The metabolic consequences of evoked spreading depolarization in brain slices

TL;DR

This study explores how brain tissue metabolism changes during spreading depolarization and finds that glucose deprivation worsens recovery, but coenzyme Q10 may help.

Contribution

The study reveals how spreading depolarization affects metabolism and shows coenzyme Q10 improves recovery under glucose deficiency.

Findings

Spreading depolarization increases mitochondrial activity and shifts metabolism to anaerobic respiration and glycolysis.

Glucose deprivation impairs recovery and disrupts central carbon metabolism.

Coenzyme Q10 supplementation improves metabolic recovery in glucose-deprived brain slices.

Abstract

Spreading depolarization is a wave of neuronal and glial depolarization that propagates through brain tissue, triggering neuropeptide release and altered blood flow. It has been observed in ischemic stroke, traumatic brain injury, subarachnoid haemorrhage, epilepsy, and migraine aura. Spreading depolarization imposes a high energetic demand, and recovery impaired under metabolic substrate deficiency. Despite its clinical relevance, metabolic responses remain poorly understood, limiting therapeutic progress. We investigated metabolic effects of spreading depolarisation using an ex vivo brain slice model, aiming to characterise changes in intracellular calcium signalling, mitochondrial function, and central carbon metabolism, and to assess the impact of glucose deprivation. We further tested whether coenzyme Q10 could improve recovery under metabolically compromised conditions. Spreading depolarization increased mitochondrial activity and shifted metabolism toward anaerobic respiration and glycolysis. Glucose deprivation impaired recovery, inducing mitochondrial dysfunction and accumulation intermediates indicative of tricarboxylic acid cycle stalling and disrupted central carbon metabolism. Supplementing glucose-deprived brain slices with coenzyme Q10 shortened spreading depolarization repolarization duration, indicating enhanced metabolic recovery. These findings demonstrate that spreading depolarization imposes a significant metabolic burden, particularly under glucose limitation, and that mitochondrial-targeted interventions such as coenzyme Q10 may enhance tissue resilience in neurological disorders.

Figures

KCl-induced widespread depolarization and increased neuronal activity across cortical slices under physiological conditions. (A) Image showing the positioning of an acute brain slice on the MEA; black dots indicate recording electrodes. (B) Example traces showing voltage amplitude changes over time, with each trace representing a channel. (C) Example spreading depolarization event, demonstrating how parameters are calculated. Heat maps illustrate spreading depolarization (D) latency, (E) repolarization duration, and (F) spreading depolarization event duration. (G) Neuronal activity as assessed

Spreading depolarization increases mitochondrial activity and alters glycolysis-associated metabolites. (A) Mean change in mitochondrial activity (RH-123 fluorescence) in physiological conditions at baseline (control, black) and following KCl stimulation (spreading depolarization, magenta) (*n* = 5 mice). Concentrations of (B) lactate, (C) malate, and (D) pyruvate were altered following spreading depolarization (control: *n* = 10 slices; spreading depolarization: *n* = 13 slices). Solid lines represent the mean; dashed lines indicate SEM. SD; spreading depolarization. * *p* < 0.05; *p* < 0.01;

Spreading depolarization characteristics differ between physiological conditions and glucose-deprivation. (A) Comparison of Ca^2+^ signalling as assessed by mean change in Ca²⁺ fluorescence between physiological conditions (magenta, *n* = 11 mice) and glucose deprivation (blue, *n* = 8 mice) at baseline and (B) following stimulation (physiological conditions, *n* = 11 mice, glucose deprivation, *n* = 7 mice). Solid lines represent the mean; dashed lines indicate SEM. (C) Example trace of a spreading depolarization event in physiological conditions. (D) Example trace of a spreading depolarizati

Spreading depolarization induced changes in central carbon metabolism in glucose-deficient slices. (A) Mitochondrial activity as measured by change in RH-123 fluorescence between physiological conditions (magenta, *n* = 9 mice) and glucose-deprivation (blue, *n* = 8 mice) at baseline and (B) following stimulation (physiological conditions, *n* = 5 mice, glucose deprivation, *n* = 4 mice). Quantities of metabolites, including (C) alanine, (D) glutamate, (E) isoleucine, (F) lactate, (G) malate, (H) aspartate, (I) proline, and (J) succinate in slices following spreading depolarization in physiolo

CoQ10 supplementation rescued delayed recovery from spreading depolarization. (A) Example trace of a spreading depolarization event in glucose deprivation. (B) Example trace of a spreading depolarization event in CoQ10 supplemented slice. (C) Latency was not impacted by CoQ10. (D) CoQ10 reduced the repolarization duration in glucose deprivation. (E) Spreading depolarization duration was also reduced by CoQ10 supplementation. Data are presented as mean ± SEM. SD: spreading depolarization. *p* < 0.01.

Summary of the main findings. Under physiological conditions, spreading depolarization is associated with an increase in intracellular Ca²⁺, enhanced mitochondrial activity, and upregulation of glycolysis and anaerobic glycolysis. In contrast, during metabolic substrate deficiency, the latency to spreading depolarization is shortened and repolarization is prolonged. Spreading depolarization under these conditions also leads to elevated intracellular Ca²⁺ without a corresponding increase in mitochondrial activity, accompanied by signs of metabolic dysfunction, including accumulation of metaboli

Workflow of experiments assessing physiological and metabolic measurements following spreading depolarization. aCSF; artificial cerebrospinal fluid, Ca^2+^; calcium, SD; spreading depolarization, GCMS; gas chromatography mass spectroscopy, MEA; multi-electrode array. Created using BioRender.

