. 2014 May 21;289(29):20200–20208. doi: 10.1074/jbc.M114.570978

Lactate Storm Marks Cerebral Metabolism following Brain Trauma^*

Sanju Lama ^‡, Roland N Auer ^§, Randy Tyson ^‡, Clare N Gallagher ^‡, Boguslaw Tomanek ^‡, Garnette R Sutherland ^‡,¹

PMCID: PMC4106336 PMID: 24849602

Background: In brain metabolism, neurons are fueled by lactate passed to them by glia in a metabolic coupling.

Results: Following brain trauma, lactate uptake into neurons from glia was impaired, producing a metabolic lactate storm.

Conclusion: Brain trauma results in neuronal-glial metabolic uncoupling, releasing free lactate.

Significance: Inhibition of lactate production or its removal may be an important therapeutic strategy for brain trauma.

Keywords: Brain Metabolism, Glial Cell, Neuron, Tau Protein (Tau), Tricarboxylic Acid Cycle (TCA Cycle) (Krebs Cycle), ¹³C-Labeled Substrates, MR Spectroscopy, Lactate, Neuronal-Glial Metabolism, Traumatic Brain Injury

Abstract

Brain metabolism is thought to be maintained by neuronal-glial metabolic coupling. Glia take up glutamate from the synaptic cleft for conversion into glutamine, triggering glial glycolysis and lactate production. This lactate is shuttled into neurons and further metabolized. The origin and role of lactate in severe traumatic brain injury (TBI) remains controversial. Using a modified weight drop model of severe TBI and magnetic resonance (MR) spectroscopy with infusion of ¹³C-labeled glucose, lactate, and acetate, the present study investigated the possibility that neuronal-glial metabolism is uncoupled following severe TBI. Histopathology of the model showed severe brain injury with subarachnoid and hemorrhage together with glial cell activation and positive staining for Tau at 90 min post-trauma. High resolution MR spectroscopy of brain metabolites revealed significant labeling of lactate at C-3 and C-2 irrespective of the infused substrates. Increased ¹³C-labeled lactate in all study groups in the absence of ischemia implied activated astrocytic glycolysis and production of lactate with failure of neuronal uptake (i.e. a loss of glial sensing for glutamate). The early increase in extracellular lactate in severe TBI with the injured neurons rendered unable to pick it up probably contributes to a rapid progression toward irreversible injury and pan-necrosis. Hence, a method to detect and scavenge the excess extracellular lactate on site or early following severe TBI may be a potential primary therapeutic measure.

Introduction

It has been well known that brain lactate levels increase following severe traumatic brain injury (TBI),² but its origin remains controversial (1). Lactate serves as an alternative energy substrate to glucose for normal or injured neurons (2 –4). Contemporary studies have also suggested its potential neuroprotective or therapeutic role in TBI (5, 6). Indeed, although a recent international clinical trial is aimed at the study of the therapeutic benefit of lactate in TBI,³ it is important to consider that the findings presented here suggest that this may not be the case.

Using isotopes of carbon, such as ¹⁴C and ¹³C, investigators have studied the effect of TBI on brain metabolism, in particular how it relates to increased brain lactate levels at and around the site of injury as compared with other regions (7 –9). This further led to the assumption that increased lactate may be a result of impaired mitochondrial function following TBI with a compensatory increase in anaerobic glycolysis for ionic homeostasis and generation of extracellular fluid lactate (10, 11). This phenomenon has been explained to be a protective mechanism in TBI by which the injured brain is able to uptake and metabolize lactate. The normal human brain already shows striking differences in lactate/pyruvate ratio from plasma, which suggest that lactate is a physiologic fuel for neurons (12). Accordingly, it has been speculated that lactate might be a useful therapy following TBI (13, 14). However, a conundrum arises as a result of the common observation that excessive lactate accumulation following TBI correlates with poor prognosis (15 –17). This is in agreement with the common notion that lactate accumulation can be detrimental to neuronal survival, despite its recent demonstration as an alternative fuel for neurons. Lactate is a strong acid with a pK_a of 3.86, with implications for tissue pH.

