Cardiovascular disease and drug-induced health side effects are frequently associated with—or even caused by—an imbalance between the concentrations of reactive oxygen and nitrogen species (RONS) and antioxidants, respectively, determining the metabolism of these harmful oxidants.According to the “kindling radical” hypothesis, the initial formation of RONS may further trigger the additional activation of RONS formation under certain pathological conditions. The present review specifically focuses on a dysfunctional, uncoupled endothelial nitric oxide synthase (eNOS) caused by RONS in the setting of transportation noise exposure or chronic treatment with organic nitrates, especially nitroglycerin (GTN). We further describe the various “redox switches” that are proposed to be involved in the uncoupling process of eNOS.l-arginine deficiency are discussed as alternative mechanisms of eNOS uncoupling.In particular, the oxidative depletion of tetrahydrobiopterin and S-glutathionylation of the eNOS reductase domain are highlighted as major pathways for eNOS uncoupling upon noise exposure or GTN treatment. In addition, oxidative disruption of the eNOS dimer, inhibitory phosphorylation of eNOS at the threonine or tyrosine residues, redox-triggered accumulation of asymmetric dimethylarginine, and The clinical consequences of eNOS dysfunction due to uncoupling on cardiovascular disease are summarized also, providing a template for future clinical studies on endothelial dysfunction caused by pharmacological or environmental risk factors. Environmental noise is a major environmental health risk factor   and reactive nitrogen species in the mitochondria and by different additional ROS-mediated processes, including endothelial nitric oxide synthase (eNOS) uncoupling, nitration/inhibition of prostacyclin synthase, and vascular NADPH oxidase activation , hydrogen peroxide, hydroxyl radicals, carbon-centered peroxides, peroxyl radicals, nitric oxide (•NO) and nitrogen dioxide radicals, peroxynitrite (ONOO−), and hypochlorite.Increased production of reactive oxygen and nitrogen species (RONS) leading to increased oxidative stress has been demonstrated to be a characteristic concerning many cardiovascular and cerebrovascular diseases Sies, , and als•NO) represents a second messenger and is probably the best-characterized radical species that plays a central role in the beneficial regulation of vascular tone and suppression of platelet activation. Xanthine oxidase (XO), NADPH oxidases, uncoupled nitric oxide synthases, and the mitochondrial respiratory chain are the most important biological sources of the superoxide anion (•O2−), an important precursor of many ROS. The detrimental biological role of •O2− was further substantiated by the discovery of superoxide dismutases , highly specialized enzymes with the sole biological function of detoxifying •O2−   that cause one-electron oxidation reactions leading to new products via hydroxylation and nitration. The biological existence of ONOO− was demonstrated by the nitration of protein tyrosine residues in atherosclerotic lesions . Upon protonation, peroxynitrous acid (ONOOH) either isomerizes spontaneously to nitrate  or it undergoes homolysis to form It should, however, be kept in mind that 3-nitrotyrosine is not only an exclusive marker of peroxynitrite reactivity but can be also formed under inflammatory conditions by the reaction of myeloperoxidase (MPO) with hydrogen peroxide in the presence of nitrite leading to the formation of nitrogen dioxide radicals that can nitrate tyrosine , nitrate tolerance  elegantly demonstrated the crucial role of ROS producing or degrading enzymes with respect to the initiation and also progression of cardiovascular and cerebral diseases . The ROS-induced ROS production concept, originally postulated for redox activation of mitochondrial reactive oxygen species (mtROS) production by ROS from neighbored dysfunctional mitochondria , the initial formation of ROS, for example, 2O2), these reactive species can activate the redox-sensitive zinc-finger-like complex in the protein kinase C (PKC)  was reported in the brain of noise-exposed mice, which was prevented by genetic deletion of •O2− instead of •NO.In addition to the classical activation mechanisms of eNOS by calcium/calmodulin, caveolin, heat shock protein 90, palmitoylation and myristoylation   is the most commonly accepted one. In the 1990s, many preclinical as well as clinical studies demonstrated that the presence of BH4 is essential for a normal functioning of eNOS  by peroxynitrite, thereby providing a concept of how RONS  via functional BH4 depletion may cause oxidative uncoupling of eNOS   , and accordingly may compete with •NO being present in the pico- to nanomolar range in most biological fluids (intra- and extracellular). The new concept postulates that the •BH4+ radicals  , a structural analog of BH4 sharing the same antioxidant properties, which, however, failed to improve endothelial dysfunction in the smokers.The essential role of BHde novo synthesis of BH4, improved endothelial function in nitrate-tolerant human subjects  in the binding region of the eNOS dimer. This will lead to a loss of eNOS dimerization, a phenomenon that has been first described by Zou et al  is probably the most potent endogenous inhibitor of eNOS Boger,  and it iHowever, higher ADMA levels in response to chronic GTN therapy could not be confirmed in a human study  is ∼2.9 μM for eNOS  in the presence of an extracellular excess of cationic amino acids such as l-arginine  with nitric oxide, thereby consuming the vasodilator , the use of eNOS inhibitors will decrease the •O2− signal in case eNOS is uncoupled and represents a significant •O2− source.According to previous reports on nitrate tolerance  and that low lucigenin concentrations (5 μM) are suitable for biological detection of •O2−  data wiped out the suspicion of artifact research by lucigenin.Methodologically, these studies used lucigenin in aortic tissue, a chemiluminescence dye that reacts with Other suitable detection methods for NOS uncoupling  that can be used include the cytochrome c assay   demonstrated also high specificity for •O2−, for example, in response to GTN treatment, although the sensitivity of these spin probes in vascular samples or cell culture is rather limited   in the presence of drugs that are known to ameliorate the function (coupling state) of eNOS . The prevention of endothelial dysfunction in isolated vessels of diseased animals (ex vivo) by the BH4 precursor sepiapterin is another approach to prove eNOS uncoupling in vascular tissue , whereas the degree of nitrate tolerance (the impaired vasodilatory potency of GTN) was not improved in the knockout mice  cells that were transfected with Nox-1, Nox-4, and the related other required subunits for full activation , all of which was completely prevented by coadministration of vitamin C  Gori et. Also, sIn summary, endothelial dysfunction, atherosclerosis, and the status of full-blown cardiovascular disease are associated with a chronic activation of the local and/or circulating renin–angiotensin–aldosterone system (RAAS) and an uncoupled eNOS. This was demonstrated in the setting of nitrate tolerance in response to chronic isosorbide-5-mononitrate therapy and GTN therapy, and also in response to transportation noise exposure, when the animals were exposed in the sleeping phase. Accordingly, in the setting of nitrate tolerance  activation of vasoconstrictor pathways including the RAAS as a consequence of GTN-induced hypotension, but this response may be also redox-driven as a consequence of increased endothelin-1 or angiotensin-II expression/release and signaling  to produce the “kindling radicals” leading to uncoupling of eNOS via the above-described “redox switches”  can be used to avoid the mentioned complications such as eNOS uncoupling, and several clinical studies confirmed that inhibitors of the RAAS can indeed develop nitrate tolerance /heme oxygenase-1 activation  , large clinical trials are not available. Wearing earplugs and noise-canceling headphones  may in theory be effective, however, large clinical studies on the protective effects of earplugs and noise-canceling headphones against (nighttime) noise exposure are currently not available. Obviously, there are still many gaps in noise research that should urgently be addressed in light of the potential additive risk of noise exposure in individuals with preexisting cardiovascular disease suggested by human (Schmidt et al, Although there is evidence for improvement of noise adverse health effects by physical activity (


Skeletal muscle has the widest range of oxygen metabolism of any organ, and regulation of oxygen supply with tissue demand is a fundamental requirement for locomotion and muscle function. During exercise, this process is exquisitely controlled and coordinated by the cardiovascular, respiratory, and autonomic systems, and also within the skeletal muscle microcirculation and mitochondria as the terminal site of oxygen exchange and utilization. This review highlights the potential contribution of the microcirculation and integrative cardiovascular physiology to the pathogenesis of ICU-AW. An overview of skeletal muscle microvascular structure and function is provided, as well as our understanding of microvascular dysfunction during the acute phase of critical illness; whether microvascular dysfunction persists after ICU discharge is currently not known. Molecular mechanisms that regulate crosstalk between endothelial cells and myocytes are discussed, including the role of the microcirculation in skeletal muscle atrophy, oxidative stress, and satellite cell biology. The concept of integrated control of oxygen delivery and utilization during exercise is introduced, with evidence of physiological dysfunction throughout the oxygen delivery pathway - from mouth to mitochondria - causing reduced exercise capacity in patients with chronic disease . We suggest that objective and perceived weakness after critical illness represents a physiological failure of oxygen supply-demand matching - both globally throughout the body and locally within skeletal muscle. Lastly, we highlight the value of standardized cardiopulmonary exercise testing protocols for evaluating fitness in ICU survivors, and the application of near-infrared spectroscopy for directly measuring skeletal muscle oxygenation, representing potential advancements in ICU-AW research and rehabilitation.Skeletal muscle dysfunction after critical illness, defined as ICU-acquired weakness (ICU-AW), is a complex and multifactorial syndrome that contributes significantly to long-term morbidity and reduced quality of life for ICU survivors and caregivers. Historically, research in this field has focused on pathological changes within the muscle itself, without much consideration for their  Millions of patients are admitted annually to the intensive care unit (ICU) , and enhin vivo physiological environment. Particularly, the role of skeletal muscle microcirculation and microvascular milieu in initiating, propagating, and sustaining ICU-AW has not been studied. Moreover, the cardiovascular control of locomotion - through matching of global and regional oxygen supply with tissue demand - has rarely been explored within the context of ICU-AW. Intact and well-regulated cardiovascular and microvascular systems are a fundamental requirements for normal skeletal muscle function, and their dysfunction contributes to weakness and impaired exercise capacity in numerous disease conditions.The biological mechanisms underlying ICU-AW are complex, commonly identified as increased protein catabolism, abnormal calcium signaling, transcriptional and post-transcriptional modification, mitochondrial dysfunction, and impaired satellite cell homeostasis–the reader is directed to recent excellent reviews for more details . The