Full text

Introduction

Spreading depolarization is a slowly propagating wave of neuronal and glial depolarization that traverses the brain, triggering the release of neuropeptides and causing changes in cerebral blood flow^1–3^. Spreading depolarization has been observed in various neurological conditions, including ischemic stroke^4^, traumatic brain injury^4^, subarachnoid haemorrhage^5^, epilepsy^6^, and migraine aura^7^. Preclinical evidence also suggests that spreading depolarization can trigger downstream signalling events that contribute to nociception^8^.

During spreading depolarization, substantial ionic shifts occur including increased extracellular potassium (K^+^), intracellular influxes of sodium (Na^+^) and chloride (Cl^−^) and the release of glutamate, all of which are key in propagating the depolarization wave^9,10^. This is followed by a phase of inhibited electrical and synaptic activity. During spreading depolarization, a massive influx of calcium (Ca^2+^) into neurons and glia acts as a key intracellular signal, driving downstream processes such as neurotransmitter release, metabolic stress, and potential excitotoxicity^11^. Spreading depolarization is an energy-intensive phenomenon that significantly increases metabolic demands with rapid oxygen consumption, potentially causing localized hypoxia^12,13^. However, the metabolic consequences of spreading depolarization remain incompletely characterized. While healthy tissue may have the resilience to recover from spreading depolarization, pathological conditions with depleted metabolic substrates severely impair recovery capacity and are linked to poorer clinical outcomes^14,15^. Limited understanding of the specific metabolic pathways involved has hindered the success of nutraceutical strategies, emphasizing the need for further investigations.

Coenzyme Q10 (CoQ10) is in the internal membrane of mitochondria and acts as an electron carrier, shuttling electrons from complex I and II to complex III and thereby directly contributes towards adenosine triphosphate (ATP) production. CoQ10 is additionally a lipid soluble antioxidant which protects the mitochondria from the products of reactive oxygen species (ROS). CoQ10 has exhibited neuroprotective properties in animal models of neurological disorders which feature spreading depolarization, including stroke^16^, traumatic brain injury^17^, epilepsy^18^, subarachnoid haemorrhage^19^, migraine aura^1^. However, the neuroprotective properties of CoQ10 have not been characterised in the context of metabolic stress and spreading depolarization.

This study aimed to investigate the metabolic response to spreading depolarization using an ex vivo brain slice model. Specifically, our objectives were to evaluate intracellular Ca^2+^ signalling, characterize mitochondrial activity and assess alterations in central carbon metabolism. Profound metabolic stress is often a feature of the acute phase of critical illness due to stroke, traumatic brain injury, status epilepsy, and subarachnoid haemorrhage^20^. Hence, we also aimed to examine the effects of metabolic substrate deficiency on spreading depolarization by employing a glucose-depletion model. Additionally, we wanted to explore the effects of CoQ10 supplementation in the setting of metabolic substrate deficiency on spreading depolarization responses. These aims were driven by the translational significance of understanding spreading depolarization energetics, particularly in conditions such as ischemia, stroke, and traumatic brain injury, where compromised glucose supply may hinder cortical tissue recovery^21–23^.

Results

Electrophysiological measurements confirmed spreading depolarization in physiological conditions

We measured the spatial and temporal changes in extracellular field potentials using MEA recordings (Fig. 1A and B). Topical application of KCl elicited a classical spreading depolarization response, as evidenced by recordings from 59 electrodes that demonstrated a wave of depolarization propagating with a delay across the cortical surface (Fig. 1B). By assessing the characteristics of spreading depolarization (Fig. 1C), we observed spatial and temporal differences in the latency to depolarization, repolarization duration, and overall event duration across the cortical regions of the slice (Fig. 1D–F).

Fig. 1KCl-induced widespread depolarization and increased neuronal activity across cortical slices under physiological conditions. (A) Image showing the positioning of an acute brain slice on the MEA; black dots indicate recording electrodes. (B) Example traces showing voltage amplitude changes over time, with each trace representing a channel. (C) Example spreading depolarization event, demonstrating how parameters are calculated. Heat maps illustrate spreading depolarization (D) latency, (E) repolarization duration, and (F) spreading depolarization event duration. (G) Neuronal activity as assessed by mean change in Ca²⁺ fluorescence at baseline (control, black) and following KCl stimulation (spreading depolarization, magenta) (n = 8 mice). Solid lines represent the mean; dashed lines indicate SEM. p < 0.001; ***. SD: spreading depolarization; Mv: millivolts; REF: reference electrode.

Ca2+ activity was upregulated during spreading depolarization under physiological conditions

Using a validated model of brain slice spreading depolarization^24–26^, we measured Ca^2+^ transients to capture intracellular responses and signalling events during spreading depolarization. Intracellular Ca^2+^ activity was assessed in brain slices before and after KCl stimulation by measuring changes in Fluo-4-AM fluorescence. In physiological conditions at baseline there were minimal changes in Ca^2+^ fluorescence (Fig. 1G). Following induction of spreading depolarization, intracellular Ca^2+^ fluorescence significantly increased (mean AUC (SEM) control = 2.26 (0.21), spreading depolarization = 11.30 (0.30) p < 0.0001 n = 8 mice Fig. 1G).