The present study is based on the understanding that neuronal and glial metabolism are coupled; the glia take up and metabolize glutamate to glutamine from the neuronal synapse, for maintenance of synaptic glutamate levels below those associated with neurotoxicity (18, 19). The uptake of glutamate is specific to glutamate transporters and requires an ionic gradient. Glutamate is converted to glutamine in an ATP-dependent reaction. The role of astrocytic metabolism can be traced as a decrease in ATP activating astrocytic glycolysis, resulting in production of lactate that is shuttled to neurons, widely described as the astrocytic neuronal lactate shuttle hypothesis model of brain metabolism (20, 21). Accordingly, the present study was designed to enable the dissection of the neuronal and glial metabolic compartments and to investigate how they behave following severe TBI. The study utilizes a modified weight drop impact acceleration model of acute severe TBI, histopathology, and ex vivo high resolution MR spectroscopy with infusion of ¹³C-labeled substrates specific to neurons and astrocytes in control and TBI rats.

MR spectroscopy provides precise metabolic mapping of neurons and glia after administration of ¹³C labeled glucose, lactate, and acetate to injured and control rats. Glucose is an ambiguous substrate to both neurons and glia, lactate is exclusively neuronal, and acetate is astrocytic. ¹³C is a non-radioactive, naturally occurring, safe and stable isotope. Its abundance is ∼1.1% of carbon in nature. Due to its half nuclear spin properties, this carbon isotope can be studied with MR spectroscopy (22, 23). The ¹³C MR spectroscopy technique provides information on the organic structure of the molecule being studied via incorporation of tissue metabolites, such as glutamate, glutamine, lactate, etc. incorporated with ¹³C isotopes following their systemic administration.

MATERIALS AND METHODS

All experiments were approved by the Animal Care Committee of the National Research Council of Canada and of the University of Calgary. Forty-five adult male Sprague- Dawley rats (Charles River Laboratories, Montreal, Canada) weighing 250–300 g (mean 280 ± 70 g) were used. Twelve rats were utilized for histopathology, and the remaining 33 were utilized for ¹³C-labeled substrate infusion, the brains being processed for high resolution ex vivo MR spectroscopy.

Anesthesia and Surgical Procedures

Each rat was fasted for 2 h and anesthetized using isoflurane (1–2%, inhalational anesthesia for induction as well as maintenance) in 100% O₂. After 10–15 min, the animals were intubated and mechanically ventilated throughout the experiment. An area of inner thigh was shaved, and the femoral artery and vein were cannulated for blood pressure monitoring and labeled substrate infusion. Body temperature was maintained at 37 ± 1 °C with the use of a circulating water bath. In TBI groups, head trauma was induced using 500-g/1.4-meter weight/height impact, modified from and based on the model described by Marmarou et al. (24, 25). The pH, pO₂, and pCO₂ in the blood were determined using an ABL 725 radiometer before and over a 2-h period after trauma and parameters set as follows: PCO₂, 35–45 mm Hg; PO₂, 120–140 mm Hg; pH, 7.30–7.58; mean arterial pressure, 90–110 mm Hg.

Trauma Device

The trauma device used was composed of a columnar Plexiglas tube and a set of brass weights falling freely by gravity onto a metallic helmet fixed by dental acrylic to the skull vertex of the rat. From a designated height the brass weight fell through the lumen of a transparent Plexiglas tube held in a ring stand. The helmet was a stainless steel disc 10 mm in diameter and 3 mm thick. The contact side of the disc was grooved concentrically to accept acrylic and secure the contact.