comWe believe these updated perspectives may provide novel insights into ICU-AW pathogenesis and opportunities for treatment. The aim of this review is to overview the significance of the microcirculation and integrative cardiovascular physiology in skeletal muscle health and disease, provide supporting evidence for their role in the pathogenesis ICU-AW using comparisons with other disease states, and bring forward unanswered questions and hypotheses for studying the microcirculation and cardiovascular system within the context of ICU-AW.The microcirculation defines the portion of the cardiovascular system comprising the smallest blood vessels throughout the body. Although there is no exact size cutoff, it is generally accepted that the microcirculation denotes vessels with internal diameter less than 100–150 microns, with upper estimates of 300 microns . BecauseMicrovascular networks share commonality throughout the body, but also manifest organ-specific structure and function to serve their specialized needs . Skeletal muscle is the largest organ by mass, accounting for approximately 40% of total body weight  and largSkeletal muscle microvascular networks can be separated into two anatomical and functional domains: arterioles/venules, and capillaries. Arterioles are organized into branching trees and looping arcades within skeletal muscle  and accovia the vascular endothelium  to a pro-inflammatory phenotype . PathogeFrom a hemodynamic perspective, endothelial activation causes vasodilation through upregulation of inducible nitric oxide synthase (iNOS) within ECs acting on arteriolar smooth muscle , which cHuman studies have also demonstrated microvascular dysfunction during the acute phase of critical illness using handheld vital microscopy (HVM) of the sublingual microcirculation, as well as skeletal muscle near-infrared spectroscopy (NIRS). De Backer and others were among the first to identify loss of microvascular perfusion in septic patients using sublingual HVM , and furHowever, microvascular research in critical illness has focused almost exclusively on observations during ICU admission. Accordingly, the persistence of microvascular dysfunction during recovery from critical illness and its potential contribution to ICU-AW remains largely unknown. Sakr and others demonstrated that PPV was lower in non-survivors vs. survivors of sepsis throughout the duration of ICU admission . De BackIntracellular events and molecular pathways within skeletal muscle cells (myocytes) that initiate and propagate ICU-AW have been well-described , but thein vitro to exogenous IL-6 upregulated mitochondrial reactive oxygen species (ROS) with negative effects on cellular stress and mitochondrial respiration  . SimilarAnother way that the skeletal muscle microcirculation may play a role in the pathogenesis of ICU-AW is through ineffective nutrient and metabolite transfer during the acute phase of critical illness, and also in the chronic phase in ICU survivors with established weakness. The two primary substrates required for adequate cellular respiration are oxygen and glucose; of these two, oxygen requires continuous resupply and cannot be stored, whereas skeletal muscle does have capacity to release glucose from glycogen .When microvascular systems lose ability to regulate blood flow and oxygen delivery, skeletal muscle dysfunction ensues due to lack of adequate energy supply. This has been studied in numerous chronic disease conditions outside of critical illness. Maladaptive arteriolar response to vasodilators contributes to skeletal muscle blood flow heterogeneity and hypoxia in preclinical models of metabolic syndrome ; 2019. IMicrovascular dysfunction in skeletal muscle is an early event in the pathogenesis of critical illness, and has been shown to occur in rodent models of sepsis within 3–4 h after administration of intraperitoneal fecal slurry . These mEx vivo models of prolonged hypoxia in murine skeletal muscle cells demonstrated increased satellite cell differentiation, which the authors speculated was an adaptive mechanism to overcome limited oxygen availability (2) as well as a decrease in the percentage of type I and an increase of type 2 fibres within the vastus lateralis  delivery to match O2 utilization and metabolic demand during locomotion requires integrated control of the autonomic, cardiovascular, respiratory, and skeletal muscle systems (2 in the ambient air to O2 delivery to the mitochondria to produce energy (ATP), there are highly coordinated, rapid, and complex adaptive responses  over large distances throughout the body, and local microvascular control and metabolism within skeletal muscle  to the O2 supply (transport and delivery)—thereby re-establishing balance between O2 delivery and O2 consumption , arterial saturation, hemoglobin concentration, vasodilation, O2 extraction, arterial and venous O2 content, and oxygen consumption (VO2) (2 consumption) during physical activity or exercise must be connected with pulmonary and cardiovascular systems. Therefore, there is a strong association between muscle work, O2 delivery, and O2 utilization, which is essentially linear during steady-state exercise, except for transitions  are needed to sustain muscle work, generating a transient O2 deficit until a new steady is reached and aerobic energy production is resumed  . This wansitions . During  resumed .2 kinetics, pulmonary VO2 responses present three phases: 1) phase 1 or cardiopulmonary phase, 2) phase 2 or fundamental component, and 3) phase 3 or the steady state of VO2  reflects the exponential increase in pulmonary VO2 due to increases in pulmonary and muscle blood flow, increased O2 extraction in skeletal muscle, and deoxygenated blood return from the active muscles to the pulmonary system. It is described by a mono-exponential response until a steady state is achieved, representing closely the oxidative phosphorylation adjustments in the active muscles. Phase 3 represents the pulmonary VO2 steady state where oxygen delivery and oxygen utilization are matched throughout the cardiorespiratory and muscular systems.The dynamic profiles of the cardiovascular, respiratory, and muscular systems during transitions from resting to moderate-intensity constant work rate exercise is known as kinetics analysis . For VO2e of VO2 . Phase 12 provides insight into the integrated mechanisms regulating muscle energetics and oxidative function  and how fast or slow the physiological systems adapt during moderate-intensity exercise, which is also related to aerobic fitness  exercised in a cycle ergometer for 6 min at a constant work rate matched by absolute and relative work rate . The authors concluded that the slower adaptations in CHF from maximal and submaximal exercise indicate the presence of exercise intolerance in this population. Borghi-Silva et al. (2 and heart rate kinetics were slowed in patients living with chronic obstructive pulmonary disease (COPD). COPD patients and healthy age and sex-matched controls underwent 6 min of a constant work rate moderate-intensity exercise on a treadmill at 70% of their maximal intensity. The authors concluded that the slower VO2 and HR responses in moderate-severe COPD patients are associated with impaired O2 delivery and O2 utilization during exercise. Thus, patients with COPD experience defects in skeletal muscle oxygenation that reduce exercise capacity independent from their pulmonary impairments . VO2 kinetics was measured breath-by-breath with a metabolic cart, and muscle oxygenation using NIRS. The authors showed that older adults had a slower pulmonary VO2 response and longer time to reach steady state than healthy young adults. The slower pulmonary VO2 kinetics in older adults was accompanied by reduced skeletal muscle blood flow and O2 delivery, and pronounced muscle deoxygenation. Gurd et al.  were measured on a breath-by-breath basis using a metabolic cart and deoxygenation by NIRS. The authors concluded that in MetS, skeletal muscle O2 supply-demand matching is impaired, and contributes to reduced aerobic power and exercise capacity. Similarly, McClatchey and others  to identify localized defects in skeletal muscle O2 supply versus tissue utilization  can also be used to continuously measure phosphocreatine (PCr) recovery in vivo as a marker of mitochondrial oxidative capacity in vivo, and variations to inspired fraction of oxygen during exercise can modulate global oxygen delivery; in addition to muscle oxygenation  that is evaluated with NIRS, arterial doppler can directly quantify blood flow and convective oxygen delivery. These multi-modal protocols have generated important insights regarding skeletal muscle exercise physiology in healthy and disease conditions. Haselar and others , and abnormal slope and recovery times. They concluded that skeletal muscle oxygen utilization may contribute to impaired exercise capacity, potentially attributed to skeletal muscle mitochondrial function. Joris et al.  as well as reduced maximal workload . Similar to previous studies, this was not attributed to cardiac or respiratory abnormalities. A hypermetabolic state with increased protein catabolism was observed at 3 months after discharge, as evidenced by a respiratory exchange ratio less than 1. Prolonged half-time of VO2 recovery after exercise and reduced metabolic efficiency (workload/peak VO2) suggest impaired oxygen extraction and utilization at the level of skeletal muscle–which they attributed to either microvascular or mitochondrial dysfunction; as discussed in the above, determinations of mitochondrial dysfunction may be better evaluated with isolated limb exercises and MRS. Longobardi and others  is an advanced diagnostic technique used clinically for numerous patients with chronic cardiorespiratory diseases , and hasd others  found a d others  who utils et al.  evaluated others  further Together, these recent studies demonstrate the increasing role of CPET for evaluating fitness in ICU survivors, providing quantifiable data to categorize and track recovery, and elucidate mechanisms of disease. Although differences may exist between COVID-19 survivors and general ICU patients, it becomes clear from these studies that residual weakness may reflect abnormalities with oxygen supply throughout the cardiovascular system in addition to intrinsic muscular dysfunction. Notably, direct interrogation of oxygen delivery and utilization at the site of skeletal muscle in ICU survivors is still lacking. As described in previous sections, use of NIRS, isolated limb exercises, and other advanced methodologies during exercise protocols may provide further mechanistic physiological information for ICU-AW by measuring skeletal muscle oxygenation, oxygen delivery and utilization–together, these are natural complements for global markers of exercise capacity that are evaluated with CPET.When considering the importance of oxygen delivery and utilization to normal muscle function, and the direct contact between microvascular networks and surrounding muscle, the contribution of the cardiovascular and microvascular systems to the pathogenesis of ICU-acquired weakness is likely under-recognized. There are plausible cellular pathways that underpin crosstalk between endothelial cells and myocytes that may drive skeletal muscle atrophy and limit capacity for regeneration. Persistent microvascular dysfunction after critical illness may cause maladaptive changes in skeletal muscle related to impaired regulation of blood flow and subtrate delivery. Physiological defects throughout the entire oxygen delivery pathway may result in reduced exercise capacity in ICU survivors, and should be considered in addition to pathological changes in the muscle. Future research should focus on elucidating these biological and physiological mechanisms, and incorporating standardized and quantitative exercise testing during post-ICU follow-up.