The central carbon metabolism response to spreading depolarization under physiological conditions

Mitochondrial activity was assessed by measuring changes in RH-123 fluorescence in brain slices following induction of spreading depolarization. Under physiological, baseline conditions, no changes in mitochondrial activity were observed (Fig. 2A). However, KCl stimulation induced a significant increase in mitochondrial activity (mean AUC (SEM) baseline = 0.16 (0.02), spreading depolarization = 1.12 (0.09), n = 5 mice, p < 0.0001 Fig. 2A).

Fig. 2. Spreading depolarization increases mitochondrial activity and alters glycolysis-associated metabolites. (A) Mean change in mitochondrial activity (RH-123 fluorescence) in physiological conditions at baseline (control, black) and following KCl stimulation (spreading depolarization, magenta) (n = 5 mice). Concentrations of (B) lactate, (C) malate, and (D) pyruvate were altered following spreading depolarization (control: n = 10 slices; spreading depolarization: n = 13 slices). Solid lines represent the mean; dashed lines indicate SEM. SD; spreading depolarization. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Quantitative changes in central carbon metabolism were assessed using GC-MS following spreading depolarization. We measured alterations in metabolites associated with key metabolic pathways, including glycolysis and oxidative phosphorylation (Table 1). Notably, lactate was found to be increased (mean (SD) control = 148.52 (52.71) n = 10 slices, spreading depolarization = 457.16 (221.13) n = 13 slices, p = 0.0006 Fig. 2B), in addition to increased pyruvate (control = 3.09 (0.93), spreading depolarization = 3.98 (0.91), p = 0.0312 Fig. 2C) and malate (control = 8.01 (1.53), spreading depolarization = 11.89 (3.90), p = 0.001 Fig. 2D). Several other metabolites appeared to increase following spreading depolarization induction, but these trends did not reach significance (Table 1).

Table 1. Central carbon metabolites quantified at baseline and following spreading depolarization in physiological conditions.MetaboliteMean (SD) P Baselinen = 10Post-spreading depolarizationn = 13Alanine12.55 (5.41)19.22 (10.17)0.075Aspartate186.46 (59.66)205.40 (88.22)0.566Citrate3.58 (1.24)5.79 (3.47)0.069Fumarate4.83 (1.41)5.48 (1.75)0.353Glutamate372.87 (131.48)511.72 (234.31)0.109Glycine37.50 (20.57)47.42 (24.14)0.318Isoleucine0.85 (0.26)1.12 (0.44)0.107Lactate148.52 (52.71)457.16 (221.13)0.001 ***Leucine0.91 (0.35)1.39 (0.86)0.111Malate8.01 (1.53)11.89 (3.90)0.010 **Pyruvate3.09 (0.93)3.98 (0.91)0.032 *Succinate11.83 (4.30)16.17 (7.00)0.100SD; standard deviation. Significance determined by unpaired t test, * p < 0.05, ** p < 0.01, ***, p < 0.001.

Spreading depolarization was altered under metabolic substrate deficiency

We next investigated how depletion of metabolic substrates affected intracellular Ca²⁺ signalling in response to spreading depolarization. Prior to stimulation, the baseline Ca^2+^ activity was higher in slices in glucose deprivation compared to those under physiological conditions (mean AUC (SEM) physiological conditions = 2.26 (0.21) n = 8 mice, glucose deprived conditions = 4.81 (0.24) p < 0.0001 n = 7 mice Fig. 3A). Moreover, in the absence of glucose, we observed higher amplitude Ca²⁺ transients, as indicated by significantly increased intracellular Ca²⁺ fluorescence after KCl stimulation (mean AUC (SEM) physiological conditions = 36.15 (0.59) n = 11 mice, glucose deprived conditions = 45.19 (0.98) p < 0.0001 n = 11 mice Fig. 3B).Following KCl stimulation, the rate of Ca^2+^ fluorescence change (ΔF/F0), was significantly faster in glucose-deprived slices compared to those under physiological conditions (physiological conditions slope = 0.00073 n = 11 mice, glucose-deprivation slope = 0.00087 n = 11 mice, p < 0.0001 Fig. 3B). Moreover, the fluorescent Ca^2+^ signal continued to increase to a higher maximum ΔF/F0 in glucose-deprivation (mean (SEM) maximum F/F0 physiological conditions = 1.27 (0.02), glucose-deprivation = 1.32 (0.03)).

Fig. 3. Spreading depolarization characteristics differ between physiological conditions and glucose-deprivation. (A) Comparison of Ca^2+^ signalling as assessed by mean change in Ca²⁺ fluorescence between physiological conditions (magenta, n = 11 mice) and glucose deprivation (blue, n = 8 mice) at baseline and (B) following stimulation (physiological conditions, n = 11 mice, glucose deprivation, n = 7 mice). Solid lines represent the mean; dashed lines indicate SEM. (C) Example trace of a spreading depolarization event in physiological conditions. (D) Example trace of a spreading depolarization event in glucose deprived slice. (E) Latency to the spreading depolarization event was significantly lower in glucose-deprived slices. (F) Repolarization duration and spreading depolarization duration (G) was significantly longer in glucose-deprivation. * p < 0.05; *** p < 0.001.