Induction of Head Trauma

The scalp of the anesthetized animal was shaved, a midline incision was made, and the periosteum covering the vertex was reflected. The metallic disc/helmet was placed centrally on the skull vault between the coronal and lambdoid sutures. The rat was placed securely in the prone position on a foam bed of known spring constant. The lower end of the Plexiglas tube was positioned directly above the helmet. The injury was delivered by dropping a 400-g weight from the predetermined height of 1.4 m. Rebound impact was prevented by moving the foam bed containing the animal away from the tube immediately following the impact.

Histopathology

At 90 min post-TBI, 12 (10 TBI, 2 controls) rats were euthanized by intraperitoneal administration of pentobarbital (200 mg/kg) and perfusion-fixed with trans-cardiac injection of formaldehyde (0.4%, 60 ml) following a saline flush (0.9%, 60 ml). The animals were decapitated, and the heads were placed in the same fixative for 2 weeks prior to sectioning. The brains were removed and cut coronally into 2.8-mm-thick slices, dehydrated in graded concentrations of ethanol, and embedded in paraffin. Serial sections were cut at 8 μm and stained with hematoxylin and eosin (H&E), Bielschowsky Ag stain, glial fibrillary acidic protein, and Tau immunohistochemistry.

Labeled Substrate Infusion

Thirty-three adult male rats were randomized for substrate infusion and brain metabolite MR spectroscopy. Control animals (n = 9, 3 rats/group) received either [1-¹³C]glucose (10%, w/v), sodium [2-¹³C]acetate (7.5%, w/v), or sodium [3-¹³C]lactate (6%, w/v) (Cambridge Isotopes, Andover, MA) for 12 min. Animals receiving TBI (n = 24, 4 rats/group) were also randomly allocated to receive one of the three isotopes. In these experiments, the infusion began immediately following TBI in 12 of the animals and at 60 min post-TBI in the other 12 animals. For all isotope infusions, animals received a bolus (0.8 ml/kg) of one of the three solutions, followed immediately by continuous infusion via syringe pump. The rate of infusion was decreased exponentially from an initial 15 ml/kg/h over 4 min to 2.9 ml/kg/h. Following the experiments, the animals were sacrificed by funnel freezing of the heads using liquid N₂ while anesthetized and ventilated (26). The brains were stored at −80 °C for metabolite extraction and high resolution MR spectroscopy.

Metabolite Extraction for High Resolution MR Spectroscopy

Metabolite extraction from frozen brain specimens was performed as described previously (27). Briefly, the funnel-frozen brain was recovered from −80 °C, and neocortical brain samples averaging 18–20 g were weighed in a cold chamber. The samples were homogenized and centrifuged three times in a 2:2:1 chloroform/methanol/H₂O mixture at 20,500 rpm for 10 min. The methanol-H₂O monolayer was retained following separation from the heavier chloroform-containing organic solvent. Excess methanol was removed using a rotary evaporator and freeze-dried for 48 h. Prior to MR spectroscopy, the sample was reconstituted in 0.6 ml of D₂O stock solution (590 μl of D₂O, 10 μl of 2,2-dimethyl-2-silapentanesulfonic acid, an internal standard for chemical shift), and the pH was adjusted to 6.9–7.1 using KOD and KCl and placed in 5-mm NMR tubes for analysis.

Metabolism of the labeled substrates was studied by obtaining the spectroscopy data, which allows both identification and position of the compounds of interest based on their known chemical shifts. An inverse detection technique called ¹H{¹³C} spin-echo difference (SED) spectroscopy (28) that allows detection of ¹³C label using ¹H (proton) MR spectroscopy through spin-spin interaction between ¹³C and their respective protons attached was utilized. The technique allows increased sensitivity for detection of the labeled metabolites, which are obtained as two subspectra, one with signals from protons attached to ¹²C-¹³C nuclei in-phase and the other from protons attached to ¹²C-¹³C nuclei anti-phase. The difference between the two provides only the signals arising from protons attached to ¹³C nuclei.

