Adora2A downregulation promotes caffeine neuroprotective effect against LPS-induced neuroinflammation in the hippocampus.

Caffeine has been extensively studied in the context of CNS pathologies as many researchers have shown that consuming it reduces pro-inflammatory biomarkers, potentially delaying the progression of neurodegenerative pathologies. Several lines of evidence suggest that adenosine receptors, especially A1 and A2A receptors, are the main targets of its neuroprotective action. We found that caffeine pretreatment 15 min before LPS administration reduced the expression of Il1b in the hippocampus and striatum. The harmful modulation of caffeine-induced inflammatory response involved the downregulation of the expression of A2A receptors, especially in the hippocampus. Caffeine treatment alone promoted the downregulation of the adenosinergic receptor Adora2A; however, this promotion effect was reversed by LPS. Although administering caffeine increased the expression of the enzymes DNA methyltransferases 1 and 3A and decreased the expression of the demethylase enzyme Tet1, this effect was reversed by LPS in the hippocampus of mice that were administered Caffeine + LPS, relative to the basal condition; no significant differences were observed in the methylation status of the promoter regions of adenosine receptors. Finally, the bioinformatics analysis of the expanded network demonstrated the following results: the Adora2B gene connects the extended networks of the adenosine receptors Adora1 and Adora2A; the Mapk3 and Esr1 genes connect the extended Adora1 network; the Mapk4 and Arrb2 genes connect the extended Adora2A network with the extended network of the proinflammatory cytokine Il1ß. These results indicated that the anti-inflammatory effects of acute caffeine administration in the hippocampus may be mediated by a complex network of interdependencies between the Adora2B and Adora2A genes.

Palmitate Compromises C6 Astrocytic Cell Viability and Mitochondrial Function.

Consumption of high-fat diets (HFD) is associated with brain alterations, including changes in feeding behavior, cognitive decline, and dementia. Astrocytes play a role in HFD-induced neuroinflammation and brain dysfunction; however, this process is not entirely understood. We hypothesized that exposure to saturated fatty acids can compromise astrocyte viability and mitochondrial function. The C6 (astrocytes) cell line was treated with palmitate or stearate (200 µM and 400 µM) for 6 h. Cell viability, morphology, inflammatory markers, and oxidative stress were evaluated. To assess mitochondrial function, various parameters were measured (membrane potential, mass, respiration, and complex activities). We observed that 6 h of treatment with 400 µM palmitate decreased cell viability, and treatment with 200 µM palmitate changed the astrocyte morphology. Palmitate increased inflammatory markers (TNF-α and IL6) but did not induce oxidative stress. Palmitate significantly decreased the mitochondrial membrane potential and mitochondrial mass. Complex I activity also decreased in palmitate-treated cells; however, no changes were observed in mitochondrial respiration. In conclusion, palmitate, a saturated fatty acid, induces inflammation and impairs mitochondrial function, leading to reduced astrocytic cell viability and changes in cellular morphology. Our study provides valuable insights into the potential mechanisms underlying the relationship between saturated fatty acids, astrocytes, and mitochondrial function in obesity-related brain dysfunction.

Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL): Report of a new family and a novel DARS2 mutation.