Adolescent idiopathic scoliosis is a coronal plane spinal deformity which most commonly presents in adolescent girls from ages 10 to 18 (A). Diagnosis is made with full-length standing posteroanterior (PA) and lateral spine radiographs. Treatment can be observation, bracing, or surgical management depending on the skeletal maturity of the patient, magnitude of deformity, and curve progression. In scoliosis cases, surgical intervention is typically considered when the curvature progresses to a certain degree or when it causes significant pain, respiratory problems, or other complications . The goal of scoliosis surgery is to correct the curvature and stabilize the spine. However, despite surgical intervention, there is a small risk of recurrence or progression of the deformity. Factors that may increase the risk of recurrence include inadequate surgical correction, incomplete fusion of the spinal segments, failure of implants or instrumentation, and continued growth of the spine in skeletally immature individuals. Here, we report a rare presentation of operated case of adolescent idiopathic scoliosis at dorsal spine with recurrence of deformity (D). Recurrence of deformity is very uncommon (<0.5 %). A 22-year-old female patient was brought to the orthopedics outpatient department (OPD) with complaints of pain and deformity in his upper back region since 5 years which were increasing over the period of time (E). On examination, there was scar of previous surgery present over midline approximately 15 cm in length at dorso lumbar level, recurrence of deformity over right side convex curve and no neurodeficit present and power in bilateral upper limb and lower limb v/v.


Both the calvarial and the cortical bones develop through intramembranous ossification, yet they have very different structures and functions. The calvaria enables the rapid while protected growth of the brain, whereas the cortical bone takes part in locomotion. Both types of bones undergo extensive modeling during embryonic and post-natal growth, while bone remodeling is the most dominant process in adults. Their shared formation mechanism and their highly distinct functions raise the fundamental question of how similar or diverse the molecular pathways that act in each bone type are.To answer this question, we aimed to compare the transcriptomes of calvaria and cortices from 21-day old mice by bulk RNA-Seq analysis.The results revealed clear differences in expression levels of genes related to bone pathologies, craniosynostosis, mechanical loading and bone-relevant signaling pathways like WNT and IHH, emphasizing the functional differences between these bones. We further discussed the less expected candidate genes and gene sets in the context of bone. Finally, we compared differences between juvenile and mature bone, highlighting commonalities and dissimilarities of gene expression between calvaria and cortices during post-natal bone growth and adult bone remodeling.Altogether, this study revealed significant differences between the transcriptome of calvaria and cortical bones in juvenile female mice, highlighting the most important pathway mediators for the development and function of two different bone types that originate both through intramembranous ossification. The calvaria, the upper structure of the skull, allows fast and protected brain growth without heavy load bearing. Conversely, the cortical bone, which is the external layer of long bones, is crucial for locomotion and is constantly remodeled under the influence of weight loading. Despite differences in structure and function, both bones develop through the mechanism of intramembranous ossification, which initiates in the embryo and extensively progresses in neonates . The shaThe process of intramembranous ossification is initiated by the condensation of mesenchymal cells in the flat bones of the skull and in the cortices of long bones. Following the differentiation of these mesenchymal cells into osteoblast , 3, a coIn mice, the calvaria is formed by several skull bones connected through cranial sutures derived from the cranial neural crest and head mesoderm . CalvariOn the other hand, the femoral cortical bone originates from the porous bone collar formed by osteoblasts, which derives from the lateral plate mesoderm during the early stages of bone development . In C57BDespite the shared process of intramembranous bone formation at early stages, differences between the extra-cellular matrix (ECM) of long bones and calvaria were observed in mature mice. For example, collagen was found to be more abundant in the calvaria and suggested to be less crosslinked. Contrary to this, the collagenous matrix of the long bones is more mineralized, probably providing an increased strength to the long bone that is necessary for weight bearing .The growth and homeostasis of the cortical and calvarial bones are regulated by various signaling pathways, hence mutations in several signaling pathway mediators lead to distinct skeletal diseases that are shared between human and mice, making the latter a useful model for research . Other gPrevious studies on adult mice, rats and macaques have compared gene expression between cortical and calvaria bones , 17. BotThis study focused on the post-natal developmental period by investigating juvenile cortical and calvarial bones. We used bulk RNA-Sequencing to analyze the transcriptome of 7 calvaria and 7 femoral cortical samples of 21 days old juvenile female mice to identify commonalities and differences in the expression level of genes associated with bone development, disease, weight loading and homeostasis. We further highlight differences between the juvenile and mature bone as well as differences in bone relevant signaling pathways observed between the juvenile murine calvaria and cortices.As described in Kalev-Altman et al. , female For RNA-Sequencing, mice were euthanized with CO2 at three weeks P21). Cortical samples (n=7) were cleaned from all adjacent tissues  and the diaphyseal bone marrow was removed by washing it out with ultra-pure water, using a small needle and syringe. Periosteum and endosteum were not removed. The left or right frontal bone was taken for the calvaria samples (n=7) without removing the periosteum or adjacent suture mesenchyme. The samples were individually flash-frozen in liquid nitrogen and were subjected to manual pulverization. Total RNA was extracted as previously described , 21. Eac. CorticaBioinformatical analysis was performed as previously described  with theThe calvarial and femoral cortical bone transcriptome data subset of 21 days old female C57BL/6J WT mice from Kalev-Altman et al.  was usedCollagen type I alpha 1 (Col1a1), Col1a2, Serpin family F member 1 (Serpinf1), Bone gamma-carboxyglutamate protein 1 (Bglap1), Bglap2, Prolyl 4-hydroxylase subunit beta (P4hb), Osteonectin (Sparc)   were det (Sparc)  is shown2 fold change indicated two clusters of genes (Homeobox (Hox) family of transcription factors, while in the bottom cluster DEGs, such as the BMP inhibitor Noggin (Nog), were higher expressed in the calvaria.Moreover, from the 10569 genes found, 602 differentially expressed genes (DEGs) were detected between the calvaria and the cortices. Of these 140 DEGs were higher expressed and 462 were lower expressed in the cortical bone compared to the calvaria Table S3As the first step of identifying DEGs that are highly relevant for bone formation, we compared our list of DEGs with the review of Brommage and Ohlsson , which sMechanical loading is a major effector in the modeling and remodeling of bones . Thus, wJuvenile and adolescent bone differ in various processes, such as bone elongation. Hence, we sought to investigate if the DEGs found between the juvenile calvaria and cortices overlap with DEGs found between adult calvaria and cortices. We compared our list of DEGs obtained from 21 days old female mice to the combined 225 DEGs found by Wang et al. , who anaFirst, we found several DEGs with a higher expression level in the calvaria as compared to the cortical bone in juvenile animals, which was also reported for mature animals. These include, among others, Insulin-like growth factor binding protein 5 (Igfbp5), Lrp4, Msh homeobox 1 (Msx1) and Nog Table S4Next, to further address commonalities between adolescent and mature bone, we focused on genes with a higher expression level in the cortical bone as compared to the calvaria. In agreement with the studies mentioned above on mature bone, we found DEGs such as Frizzled-related protein (Frzb), Short stature homeobox 2 (Shox2) and several HOX genes having a significantly higher expression level in the juvenile cortical bone compared to the calvaria. Furthermore, our analysis of juvenile bone revealed only a non-significant higher expression level of genes such as Cartilage oligomeric matrix protein (Comp) and Catenin beta 1 (Ctnnb1) in the cortical bone compared to the calvaria. Their expression levels were found to be significantly higher in Wang et al.  and RawlAt variance with the expression levels observed by Rawlinson et al.  investigMoreover, we detected additional bone or ECM related DEGs that were not detected or categorized as being differentially expressed by Wang et al.  and RawlIn summary, we found over 600 DEGs between calvaria and cortical bone of femur in juvenile female mice, including DEGs that were detected in previous studies that focused on adult animals and differed in experimental