNγ, TNFα, and MCP-1 (Palafox-Sanchez et al., 2021).At later time points, sterol biosynthesis genes were down-regulated in injured tissue, and were accompanied by down-regulation of microglial immunoregulatory genes (e.g., P2ry12) and the up-regulation of pro-inflammatory genes (e.g., Nlrp3 and Tlr4) (Catta-Preta et al., 2021).Altogether, this suggests that changes in cholesterol metabolism after TBI may contribute to persistent neuroinflammation that can promote chronic neurodegeneration.
Pre-clinical studies have investigated ketone bodies as alternative fuels for the injured brain, including interventions using BHB and varied nutritional supplements (Maalouf et al., 2009;McKenna et al., 2015;Prins, 2008).Perivascular MCT2, a transporter protein for BHB, is increased acutely after TBI (Prins & Giza, 2006), while 13 C-labeled BHB tracing experiments demonstrate increased ketone bodies incorporation into the TCA cycle in the injured cortex of rats (Scafidi et al., 2022).These tracing studies indicate that the metabolic rate of the TCA cycle stimulated by BHB administration is unaltered following TBI, but that there is increased pyruvate recycling from malate during acute brain injury (Scafidi et al., 2022).This is consistent with the observed changes in TCA cycle and glucose metabolism acutely after TBI (Bernini et al., 2020;O'Connell et al., 2005;Singh et al., 2006), and allows ketone body metabolism to support oxidative phosphorylation and drive malate cytosolic NADPH production and NOX2 activity.
In clinical microdialysates of severely injured TBI patients, high ketone body levels correlate with higher pyruvate and lactate levels, which are known to be increased in pro-inflammatory microglia (Bernini et al., 2020).On the other hand, ketone body supplementation in TBI rats results in reduced microglia/macrophage activation and improved sensorimotor function in injured animals (Almeida-Suhett et al., 2022).This Janus-faced effect of ketone bodies may be caused by signaling pathways modulated by ketone bodies, including GPR109a and downstream signaling pathways (Fu et al., 2015;Viatchenko-Karpinski et al., 2022), which suggests that their metabolic fate is more influenced by the inflammatory state.

| TECHNOLOG I C AL LIMITS AND PER S PEC TIVE S FOR MICROG LIAL ME TABOLI C PROFILING IN INJ URED B R AIN
The metabolic profile of microglia has been understudied when compared to metabolism in other brain cells (e.g., neurons and astrocytes).
Unique characteristics of microglia mean that the use of generic tools for metabolic profiling are unsuitable to investigate metabolism in these highly plastic brain-resident immune cells.Although we outline key metabolic features of microglia in TBI (Figure 6

| RE S PIROME TRY
The state-of-the-art in metabolic profiling involves the assessment of ETS complex-specific activity using highly sensitive Clark-type Furthermore, guidelines for respirometry use and analysis (Connolly et al., 2018;Divakaruni & Jastroch, 2022;Jaber et al., 2020), which are established but have yet to be widely adopted, should be used to improve the quality and rigor of metabolic profiling of microglia in the field.Further advances in respirometric analysis, using scanning electrochemical microscopy, also allow single-cell measurements of oxygen consumption to be assessed (Alavian et al., 2011;Land et al., 1999;Santos et al., 2017).This could be applied in brain slices to further uncover region-specific microglial immunometabolic signatures following TBI.