We also explored how metabolic depletion affected the characteristics and energetics of spreading depolarization by comparing responses under physiological conditions (Fig. 3C) to those observed under restricted glucose availability (Fig. 3D). The latency to depolarization was significantly reduced in glucose-deprived slices (mean (SD) physiological conditions = 15.92s (9.52) n = 6 mice, glucose deprivation = 6.93s (4.48) n = 7 mice, p = 0.047 Fig. 3E), suggesting increased susceptibility to spreading depolarization, evidenced by a more rapid depolarization in the absence of glucose. It took longer for slices to recover, as evidenced by prolonged repolarization durations observed in the glucose-deprived slices (physiological conditions = 27.51s (8.71) n = 6 mice, glucose deprivation = 47.84s (15.23) n = 7 mice, p = 0.015 Fig. 3F). The spreading depolarization event duration was also significantly longer in glucose-deprived slices (physiological conditions 46.65s (14.86) n = 6 mice, glucose deprivation 81.07 (25.19) n = 7 mice, p = 0.014 Fig. 3G).

Glucose deprivation suppresses mitochondrial activity during spreading depolarization

We evaluated mitochondrial activity following spreading depolarization in physiological conditions compared to conditions of glucose-deprivation. Prior to stimulation, the baseline mitochondrial activity was higher in slices in glucose deprivation (mean AUC (SEM) physiological conditions = 18.07 (0.66) n = 5 mice, glucose deprived conditions = 20.61 (0.70) p = 0.009 n = 4 mice Fig. 4A). The increase in mitochondrial activity observed in physiological conditions was not observed in glucose-deprived brain slices (mean AUC RH-123 fluorescence (SEM) physiological conditions = 780.00 (22.2) n = 9 mice, glucose-deprivation = 20.4 (5.84) n = 8 mice p < 0.0001 Fig. 4B).

Fig. 4. Spreading depolarization induced changes in central carbon metabolism in glucose-deficient slices. (A) Mitochondrial activity as measured by change in RH-123 fluorescence between physiological conditions (magenta, n = 9 mice) and glucose-deprivation (blue, n = 8 mice) at baseline and (B) following stimulation (physiological conditions, n = 5 mice, glucose deprivation, n = 4 mice). Quantities of metabolites, including (C) alanine, (D) glutamate, (E) isoleucine, (F) lactate, (G) malate, (H) aspartate, (I) proline, and (J) succinate in slices following spreading depolarization in physiological conditions and glucose-deprivation. Data are presented as mean ± SEM. AU: arbitrary units. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Glucose deprivation alters central carbon metabolism after spreading depolarization

Metabolite profiles were compared following spreading depolarization under physiological and glucose-deprived conditions. The central carbon metabolism of brain slices differed markedly depending on glucose availability. Notably alanine, glutamate, isoleucine, lactate and malate were significantly lower in glucose deprivation compared to physiological conditions after spreading depolarization (Table 2; Fig. 4C-H). Aspartate, proline and succinate, however, were found to be significantly higher in glucose-deprived slices compared to physiological conditions (Table 2; Fig. 4I-J). Decreased levels of lactate, alanine, glutamate, isoleucine, and malate indicate suppressed glycolysis and impaired tricarboxylic acid (TCA) cycle activity. In contrast, elevated levels of succinate, aspartate, and proline may suggest mitochondrial stress, incomplete TCA cycling, and a compensatory reliance on amino acid metabolism.

Table 2. Central carbon metabolites quantified in slices following spreading depolarization in physiological conditions and glucose-deprivation.MetaboliteMean (SD) P Physiological conditionsn = 13Glucose-deprivationn = 13Alanine19.22 (10.17)6.83 (3.30)0.0003 ***Aspartate205.40 (88.22)285.35 (103.69)0.045 *Citrate5.79 (3.47)5.99 (3.92)0.894Fumarate5.48 (1.75)5.03 (1.54)0.498Glutamate511.72 (234.31)213.74 (78.99)0.0002 ***Glycine47.42 (24.14)32.88 (8.60)0.053Isoleucine1.12 (0.44)0.83 (0.19)0.042 *Lactate457.16 (221.13)98.34 (67.02)< 0.0001 ****Leucine1.39 (0.86)0.92 (0.33)0.079Malate11.89 (3.90)9.06 (2.32)0.050 *Pyruvate3.98 (0.91)3.81 (1.39)0.712Succinate16.17 (7.00)38.96 (14.12)< 0.0001 ****SD; standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Coenzyme Q10 prevents alterations in spreading depolarization responses under metabolic substrate deficiency

To address deficits induced by metabolic substrate deprivation, we supplemented glucose-deprived brain slices with CoQ10, a key component of the electron transport chain with antioxidant properties. We assessed spreading depolarization responses in slices in glucose deprivation (Fig. 5A), compared to those in glucose deprivation but supplemented with CoQ10 (Fig. 5B).

Fig. 5. CoQ10 supplementation rescued delayed recovery from spreading depolarization. (A) Example trace of a spreading depolarization event in glucose deprivation. (B) Example trace of a spreading depolarization event in CoQ10 supplemented slice. (C) Latency was not impacted by CoQ10. (D) CoQ10 reduced the repolarization duration in glucose deprivation. (E) Spreading depolarization duration was also reduced by CoQ10 supplementation. Data are presented as mean ± SEM. SD: spreading depolarization. **p < 0.01.

