Cerebrospinal fluid purinomics as a biomarker approach to predict outcome after severe traumatic brain injury.

Severe traumatic brain injury (TBI) is associated with high rates of mortality and long-term disability linked to neurochemical abnormalities. Although purine derivatives play important roles in TBI pathogenesis in preclinical models, little is known about potential changes in purine levels and their implications in human TBI. We assessed cerebrospinal fluid (CSF) levels of purines in severe TBI patients as potential biomarkers that predict mortality and long-term dysfunction. This was a cross-sectional study performed in 17 severe TBI patients (Glasgow Coma Scale <8) and 51 controls. Two to 4 h after admission to ICU, patients were submitted to ventricular drainage and CSF collection for quantification of adenine and guanine purine derivatives by HPLC. TBI patients' survival was followed up to 3 days from admission. A neurofunctional assessment was performed through the modified Rankin Scale (mRS) 2 years after ICU admission. Purine levels were compared between control and TBI patients, and between surviving and non-surviving patients. Relative to controls, TBI patients presented increased CSF levels of GDP, guanosine, adenosine, inosine, hypoxanthine, and xanthine. Further, GTP, GDP, IMP, and xanthine levels were different between surviving and non-surviving patients. Among the purines, guanosine was associated with improved mRS (p = 0.042; r = -0.506). Remarkably, GTP displayed predictive value (AUC = 0.841, p = 0.024) for discriminating survival versus non-survival patients up to 3 days from admission. These results support TBI-specific purine signatures, suggesting GTP as a promising biomarker of mortality and guanosine as an indicator of long-term functional disability.

Glutamate transporter-1 link astrocytes with heightened aggressive behavior induced by steroid abuse in male CF1 mice.

The astrocytic glutamate transporter GLT-1 performs glutamate uptake thereby mediating NMDAr responses in neurons. Ceftriaxone (CEF) upregulates astrocytic GLT-1 expression/activity, which could counteract excessive glutamate levels and aggressive behavior induced by anabolic synthetic steroids such as nandrolone decanoate (ND). Here, adult male CF-1 mice were allocated to oil (VEH), ND, CEF, and ND/CEF groups. Mice were subcutaneously (s.c.) injected with ND (15 mg/kg) or VEH for 19 days, and received intraperitoneal (i.p.) injections of CEF (200 mg/kg) or saline for 5 days. The ND/CEF group received ND for 19 days plus coadministration of CEF in the last 5 days. On the 19th day, the aggressive phenotypes were evaluated through the resident-intruder test. After 24 h, cerebrospinal fluid was collected to measure glutamate levels, and the pre-frontal cortex was used to assess GLT-1, pGluN2BTyr1472, and pGluN2ATyr1246 by Western blot. Synaptosomes from the left brain hemisphere was used to evaluate mitochondrial function including complex II-succinate dehydrogenase (SDH), Ca2+ handling, membrane potential (ΔÑ°m), and H2O2 production. ND decreased the latency for the first attack and increased the number of attacks by the resident mice against the intruder, mechanistically associated with an increase in glutamate levels and pGluN2BTyr1472 but not pGluN2ATyr1244, and GLT-1 downregulation. The abnormalities in mitochondrial Ca2+ influx, SDH, ΔÑ°m, and H2O2 implies in deficient energy support to the synaptic machinery. The ND/CEF group displayed a decreased aggressive behavior, normalization of glutamate and pGluN2BTyr1472levels, and mitochondrial function at synaptic terminals. In conclusion, the pharmacological modulation of GLT-1 highlights its relevance as an astrocytic target against highly impulsive and aggressive phenotypes.

SHORT-TERM INFLAMMATORY BIOMARKER PROFILES ARE ASSOCIATED WITH DEFICIENT MITOCHONDRIAL BIOENERGETICS IN LYMPHOCYTES OF SEPTIC SHOCK PATIENTS-A PROSPECTIVE COHORT STUDY.