| ME TABOLIC IMAG ING
Fluorescence lifetime imaging microscopy (FLIM) is a metabolic imaging technology that can be used to assess microglial immunometabolic responses in situ in brain tissues (Bernier et al., 2020;York et al., 2021).FLIM is based on the intrinsic differences in the fluorescence lifetime of glycolysis-derived cytosolic NADH, which has a relatively short lifetime in the free state, and mitochondrial enzyme-associated NADH, which has a much longer lifetime in the bound state (Bernier et al., 2020;Okkelman, Papkovsky, & Dmitriev, 2020;Shen et al., 2018;York et al., 2021).This difference in fluorescence lifetime allows for the qualitative discrimination of the contribution of glycolysis and aerobic metabolism for energy supply.The intrinsic NADH fluorescence cannot be distinguished from that of NADPH, and therefore it is referred to as NAD(P) H in FLIM measurements.When FLIM is coupled with genetically encoded microglial fluorescent reporter mice (e.g., Cx3cr1 GFP/GFP ), microglial metabolism may be investigated in a cell-specific manner; without the requirement for major ex vivo isolation procedures or extensive tissue manipulation.An advantage of FLIM is the ability to perform spatially defined assessments, which may provide important information about regional differences in the  (Okkelman, Puschhof, et al., 2020;Perottoni et al., 2021;Shen et al., 2018).
FLIM has been used to demonstrate microglial metabolic flexibility, whereby under conditions of reduced glucose availability, microglial energy metabolism in mouse brain slices is sustained by glutaminolysis, as revealed by an increase in the average fluorescence lifetime of NAD(P)H that is characteristic of increased mitochondrial NAD(P)H pool and respiration (Bernier et al., 2020).
Furthermore, the pro-glycolytic metabolic shift in microglia in response to LPS stimulation has been confirmed in freshly isolated brain slices using FLIM (York et al., 2021).Importantly, FLIM does not discriminate cellular NADH and NADPH levels, and it may lead to an over-estimation of metabolic responses because NADPH produced to drive respiratory burst.Further, it may underestimate mitochondrial respiration as it does not allow the assessment of energy transfer through the ETS from substrates that do not produce NADH.Also, it does not allow the estimation of how much NADH levels in the mitochondria actually reflect ATP production.
Nonetheless, FLIM offers an important additional approach to investigate microglial immunometabolism in situ, which includes intercellular interactions between microglia and adjacent neurons and astrocytes in injured brain tissues.

| CELL-S PECIFI C AND S PATIAL MULTIOMIC S
An in-depth and integrated analysis of the microglial transcriptome, proteome, and metabolome is needed to provide a comprehensive view of metabolic alterations following TBI and provide significant insights into microglial immunometabolic transitions after acute and chronic TBI (Sankowski et al., 2022).This approach has been used to demonstrate key metabolic changes underlying inflammatory function in different neurological diseases.For example, microglia isolated from AD mice have enriched expression of genes related to glycolysis and lipid oxidation, concomitant to an increase in lipid metabolites, when compared to levels in age-matched control mice (Xia et al., 2022).As another example, cell-specific multiomic analyses demonstrate a reduction in TCA cycle metabolites in microglia in diabetic retinopathy (Lv et al., 2022), coexistent with an increase in glycolytic metabolites and genes, thereby providing important insights into orchestrated metabolic shifts associated with the diseased state.
Imaging methods also allow for the assessment of microglial immunometabolic features in situ, reducing tissue processing and allowing a more accurate representation of in vivo microglial phenotypes.Fourier transform infrared spectroscopy (FITR) allows the quantification of biomolecules in brain slices, providing insight into composition changes that could underlie microglial metabolic adaptations following TBI.This tool may be combined with fluorescent markers to provide cell-type specificity and has enough resolution to identify an increase in β-sheet structures within microglia adjacent to amyloid plaques (Prater et al., 2023).Furthermore, Raman spectroscopy and radiotracer-labeled substrates can be used to estimate metabolite flow in biochemical pathways.This has been used to dissect glucose metabolism in multiple tissues (Zhang, Shi, et al., 2019) and could provide clarity to the major metabolic fates of key substrates in microglia following TBI.
Other spatial metabolomic analyses can be performed using mass spectroscopic imaging (MSI) in brain slices.Matrix-assisted laser desorption/ionization (MALDI) spectroscopic imaging allows a metabolomic profile to be obtained with a near single-cell resolution that can provide information on spatial distribution of immunometabolic features in microglia.For example, in spinal cord injury models in rats, MALDI identified increased acylcarnitines levels in microglia and macrophages in the perilesional zone (Quanico et al., 2018).Microfluidics technologies provide other opportunities.Methods such as microfluidic deterministic barcoding (DBiT-seq) allow for an integrated multiomics assessment of protein and transcript levels using high-resolution next-gen-seq (NGS), a powerful tool that has been employed to identify spatial proteome and transcriptome patterns in whole mouse embryos (Liu et al., 2020).These multiomics approaches could be used to characterize immunometabolic changes at the metabolite, protein, and transcript levels following TBI, and could assist in the development of immunotherapies that target specific subpopulations of microglia, preserving neuroreparative responses to accelerate recovery after TBI (Vandereyken et al., 2023).