CoQ10 did not significantly impact latency (Fig. 5C), indicating supplementation did not influence susceptibility to spreading depolarization. However, repolarization duration was significantly reduced in CoQ10 supplementation compared to glucose deprivation (mean (SD) CoQ10 = 19.27s (5.27) n = 7 mice, glucose deprivation = 47.84s (15.23) n = 7 mice, p = 0.003, Fig. 5D), meaning slices receiving CoQ10 recovered quicker. The spreading depolarization duration was also reduced in slices supplemented with CoQ10 compared to those in glucose-deprivation (CoQ10 = 34.50s (11.25) n = 7 mice, glucose deprivation = 81.07s (25.19) n = 7 mice, p = 0.003 Fig. 5E).

Discussion

Spreading depolarization has been observed in conditions including ischemic stroke^4^, traumatic brain injury^4,27^, subarachnoid haemorrhage^5,6^, epilepsy^6^, and migraine aura^7^. We suggest that central carbon metabolism is abrogated after spreading depolarization and that metabolic pathways may play a role in recovery. We first characterized mitochondrial activity and central carbon metabolism following spreading depolarization under physiological conditions. We then assessed the effects of glucose deprivation on spreading depolarization dynamics to model metabolic substrate deficiency, which is often a feature of the acute phase of critical illness due to stroke, traumatic brain injury, status epilepsy, and subarachnoid haemorrhage^20^. We also tested whether CoQ10 supplementation, an electron carrier and antioxidant, could enhance recovery under metabolic stress.

Using an ex vivo brain slice model, we showed that spreading depolarization led to intracellular Ca^2+^ transients, boosts mitochondrial activity, and shifts central carbon metabolism toward glycolysis and anaerobic respiration (Fig. 6). Glucose deprivation reduced spreading depolarization onset latency and delayed recovery, with prolonged depolarization and repolarization durations. This was accompanied by reduced mitochondrial activity and metabolic disruption, including markers of stalled TCA cycle function.

Fig. 6. Summary of the main findings. Under physiological conditions, spreading depolarization is associated with an increase in intracellular Ca²⁺, enhanced mitochondrial activity, and upregulation of glycolysis and anaerobic glycolysis. In contrast, during metabolic substrate deficiency, the latency to spreading depolarization is shortened and repolarization is prolonged. Spreading depolarization under these conditions also leads to elevated intracellular Ca²⁺ without a corresponding increase in mitochondrial activity, accompanied by signs of metabolic dysfunction, including accumulation of metabolites indicative of impaired TCA cycle function. Treatment with CoQ10 significantly reduced both the duration of spreading depolarization and repolarization, suggesting improved metabolic recovery. Ca²⁺, calcium; ROS, reactive oxygen species. Created using BioRender.

To address these deficits, we supplemented glucose-deprived brain slices with CoQ10. CoQ10 significantly reduced spreading depolarization and repolarization durations, indicating improved recovery. These findings suggest that spreading depolarization induces profound metabolic stress, particularly under energy substrate deficiency, and mitochondrial therapies like CoQ10 can support recovery from spreading depolarization.

Spreading depolarization was observed in glucose-deficient slices; but with significantly shorter latency compared to physiological conditions, indicating faster tissue depolarization. In a cortical neuron model, oxygen-glucose deprivation impaired mitochondrial ATP production, disrupting Na⁺/K⁺-ATPase function and accelerating neuronal depolarization^28^. Therefore, in the absence of glucose, the compromised energy state of the tissue may have lowered the threshold for initiating spreading depolarization, accounting for the reduced latency observed in our model.

Glucose deficiency significantly prolonged both spreading depolarization and repolarization, likely due to limited energy availability impairing restoration of ionic gradients. Prolonged depolarized states have been described in hippocampal slices exposed to oxygen-glucose deprivation, where neurons fail to repolarize effectively^29^. This failure is often associated with a sustained loss of membrane potential^29,30^. Together, these findings suggest that insufficient metabolic support exacerbates the vulnerability of neural tissue to spreading depolarization by impairing recovery and prolonging depolarization.

Under physiological conditions, brain slices demonstrated typical Ca²⁺ transients following stimulation. Spreading depolarization triggers intracellular Ca²⁺ signalling in both neurons and glia^31,32^, making Ca²⁺ a marker of widespread activation. While Fluo-4 AM is commonly used for astrocytic Ca²⁺ imaging, it additionally captures electrically evoked neuronal signalling and activity in brain slices^33^. While specific cell types were not identified in this study, the Ca²⁺ transients reflect broad intracellular network signalling.

Under glucose-deprived conditions, brain slices demonstrated an increased Ca²⁺ fluorescent signal compared to physiological conditions. These results align with oxygen-glucose deprivation models, where Ca²⁺ rapidly accumulates due of NMDA receptor overactivation, impaired mitochondrial buffering, and ATP-dependent Na⁺/Ca²⁺ exchanger failure^28^. Therefore, our results may indicate compromised cell health and stress due to loss of ionic regulation or excitotoxicity due to the lack of metabolic substrates. Heightened Ca²⁺ observed in our model may contribute to reduced latency to depolarization, as elevated Ca²⁺ can lower the threshold for neuronal excitability and accelerate the onset of spreading depolarization^32^, potentially due to this stressed state. Ca²⁺ and redox signalling interact bidirectionally to regulate cellular homeostasis, however under metabolic stress this can lead to excitotoxicity and oxidative damage^34^. The amplified Ca²⁺ signal observed in glucose-deprived slices may reflect early dysregulation, consistent with observations in ischaemic models^35,36^.