All ¹H{¹³C} SED MR Spectroscopy experiments were performed using a 600-MHz Bruker spectrometer (AMX 600; Bruker Spectrospin Ltd., Ettlingen, Germany) at 298 K. The acquisition parameters, as described previously (29), were as follows: 8000 complex data points of the free induction decay, 600-MHz spectral width (centered on the residual H₂O resonance), 0.41-s acquisition time, 6.13-s relaxation delay, and a ¹H 90º pulse length lasting 10.8 ± 0.4 μs. The spin-echo time was set to 1/JHC = 7.2 ms, where JHC is the mean ¹H-¹³C spin-spin coupling constant (140 Hz). Composite pulse decoupling was used with a ¹³C 90° pulse length of 20.0 ms. The residual H₂O resonance was diminished using an on-resonance presaturation pulse. At least 512 scans were obtained for each subspectrum. Before the Fourier transformation of the free induction decay, the time domain data were zero-filled to 32,000 data points and multiplied with a line-broadening parameter of 0.5 Hz. All chemical shifts are shown in parts per million (ppm) relative to the 2,2-dimethyl-2-silapentanesulfonic acid resonance, which is defined to be 0 ppm.

The ¹H{¹³C} SED NMR spectra were analyzed using a commercially available NMR analysis software package (NUTS; Acorn NMR Inc., Fremont, CA) to determine the fractional enrichment of the label in glutamate (C-4 and C-3 complement positions), γ-aminobutyric acid (GABA) (C-2 and C-4), glutamine (C-4 and C-3), lactate (C-3), aspartate (C-3), alanine (C-3), and succinate (C-2,3). One advantage of using ¹H{¹³C} SED MR spectroscopy over direct detection of the label using ¹³C MR spectroscopy, aside from the increased sensitivity, is that the fractionated enrichment can be directly obtained from the difference spectrum, containing only signals from protons attached to ¹³C nuclei, and the total proton subspectrum. Any differences in line width between the resonance from protons bonded to ¹²C and ¹³C nuclei were assumed to be small compared with the applied line broadening parameter. The percentages of fractional enrichment data were compared between groups for each metabolite by using pooled or separate Student's t tests, depending on the absence or presence of heterogeneity of variance.

RESULTS

Histopathology

In histopathological analysis using H&E staining of paraffin-embedded brain sections, all TBI animals showed evidence of severe brain injury (Table 1 and Fig. 1). Subarachnoid hemorrhage and intraventricular hemorrhage was evident in all TBI animals, 89% had neocortical hemorrhage, 80% demonstrated shear injury in the form of dehiscence of the septal nuclei from the inferior aspect of the corpus callosum and with Bielschowsky stain a few early neuroaxonal spheroids. More than 50% showed dark neurons in the CA4 sector and the dentate, and 30% had brain stem hemorrhage. The two control brains were unremarkable.

TABLE 1.

Gross injury pattern and locations following TBI based on histopathological assessment (percentage of animals, n = 10).

SAH, subarachnoid hemorrhage; IVH, intraventricular hemorrhage.

	Traumatic SAH	Traumatic IVH	Parenchymal hemorrhage	Shear injury	Abnormal hippocampal neurons (CA4)	Abnormal neurons (dentate blade)	Brain stem hemorrhage
	%	%	%	%	%	%	%
Lesions (n = 10)	100	100	90	80	60	60	30

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FIGURE 1. — Hematoxylin and eosin stain demonstrating evidence of severe TBI, with (*clockwise* from *left*) subarachnoid hemorrhage, shear injury, intraparenchymal hemorrhage (*top right*) with blood extending to the fourth ventricle and into the subarachnoid space around the cerebellum (*bottom right*).