Background: LBSL is a mitochondrial disorder caused by mutations in the mitochondrial aspartyl-tRNA synthetase gene DARS2, resulting in a distinctive pattern on brain magnetic resonance imaging (MRI) and spectroscopy. Clinical presentation varies from severe infantile to chronic, slowly progressive neuronal deterioration in adolescents or adults. Most individuals with LBSL are compound heterozygous for one splicing defect in an intron 2 mutational hotspot and a second defect that could be a missense, non-sense, or splice site mutation or deletion resulting in decreased expression of the full-length protein. Aim: To present a new family with two affected members with LBSL and report a novel DARS2 mutation. Results: An 8-year-old boy (Patient 1) was referred due to headaches and abnormal MRI, suggestive of LBSL. Genetic testing revealed a previously reported c.492 + 2 T > C mutation in the DARS2 gene. Sanger sequencing uncovered a novel variant c.228-17C > G in the intron 2 hotspot. Family studies found the same genetic changes in an asymptomatic 4-year-old younger brother (Patient 2), who was found on follow-up to have an abnormal MRI. mRNA extracted from patients' fibroblasts showed that the c.228-17C > G mutation caused skipping of exon 3 resulting in lower DARS2 mRNA level. Complete absence of DARS2 protein was also found in both patients. Summary: We present a new family with two children affected with LBSL and describe a novel mutation in the DARS2 intron 2 hotspot. Despite findings of extensive white matter disease in the brain and spine, the proband in this family presented only with headaches, while the younger sibling, who also had extensive white matter changes, was asymptomatic. Our in-vitro results confirmed skipping of exon 3 in patients and family members carrying the intron 2 variant, which is consistent with previous reported mutations in intron 2 hotspots. DARS2 mRNA and protein levels were also reduced in both patients, further supporting the pathogenicity of the novel variant.

MicroRNA biogenesis machinery activation and lncRNA and REST overexpression as neuroprotective responses to fight inflammation in the hippocampus.

Brain Long non-coding RNA (lncRNA) and microRNAs (miRs) play essential roles in the regulation of several important biological processes, including neuronal activity, cognitive processes, neurogenesis, angiogenesis, and neuroinflammation. In this context, the transcriptional repressor, RE1 silencing transcription factor (Rest), acts regulating the expression of neuronal genes as well as of lncRNAs and multiple miRNAs in the central nervous system. Nevertheless, its role in neuroinflammation was less explored. Here, we demonstrate, using an in vivo model of neuroinflammation induced by i.p. injection of LPS (0.33 mg/kg), that neuroinflammation increases gene expression of pro-inflammatory cytokines concomitant with the native and truncated forms of Rest and of non-coding RNAs. Additionally, the increased expression of enzymes Drosha ribonuclease III) (Drosha), Exportin 5 (Xpo5) and Endoribonuclease dicer (Dicer), associated with high expression of neuroprotective miRs 22 and 132 are indicative that the activation of biogenesis of miRs in the hippocampal region is a Central Nervous System (CNS) protective mechanism for the deleterious effects of neuroinflammation. Our results indicate that positive regulation of Rest gene expression in the hippocampal region by neuroinflammation correlates directly with the expression of miRs 22 and 132 and inversely with miR 335. In parallel, the confirmation of the possible alignment between the lncRNAs with miR 335 by bioinformatics corroborates with the sponge effect of Hottip and Hotair hybridizing and inhibiting the pro-inflammatory action of miR 335. This suggests the existence of a possible correlation between the activation of miR biogenesis machinery with increased expression of the transcription factor Rest, contributing to neuroprotection.

Tetrahydrobiopterin: Beyond Its Traditional Role as a Cofactor.

Tetrahydrobiopterin (BH4) is an endogenous cofactor for some enzymatic conversions of essential biomolecules, including nitric oxide, and monoamine neurotransmitters, and for the metabolism of phenylalanine and lipid esters. Over the last decade, BH4 metabolism has emerged as a promising metabolic target for negatively modulating toxic pathways that may result in cell death. Strong preclinical evidence has shown that BH4 metabolism has multiple biological roles beyond its traditional cofactor activity. We have shown that BH4 supports essential pathways, e.g., to generate energy, to enhance the antioxidant resistance of cells against stressful conditions, and to protect from sustained inflammation, among others. Therefore, BH4 should not be understood solely as an enzyme cofactor, but should instead be depicted as a cytoprotective pathway that is finely regulated by the interaction of three different metabolic pathways, thus assuring specific intracellular concentrations. Here, we bring state-of-the-art information about the dependency of mitochondrial activity upon the availability of BH4, as well as the cytoprotective pathways that are enhanced after BH4 exposure. We also bring evidence about the potential use of BH4 as a new pharmacological option for diseases in which mitochondrial disfunction has been implicated, including chronic metabolic disorders, neurodegenerative diseases, and primary mitochondriopathies.