procedures. Many of our bone related DEGs, like several WNT signaling mediators, were not categorized as DEGs in these studies, and many DEGs reported in these studies were not confirmed by us in juvenile animals.Next, to check for enriched gene ontology (GO) Biological Process terms, we used GOrilla on the DEGs detected in this study. The resulting top 20 terms ranked by the enrichment score are shown in In the next step, we performed GSEA using the KEGG database to identify enriched data sets by ranking all detected genes, including DEGs Table S5Gene sets positively enriched in the cortical bone focused on cellular pathways and included nucleotide metabolism, homologous recombination, DNA replication, mismatch repair, spliceosome and ribosome-related terms. Moreover, the calvaria demonstrated a positive enrichment for terms focusing on signaling pathways such as calcium, Hedgehog, MAPK and WNT signaling as well as related terms like ECM-receptor interaction and Focal adhesion.For instance, in vitro. Finally, the calvaria showed a positive enrichment for gene sets related to cardiovascular-related terms compared to the cortices. This is not surprising as the enriched gene sets included integrins as well as DEGs like Lamin A/C (Lmna)   and TenaC (Lmna) , both tain-vitro before RNA-Seq analysis was reported to affect gene expression levels , Col1a1, Col1a2, Dentin matrix protein 1 (Dmp), Integrin binding sialoprotein (Ibsp), Runx1, Runx2, Runx3, Osterix (Sp7), Serpinf1 or Sparc. It seems likely that the expression levels of these universal bone markers are bone type independent and bone-specific differences occur in the subsequent processes, such as posttranscriptional regulation. At variance from the classical bone genes described above, many genes displayed significant differences in expression level between both bone types. For instance, the expression level of Lox was significantly higher in the cortical bone compared to the skull. This finding is in line with the increased cross-linked collagen in the cortical bone reported by van den Bos et al. . Furthervia WNT signaling  or the nuclear matrix protein Lmna , Dkk1, Sost and a few Hox genes were not found to be differentially expressed in juvenile bones in our study. In addition to the age differences, this may be attributed to our samples’ origin from female mice compared to male rats being investigated by Rawlinson et al. modeling of cortical and calvarial bone could primarily be mediated by a key gene set related to pathways like BMP, HH and WNT signaling. Many additional genes related to these pathways only show a differential expression between the cortices and calvaria either in juvenile animals during bone modeling or mature animals during remodeling. In line with this is our finding of only four genes Lox in the cortical bone provide the bone’s stiffness for locomotion in juvenile and mature mice, as locomotion is essential age-independently. This is complemented by age-dependent DEGs as demonstrated in this study or reported by Wang et al.  can be found below: The animal study was reviewed and approved by the Hebrew University Animal Care Committee (license number 21-16657-3).JJ, RK-A, DS-D and EM-O contributed to conception and design of the study. JJ and TS organized the database. JJ and TS performed the statistical analysis. JJ wrote the first draft of the manuscript. JJ and RK-A wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.


Multiple sclerosis (MS) is a complex and long-lasting neurodegenerative disease of the central nervous system (CNS), characterized by the loss of myelin within the white matter and cortical fibers, axonopathy, and inflammatory responses leading to consequent sensory-motor and cognitive deficits of patients. While complete resolution of the disease is not yet a reality, partial tissue repair has been observed in patients which offers hope for therapeutic strategies. To address the molecular and cellular events of the pathomechanisms, a variety of animal models have been developed to investigate distinct aspects of MS disease. Recent advances of multiscale intravital imaging facilitated the direct in vivo analysis of MS in the animal models with perspective of clinical transfer to patients. This review gives an overview of MS animal models, focusing on the current imaging modalities at the microscopic and macroscopic levels and emphasizing the importance of multimodal approaches to improve our understanding of the disease and minimize the use of animals.The online version contains supplementary material available at 10.1007/s13311-022-01324-6. With 2.5 millions of people affected with multiple sclerosis (MS) worldwide, MS is one of the most common neurodegenerative diseases . There iSince more than 70 years, animal models have been essential to study the pathophysiology of MS. Even if they are not equivalent to the human disease, they are able to partially recapitulate the events associated with MS at a high level of reproducibility and thus helpful to address MS-related questions. Thereby, we obtained a detailed knowledge of cellular interactions and mechanisms underlying CNS demyelination or used them to test potential therapies. However, simultaneously, each model is restricted to distinct processes: the models of experimental autoimmune encephalomyelitis (EAE) exhibit neuroinflammation and immune system activation toward myelin, toxin-induced demyelination models are relevant to decipher de- and remyelination processes selectively targeting oligodendrocytes and virus-induced MS models start with axonal damage while induce inflammation leading to demyelination.EAE models mimic clinical symptoms of MS patients as well as the time course of the disease. EAE is based on immunizing mice by a subcutaneous injection of a water-in-oil emulsion of myelin-associated peptides together with or without inactivated mycobacteria . The choice of the adjuvant has its relevance: IFA implies Th2 humoral response whereas CFA triggers Th1 cells mediated immunity with microglia activation releasing pro-inflammatory chemokines or reactive oxygen species (ROS) and subsequent activation of astrocytes , 5. The The ability of lysolecithin or lysophosphatidylcholine (LPC) to produce demyelination was first described in 1972 by Hall . Due to Involvement of Schwann cells strongly depends on the model used to induce the death of OLs. Ethidium bromide (EtBr) is a DNA intercalator that inhibits transcription and replication in the nucleus of all living cells causing their death . Unlike While LPC and EtBr must be applied topically or injected into the tissue to induce focal lesions, cuprizone (CPZ) is instead a systemically administered drug that produces reversible demyelination in relatively predictable brain regions such as CC, hippocampus, or cerebral and cerebellar cortex –28. The + and CD8+ cytotoxic T cells, B cells, and even macrophages MeDAS and [11C]-PIB were thus proven to reliably report myelin status during demyelination and remyelination in both LPC and EAE  was thus used to label activated macrophages [11C]-labeled antibodies [11C]-SMW139 ligand [18F]-VC701 or [18F]-DPA-714, two different ligands of the 18kD-translocator protein (TSPO) whose expression is increased in glial cells upon parenchymal infiltration of peripheral inflammatory cells. Although [18F]-VC701 and [18F]-DPA-714 indeed accumulated into the demyelinated lesions previously determined by T2*-weighted MRI into the cortex, hippocampus, cerebellum or cervical SC 9 ligand  to monitvical SC , 122, thvical SC .Because radioactive tracing can be implemented without significantly affecting the 3D structure of the molecule of interest, PET scan imaging can also be used to evaluate in situ the distribution and function of enzyme or bioactive metabolites controlling neuroinflammation . RadiolaBecause both T cells  and B ceIn this prospect, NHP models of MS  will be Due to the complex nature of the technique and the high costs involved, PET imaging will probably never replace MR imaging to study MS, but it can excellently complement MR imaging by bringing molecular specificity to in vivo evaluation of CNS inflammatory plaques, in particular when therapeutic drug development is concerned. The best option to really characterize inflammatory cell recruitment with respect to demyelination at the scale of the entire brain or SC is certainly to perform multimodal PET-MRI Fig. a.18F]DPA-714 PET gave access to the corresponding activation status of resident glial cells in the same regions by PET [Besides giving access to detailed anatomical structures and to the myelination status through endogenous MRI contrast, the use of SPIO allowed quantifying the differential recruitment of peripheral macrophages in the hippocampus, thalamus, and cerebellum by MRI, while the use of [s by PET . These fs by PET  and mults by PET  to simuls by PET . On top s by PET  or to trs by PET  or geners by PET . Notewor2+ indicators and/or intraveinous injection of fluorescent contrast agents [In this prospect, recent methodological studies indicated that the cellular origin of the recorded signals can be detected in the same animals using IVM along with viral transgenic expression of fluorescent genetically encoded Cat agents , 148. Int agents . Alternat agents  Supplementary file2 (PDF 517 kb)Supplementary file3 (PDF 525 kb)Supplementary file4 (PDF 508 kb)Supplementary file5 (PDF 517 kb)Below is the link to the electronic supplementary material.