Brain sections of all TBI animals stained with glial fibrillary acidic protein showed astrocytic activation in the CA3 zone of the hippocampus, more than the CA1 zone (Fig. 2). There was also significant activation in the CA4 zone and the hilus of the dentate gyrus. The dentate gyrus itself showed astrocyte activation throughout the granule cell layer, mostly toward the inner surface that interfaced with the CA4 zone. The neocortex showed both superficial glial activation in layers 1–3 and deep activation in layer 6. Activated astrocytes were not present in layers 4 and 5. The control brains showed minimal glial activation.

FIGURE 2. — **Glial fibrillary acidic protein staining showing activated astrocytes in the hippocampus (*four left panels*).** In the neocortex, glial activation was present in layers 1–3 (*top right*) and in layer 6 (*bottom right*).

Fifty percent of the animals showed positive staining for Tau (Fig. 3). Staining was present in the mossy fibers or axons of the dentate granule cells. The CA3 pyramidal cell band showed positive staining of mossy fibers. Within the neocortex, Tau staining of large neurons was not evident. Lesser staining anatomically restricted to the mossy fibers and subpial/periventricular zones was seen in the two control brains.

FIGURE 3. — **Tau immunohistochemistry showing tau staining mossy fibers or axons in the dentate granular cell (*top left*), arc of CA3 (*bottom left*), and large cortical neurons (*right*).**

MR Spectroscopy

From the acquired ¹H{¹³C} MR spectra, a number of metabolites (lactate, alanine, acetate, N-acetylaspartate, GABA, glutamate, succinate, glutamine, aspartate, ino sitol, creatine, and taurine) could be compared between groups. Labeling of metabolites following [1-¹³C]glucose and [3-¹³C]lactate was similar. The peaks for C-3 lactate and C-4 glutamate show similar chemical shifts and peak height. Following [2-¹³C] acetate infusion, however, a small amount of labeled lactate and alanine at C-3 position was observed. Representative spectra for each substrate infusion immediately post-TBI and 60 min post-TBI are shown in Fig. 4.

FIGURE 4. — 600-MHz one-dimensional ¹H NMR spectrum of rat forebrain, chloroform/methanol/H₂O extract (*bottom*), and ¹H-¹³C spin-echo difference spectrum from control, immediately post-TBI, and 60-min post-TBI rats, showing incorporation of the ¹³C label from [1-¹³C]glucose, [3-¹³C]lactate, and [2-¹³C]acetate. The major resonances were measured and are shown as follows. *Lac*, lactate; *Ala*, alanine; *Ace*, acetate; *NAA*, N-acetylaspartate; *GABA*, γ-aminobutyric acid; *Glu*, glutamate; *Suc*, succinate; *Gln*, glutamine; *Asp*, aspartate; Cr, creatine.

Following severe TBI and immediate infusion of ¹³C-labeled substrates, C-3 lactate was significantly increased in all three infusion groups (i.e. [1-¹³C]glucose (p < 0.01), [3-¹³C]lactate (p < 0.01), and [2-¹³C]acetate (p < 0.01)) compared with their control counterparts, as shown by percentage enrichment in the subtraction spectra (Fig. 5). It was also observed that percentage enrichment of 3-C alanine was significantly increased immediately following TBI in the [3-¹³C]lactate infusion group (p < 0.05). Interestingly, 4-C succinate was significantly increased in the [2-¹³C]acetate infusion group (p < 0.05). Glutamate and aspartate showed a decreased labeling pattern and percentage enrichment immediately following severe TBI, reaching a significance level for aspartate only in the [1-¹³C] glucose infusion group (p ≤ 0.005).

FIGURE 5. — Metabolite concentration (mean ± S.D. (*error bars*)) from subtraction spectra following infusion of labeled substrates ([1-¹³C]glucose, [3-¹³C]lactate, and [2-¹³C]acetate) for control, immediately after, and 60 min after severe TBI. *Lac*, lactate; *Ala*, alanine; *Ace*, acetate; *NAA*, N-acetylaspartate; *GABA*, γ-aminobutyric acid; *Glu*, glutamate; *Suc*, succinate; *Gln*, glutamine; *Asp*, aspartate; *Ino*, inositol; Cr, creatine; *Tau*, taurine. *, p < 0.05; **, p < 0.01.