ALDH2 inhibition by lead and ethanol elicits redox imbalance and mitochondrial dysfunction in SH-SY5Y human neuroblastoma cell line: Reversion by Alda-1.

Lead (Pb), a common environmental contaminant, and ethanol (EtOH), a widely available drug of abuse, are well-known neurotoxicants. In vivo, experimental evidence indicates that Pb exposure affects oxidative EtOH metabolism with a high impact on living organisms. On these bases, we evaluated the consequences of combined Pb and EtOH exposure on aldehyde dehydrogenase 2 (ALDH2) functionality. In vitro exposure to 10 µM Pb, 200 mM EtOH, or their combination for 24 h reduced ALDH2 activity and content in SH-SY5Y human neuroblastoma cells. In this scenario, we observed mitochondrial dysfunction characterized by reduced mass and membrane potential, decreased maximal respiration, and spare capacity. We also evaluated the oxidative balance in these cells finding a significant increase in reactive oxygen species (ROS) production and lipid peroxidation products under all treatments accompanied by an increase in catalase (CAT) activity and content. These data suggest that ALDH2 inhibition induces the activation of converging cytotoxic mechanisms resulting in an interplay between mitochondrial dysfunction and oxidative stress. Notably, NAD+ (1 mM for 24 h) restored ALDH2 activity in all groups, while an ALDH2 enhancer (Alda-1, 20 µM for 24 h) also reversed some of the deleterious effects resulting from impaired ALDH2 function. Overall, these results reveal the crucial role of this enzyme on the Pb and EtOH interaction and the potential of activators such as Alda-1 as therapeutic approaches against several conditions involving aldehydes accumulation.

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons.

Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

Anti-Inflammatory Effect of Caffeine on Muscle under Lipopolysaccharide-Induced Inflammation.

Evidence has shown that caffeine administration reduces pro-inflammatory biomarkers, delaying fatigue and improving endurance performance. This study examined the effects of caffeine administration on the expression of inflammatory-, adenosine receptor- (the targets of caffeine), epigenetic-, and oxidative metabolism-linked genes in the vastus lateralis muscle of mice submitted to lipopolysaccharide (LPS)-induced inflammation. We showed that caffeine pre-treatment before LPS administration reduced the expression of Il1b, Il6, and Tnfa, and increased Il10 and Il13. The negative modulation of the inflammatory response induced by caffeine involved the reduction of inflammasome components, Asc and Casp1, promoting an anti-inflammatory scenario. Caffeine treatment per se promoted the upregulation of adenosinergic receptors, Adora1 and Adora2A, an effect that was counterbalanced by LPS. Moreover, there was observed a marked Adora2A promoter hypermethylation, which could represent a compensatory response towards the increased Adora2A expression. Though caffeine administration did not alter DNA methylation patterns, the expression of DNA demethylating enzymes, Tet1 and Tet2, was increased in mice receiving Caffeine+LPS, when compared with the basal condition. Finally, caffeine administration attenuated the LPS-induced catabolic state, by rescuing basal levels of Ampk expression. Altogether, the anti-inflammatory effects of caffeine in the muscle can be mediated by modifications on the epigenetic landscape.

Hyperglycemic microenvironment compromises the homeostasis of communication between the bone-brain axis by the epigenetic repression of the osteocalcin receptor, Gpr158 in the hippocampus.