ABSTRACT: Introduction: A biomarker strategy based on the quantification of an immune profile could provide a clinical understanding of the inflammatory state in patients with sepsis and its potential implications for the bioenergetic state of lymphocytes, whose metabolism is associated with altered outcomes in sepsis. The objective of this study is to investigate the association between mitochondrial respiratory states and inflammatory biomarkers in patients with septic shock. Methods: This prospective cohort study included patients with septic shock. Routine, complex I, complex II respiration, and biochemical coupling efficiency were measured to evaluate mitochondrial activity. We measured IL-1ß, IL-6, IL-10, total lymphocyte count, and C-reactive protein levels on days 1 and 3 of septic shock management as well as mitochondrial variables. The variability of these measurements was evaluated using delta counts (days 3-1 counts). Results: Sixty-four patients were included in this analysis. There was a negative correlation between complex II respiration and IL-1ß (Spearman ρ, -0.275; P = 0.028). Biochemical coupling efficiency at day 1 was negative correlated with IL-6: Spearman ρ, -0.247; P = 0.05. Delta complex II respiration was negatively correlated with delta IL-6 (Spearman ρ, -0.261; P = 0.042). Delta complex I respiration was negatively correlated with delta IL-6 (Spearman ρ, -0.346; P = 0.006), and delta routine respiration was also negatively correlated with both delta IL-10 (Spearman ρ, -0.257; P = 0.046) and delta IL-6 (Spearman ρ, -0.32; P = 0.012). Conclusions: The metabolic change observed in mitochondrial complex I and complex II of lymphocytes is associated with a decrease in IL-6 levels, which can signal a decrease in global inflammatory activity.

Lower and higher volumes of physical exercise build up brain reserves against memory deficits triggered by a head injury in mice.

Decreasing neurotrophic support and impaired mitochondrial bioenergetics are key mechanisms for long-term neurodegeneration and cognitive decline after traumatic brain injury (TBI). We hypothesize that preconditioning with lower and higher volumes of physical exercise upregulates the CREB-BDNF axis and bioenergetic capability, which might serve as neural reserves against cognitive impairment after severe TBI. Using a running wheel mounted in the home cage, mice were engaged in lower (LV, 48 h free access, and 48 h locked) and higher (HV, daily free access) exercise volumes for thirty days. Subsequently, LV and HV mice remained for additional thirty days in the home cage with the running wheel locked and were euthanized. The sedentary group had the running wheel always locked. For the same type of exercise stimulus in a given time, daily workout presents higher volume than alternate days workout. The total distance ran in the wheel was the reference parameter to confirm distinct exercise volumes. On average, LV exercise ran 27.522 m and HV exercise ran 52.076 m. Primarily, we investigate whether LV and HV protocols increase neurotrophic and bioenergetic support in the hippocampus thirty days after exercise ceased. Regardless of volume, exercise increased hippocampal pCREBSer133-CREB-proBDNF-BDNF signaling and mitochondrial coupling efficiency, excess capacity, and leak control, that may compose the neurobiological basis for neural reserves. Further, we challenge these neural reserves against secondary memory deficits triggered by a severe TBI. After thirty days of exercise LV and HV, and sedentary (SED) mice were submitted to the CCI model. Mice remained for additional thirty days in the home cage with the running wheel locked. The mortality after severe TBI was approximately 20% in LV and HV, while in the SED was 40%. Also, LV and HV exercise sustained hippocampal pCREBSer133-CREB-proBDNF-BDNF signaling, mitochondrial coupling efficiency, excess capacity, and leak control for thirty days after severe TBI. Corroborating these benefits, the mitochondrial H2O2 production linked to complexes I and II was attenuated by exercise regardless of the volume. These adaptations attenuated spatial learning and memory deficits caused by TBI. In summary, preconditioning with LV and HV exercise builds up long-lasting CREB-BDNF and bioenergetic neural reserves that preserve memory fitness after severe TBI.

Association Between Hyperlactatemia, Perfusional Parameters, and Lymphocyte Mitochondrial Dysfunction in Septic Shock Patients.