| OUTS TANDING QUE S TIONS AND FUTURE PER S PEC TIVE S
The growing literature on immunometabolism provides new insights into the crosstalk between metabolites and the regulation of microglia following TBI.Key advances in the field can push boundaries in TBI immunometabolism and lead to important new mechanisms and therapeutic targets.Here, we address what we believe are key outstanding questions.
• It is likely that microglia and infiltrating immune cells undergo immunometabolic rewiring during the acute and chronic stages of TBI.However, detailed metabolic phenotyping studies are lacking.Would different immunometabolic signatures in microglia be observed following injury, and could they be biomarkers of functional recovery?
• The spatial distribution of immunometabolic alterations after TBI is still poorly understood and may be required to identify and therapeutically target-specific subpopulations of dysfunctional or pro-reparative microglia based on immunometabolic profile.
• Microglial phenotypes have expanded from pro-and antiinflammatory activation signatures, but the metabolic profiles for this cell type still rely on simplistic dichotomized M1 and M2 classifications, which is not particularly useful (Paolicelli et al., 2022).
Do microglial phenotypes translate to metabolically distinct profiles, and how can they be classified?
• Could the abnormal immune response following TBI be modulated by metabolic disruptors, or drugs that modulate energy metabolism, such as the clinically relevant metformin?
A deeper understanding of fundamental mechanisms of secondary injury is needed to develop TBI-specific therapies and interventions.Further investigation into the interactions between immune and metabolic pathways after TBI may provide a broader integrative view of secondary damage and may also lead to novel immunometabolic therapies for this devastating neurological disorder.

à
Pesquisa do Estado do Rio Grande do Sul, Grant/Award Number: 1010267, 16/2551-0000499-4 and 17/2551-0001; Irish Research Council, Grant/Award Number: GOIPD/2022/792; National Institutes of Health, Grant/Award Number: NS112212 and NS122777; Science Foundation Ireland, Grant/Award Number: SFI17/FRL/4860 cortical vs. hippocampal), as well as between equivalent ipsilateral and contralateral regions(Hill et al., 2017;Pandya et al., 2021;Singh et al., 2006;Wofford et al., 2017).Such differences could impact functional responses, such as vascularization and oxygen perfusion, or peripheral immune cell infiltration and resident microglial proliferation, in distinct regions of the injured brain.The metabolic environment of the injured brain may provide important insights into microglial immunometabolic function.While increased succinate levels in LPS-stimulated immune cells promote increased glycolytic flux, HIF1α stabilization, and pro-inflammatory cytokine production(Tannahill et al., 2013), following TBI, there is a reliance on complex II driven respiration(Kilbaugh et al., 2015;Stovell et al., 2018).Microglia co-cultured with needle scratchinjured neurons have increased glutamine metabolism, which could feed into the pool of mitochondrial succinate in injured brain tissue(Liu et al., 2023).Surprisingly, although succinate accumulation is a hallmark of pro-inflammatory activation in immune cells, and proinflammatory microglia are associated with secondary brain damage, acute succinate supplementation following TBI is neuroprotective in clinical studies and pre-clinical models(Giorgi-Coll et al., 2017;Khellaf et al., 2022).Therefore, succinate accumulation in microglia may be part of an adaptive response to boost acute pro-inflammatory activation required for coordinated tissue repair mechanisms following TBI(Loane & Kumar, 2016)  (Figure3).

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I G U R E 3 TCA cycle and ETS changes during pro-and anti-inflammatory signaling in microglia, and following TBI.Pro-inflammatory microglial polarization (purple arrows) is accompanied by increased glutaminolysis and a reduction in Complex II (CII) activity.This reduces mitochondrial membrane potential (Δψm) and ATP production and causes succinate accumulation.Succinate accretion from CII inhibition and GABA shunt may also stimulate glycolytic metabolism through increased HIF1α.TBI (yellow arrows) replicates many biochemical pathway changes observed in pro-inflammatory microglia, except for succinate, which undergoes increased succinate oxidation following TBI.Anti-inflammatory microglial polarization (green arrows) results in increased mitochondrial respiratory rates, and mitochondrial pyruvate and lactate oxidation.In addition, increased oxidative phosphorylation is accompanied by increased citrate, aconitate, isocitrate and itaconate synthesis.Broken arrows represent reduced flux of substrates.