Diabetes mellitus (DM) is a chronic metabolic disease, mainly characterized by increased blood glucose and insulin dysfunction. In response to the persistent systemic hyperglycemic state, numerous metabolic and physiological complications have already been well characterized. However, its relationship to bone fragility, cognitive deficits and increased risk of dementia still needs to be better understood. The impact of chronic hyperglycemia on bone physiology and architecture was assessed in a model of chronic hyperglycemia induced by a single intraperitoneal administration of streptozotocin (STZ; 55 mg/kg) in Wistar rats. In addition, the bone-to-brain communication was investigated by analyzing the gene expression and methylation status of genes that encode the main osteokines released by the bone [Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) and their receptors in both, the bone and the brain [Fgfr1 (fibroblast growth factor receptor 1), Gpr6A (G-protein coupled receptor family C group 6 member A), Gpr158 (G protein-coupled receptor 158) and Slc22a17 (Solute carrier family 22 member 17)]. It was observed that chronic hyperglycemia negatively impacted on bone biology and compromised the balance of the bone-brain endocrine axis. Ultrastructural disorganization was accompanied by global DNA hypomethylation and changes in gene expression of DNA-modifying enzymes that were accompanied by changes in the methylation status of the osteokine promoter region Bglap and Lcn2 (lipocalin 2) in the femur. Additionally, the chronic hyperglycemic state was accompanied by modulation of gene expression of the osteokines Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) in the different brain regions. However, transcriptional regulation mediated by DNA methylation was observed only for the osteokine receptors, Fgfr1(fibroblast growth factor receptor 1) in the striatum and Gpr158 (G protein-coupled receptor 158) in the hippocampus. This is a pioneer study demonstrating that the chronic hyperglycemic state compromises the crosstalk between bone tissue and the brain, mainly affecting the hippocampus, through transcriptional silencing of the Bglap receptor by hypermethylation of Gpr158 gene.

Scavenging of reactive species probed by EPR and ex-vivo nanomolar reduction of lipid peroxidation of manganese complexes.

Antioxidant activity toward H2O2, anion radical superoxide, hydroxyl and DPPH (2,2-diphenyl-1-picrylhydrazyl) of two manganese complexes [Mn(III)(bpa)2]Cl.H2O (1) and [(Cl)Mn(µ-hbpclnol)(µ-bpclnol)Mn](ClO4).3H2O (2) (hbpa = (2-hydroxybenzyl-2-pyridylmethyl)amine and h2bpclnol = (N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)[(3-chloro)(2-hydroxy)]propylamine) are presented. X-ray diffraction studies were performed for complex (1). Both complexes presented similar or better activities than reference complex [Mn(salen)Cl], when the interaction between them and ROS (H2O2, O2•- and •OH), was monitored, by EPR (Electron Paramagnetic Resonance), in PBS, DMSO and water. The antioxidant activity rank of complexes toward •OH, generated by Fenton reaction and monitored by EPR, is (2) > (1) > [Mn(salen)Cl], in water (0.1% of DMSO for each complex), with the values of the IC50 of 7.2 (±1.6), 15.5 (±1.8) and 29.1 (±2.01) µM respectively. EPR data presented herein suggest that complex (2) presents the better scavenging activity toward hydroxyl, being in good agreement with TBARS assay results, in which complex (2) presented the best inhibitory activity toward lipid peroxidation, employing Swiss mice liver homogenate tissue model. IC50 values obtained from the interaction between these complexes and hydroxyl, using TBARS method, were: 0.88 (± 0.029); 0.73 (± 0.01) and 42.7 (± 3.5) nM, respectively for (1), (2) and [Mn(salen)Cl]. Complexes (1) and (2) are regulating the lipid homeostasis, protecting the tissue from the lipid peroxidation, in nanomolar scale, motivating in vivo studies. Redox properties and radical scavenging activity of complexes toward DPPH are non-linear and solvent dependent. Furthermore, the monitoring of antioxidant activity probed by EPR could be a fair and appropriate study to guide more advanced investigations.

ROS scavenging of SOD/CAT mimics probed by EPR and reduction of lipid peroxidation in S. cerevisiae and mouse liver, under severe hydroxyl radical stress condition.