INTRODUCTION: In septic shock, mitochondrial dysfunction, and hypoperfusion are the main triggers of multi-organ failure. Little is known about the crosstalk between mitochondrial dysfunction and hemodynamic alterations, especially in the post-resuscitation phase. Here, we assess whether hypoperfusion and lactate levels are associated with oxygen consumption linked to mitochondrial bioenergetic activity in lymphocytes of patients admitted with septic shock. PATIENTS AND METHODS: Prospective cohort study in patients with septic shock defined as the requirement of vasopressors to maintain a mean arterial pressure 65 mm Hg after initial fluid administration. Basal mitochondrial and Complex I respiration was measured to evaluate mitochondrial activity. Both variables and capillary refill time were compared with arterial lactate post-fluid resuscitation. We also compared mitochondrial activity measurements between patients with and without hypoperfusion status. RESULTS: A total of 90 patients were included in analysis. The median arterial lactate at the time of septic shock diagnosis was 2.0 mmol/Dl (IQR 1.3-3.0). Baseline respiration at the time of septic shock diagnosis was correlated with lactate (Spearman -0.388, 95% CI -0.4893 to -0.1021; P = 0.003), as well as Complex I respiration (Spearman -0.403, 95% CI -0.567 to -0.208; P < 0.001). Patients with hypoperfusion status had no difference in basal respiration when compared with patients who did not have hypoperfusion status (P = 0.22) nor in Complex I respiration (P = 0.09). CONCLUSION: Changes in lymphocytic mitochondrial metabolism are associated with post-resuscitation arterial lactate in septic shock; however, they are not associated with the presence of a hypoperfusional status. In this scenario, it is therefore suggested that systemic perfusion and mitochondrial metabolism have different courses.

Antibiotic therapy does not alter mitochondrial bioenergetics in lymphocytes of patients with septic shock - A prospective cohort study.

Antibiotics may trigger alterations in mitochondrial function, which has been explored in cells culture, and in animal model of sepsis. This study sought to evaluate whether antibiotic therapy affects mitochondrial bioenergetics in a 68-patients clinical study. We studied mitochondrial respiratory rates at two time points: the first day of antibiotic administration and three days after. The Δbasal, ΔCI, ΔCII respiration, and ΔBCE respiratory rates were not different between patients administered with polymyxin, vancomycin, amoxicillin-clavulanate, and azithromycin compared to those who were not administered. Specific beta-lactams are associated with specific modifications in mitochondrial respiratory endpoints - patients who used meropenem had higher delta C2 values compared to those who did not (p = 0.03). Patients who used piperacillin-tazobactam had lower delta C1 (p = 0.03) values than those who did not, but higher delta C2 values (p = 0.02). These mitochondrial metabolic signatures in isolated lymphocytes challenges the proposed effects of antibiotics in mitochondrial bioenergetics of cell cultures, but at current status have an uncertain clinical significance.

Mortality of septic shock patients is associated with impaired mitochondrial oxidative coupling efficiency in lymphocytes: a prospective cohort study.

BACKGROUND: Septic shock is a life-threatening condition that challenges immune cells to reprogram their mitochondrial metabolism towards to increase ATP synthesis for building an appropriate immunity. This could print metabolic signatures in mitochondria whose association with disease progression and clinical outcomes remain elusive. METHOD: This is a single-center prospective cohort study performed in the ICU of one tertiary referral hospital in Brazil. Between November 2017 and July 2018, 90 consecutive patients, aged 18 years or older, admitted to the ICU with septic shock were enrolled. Seventy-five patients had Simplified Acute Physiology Score (SAPS 3) assessed at admission, and Sequential Organ Failure Assessment (SOFA) assessed on the first (D1) and third (D3) days after admission. Mitochondrial respiration linked to complexes I, II, V, and biochemical coupling efficiency (BCE) were assessed at D1 and D3 and Δ (D3-D1) in isolated lymphocytes. Clinical and mitochondrial endpoints were used to dichotomize the survival and death outcomes. Our primary outcome was 6-month mortality, and secondary outcomes were ICU and hospital ward mortality. RESULTS: The mean SAPS 3 and SOFA scores at septic shock diagnosis were 75.8 (± 12.9) and 8 (± 3) points, respectively. The cumulative ICU, hospital ward, and 6-month mortality were 32 (45%), 43 (57%), and 50 (66%), respectively. At the ICU, non-surviving patients presented elevated arterial lactate (2.8 mmol/L, IQR, 2-4), C-reactive protein (220 mg/L, IQR, 119-284), and capillary refill time (5.5 s, IQR, 3-8). Respiratory rates linked to CII at D1 and D3, and ΔCII were decreased in non-surviving patients. Also, the BCE at D1 and D3 and the ΔBCE discriminated patients who would evolve to death in the ICU, hospital ward, and 6 months after admission. After adjusting for possible confounders, the ΔBCE value but not SOFA scores was independently associated with 6-month mortality (RR 0.38, CI 95% 0.18-0.78; P = 0.009). At a cut-off of - 0.002, ΔBCE displayed 100% sensitivity and 73% specificity for predicting 6-month mortality CONCLUSIONS: The ΔBCE signature in lymphocytes provided an earlier recognition of septic shock patients in the ICU at risk of long-term deterioration of health status.