Figure 5 )
Figure5).The mechanisms underlying cholesterol and APOEmediated microglial regulation are still under scrutiny, but impaired cholesterol transport driven by either TREM2 loss or APOE4 expression can be rescued by inhibition of liver X receptors (LXR) or stimulation of acetyl-CoA acyltransferase 1 (ACAT1), two key regulators low-carbohydrate content in ketogenic diets could lead to reduced glycolytic flux, and increased mitochondrial respiration; pathways known to promote anti-inflammatory microglial phenotypic transitions(Cheng et al., 2021;Liu et al., 2017;McManus et al., 2022; York F I G U R E 5 Cholesterol inhibits lipid oxidation and stimulates glycolysis and lipid de novo biosynthesis in microglia.Despite not contributing to energy production, cholesterol promotes broad metabolic adaptations in microglia.Poor brain cholesterol clearance in the brain, observed in APOE4 carriers, is associated with increased lipid biosynthesis and accumulation of pro-inflammatory lipid-laden dysmorphic microglia (purple arrows).Loss-of-function mutations in cholesterol-related protein TREM2 recapitulate lipid metabolism alterations observed in APOE4 carriers.Cholesterol accumulation is associated with increases in glycolytic flux-limiting enzyme hexokinase 2 (HK2) and glycolytic regulator hypoxia-inducible factor alpha-1 (HIF-1α) and inhibits anti-inflammatory lipid oxidation pathway (green arrows) through regulatory enzyme lipoprotein lipase (LPL).15-HETE, 15-Hydroxyeicosatetraenoic acid; Ac-CoA, Acetyl-CoA; APOE4, Apolipoprotein epsilon 4; FA-CoA, Fatty acid-coenzyme A; HIF1α, Hypoxia-inducible factor alpha-1; HK2, Hexokinase 2; TREM2, Triggering receptor expressed on myeloid cells 2. Created with BioRe nder.com.

FA
, particularly products of lipoxygenation by LOX-15, whereas at 24 h post-injury, only anti-inflammatory FA are observed(Anthonymuthu et al., 2017).Another pre-clinical study demonstrated persistent lipidomic alterations through 7 days post-injury, with increases in unsaturated FA (18:0) and PUFA, such as arachidonic acid and DHA(Hogan et al., 2018), which are known targets of lipoxygenase enzymes.Interestingly, increased post-traumatic lipoxygenation is associated with macrophage and granulocyte recruitment to the injured brain of rats, such that administration of selective LOX inhibitors reduces peripheral immune cell infiltration and limits proinflammatory responses(Hartig et al., 2013).PUFA are involved in the crosstalk between brain lipid metabolism and post-traumatic neuroinflammation.In mice expressing the C. elegans transgene, fat1 + , which endogenously produces ϖ-3 PUFA, there are reduced numbers of Iba1+ microglia/macrophages within the cortical lesion and reduced expression of proinflammatory mediators in TBI tissue (e.g., Cxcl1, Tnfa, Il6; Mondal ), a fundamental understanding of their immunometabolic functions following CNS injury remains incomplete.New technologies have pushed the boundaries of immunometabolism research and expanded interest in this important topic.Next, we present the challenges and pitfalls in microglial metabolism profiling and showcase opportunities to use cutting-edge technologies for microglial immunometabolism research within the context of acute and chronic TBI.

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Immunometabolic features of microglia following TBI.After TBI, microglia/macrophages display increased glucose metabolism through both glycolysis and PPP.The PPP increase feeds the NADPH pool, which serves as substrate for pro-inflammatory enzyme NADPH oxidase 2 (NOX2), a driver of chronic neuroinflammation after TBI.Increased glycolytic flux is associated with accumulation of lactate following injury, indicative of anaerobic glucose oxidation.Oxygen consumption and ATP production by the electron transfer system (ETS) are reduced, particularly that driven by Complex I substrates.Complex II contribution to ATP synthesis increases.Lipid catabolism is inhibited after TBI and lipid biosynthesis is increased in microglia.This increase in lipid accumulation is sustained by the increase in NADPH production by PPP.Lipid accumulation aggravates the pro-inflammatory phenotype and could contribute to chronic neuroinflammation.Orange and blue arrows indicate pathways that are, respectively, decreased and increased following TBI.ETS, Electron transfer system; GLUT1, Glucose transporter; HK, Hexokinase; NOX2, NADPH oxidase 2; PFKB3, Phosphofructokinase B3; PKM2, Pyruvate kinase M2; PPP, Pentose phosphate pathway; α-KG, Alpha-ketoglutarate. Created with BioRe nder.com.immunometabolic response throughout the brain.As a result of the progressive nature of secondary brain injury, the capacity to investigate regional differences in microglial metabolism may provide novel insight into immunometabolic dysfunction and spreading neurotoxicity and damage following TBI.FLIM also offers the possibility of performing real-time metabolic challenge studies in microglia by the sequential titration of pathway-specific substrates or inhibitors.This approach combines more complex metabolic-profiling techniques (e.g., respirometry) with in situ metabolic imaging to identify specific contributions of metabolites and enzymes in the regulation of immunometabolic responses following TBI, and spatiotemporal dynamics throughout the various stages of neurological impairment and/or recovery