The interaction between CuII, FeIII and MnII complexes, derived from the ligands 1-[bis(pyridine-2-ylmethyl)amino]-3-chloropropan-2-ol (hpclnol) and bis(pyridine-2-ylmethyl)amine (bpma), and the free radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) and reactive oxygen species (ROS), was investigated by colorimetric and EPR (Electron Paramagnetic Resonance) techniques. A comparison between these results and those reported to [Mn(salen)Cl] or EUK-8 was also addressed. EPR studies allowed us the identification of intermediates species such as superoxide­copper(I) and superoxide­copper(II), a mixed-valence FeIIIFeII species and a 16-line feature attributed to MnIII-oxo-MnIV species. The biomarker malondialdehyde (MDA) was determined by TBARS assay in S. cerevisiae cells, and the determination of the IC50 indicate that the antioxidant activity shown dependence on the metal center (CuII ≈ FeIII > MnII ≈ [Mn(salen)Cl]. The lipid peroxidation attenuation was also investigated in liver homogenates obtained from Swiss mice and the IC50 values were in the nanomolar concentrations. We demonstrated here that all the complexes interact with the free radical DPPH and with ROS (H2O2, O2•- and hydroxyl radical), enhancing the cellular protection against oxidative stress generated by hydroxyl radical, employing two experimental model systems, S. cerevisiae (in vivo) and mouse liver (ex vivo).

Impact of neuroinflammation on epigenetic transcriptional control of Sonic Hedgehog members in the central nervous system.

Sonic Hedgehog (Shh) signaling plays a critical role during central nervous system (CNS) development, and its dysregulation leads to neurological disorders. Nevertheless, little is known about Shh signaling regulation in the adult brain. Here, we investigated the contribution of DNA methylation on the transcriptional control of Shh signaling pathway members and its basal distribution impact on the brain, as well as its modulation by inflammation. The methylation status of the promoter regions of these members and the transcriptional profile of DNA-modifying enzymes (DNA Methyltransferases - DNMTs and Tet Methylcytosine Dioxygenase - TETs) were investigated in a murine model of neuroinflammation by qPCR. We showed that, in the adult brain, methylation in the CpG promoter regions of the Shh signaling pathway members was critical to determine the endogenous differential transcriptional pattern observed between distinct brain regions. We also found that neuroinflammation differentially modulates gene expression of DNA-modifying enzymes. This study reveals the basal transcriptional profile of DNMTs and TETs enzymes in the CNS and demonstrates the effect of neuroinflammation on the transcriptional control of members of the Shh Signaling pathway in the adult brain.

Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer.

Increased tetrahydrobiopterin (BH4) generated in injured sensory neurons contributes to increased pain sensitivity and its persistence. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme in the de novo BH4 synthetic pathway, and human single-nucleotide polymorphism studies, together with mouse genetic modeling, have demonstrated that decreased GCH1 leads to both reduced BH4 and pain. However, little is known about the regulation of Gch1 expression upon nerve injury and whether this could be modulated as an analgesic therapeutic intervention. We performed a phenotypic screen using about 1000 bioactive compounds, many of which are target-annotated FDA-approved drugs, for their effect on regulating Gch1 expression in rodent injured dorsal root ganglion neurons. From this approach, we uncovered relevant pathways that regulate Gch1 expression in sensory neurons. We report that EGFR/KRAS signaling triggers increased Gch1 expression and contributes to neuropathic pain; conversely, inhibiting EGFR suppressed GCH1 and BH4 and exerted analgesic effects, suggesting a molecular link between EGFR/KRAS and pain perception. We also show that GCH1/BH4 acts downstream of KRAS to drive lung cancer, identifying a potentially druggable pathway. Our screen shows that pharmacologic modulation of GCH1 expression and BH4 could be used to develop pharmacological treatments to alleviate pain and identified a critical role for EGFR-regulated GCH1/BH4 expression in neuropathic pain and cancer in rodents.

Gentle Touch Therapy, Pain Relief and Neuroplasticity at Baseline in Fibromyalgia Syndrome: A Randomized, Multicenter Trial with Six-Month Follow-Up.