Intermittent fasting promotes anxiolytic-like effects unrelated to synaptic mitochondrial function and BDNF support.

Anxiety disorders are linked to mitochondrial dysfunction and decreased neurotrophic support. Since anxiolytic drugs target mitochondria, non-pharmacological approaches to improve mitochondrial metabolism such as intermittent fasting (IF) may cause parallel behavioral benefits against anxiety disorders. Here, we investigated whether a chronic IF regimen could induce anxiolytic-like effects concomitantly to modulation in mitochondrial bioenergetics and trophic signaling in mice brain. A total of 44 Male C57BL/6 J mice (180 days old) were assigned to two dietary regimens: a normal, ad libitum diet (AL group) and an alternate-day fasting (IF group), where animals underwent 10 cycles of 24 h food restriction followed by 24 h ad libitum access. Animals underwent the open field test, dark/light box and elevated plus maze tasks. Isolated nerve terminals were obtained from mice brain and used for mitochondrial respirometry, hydrogen peroxide production and assessment of membrane potential dynamics, calcium handling and western blotting. We showed that IF significantly alters total daily food intake and food consumption patterns but not body weight. There were no differences in the exploratory and locomotory parameters. Remarkably, animals from IF showed decreased anxiety-like behavior. Mitochondrial metabolic responses in different coupling states and parameters linked with H2O2 production, Ca2+ buffering and electric gradient were not different between groups. Finally, no alterations in molecular indicators of apoptotic death (Bax/Bcl-2 ratio) and neuroplasticity (proBDNF/BDNF and synaptophysin were observed). In conclusion, IF exerts anxiolytic-like effect not associated with modulation in synaptic neuronergetics or expression of neurotrophic proteins. These results highlight a potential benefit of intermittent fasting as a nutritional intervention in anxiety-related disorders.

Anabolic-androgenic steroids impair mitochondrial function and redox status in the heart and liver of mice.

Supraphysiological doses of anabolic-androgenic steroids (AAS) may cause long-term functional abnormalities, particularly in the heart and liver, which may only represent the later-stage of the cumulative damage caused by dysfunctional organelles. We investigated whether mid-term supraphysiological doses of Testosterone and Nandrolone impair mitochondrial Ca2+ and membrane potential (ΔΨm) dynamics, and redox machinery in the heart and liver of mice. CF1 albino mice were treated daily with 15 mg/kg of Nandrolone (ND) or Testosterone (T), or oil (vehicle) for 19 days. Preparations enriched in mitochondria from the heart or liver were used to perform assays of Ca2+ influx/efflux, ΔΨm, and H2O2 production. ND significantly impaired mitochondrial Ca2+ influx in the heart, and ΔΨm in both organs. ND and T increased H2O2 levels in the heart and liver relative to controls. Also, ND increased oxidative damage to lipids and proteins (TBARS and carbonyls) in the heart, and both AAS decreased glutathione peroxidase activity in the heart and liver. In summary, supraphysiological doses of ND, and in a lesser extend T, impaired mitochondrial Ca2+ influx and ΔΨm, and redox homeostasis being early mechanistic substrates for inducing heart and liver tissue damage.

Anabolic-androgen steroids effects on bioenergetics responsiveness of synaptic and extrasynaptic mitochondria.