NAFLD, or metabolic dysfunction–associated steatotic liver disease, has increased in prevalence hand in hand with the rise in obesity and increased free sugars in the food supply. The causes of NAFLD are genetic in origin combined with environmental drivers of the disease phenotype. Dietary intake of added sugars has been shown to have a major role in the phenotypic onset and progression of the disease. Simple sugars are key drivers of steatosis, likely through fueling de novo lipogenesis, the conversion of excess carbohydrates into fatty acids, but also appear to upregulate lipogenic metabolism and trigger hyperinsulinemia, another driver. NAFLD carries a clinical burden as it is associated with obesity, type 2 diabetes, metabolic syndrome, and cardiovascular disease. Patient quality of life is also impacted, and there is an enormous economic burden due to healthcare use, which is likely to increase in the coming years. This review aims to discuss the role of dietary sugar in NAFLD pathogenesis, the health and economic burden, and the promising potential of sugar reduction to improve health outcomes for patients with this chronic liver disease. These differences are largely due to socioeconomic factors, sugar consumption, and, in part, due to the population prevalence of genetic polymorphisms underlying the susceptibility for developing NAFLD.13 In the United States, the highest prevalence of NAFLD is seen in those of Hispanic heritage, possibly driven by the inheritance of the most prevalent polymorphism associated with NAFLD, patatin-like phospholipase domain–containing protein three (PNPLA3).14In association with the obesity epidemic and the major changes in the food supply over the past few decades, the global prevalence of NAFLD has risen markedly to an estimated 25.2%. By 2040, it is projected that over half the adult population will have NAFLD.15 the US annual direct medical costs of NAFLD are ~$103 billion, and in the European countries , the annual cost is about $35 billion, with total costs highest in patients aged 45–65.15 Furthermore, in the US, the annual hospitalization rate due to NAFLD has tripled from 2007 to 2014, with a greater increase in males versus females and Latino/Hispanics versus other ethnicities.16 Recent data suggest that NAFLD will become the most common indication for liver transplantation in the near future.17 NAFLD accounts for the highest increase in disability-adjusted life years compared to other liver-related chronic diseases.18 At the individual level, the cost of medical care  for a patient with NAFLD is estimated to be nearly twice as high compared with healthy individuals.15 In addition, there is an indirect societal impact due to absenteeism, caregiver burden, and reduced health-related quality of life.19NAFLD and its complications cause a considerable healthcare burden worldwide. According to Younossi et al,20 The link between steatosis in the liver and fructose consumption has been attributed to Pliny, the Elder, who wrote that Marcus Apicius, the famous Roman chef, would make fatty liver (foie gras) by overfeeding geese dried figs (a rich source of fructose). The process of force-feeding geese originally comes from ancient Egypt, with depictions of the practice found in the tomb of Mereruka dated 2500 BC21  of sugar on a 2000-calorie/day diet. A further reduction to less than 5% of total energy intake is recommended for additional health benefits.25 While total sugar consumption in the United States has decreased over recent years,26 current intakes remain above these guidelines, with sugar-sweetened beverages (SSB) as the top source, followed by desserts and sweet snacks.27 The average American adult and child consume about 17 teaspoons (68 gm) of added sugar per day,28 and the most common added sugars in the contemporary human diet include sucrose (table sugar) and high fructose corn syrup.29Excessive dietary sugar intake is thought to hold a major role in the onset and progression of NAFLD. The Dietary Guidelines for Americans 2020–2025 and the World Health Organization recommend that the intake of added sugars should not exceed 10% of total energy intake.13 , they are absorbed and metabolized differently in the gut.36 Glucose is transported from the intestinal lumen into the enterocytes through an energy-requiring process mediated by the sodium-glucose cotransporter 1, whereas fructose is absorbed through a facilitated passive transport mechanism by GLUT5 on the apical border of enterocytes.38 At the basolateral membrane, GLUT2 facilitates the passive absorption of both glucose and fructose into the circulation.36 The small intestine plays a key role in dietary fructose metabolism. Studies in mice have shown that within the enterocyte, the majority of fructose is converted to glucose and other organic acids. Thus, the small intestine acts as a shield, protecting the liver from the lipogenic effects of fructose. However, excessive fructose intake can overwhelm intestinal fructose absorption, which is transported to the liver through the portal vein.39 A recent study measured fructose absorption/metabolism in 9 children with biopsy-proven NAFLD compared with 6 obese and 9 lean non-NAFLD controls, ages 8–18 years. The subjects with NAFLD demonstrated increased absorption and exaggerated metabolic response  to fructose administration compared to lean children, while obese children without NAFLD had an intermediate response.40It is important to note that although fructose and glucose share the same molecular formula .30 In both adults and children with NAFLD, plasma endotoxin levels are elevated, suggesting either a failure in endotoxin removal or an increase in production that surpasses the liver's cleanup mechanisms.44The human gut microbiome plays a significant role in human health and disease.45 and the majority are cleared by the liver (~80%).47 Although the mechanisms are still being investigated, recent evidence in animal models revealed that LPS disappears rapidly from the circulation  and is scavenged primarily by the liver sinusoidal endothelial cells, which possess a high endocytic ability, and to a lesser extent by the Kupffer cells.48 In patients with NAFLD, the function of the Kupffer cells is impaired, which may result in disturbed hepatic clearance and increased levels of circulating LPS, leading to accelerated liver injury.49 Endotoxemia activates the innate immune system and stimulates hepatic toll-like receptor 4, which induces inflammasomes and proinflammatory cytokines, promoting hepatic inflammation and insulin resistance (IR)  to intrahepatic triglyceride formation, suggesting that elevated de novo lipogenesis is a distinct feature of NAFLD pathophysiology.62 They also demonstrated that increases in circulating insulin stimulate hepatic de novo lipogenesis in individuals with NAFLD.62 Further, they explored the concept that insulin mechanisms in the liver diverge, whereas the insulin receptors for glucose metabolism are resistant, leading to increased glycemia; the lipogenic mechanisms are intact and are overstimulated by excess insulin action lipogenesis.64 On the other hand, a study by Ter Horst et al revealed that patients with NAFLD and ce novo lipogenesis due to increased availability of lipogenic substrates like dietary fructose, while glucose-stimulated/insulin action lipogenesis was attenuated. However, the authors also stated that residual insulin-driven expression of lipogenic enzymes through SREBP-1c activation likely still plays a role.  and carbohydrate-responsive element–binding protein (ChREBP), which results in increased hepatic steatosis and directly affects insulin signaling and lipotoxicity.67A recent study of 40 adolescent boys aged 11–16 years with NAFLD, undergoing an 8-week feeding protocol, demonstrated that the rate of de novo lipogenesis decreased from 25% to 17% in parallel to decreases in alanine aminotransferase and hepatic steatosis, consistent with the theory that de novo lipogenesis is a critical metabolic function linking dietary sugars and NAFLD.68 Fructose metabolism is considered a major contributor to de novo lipogenesis in that it bypasses the rate-limiting enzyme of glycolysis (phosphofructokinase-1) and is rapidly phosphorylated by fructokinase C  to fructose-1-phosphate without any negative feedback control.69 For this reason, fructose is considered more lipogenic than glucose. This reaction also requires ATP consumption, producing a drop in intracellular phosphate, ultimately resulting in purine nucleotide turnover and elevated uric acid formation  is triggered by insulin-mediated glucose metabolism, while fructose bypasses leptin secretion and attenuates the suppression of ghrelin (the hunger hormone), increasing appetite.83 Shapiro et al demonstrated that high fructose intake also induced leptin resistance in rats.84 These changes can eventually lead to obesity and peripheral insulin resistance, promoting hepatic steatosis since hyperinsulinemia triggers adipose tissue lipolysis, causing an increase in free fatty acid delivery to the liver.57 Additionally, insulin resistance in skeletal muscle can promote increased hepatic de novo lipogenesis by diverting ingested glucose away from muscle glycogen synthesis toward the liver for hepatic triglyceride synthesis.87Overconsumption of added sugars can cause adaptations beyond the liver in the brain, adipose tissue, skeletal muscle, and pancreas. These extrahepatic and central responses to high sugar intake produce downstream changes that can indirectly contribute to hepatic steatosis. Added sugar intake triggers neuroadaptations in the mesolimbic reward pathway, promoting hedonic caloric intake above energy needs, leading to increased adiposity.89 This polymorphism promotes hepatic steatosis by inhibiting the activity of lipases on the surface of lipid droplets, reducing triglyceride mobilization.90 Individuals who carry this variant are more susceptible to increased hepatic fat accumulation when sugar consumption is high.91 In the United States, the highest frequency of PNPLA3 rs738409 was found among those of Latino/Hispanic origin (0.49) compared to European ancestry (0.23) and African ancestry (0.17).88 A nutrigenetic analysis in Latino/Hispanic children demonstrated that triglyceride accumulation in the liver was dependent on sugar intake in those with the homozygous GG substitution of the PNPLA3 genotype.91 This evidence suggests that added sugar consumption may exacerbate the effects of this polymorphism by increasing intrahepatic lipid volume in the setting of a decreased ability to mobilize and remove lipids. Little is known about the impact of dietary sugars on other genes, including the glucokinase regulatory gene, which regulates de novo lipogenesis by controlling hepatocyte glucose influx, and the transmembrane 6-superfamily member 2, which regulates VLDL secretion.92 Therefore, more research is needed to investigate the interaction between dietary sugars and these genetic polymorphisms in NAFLD pathogenesis.13While the addition of high levels of simple sugars to the diet is a relatively modern phenomenon, the genetic underpinnings driving biological responses to this diet variant are not. There are a number of genetic polymorphisms linked to NAFLD in humans, but one of the most common appears to further explain the sugar–NAFLD relationship. The single nucleotide polymorphism in the PNPLA3 gene , which encodes a 481 amino acid protein called adiponutrin, is strongly associated with NAFLD in the setting of increased insulin resistance and body mass index.94 This movement is now increasingly apparent in low-income and middle-income countries.95 In food production, sugar is not only used as a caloric sweetener but also as a bulking and browning agent, humectant, texture modifier, fermentation substrate, and preservative.96 As a result, added sugars are a ubiquitous component of processed foods and beverages in the food supply.97 With the myriad of synonyms for sugar used on nutrition labels, manufacturers disguise