BACKGROUND: Fibromyalgia (FM) is considered a stress-related disorder characterized mainly by chronic widespread pain. Its pathogenesis is unknown, but cumulative evidence points at dysfunctional transmitter systems and inflammatory biomarkers that may underlie the major symptoms of the condition. This study aimed to evaluate pain scores (primary outcome), quality of life, inflammatory biomarkers and neurotransmitter systems in women with FM (secondary outcomes) subjected to gentle touch therapy (GTT) or placebo. METHODS: A total of 64 female patients with FM were randomly assigned to two groups, namely GTT (n = 32) or Placebo (n = 32). Clinical assessments were conducted at baseline and post-intervention with six-month follow-up. We measured serum catecholamines (dopamine), indolamines and intermediary metabolites (serotonin or 5-hydroxyindolacetic acid (5-HIAA)), as well as tetrahydrobiopterin (BH4), which is a cofactor for the synthesis of neurotransmitters and inflammatory biomarkers in women with FM. A group of healthy individuals with no intervention (control group) was used to compare biochemical measurements. Intervention effects were analyzed using repeated measures (RM) two-way ANOVA followed by Bonferroni post hoc test and mixed ANCOVA model with intention to treat. RESULTS: Compared to placebo, the GTT group presented lower pain scores and brain-derived neurotrophic factor (BDNF) levels without altering the quality of life of women with FM. Changes in BDNF had a mediating role in pain. Higher baseline serum BDNF and 5-HIAA or those with a history of anxiety disorder showed a higher reduction in pain scores across time. However, women with higher serum dopamine levels at baseline showed a lower effect of the intervention across the observation period revealed by an ANCOVA mixed model. CONCLUSIONS: In conclusion, lower pain scores were observed in the GTT group compared to the placebo group without altering the quality of life in women with FM. Reductions in BDNF levels could be a mechanism of FM pain status improvement. In this sense, the present study encourages the use of these GTT techniques as an integrative and complementary treatment of FM.

Muscle Fatigue Is Attenuated When Applying Intermittent Compared With Continuous Blood Flow Restriction During Endurance Cycling.

PURPOSE: The aim of this study was to identify a blood-flow-restriction (BFR) endurance exercise protocol that maximizes metabolic strain and minimizes muscle fatigue. METHODS: Twelve healthy participants accomplished 5 different interval cycling endurance exercises (2-min work, 1-min rest) in a randomized order: (1) control, low intensity with unrestricted blood flow (CON30); (2) low intensity with intermittent BFR (i-BFR30, ∼150 mm Hg); (3) low intensity with continuous BFR (c-BFR, ∼100 mm Hg); (4) unloaded cycling with i-BFR0 (∼150 mm Hg); and (5) high intensity (HI) with unrestricted blood flow. Force production, creatine kinase activity, antioxidant markers, blood pH, and potassium (K+) were measured in a range of 5 minutes before and after each cycling exercise protocol. RESULTS: HI showed the highest reduction (Δ = -0.26 [0.05], d = 5.6) on blood pH. Delta pH for c-BRF30 (Δ = -0.02 [0.03], d = 0.8) and Δ pH for i-BRF30 (Δ = -0.04 [0.03], d = 1.6) were different from each other, and both were higher compared with CON30 (Δ = 0.03 [0.03]). There was significant before-to-after force loss following HI (Δ = 55 [40] N·m-1, d = 1.5) and c-BFR30 (Δ = 27 [21] N·m-1, d = 0.7) protocols only, which were accompanied by significant increases in K+ (HI: Δ = 0.94 [0.65] mmol·L-1, d = 1.8; c-BFR30: Δ = 0.72 [0.85] mmol·L-1, d = 1.2). Moreover, all BFR conditions elicited slight increases in plasma creatine kinase, but not for HI and CON30. Glutathione changes from before to after were significant for all BFR conditions and HI, but not for CON30. CONCLUSIONS: The attenuation in fatigue-induced reductions in maximal force suggests that i-BFR exercise could be preferable to c-BFR in improving exercise capacity, with considerably less biologic stress elicited from HI exercises.