We hypothesized that supraphysiological administration of the anabolic-androgenic steroids (AAS) like testosterone (TEST) and nandrolone decanoate (NAND) might differentially affect synaptic and extrasynaptic components of mitochondrial bioenergetics, thereby resulting in memory impairment. Oil (VEH), NAND or TEST (15 mg/Kg) were daily administered to male CF-1 albino mice for 19-days. We evaluated in the synaptosomes and extrasynaptic mitochondria, Ca2+ influx/efflux, membrane potential ΔÑ°m, oxidative respiratory states, dehydrogenases activity, H2O2 production, Tau phosphorylation, and spatial memory in the Morris water maze (MWM). In synaptosomes, both AAS increased Ca2+ influx and Na+ dependent efflux. In extrasynaptic mitochondria, NAND increased the Ca2+ influx. NAND prominently impaired ΔÑ°m formation and dissipation in synaptosomal and extrasynaptic mitochondria, while the effect of TEST was less pronounced. TEST increased the Reserve Respiratory Capacity in synaptosomes, and NAND decreased dehydrogenases activity in synaptic and extrasynaptic mitochondria. Also, NAND increased H2O2 production by synaptosomes and extrasynaptic mitochondria. NAND increased pTauSer396 in synaptosomes. Both AAS did not impair memory performance on MWM. We highlight that high doses of NAND cause neurotoxic effects to components of synaptic and extrasynaptic mitochondrial bioenergetics, like calcium influx, membrane potential and H2O2 production. TEST was less neurotoxic to synaptic and extrasynaptic mitochondrial bioenergetics responses.

Sustained elevation of cerebrospinal fluid glucose and lactate after a single seizure does not parallel with mitochondria energy production.

Generalized seizures trigger excessive neuronal firing that imposes large demands on the brain glucose/lactate availability and utilization, which synchronization requires an integral mitochondrial oxidative capability. We investigated whether a single convulsive crisis affects brain glucose/lactate availability and mitochondrial energy production. Adult male Wistar rats received a single injection of pentylentetrazol (PTZ, 60 mg/kg, i.p.) or saline. The cerebrospinal fluid (CSF) levels of glucose and lactate, mitochondrial respirometry, [14C]-2-deoxy-D-glucose uptake, glycogen content and cell viability in hippocampus were measured. CSF levels of glucose and lactate (mean ± SD) in control animals were 68.08 ± 11.62 mg/dL and 1.17 ± 0.32 mmol/L, respectively. Tonic-clonic seizures increased glucose levels at 10 min (96.25 ± 13.19) peaking at 60 min (113.03 ± 16.34) returning to control levels at 24 h (50.12 ± 12.81), while lactate increased at 10 min (3.23 ± 1.57) but returned to control levels at 360 min after seizures (1.58 ± 0.21). The hippocampal [14C]-2-deoxy-D-glucose uptake, glycogen content, and cell viability decreased up to 60 min after the seizures onset. Also, an uncoupling between mitochondrial oxygen consumption and ATP synthesis via FoF1-ATP synthase was observed at 10 min, 60 min and 24 h after seizures. In summary, after a convulsive seizure glucose and lactate levels immediately rise within the brain, however, considering the acute impact of this metabolic crisis, mitochondria are not able to increase energy production thereby affecting cell viability.

Testosterone Administration after Traumatic Brain Injury Reduces Mitochondrial Dysfunction and Neurodegeneration.

Traumatic brain injury (TBI) increases Ca2+ influx into neurons and desynchronizes mitochondrial function leading to energy depletion and apoptosis. This process may be influenced by brain testosterone (TS) levels, which are known to decrease after TBI. We hypothesized that a TS-based therapy could preserve mitochondrial neuroenergetics after TBI, thereby reducing neurodegeneration. C57BL/6J mice were submitted to sham treatment or severe parasagittal controlled cortical impact (CCI) and were subcutaneously injected with either vehicle (VEH-SHAM and VEH-CCI) or testosterone cypionate (15 mg/kg, TS-CCI) for 10 days. Cortical tissue homogenates ipsilateral to injury were used for neurochemical analysis. The VEH-CCI group displayed an increased Ca2+-induced mitochondrial swelling after the addition of metabolic substrates (pyruvate, malate, glutamate, succinate, and adenosine diphosphate [PMGSA]). The addition of Na+ stimulated mitochondrial Ca2+ extrusion through Na+/Ca2+/Li+ exchanger (NCLX) in VEH-SHAM and TS-CCI, but not in the VEH-CCI group. Reduction in Ca2+ efflux post-injury was associated with impaired mitochondrial membrane potential formation/dissipation, and decreased mitochondrial adenosine triphosphate (ATP)-synthase coupling efficiency. Corroborating evidence of mitochondrial uncoupling was observed with an increase in H2O2 production post-injury, but not in superoxide dismutase (SOD2) protein levels. TS administration significantly reduced these neuroenergetic alterations. At molecular level, TS prevented the increase in pTauSer396 and alpha-Spectrin fragmentation by the Ca2+dependent calpain-2 activation, and decreased both caspase-3 activation and Bax/BCL-2 ratio, which suggests a downregulation of mitochondrial apoptotic signals. Search Tool for the Retrieval of Interacting Genes/Proteins database provided two distinct gene/protein clusters, "upregulated and downregulated," interconnected through SOD2. Therefore, TS administration after a severe CCI improves the mitochondrial Ca2+extrusion through NCLX exchanger and ATP synthesis efficiency, ultimately downregulating the overexpression of molecular drivers of neurodegeneration.