Under physiological conditions, spreading depolarization increased mitochondrial activity, indicating that adequate substrates allow upregulation of oxidative phosphorylation to meet elevated energy demands^37,38^. Under metabolic substrate deficiency, mitochondrial activity failed to rise after spreading depolarization, reflecting impaired oxidative phosphorylation due to lack of Krebs cycle donors. This mitochondrial failure may reflect severe stress or loss of electrochemical gradient maintenance. In glucose deprivation, increased Ca²⁺ influx may worsen dysfunction, as mitochondrial Ca²⁺ overload impairs function and promotes cell death in ischemia and brain injury models^39,40^. Clinical studies further support the relevance of mitochondrial health, with mitochondrial DNA copy number correlating with baseline severity and prognosis in stroke patients^41^. These findings may suggest impaired mitochondrial responses to spreading depolarization under metabolic stress may contribute to poor neurological outcomes.

We measured changes in metabolites following spreading depolarization under physiological conditions and found increases in pyruvate, lactate and malate. Pyruvate is a product of glycolysis, correlating with findings of increased glycolytic activity during spreading depolarization^42,43^. Lactate, an anaerobic glycolytic byproduct, was also elevated, consistent with rodent^42^ and human studies^44^ showing rapid glucose utilization and lactate accumulation following stimulation. In vivo, spreading depolarization can trigger neurovascular changes that limit oxygen supply to metabolically active tissue, causing hypoxia^13,37^. This may cause a shift in metabolism toward anaerobic respiration, evidenced by our findings of increased pyruvate and lactate. Moreover, in conditions of metabolic stress, neuronal tissue may resort to lactate as an alternative energy substrate^45^.

In glucose-deprived slices, spreading depolarization led to significant alterations in metabolic profiles. Notably alanine, a metabolite involved in antioxidant defence^46,47^, was significantly reduced, suggesting a diminished capacity to counteract ROS. Succinate concentrations were markedly elevated, supporting increased oxidative stress, as succinate accumulation is a recognised marker of ischemia^48^ and driver of mitochondrial ROS during reperfusion^49^. While ROS were not directly measured, decreased alanine and increased succinate suggest elevated ROS in glucose-deficient tissue. We observed reduced glutamate in glucose-deprived slices. This suggests a compensatory mechanism, switching to the use of glutamate as an alternative TCA cycle substrate when glucose is unavailable, a mechanism previously described under metabolic stress in brain slices^50^. This metabolic shift could also serve a protective role by limiting excitotoxic accumulation of extracellular glutamate.

These findings suggest that without glucose, brain slices undergo metabolic reprogramming during spreading depolarization, shifting to alternative, less efficient pathways aimed at maintaining cellular function under metabolic stress. This is characterised by impaired mitochondrial responsiveness, compromised antioxidant defences, and potential use of alternative substrates to sustain oxidative metabolism.

Due to impaired mitochondrial function in glucose-deprived slices, we tested whether CoQ10 could aid recovery. As a mitochondrial electron carrier and antioxidant, CoQ10 enhances complex II activity, supports ATP production from non-glucose substrates^51^, stabilizes membranes^52^, and reduces oxidative injury in stroke and brain injury^17,53^.

In our model, CoQ10 supplementation significantly reduced spreading depolarization duration and repolarization duration in glucose-deprived slices, indicating improved recovery dynamics. CoQ10 did not alter the latency to depolarization, suggesting it did not affect the initial threshold for depolarization onset. These findings suggest that while CoQ10 does not prevent the initiation of spreading depolarization under metabolic stress but may enhance the tissue’s ability to recover from the depolarized state. Future experiments examining mitochondrial activity and metabolic pathway alterations in the presence of CoQ10 following spreading depolarization would clarify whether its effects are mediated by restored mitochondrial function and reduced oxidative burden. These initial findings support the potential of CoQ10 as a therapeutic strategy to improve recovery following spreading depolarization in metabolically compromised brain tissue.

Limitations

Although total glucose deprivation is not physiologically representative, it provides a useful model to investigate spreading depolarization under severe metabolic stress and yields key proof-of-concept insights. A model such as oxygen-glucose deprivation, for instance, may have resulted in spontaneous spreading depolarization rather than evoked. It is also important to note that this study employed both male and female animals to create acute brain slices. Recent evidence has highlighted sex-related differences in spreading depolarization, particularly demonstrating increased susceptibility in females^54,55^. However, there were no overt differences found in metabolic responses between slices from sexes within our study. Finally, this investigation focused exclusively on CoQ10 supplementation as a mitochondrial-targeted intervention. Our ex vivo brain slice model does not fully capture the translational context of coenzyme Q10 administration, which in clinical settings involves repeated systemic dosing that may influence central metabolism over time. Future studies would involve administration in vivo models and should explore a broader range of metabolic therapies to fully assess their potential in mitigating metabolic stress associated with spreading depolarization.