Following severe TBI and infusion of ¹³C labeled substrates 60 min postinjury, again C-3 lactate was significantly increased in all three infusion groups in comparison with the corresponding controls (p < 0.005 for 1-¹³C glucose and [3-¹³C]lactate infusion groups and p < 0.05 for [2-¹³C]acetate infusion group) (Fig. 5). A decreased C-4 glutamate was again observed in all three groups 60 min following severe TBI, reaching a significance level only for the [3-¹³C]lactate infusion group. A number of metabolites (i.e. alanine, acetate, and GABA) in the [2-¹³C]acetate infusion group showed a significantly increased labeling pattern (p < 0.05 for all four metabolites), whereas in the lactate and glucose infusion groups, this was not the case.

It was of interest to note that GABA was decreased following [1-¹³C]glucose infusion both immediately and 60 min following acute severe TBI. However, in the [3-¹³C]lactate and [2-¹³C]acetate infusion groups, GABA was increased following severe TBI and infusion of substrates immediately and 60 min after the impact.

The significant increase in C-3 lactate following severe TBI and infusion of ¹³C-labeled substrates prompted questions as to the origin of this metabolite. Specifically, infusion of [2-¹³C]acetate showed increased labeling of 3-C lactate, a phenomenon not usually expected. The origin of this was investigated by testing whether lactate was also labeled at the C-2 position, and it was found that regardless of the type of substrate infusion, C-2 lactate was labeled for both the immediate post-TBI and 60-min post-TBI group of animals. Again, the TBI groups showed an increased percentage enrichment for C-2 lactate, but the difference was not statistically significant.

DISCUSSION

This study demonstrates that the injured brain continues to produce lactate within minutes following severe TBI, which can be toxic and incompatible with life (16, 30). Furthermore, the injured neurons early following severe TBI are unable to pick up and metabolize the increased lactate, resulting in a lactate storm in the failing metabolic environment. The findings have significant clinical implications because perhaps a mechanism to safely scavenge and/or chelate the excess lactate (31), buffer the pH effect (32), or inhibit glial metabolism (33, 34) may be of benefit to the dying brain as opposed to further administering lactate, as some of the contemporary research has been suggesting (5, 6). Given that uncoupled metabolism occurs rapidly following trauma, such therapeutic measures would best be administered at the accident site, taking advantage of the known blood-brain barrier disruption and understanding of the neurovascular unit (35 –37). The efficacy of these pharmacological strategies may be augmented by methods that improve their transport across the blood-brain barrier, such as membrane transporters, receptors, nanosized molecular chaperones, and vectors (38 –40).

The present study examined neuronal-glial metabolic coupling following acute severe TBI. Utilizing established techniques of ¹³C-labeled substrate infusion specific to each of the cell types, the study demonstrates a unique observation: a significantly increased C-3 lactate following severe TBI and infusion of [1-¹³C]glucose, [3-¹³C]lactate, and, interestingly, [2-¹³C]acetate. The metabolic fate of each of the substrates following systemic administration has been well outlined previously. It is important to recognize the divergence that can occur between the C-3 and C-4 carbons in glutamine and glutamate as the metabolites between neuronal and glial pools are exchanged. Specifically, following 1-¹³C glucose and 3-¹³C lactate infusion, it is expected that products of glycolysis are labeled at precisely the same carbon skeletons, both being taken up by neurons and metabolized, recognizing that astrocytes also take up glucose. Astrocytic metabolism has been studied by some using [2-¹³C]acetate, a purely astroglial substrate due to their selective transport activity. Incorporation of ¹³C label is expected in the glial glutamine and glutamate pool. C-4 glutamate, often observed in acetate metabolism, is attributed to the C-4 glutamine produced by astrocytic oxidative phosphorylation that is picked up by neurons to replenish its C-4 glutamate pool. Although proposing a metabolic modeling is beyond the scope of this report, a similar mechanism as outlined by many is a likely proposition.