Serum Biomarkers and Clinical Outcomes in Traumatic Spinal Cord Injury: Prospective Cohort Study.

BACKGROUND: A plethora of reactive cellular responses emerge immediately after a traumatic spinal cord injury (SCI) and may influence the patient's outcomes. We investigated whether serum concentrations of neuron-specific enolase, interleukin-6, glial-derived neurotrophic factor, and neurotrophic growth factor reflect the acute-phase responses to different etiologies of SCI and may serve as predictive biomarkers of neurologic and functional outcomes. METHODS: Fifty-two patients were admitted to the intensive care unit after SCI due to traffic accidents, falls, and firearm wounds and had blood samples collected within 48 hours and 7 days after SCI. Thirty-six healthy subjects with no history of SCI were included as controls. Neurologic and functional status was evaluated on the basis of American Spinal Injury Association and Functional Independence Measure scores over a period of 48 hours and 6 months after SCI. RESULTS: Serum NSE increased significantly 48 hours and 7 days after SCI compared with controls, while interleukin-6 increased only at 48 hours. In contrast, the neurotrophic growth factor level significantly decreased 48 hours and 7 days after SCI. Serum glial-derived neurotrophic factor level did not differ from control at any time point. Also, there was no significant difference in biomarker concentrations between the etiologies of SCI or the level of spinal injury. There were no correlations between biomarker levels at 48 hours with neurologic or functional outcomes 7 days and 6 months after SCI. CONCLUSIONS: Our results suggest expansive axonal damage coupled with an acute proinflammatory response after SCI. However, in our study biomarker concentration did not correlate with short- or long-term prognosis, such as survival rate or sensory and motor function.

Elevated glutamate and lactate predict brain death after severe head trauma.

OBJECTIVE: Clinical neurological assessment is challenging for severe traumatic brain injury (TBI) patients in the acute setting. Waves of neurochemical abnormalities that follow TBI may serve as fluid biomarkers of neurological status. We assessed the cerebrospinal fluid (CSF) levels of glutamate, lactate, BDNF, and GDNF, to identify potential prognostic biomarkers of neurological outcome. METHODS: This cross-sectional study was carried out in a total of 20 consecutive patients (mean [SD] age, 29 [13] years; M/F, 9:1) with severe TBI Glasgow Coma Scale ≤ 8 and abnormal computed tomography scan on admission. Patients were submitted to ventricular drainage and had CSF collected between 2 and 4 h after hospital admission. Patients were then stratified according to two clinical outcomes: deterioration to brain death (nonsurvival, n = 6) or survival (survival, n = 14), within 3 days after hospital admission. CSF levels of brain-derived substances were compared between nonsurvival and survival groups. Clinical and neurological parameters were also assessed. RESULTS: Glutamate and lactate are significantly increased in nonsurvival relative to survival patients. We tested the accuracy of both biomarkers to discriminate patient outcome. Setting a cutoff of >57.75, glutamate provides 80.0% of sensitivity and 84.62% of specificity (AUC: 0.8214, 95% CL: 54.55-98.08%; and a cutoff of >4.65, lactate has 100% of sensitivity and 85.71% of specificity (AUC: 0.8810, 95% CL: 54.55-98.08%). BDNF and GDNF did not discriminate poor outcome. INTERPRETATION: This early study suggests that glutamate and lactate concentrations at hospital admission accurately predict death within 3 days after severe TBI.