the total sugar content in food products, making efforts to reduce consumption challenging.98Globally, the food industry in high-income countries has become saturated with appetizing and potentially addictive products containing added sugars.100 When food availability was scarce, sweet foods were likely a vital energy source. Unfortunately, this biological preference makes humans vulnerable to today’s food environment, which is abundant in processed foods rich in refined sugars.101 Fructose is particularly problematic since it is the sweetest tasting natural sugar, 1.2–2.0 times sweeter than table sugar.102 The consumption of fructose has increased by 30% in the last 40 years and by 500% over the last century due to the increased consumption of processed foods and SSB.103Human sensory systems have evolved to detect and prefer the once rare, calorie-rich, sweet-tasting foods.105 Excessive consumption of sugars can stimulate the reward pathways in the brain through an intense dopamine release.82 According to DiNicolantonio et al, chronic sugar intake can lead to “dopamine deficiency” in the brain due to the downregulation of the dopamine D2 receptors. Over time, this can lead to withdrawals, promoting the drive for perpetual sugar intake and addiction.106Recent studies in animal models and humans have shown that added sugars have habit-forming properties similar to alcohol, tobacco, cocaine, and caffeine.107 Given that food preferences are established in early childhood, this may drive lifelong diet preferences.108 The distinction between fruits, which naturally contain glucose and fructose, and processed foods with added sugar should be noted. Despite their sweet taste, fruits contain relatively low amounts of simple sugars compared to processed foods and SSB. Additionally, whole fruits contain fiber and antioxidants, which may protect against the harmful metabolic effects of sugar consumption.109People of all ages are susceptible to the pervasiveness of added sugar. Today, an American child as early as 2 years of age is more likely to consume a sugar-sweetened product than a fruit or vegetable.111 In children and adolescents, fructose has a dose-dependent correlation with NAFLD onset and development of fibrosis.13 A recent meta-analysis revealed a significant positive association between higher consumption of SSB and the risk of NAFLD.112 According to Geidl-Flueck et al, even modest consumption of added sugars from beverages sweetened with fructose or sucrose over several weeks led to increased hepatic fatty acid synthesis in healthy men.113 Additionally, a systematic review of 7 studies including 4639 subjects showed that SSB consumers had a 53% increased risk of developing NAFLD compared with nonconsumers.114 In a group of otherwise healthy, overweight adults, the consumption of SSB for 6 months significantly increased hepatic steatosis, skeletal muscle fat, and visceral fat.115 This suggests that the daily consumption of SSB leads to an increased risk of metabolic and CVDs, including NAFLD. While earlier human studies are confounded by the fact that fructose was provided in the context of overfeeding and weight gain, more recent studies show that the effects of dietary fructose are independent of caloric intake and body mass index.118Excessive added sugar intake, especially fructose, is linked with metabolic abnormalities that can cause NAFLD, which can progress to advanced liver disease.8 Eventually, extrahepatic manifestations of NAFLD can occur, including chronic kidney disease and CVD,119 the leading cause of death among NAFLD patients.120 Additionally, NAFLD may play a role in the onset and progression of T2D and metabolic syndrome rather than just being an outcome of these conditions.122 A recent meta-analysis revealed that NAFLD was associated with about a 2-fold increase in the risk of incident T2D and metabolic syndrome over a median follow-up of 5 years.123With excessive sugar consumption, NAFLD typically occurs in the setting of obesity, dysglycemia, atherogenic dyslipidemia, and hypertension.124 To prevent the development of NAFLD and its complications, it is imperative for our society to adopt interventions to support sugar reduction in the population, especially in children who are most vulnerable.125 Potential strategies include food education, the distribution of fruits and vegetables at school, and increased taxes on foods that contain any form of added sugar.125Lifestyle change is the first-line therapy for NAFLD.129 A recent systematic review revealed that sugar taxation is a cost-effective policy option to reduce the health and economic burden related to the overconsumption of added sugars. These savings were driven by avoided healthcare costs and tax revenue exceeding intervention costs.130 Additionally, a microsimulation model developed by Vreman et al assessed the health and economic benefits of interventions aimed at reducing the intake of added sugars and estimated that a 20% reduction in added sugar intake would significantly reduce the prevalence of hepatic steatosis, NASH, cirrhosis, HCC, obesity, T2D, and CVD. Direct medical costs and disease-attributable disability-adjusted life years would also be reduced; these effects increased proportionally when added sugar intake was reduced by 50%.131Sugar taxation has recently emerged as a viable strategy in many countries  and US jurisdictions .35 Aligning with this, Schwimmer et al, recently conducted an 8-week, randomized, controlled intervention study in 40 adolescent boys with NAFLD (11–16 y.) to test the effect of dietary free sugar restriction on hepatic fat and found that the diet treatment group achieved a significantly greater reduction in hepatic fat (from 25% at baseline to 17% at week eight) compared to the usual control diet (21%–20%).33 In this study, meals were provided for the whole family along with individualized menu planning to restrict free sugars to less than 3% of total energy needs in the intervention diet group. A subsequent analysis of the same study participants by Cohen et al evaluated the effects of the 8-week sugar restriction on hepatic de novo lipogenesis, measured as the percentage contribution to plasma triglyceride palmitate using a 7-day metabolic labeling protocol with heavy water. Results revealed that the dietary sugar restriction significantly reduced hepatic de novo lipogenesis and fasting insulin among adolescents with NAFLD.67 Furthermore, plasma metabolomics and metagenomics were performed to investigate the systemic changes that occurred with the reduction in steatosis and de novo lipogenesis. The analyses revealed that the low free sugar diet, compared to the usual diet, was associated with metabolome and microbiome changes that may reflect biological mechanisms linking dietary sugar restriction to a therapeutic decrease in hepatic fat.132Clinical trials have demonstrated that dietary sugar restriction interventions that include meal provision and instruction are particularly effective therapeutic strategies for NAFLD. One short-term intervention study in 41 children (9–18 y) with obesity showed that dietary restriction of the free sugar fructose  via meal provision was associated with improvements in hepatic steatosis (from 7.2% to 3.8%), hepatic de novo lipogenesis, and insulin kinetics.133 However, this study only involved nutrition education without meal provision, and the goal for sugar restriction was only ≤ 10% of total calories.On the other hand, a recent study by Schmidt et al, found that a dietitian-led sugar reduction intervention did not improve liver outcomes in Latino youth with obesity (11–18 y of age).134 Another study in overweight or obese adults with NAFLD showed that a low free sugar diet intervention over 12 weeks resulted in reduced fibrosis scores and steatosis scores with improved glycemic indices and decreased concentrations of biomarkers of inflammation, triglycerides, and total cholesterol levels.135 Moreover, a study in children and adolescents (7–18 y.) with NAFLD found that a low fructose and low glycemic index/load dietary intervention over 6 months resulted in improvements in body composition and plasma markers of liver dysfunction and cardiometabolic risk.136The combination of a low-sugar diet and time-restricted feeding (16 consecutive hours of fasting and 8 hours of eating time daily) has also been shown to reduce adiposity and improve markers of liver function, dyslipidemia, and inflammation in adult patients with NAFLD.Further research is needed to confirm the long-term effectiveness of sugar reduction for NAFLD prevention and treatment, along with continued public health initiatives to decrease added sugars in our food system. Please see Table 138 This strategy has shown only moderate success, and some recommended strategies for generalized weight loss may not be the most effective in the setting of elevated hepatic fat and alanine aminotransferase.145 Recent studies have demonstrated that personalized nutrition interventions tailored to individuals or subgroups of individuals are more effective than conventional dietary advice.146 Precision nutrition  utilizes information on individual characteristics or phenotypes of disease to develop targeted nutritional recommendations.148Dietary recommendations for obesity issued over the past decade have aimed to mitigate disease progression using a one-size-fits-all approach.151 This heterogeneity is thought to arise from multiple factors, including sex, hormonal status, genetics, gut microbiota composition, other comorbidities, and certain exposures, including diet and physical activity.150 Carrillo-Larco et al identified 3 phenotypes in adults with NAFLD using a machine-learning approach. The majority of subjects fell into the average NAFLD phenotype, whereas 6% and 10% of the remaining subjects fell into phenotypes characterized by (1) high levels of anthropometrics, systolic blood pressure, and glucose , or (2) high levels of liver biomarkers and cholesterol, respectively.152 Furthermore, recent studies have demonstrated that diet and lifestyle modifications, including sugar reduction, are more effective in decreasing intrahepatic fat in NAFLD patients who are carriers of the PNPLA3 I148M gene polymorphism versus noncarriers.154 A tailored dietary approach that accounts for differences in clinical, genetic, and metabolic characteristics between responders and nonresponders to nutrition therapies for NAFLD, including sugar reduction, may be more effective than traditional lifestyle recommendations. Indeed, Zelber-Sagi et al suggested that current dietary regimens for NAFLD, such as low carbohydrate or reduced refined sugar interventions, could be improved by tailoring nutrition recommendations to meet individual preferences and goals, therefore improving long-term adherence and health outcomes.138 Although precision nutrition interventions have shown promise in several chronic disease populations,155 there is limited research in patients with NAFLD. Further research is needed to determine subphenotypes of NAFLD and tailor nutrition advice to individuals or subgroups of similar individuals with NAFLD to improve health outcomes.NAFLD is a heterogeneous condition with a broad spectrum of clinical manifestations, pathogenesis, and response to treatment.In conclusion, NAFLD is the most common cause of chronic liver disease and is strongly associated with the metabolic conditions of insulin resistance, T2D, and obesity. The increasing prevalence of NAFLD is linked to the rise of sugar consumption; therefore, dietary strategies incorporating restriction may provide an effective disease prevention and treatment solution. While the role of dietary sugar in NAFLD pathogenesis is still being elucidated, current consumption levels surpass the World Health Organization's guideline of no more than 10% of total energy intake. Given the health and economic impact of NAFLD, it's crucial to reduce free sugar intake to alleviate the current burden and prevent future obesity-related comorbidities.