Production of lactate in the brain following acute severe TBI may be attributed to any of the following key mechanisms. 1) Increased astrocytic glycolysis as a result of increased excitatory amino acid, chiefly glutamate, necessitates rapid uptake from the ECF via Na⁺-dependent co-transport. The resultant intracellular glutamate and Na⁺ increase activates Na⁺/K⁺-ATPase, which stimulates astrocytic glycolysis and production of lactate (20, 41 –43). 2) Although less likely in the present study (i.e. controlled for normoxia), in certain cases, when accompanied by tissue hypoxia, increased lactate and H⁺ accumulation occur via accelerated anaerobic glycolysis (44, 45). 3) A less commonly described but suggested mechanism is the role of astroglial cytosolic malic enzyme, which may be activated to an overdrive in its function of degrading excessive Krebs cycle intermediates, such as malate, helping them exit the tricarboxylic acid cycle to generate pyruvate, which ultimately is reduced to form lactate (46 –48). This malic enzyme-catalyzed reaction is deemed essential for complete oxidative metabolism of tricarboxylic acid cycle products, substrates, and their many derivatives, such as glutamate, glutamine, GABA, aspartate, etc. 4) A recent understanding of neuronal-astrocytic metabolic coupling emphasizes the role of a newly cloned soluble adenylyl cyclase, an HCO₃⁻ sensor expressed abundantly in astrocytes, that get activated in response to small increases in extracellular K⁺, allowing HCO₃⁻ entry into the astrocytes (49). The increased HCO₃⁻ content triggers the cyclic AMP pathway, which further initiates astrocytic glycolysis and production of lactate that is taken up by neurons. In accordance with this explanation and the known increase in extracellular K⁺ following TBI, it is possible that this pathway is responsible for increased lactate production, which injured neurons are unable to metabolize and clear from the extracellular fluid compartment.

Thus, it is suggested that the latter two mechanisms may be responsible for the astroglial overactivity within the first few minutes to hours following acute severe TBI, resulting in excessive lactate accumulation in the brain. Indeed, this may have already determined the poor outcome (i.e. a seemingly inevitable irreversible brain injury and cellular death). These mechanisms, although not well studied in TBI, could help to explain the possible mechanism of accelerated production of lactate, perhaps initiated as a defense mechanism that fails to serve any useful purpose in severe TBI.

From the present study, it can be inferred that significantly increased lactate with infusion of [2-¹³C]acetate and [1-¹³C] glucose implies ongoing glial metabolism. High lactate levels and increased alanine with [3-¹³C]lactate infusion imply decreased neuronal metabolism with likely continued glial metabolism and production of lactate, which the neurons are unable to pick up and utilize. Increased lactate, alanine, GABA, and succinate following [2-¹³C]acetate infusion further support ongoing glial metabolism, with the latter diverting to an activated malic enzyme pathway. High lactate levels, as seen in [3-¹³C]lactate infusion animals, imply impaired neuronal metabolism that fails to utilize lactate that the glial cells continue to produce. In other words, it appears that following severe TBI, as early as minutes to an hour after injury, the neurons and glial metabolism become uncoupled (i.e. the glial cells are in overdrive to produce lactate, perhaps to feed the injured neurons). However, this phenomenon seems to happen irrespective of glutamate uptake for conversion to glutamine, which in turn activates the astrocytic glycolysis to produce more lactate for neurons, because both glutamate and glutamate labeling are found to be decreased in the post-TBI brain compared with controls. In what appears to be an autonomous astrocytic activity that fails to sense any feedback mechanism for metabolic demand and supply, the excessive lactate that bathes the neurons following severe TBI may be the lethal metabolic blow to the tissue. The combined effects of direct traumatic injury together with such severe metabolic distress could lead to unsalvageable brain.