Conclusions

In summary, our findings demonstrate that spreading depolarization imposes substantial metabolic stress, disrupting mitochondrial function and central carbon metabolism (Fig. 6). Under physiological conditions, increased mitochondrial activity and glycolysis compensate for this demand, but without glucose, this capacity is lost, causing earlier onset and prolonged recovery due to impaired ATP production and oxidative stress. CoQ10 supplementation significantly improved repolarization dynamics (Fig. 6), highlighting its ability to support spreading depolarization recovery in energy-compromised brain states. These results underscore the importance of intact central carbon metabolism for neuronal recovery and suggest a therapeutic avenue for conditions where metabolic substrate availability is limited.

Materials and methods

Animal husbandry

Thy1-ChR2-YFP mice (B6.Cg-Tg(Thy1-COP4/EYFP)18Gfng/J, Jackson Laboratories) of both sexes, aged 9–20 weeks and weighing 20–30 g were used for these studies. Both wild-type and transgenic mice (expressing CHR2-YFP) were used, although the CHR2-YFP transgene was not activated in this study. Importantly, the presence of CHR2-YFP did not influence the observed metabolic outcomes.

Mice were housed in sex and litter matched groups in standard individually ventilated cages, at 22 °C with a 12:12 light/dark cycle, with standard rodent chow (EURodent Diet 14%, Labdiet). All animal handling and procedures were performed according to the guidelines specified by the UK Home Office and Animals (Scientific Procedures) Act 1986 and approved by the University Animal Welfare and Ethical Review Body (P9BA52278 and P70/8516). Animal studies are reported in compliance with ARRIVE 2.0 guidelines.

Acute brain slice preparation

Animals were euthanized by cervical dislocation without anaesthetic use. The brain was removed and placed in ice cold modified artificial cerebrospinal fluid (aCSF containing (mM); NaHCO_3_ (26), NaH_2_PO_4_ (1.25), NaCl (125), KCl (3), CaCl_2_ (2), glucose (10), while aCSF used for slicing also contained MgCl_2_ (5). Coronal slices of 350 μm thickness were generated with a vibratome (Campden Instruments, 7000SMZ-2 Vibratome) using a vibration frequency 1.25 and amplitude 80 Hz. Slices were collected in the vibratome tissue bath in ice-cold aCSF bubbled with 95% O_2_ and 5% CO_2_. Slices were then immediately transferred to a submerged slice recovery chamber (tissue slice recovery chamber Model 7470, Campden Instruments) maintained at 35 °C in aCSF with an integrated gas bubbler supplying 95% O_2_ and 5% CO_2_. Slices were allowed to recover for 1 h before proceeding (Fig. 7).

Fig. 7. Workflow of experiments assessing physiological and metabolic measurements following spreading depolarization. aCSF; artificial cerebrospinal fluid, Ca^2+^; calcium, SD; spreading depolarization, GCMS; gas chromatography mass spectroscopy, MEA; multi-electrode array. Created using BioRender.

Incubation conditions of brain slices

To assess how metabolic substrate deficiency influences the metabolic consequences of spreading depolarization, brain slices were initially incubated for 1 h in aCSF containing 10 mM glucose (physiological conditions) immediately after preparation. They were then incubated for an additional hour under physiological conditions (aCSF 10 mM glucose) or switched to glucose-deprived conditions (aCSF 0 mM glucose) (Fig. 1).

To investigate the potential protective effects of CoQ10 supplementation under glucose-deprived conditions, a separate group of slices underwent the same initial 1-hour incubation in aCSF 10 mM glucose, followed by a 1-hour incubation in aCSF 0mM glucose containing 100 µM CoQ10. This concentration was selected based on prior evidence of its neuroprotective efficacy in ex vivo models^56^.

Induction of spreading depolarization

Spreading depolarization induction was accomplished by topical application of 1µL KCl (2 M) to the cortical region of brain slices, a commonly used method in both ex vivo and in vivo animal models^6,57,58^.

Multi electrode array recording of neuronal activity and spreading depolarization

A multi-electrode array (MEA) (MEA2100-system (Multi Channel Systems, Reutlingen, Germany) was used to record spreading depolarization in brain slices. The MEA chamber (60MEA200/30iR-Ti, Multi Channel Systems GmbH, Reutlingen, Germany) is composed of 59 electrodes and 1 internal reference electrode. The brain slice was transferred to the MEA chamber perfused with oxygenated aCSF at a rate of 2-3 ml/min using a peristaltic perfusion system (Multichannel Systems) and maintained at 35 °C via a heated perfusion cannula (Multi Channel Systems) for 10 min before recording. Data was collected using the Multi Channel Experimenter software (Multi Channel Systems) at a sampling rate of 1 kHz. Baseline activity was recorded for a minimum of 30 s before 1uL KCl (2 M) was added to the cortical edge of the slice using a P2 pipette, following which activity was measured for 10 min. Data were filtered using the Multichannel Systems Filter Config software (Multi Channel Systems) without high pass filtering to allow for direct current recordings. Data was collected using the Multi Channel Experimenter software (Multi Channel Systems).

To analyse spreading depolarization parameters, recordings were converted to CED files using the Data Manager application (Multi Channel Systems), allowing them to be analysed in Spike2 software (Cambridge Electronic Design). Spreading depolarization parameters included latency (time, seconds between KCl application and depolarization induction) and repolarization duration (time, seconds between most negative apex and return to baseline) as previously described^59^. The duration of the entire depolarization event (time, seconds from depolarization initiation to return to baseline) was also calculated.