Oxidative stress may affect various signaling pathways and biological processes, promoting dysfunctional immune responses and inflammation, inducing apoptosis, deregulating autophagy and impairing mitochondrial function. The chronic oxidative stress and the dysregulation of the immune system leads to the loss of tolerance, which drives autoantibody production and inflammation with the development of endothelial dysfunction. In particular, anti-phospholipid antibodies (aPL), which target phospholipids and/or phospholipid binding proteins, mainly β-glycoprotein I (β-GPI), play a functional role in the cell signal transduction pathway(s), thus contributing to oxidative stress and thrombotic events. An oxidation–antioxidant imbalance may be detected in the blood of patients with APS as a reflection of disease progression. This review focuses on functional evidence highlighting the role of oxidative stress in the initiation and progression of APS. The protective role of food supplements and Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) activators in APS patients will be summarized to point out the potential of these therapeutic approaches to reduce APS-related clinical complications. Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by arterial and venous thrombosis and/or pregnancy morbidity, associated with circulating “anti-phospholipid antibodies” (aPLs), such as lupus anticoagulant (LAC), anticardiolipin antibodies (aCL) and anti-β2-glycoprotein I antibodies (aβ2GPI). Such manifestations can be present in primary APS or associated with an autoimmune systemic disease, such as systemic lupus erythematosus, SLE (secondary APS) ,2,3. OthA ‘two hit hypothesis’ has been suggested to explain the pathogenesis of APS. The presence of circulating aPLs that destroy the integrity of the endothelium inducing a procoagulant phenotype represents the “first hit”, but aPLs alone are not enough to cause thrombosis, which takes place only in the presence of the “second hit”, a triggering factor which is usually represented by smoking, acute infections, oxidative stress (OS) or inflammation ,7.Oxidative stress is considered a key element driving pathophysiological processes that play a role in the onset and progression of various non-communicable diseases. According to its widely endorsed definition, OS arises from an imbalance between oxidants and antioxidants in favor of the oxidants.When ROS (Reactive Oxygen Species) production increases or their scavenging by antioxidants decreases, cells undergo a process of oxidative stress. ROS are oxygen-containing molecules formed by reduction/oxidation reactions (redox reactions) or electronic excitation. Key ROS molecules include hydroxyl and superoxide free radicals and nonradical molecules, such as hydrogen peroxide. Several cytokines and growth factors regulate the ROS production in the mitochondria, mainly via the electron transport chain, where oxygen is reduced to form superoxide anion  peroxisoOxidative stress largely contributes to APS pathogenesis and its complications. In APS patients, OS favors endothelial dysfunction, mainly associated with the alteration of the NO metabolism, stimulating a prothrombotic and proinflammatory status. Several mechanisms have been reported to explain the role of aPLs as a key promoter of oxidative stress and mitochondrial dysfunction. Several studies describe oxidative stress as a possible source of antigenic epitopes responsible for aPL’s subpopulation appearance. The pathogenic role of oxidative stress in APS is also related to its ability to determine protein structural modifications  that, caIn this review, we examine the available evidence for the involvement of aPLs in the induction of a pro-oxidative state in APS patients and the possible use of oxidative stress biomarkers in the patient management. Furthermore, we will discuss the in vivo and in vitro evidence on the role of oxidative stress in the post-translational modifications of antigens associated with APS that suggest oxidative stress as a regulatory checkpoint in antiphospholipid autoantibody production. Moreover, the importance of oxidative stress in the activation of multiple signaling pathways that accelerate the progression and exacerbation of the symptoms of APS will be examined. The protective role of food supplements and NRF2 activators in APS patients will be summarized.The role of oxidative stress in the pathogenesis of APS has been widely demonstrated by experimental and clinical studies. It is considered a “second hit” contributing to the induction of the procoagulant phenotype and to the occurrence of thrombosis ,7,24. RePatients with primary APS have increased levels of circulating oxidative stress-related markers when compared to healthy subjects. Of interest, APS patients with triple positivity for aPL, i.e., positive for LAC, aCL and aβ2GPI antibodies, show significantly higher levels of these markers than patients with single or double aPL positivity . DiffereA recent study demonstrated a significant association of anti-phosphatidylethanolamine autoantibodies (aPEs) with systemic ROS levels in APS patients with unexplained deep vein thrombosis . These aAnother mechanism implicated in oxidative stress is related to the interactions between aCL antibodies and circulating antioxidant enzymes, such as the paraoxonase-1 (PON1), an antioxidant enzyme that inhibits LDL oxidation. Patients positive for aCL autoantibodies have been demonstrated to show reduced activity of PON1 . A crossData obtained in vitro demonstrated that aPLs also promote oxidative stress by modulating the activity of the pro-oxidant enzymes of immune cells. In particular, it has been demonstrated that human monoclonal aPLs and IgG fractions from APS patients activate endosomal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the generation of superoxide in plasmacytoid dendritic cells and monocytes. This activation determines the increased expression of toll-like receptor (TLR) 7 and TLR8 mRNA in plasmacytoid dendritic cells and in monocytes, respectively, and promotes inflammation, which is closely linked to pro-oxidant pathways .A further mechanism by which aPLs promote oxidative stress is the direct activation of pro-inflammatory pathways. Autoantibodies specific for β2GPI are able to activate endothelial cells , plateleAll these observations sustain the main role of autoantibodies in the induction of a pro-oxidant status in APS patients which, in turn, acts to promote atherothrombosis .Low-to-moderate levels of ROS have important biological functions, as they contribute to the regulation of signal transduction and normal physiological processes. Endogenous antioxidant defense mechanisms play a role in maintaining redox homeostasis . OxidatiSimilar results were obtained in clinical studies. In particular, the presence of high levels of prostaglandin F2-isoprostanes have been detected in plasma from APS patients. These compounds derive from ROS-mediated arachidonic acid oxidation. As F2-isoprostanes are related to lipid peroxidation, they represent an endogenous marker of oxidative stress and have the ability to predict cardiovascular events . A crossAs oxidative stress has a pivotal role in the pathogenic mechanisms of APS, particularly those related to atherothrombosis, the determination of circulating oxidative stress biomarkers could be useful for the risk assessment of vascular complications in patients with APS. A clinical study conducted in 140 patients with primary and secondary APS aimed to evaluate the relationship between oxidative stress and endothelial damage as a risk factor of thrombosis revealed altered levels of different oxidative stress markers in patients compared to controls. Lipid hydroperoxydes levels were predictive for the impaired flow-mediated dilation of the brachial artery, thus suggesting their possible role as markers of endothelial damage . HoweverThese conflicting results call for further studies to determine the association of circulating markers of oxidative stress with the occurrence of thrombosis and their possible usefulness as predictive markers of risk.The production of ROS at physiological levels cooperates with the resolution of inflammation and the maintenance of homeostasis in tissues . An overPTMs are reversible or irreversible chemical reactions, mainly catalyzed by enzymes that occur in specific amino acids of a certain amount of proteins after their biosynthesis. These processes have a significant impact on the structure and function of proteins; indeed, these modifications play a key role in regulating the folding of proteins, their targeting to specific subcellular compartments, their interaction with ligands or other proteins and, eventually, their immunogenic properties ,49. PTMsThe known PTMs in APS mainly involve β2GPI. β2GPI is a plasma glycoprotein that consists of five domains rich in cysteines, the V domain is critical for the binding of β2GPI to anionic phospholipids . β2GPI pIn particular, PTMs of cysteines include the addition of oxygen. The oxidative/reductive conversion of β2GPI may contribute to the regulation of its biological activity. One of the most functional groups in various proteins is the free sulfhydryl group (SH) contained in the amino acid cysteine. Indeed, the majority of circulating β2GPI is present in a form containing unpaired cysteines (free thiols), which constitutes the reduced form of β2GPI. Sulfhydryl modifications can alter the function of proteins containing cysteines within their catalytic centers or at the protein–protein