Severity of injury was established by a graduated weight-height interface⁴ with histopathological evidence of severe brain injury yet minimal animal mortality. Upon determination of the weight-height interface, a subgroup of animals receiving TBI sustained the spectrum of intracerebral injury commonly observed in patients of severe TBI (50, 51). Significant activation of astrocytes (glial fibrillary acidic protein), neuronal injury/inflammation with the presence of dark neurons (H&E), and tauopathy (Tau immunohistology) support the observation of neuronal and glial pathology, which in turn would explain perturbed metabolism of the cell population as shown by the MR spectroscopy data. Severe head trauma results in oxidative stress reflected by the rapid accumulation of oxidative stress markers with a decrease in antioxidant defense enzymes (52 –54). Oxidative stress is known to induce the formation of hyperphosphorylated Tau, consistent with the results reported here (55, 56).

The pronounced increase in lactate within the first few min to 1 h following acute severe TBI, as demonstrated by labeling at C-3 position and (also) C-2, is indicative of deranged cerebral metabolism in response to injury. The reason remains speculative. Activation of an enzymatic pathway, such as malic enzyme, or an astrocyte-specific HCO₃⁻ sensor is stipulated to be a possible underlying mechanism. With blood gas parameters well controlled and showing minimal variation within predetermined normal limits, hypoxia was ruled out as playing a significant role (i.e. anaerobic lactate production is unlikely). Furthermore, it has been shown that extracellular lactate increase is independent of brain hypoxia-ischemia in severe TBI (57, 58). Although future investigations of the enzymatic pathway and other possible mechanisms may help to dissect this further, it is evident from this study that uncoupled neuronal-glial metabolism leads to excessively increased brain lactate following acute severe TBI. Rather than the well formulated anaerobic mechanism being responsible, metabolic mismatch between neurons and glia and the uncoupling of the symbiotic balance between the two may be the most probable explanation. Complex as this mechanism may appear, it may be as simple as loss of the feedback mechanism for lactate sensing by glia, thereby causing them to become autonomous lactate producers perilous to the neurons, neuropil, and overall tissue survival.

CONCLUSION

The present study emphasizes the uncoupling of metabolism between neurons and glial cells following severe TBI within the first few min to 1 h of sustaining injury. It is of considerable interest to observe significantly high levels of lactate in animals irrespective of substrate infused. Although the study is acute and did not allow for survival as an outcome, the early metabolic injury sustained is comparable with those observed clinically in severe TBI with poor prognosis. Although some investigators have indicated lactate to be of benefit and as the controversy surrounding its role in brain metabolism prevails, observations from the present study suggest that lactate is both a culprit and a product of an aftermath of uncoupled neuronal-glial metabolism. Because lactate originates from the glial metabolic compartment, inhibiting its production, and not administration of it, could be considered a potential therapeutic strategy for TBI.

Acknowledgments

We thank Dr. Fangwei Yang (University of Calgary) for technical support and Dr. Hans Vogel for the use of the high resolution NMR research laboratory located at the University of Calgary.

This work was supported by the Canadian Institutes for Health Research grant entitled Cerebral Metabolism in Traumatic Brain Injury RT733596 (to G. R. S.).

ClinicalTrials.gov Identifier: NCT01573507.

⁴

S. Lama, R. N. Auer, R. Tyson, C. N. Gallagher, B. Tomanek, and G. R. Sutherland, unpublished data.

The abbreviations used are:

TBI: traumatic brain injury
MR: magnetic resonance
SED: spin echo difference.

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PERMALINK

Lactate Storm Marks Cerebral Metabolism following Brain Trauma^*

Sanju Lama

Roland N Auer

Randy Tyson

Clare N Gallagher

Boguslaw Tomanek

Garnette R Sutherland

Abstract

Introduction