Fluo-4-AM fluorescent calcium indicator

Fluo-4-AM (Invitrogen™), a fluorescent Ca^2+^ indicator, was used to measure intracellular Ca^2+^ flux as a proxy for neuronal activity during spreading depolarization. Fluo-4-AM was reconstituted with pluronic acid (Pluronic™ F-127 Invitrogen™) to aid entry into cells. Slices were incubated in 2.5µM/ml Fluo-4-AM for 30 min in oxygenated aCSF (35 °C, 95% O_2_ 5% CO_2_). Slices were then washed in fresh warmed aCSF and transferred to a 35 mm imaging dish (MatTek, P35GC-0-14-C). Slices were imaged using an inverted microscope (Zeiss Axiovert200) equipped with a differential spinning disk module (Andor Technology, DSD Revolution) and a 10 × 0.25NA dry objective and images captured with a monochrome CCD camera (Andor Technologies). Time-lapse images were captured at 2 Hz. Following a baseline of 200 frames, KCl was added at the cortical edge and imaging was repeated for 400 frames. ImageJ Fiji was used to analyse signals^60^. Ten regions of interest were manually selected approximately 200 μm from the slice edge, and the fluorescent intensity was averaged at each frame. The first five frames of both control and spreading depolarization recordings were averaged to calculate a baseline fluorescence (F0), and F/F0 was calculated to represent the change in fluorescence over time.

Rhodamine-123 mitochondrial potential dye

Rhodamine-123 (RH-123, Sigma) is a cell permeant fluorescent dye that is sequestered into active mitochondria. Brain slices were incubated in 1µM RH-123 in aCSF (35 °C, 95% O_2_ 5% CO_2_) for 15 min then washed three times in fresh aCSF. A single baseline image was taken, following which KCl was added to the cortical edge. Changes in fluorescence were recorded in slices using a Zeiss LSM780 Confocal Microscope using a 10 × 0.45NA, Water Immersed C-ACHROMAT Lens. A stack of 230 images were captured with one frame per 3.87s. ImageJ Fiji was used to analyse image stacks^60^. Ten regions of interest were manually selected approximately 200 μm from the area of stimulation. The fluorescent intensity of all ten regions of interest were averaged at each frame. The mean of the first three frames was calculated to provide a baseline fluorescence (F0), and F/F0 was calculated to represent the change in fluorescence over time compared to baseline.

Gas-chromatography mass spectroscopy

Sample preparation

Following Ca^2+^ imaging, brain slices were snap frozen in liquid nitrogen and stored at -80 °C. Approximately 30-40 mg of pulverized tissue was weighed out for metabolomic analysis, sometimes necessitating the combination of multiple brain slices from the same animal to meet this minimum weight requirement. High-performance liquid chromatography (HPLC) grade methanol, 500 µl at -20 °C, was added to quench metabolism, followed by 200 µl 2.5 µg/ml glutaric acid in dH_2_O as an internal standard. Samples were then homogenised at -20 °C using a TissueLyser II (QIAGEN), then centrifuged at maximum speed for five minutes and the supernatant moved to glass tubes. The remaining tissue pellet was dried overnight and weighed for result normalisation.

Protein contaminants were precipitated by adding 1.4 ml 2:1 acetone: isopropanol solution at -20 °C, then centrifuged to pellet protein. Supernatant was transferred to a fresh glass tube, and 1 ml HPLC grade water with 500 µl chloroform at -20 °C was added to aid separation of non-polar metabolites. Samples were agitated and centrifuged at 4000 rpm for five minutes to separate fractions. 200µL of the polar fraction was transferred to a new tube and evaporated to dryness using a SpeedVac (ThermoFisher Scientific), then stored at -80 °C.

Chemical derivatisation and data acquisition

Dried extracts were derivatized using a two-step protocol. Samples were first treated with 2% methoxamine in pyridine (40µL 1 h at 60 °C), followed by addition of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide, with 1% tert-butyldimethylchlorosilan (50µL, 1 h at 60 °C). Samples were transferred to glass vials for Gas-Chromatography Mass Spectroscopy (GC-MS) analysis using an Agilent 8890 GC and 5977B MSD system. 1µL of sample was injected in splitless mode with helium carrier gas at a rate of 1.0 mL.min − 1. Initial GC oven temperature was held at 100 °C for 1 min before ramping to 160 °C at a rate of 10 °C.min − 1, followed by a ramp to 200 °C at a rate of 5 °C.min − 1 and a final ramp to 320 °C at a rate of 10 °C.min − 1 with a five minute hold. Compound detection was carried out in scan mode. Total ion counts of each metabolite were normalized to the internal standard D6-Glutaric acid.

Experimental design and statistical analysis

All statistical analysis and graphs were made using Graphpad Prism (GraphPad Software V8, USA). The normality of data was assessed using the Shapiro-Wilk test. Data that were normally distributed were analysed using parametric tests (t tests) and reported as (mean and standard deviation (SD)). Non-normally distributed data were analysed using non-parametric tests (Mann-Whitney test) and reported as [median and (range)]. To compare changes in fluorescent Ca^2+^ and mitochondrial activity, the area under the curve (AUC) was calculated and compared between conditions using unpaired two tailed t test. Results were considered statistically significant when p values were * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.