interaction interface. In an oxidative stress condition, an overproduction of ROS quickly reacts with cysteine residues, especially redox active cysteines, to form reversible or irreversible oxidized forms. The total amount of β2GPI and the relative amount of oxidized β2GPI is increased in APS patients with a thrombotic history ,51.Oxidative stress, together with inflammation, is also involved in nonenzymatic glycosylation (glycation of lysine residues) which is a non-enzymatic process that leads to the formation of early, intermediate and advanced glycation end products; these products can modify the structure and function of self-molecules. Elevated levels of antibodies to glucose-modified β2GPI were reported in APS patients. Several factors can modulate this process . These p2O2 to cyanate, thereby further increasing carbamylation [Another important PTM, related to inflammation and oxidative stress, is carbamylation, a non-enzymatic reaction of cyanate with the primary amine of lysine residues from proteins that generate homocitrulline . Particumylation . In APS mylation . In a remylation .Although β2GPI is the most well-known protein in APS, several other proteins were described as antigenic targets and found to play a pathogenetic role in the syndrome. In particular, vimentin contains arginine residue that can undergo PTM to citrulline, catalyzed by the enzyme peptidyl arginine deiminase (PAD) which is active in different cell processes such as in NETosis. Recently, Alessandri et al. have identified citrullinated vimentin as a new autoantigen for aPL. They hypothesized that citrullination influences the production of specific autoantibodies by inducing conformational changes in vimentin. Antibodies specific for mutated citrullinated vimentin have been found in 26.6% of patients with APS. These results indicate a role for citrullination in the pathogenesis of APS and in association with the development of joint involvement .Other PTMs, including phosphorylation and acetylation, may affect β2GPI antigenicity , in partOxidative stress is the consequence of the balance alteration between ROS production and a defective detoxification, leading to the alteration of various signaling pathways and multiple biological processes through modifying proteins. These changes in the cellular functions may promote a dysfunctional immune response, inflammation, the induction of apoptosis, deregulation of autophagy, impairment of mitochondrial functions and many other mechanisms. Oxidative imbalance plays a role in the breakdown of immunological tolerance contributing to pathogenesis, pathological progression and the exacerbation of the symptoms in several autoimmune diseases ,60 Figu.One of the mechanisms by which oxidative stress can trigger an inflammatory response in the pathogenesis of autoimmune diseases concerns the activation of the enzyme poly (ADP-ribose) polymerase-1 (PARP-1) . PARP-1 PARP-1, through NF-κB activation, may induce the production of inflammatory  and effector T cell cytokines , as well as of inflammatory mediators such as metalloproteinases (MMP9), inducible nitric-oxide synthase (iNOS), several chemokines, prostaglandins (PGE2) and alarmins (HMGB1) ,64,65. OMetabolic cues such as oxidative stress trigger, in most cells, the redox-dependent activation of a mammalian target of rapamycin (mTOR), using a process that involves the cysteine oxidation of Rheb78 and raptor (regulatory-associated protein of mTOR) ,69. In tRegarding this, oxidative stress plays a role as a regulatory checkpoint in the pathogenesis of systemic lupus erythematosus (SLE) and other autoimmune diseases, where the activation of mTORC1 has been described as a central pathway. In fact, the use of N-acetylcysteine (NAC), an amino acid precursor of glutathione treatment, also reversed the prominent activation of mTORC1 in double-negative T cells ,74,75.Oxidatively modified proteins, which act as neoantigens, can trigger pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) of the immune system that represent an important link between some environmental factors such as oxidative stress and autoimmune diseases . In thisA condition of oxidative stress can also activate heat shock factors (HSF) and induce a subsequent biosynthesis of heat shock proteins (HSPs), stress-induced cellular molecules that cooperate with the antioxidant system to inhibit or neutralize the cellular effects of ROS . MoreoveAPS is characterized by uncontrolled inflammation and the over-production of ROS. Increasing evidence supports a link between OS and inflammation in autoimmune diseases . Kadl anIn addition, the NRF2/ARE signaling pathway may be considered the primary pathway for intracellular redox balance and the molecules that it induces exert antioxidant and anti-inflammatory effects by upregulating various cytoprotective enzymes and proteins. NRF2 is physiologically localized in the cytoplasm and anchored to the intracytoplasmic actin cytoskeleton in the form of the Keap1–NRF2 complex. E3 ubiquitin ligase promotes the ubiquitin-mediated degradation of NRF2 and the maintenance of a basal steady-state level of NRF2 activity. Otherwise, under oxidative or electrophilic stress conditions, NRF2 dissociates from Keap1 and translocates to the nucleus forming a heterodimer with the small musculoaponeurotic fibrosarcoma (sMAF) proteins and binds to an Antioxidant Response Element (ARE) in the promoter region of cytoprotective genes . In addiInterest in the cytoprotective role of NRF2 is growing as a potential drug target in APS, in which OS and inflammation underlie the disease’s pathogenesis. In patients with APS, the NRF2 activation was repressed in monocytes, thus suggesting a defect in the NRF2 signaling pathway . In an aKeeping in mind that an unbalanced accumulation of ROS has been linked to the occurrence and development of many acute and chronic diseases, including APS, pharmacological regulation of NRF2 pathways is an emerging treatment strategy. However, APS is a complex pathological process, in which multiple signaling pathways are involved in the immunopathogenesis of the disease. Thus, a thorough knowledge of the interplay among oxidative stress-responsive redox signaling pathways, including TLRs signaling, NRF2 and NF-κB, may contribute to a better comprehension of the immunopathogenesis of APS, driving towards a personalized medicine.Nowadays, there is an increasing interest in nutritional interventions because of their potential role in preventing tissue damage related to uncontrolled inflammation and oxidative stress. The presence of aPLs is often associated with tissue damage, such as vascular events, miscarriages, cerebral or dermatologic manifestations, probably because of the direct interaction of aPLs with tissues . More soMany natural compounds have been reported to control oxidative stress by regulating redox homeostasis and inflammation and leading to cell survival, neovascularization and the repair of damaged tissues by promoting the activation of the transcription factor NRF2 . More thTo date, several bioactive compounds have been identified as “NRF2 activators” and they mediate protection against oxidative stress and inflammation-derived injury, thus representing new therapeutic strategies for tissue healing and the prevention of the recurrence of tissue damage. Since, in APS patients, the physiological response to ROS and inflammation is not always sufficient to provide full protection, and results show that the administration of redox-targeted supplements  could improve remission in terms of circulating prothrombotic markers, an adjunctive therapy addressing the NRF2 pathway could be a promising therapy . HoweverAmong various pathophysiological processes that play a role, oxidative stress is considered a key mechanism driving the onset and progression of APS. Oxidative stress is defined as an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage. Patients with APS are characterized by disturbances of redox signaling pathways, which can be quantified by measuring components of the systemic redox status, i.e., redox biomarkers. The discovery and validation of redox biomarkers, particularly those related to thrombosis, may represent a useful tool in the clinical management of patients with APS, but as of yet, it is not always clear what all these biomarkers exactly represent and what their relative contributions are in relation to the whole-body redox status. Considering that supplementation with natural compounds have shown their role as possible supporting therapies for their numerous positive pleiotropic effects, personalized redox medicine approaches are emerging in the context of APS. A broad range of NRF2 activators or the dietary intake of antioxidant-rich food with the aims of activating endogenous antioxidant defense systems, represent an adjunctive therapy for restoring redox homeostasis and for improving remission in terms of circulating prothrombotic markers.This review deals with the role of oxidative stress in the initiation and progression of APS. Large evidence points out the role of oxidation as a key regulatory checkpoint of “antiphospholipid antibody” production, by promoting the post-translational modifications of proteins and/or triggering multiple signaling transduction pathways. Among these, the protective role of the NRF2 pathway has been elucidated. This knowledge provides new insights into the pathogenesis of APS and introduces new hypotheses for valuable therapeutic targets, including personalized redox nutraceutical medicine approaches.