Epilepsies are common chronic neurological diseases characterized by recurrent unprovoked seizures of central origin. The mainstay of treatment involves symptomatic suppression of seizures with systemically applied antiseizure drugs (ASDs). Systemic pharmacotherapies for epilepsies are facing two main challenges. First, adverse effects from (often life-long) systemic drug treatment are common, and second, about one-third of patients with epilepsy have seizures refractory to systemic pharmacotherapy. Especially the drug resistance in epilepsies remains an unmet clinical need despite the recent introduction of new ASDs. Apart from other hypotheses, epilepsy-induced alterations of the blood–brain barrier (BBB) are thought to prevent ASDs from entering the brain parenchyma in necessary amounts, thereby being involved in causing drug-resistant epilepsy. Although an invasive procedure, bypassing the BBB by targeted intracranial drug delivery is an attractive approach to circumvent BBB-associated drug resistance mechanisms and to lower the risk of systemic and neurologic adverse effects. Additionally, it offers the possibility of reaching higher local drug concentrations in appropriate target regions while minimizing them in other brain or peripheral areas, as well as using otherwise toxic drugs not suitable for systemic administration. In our review, we give an overview of experimental and clinical studies conducted on direct intracranial drug delivery in epilepsies. We also discuss challenges associated with intracranial pharmacotherapy for epilepsies. Direct drug delivery to brain structures by bypassing the blood–brain barrier (BBB) is an attractive therapeutic strategy for patients suffering from primary brain tumors and neurological disorders such as advanced Parkinson’s disease and intractable epilepsies. In our present review, we give an overview of the numerous experimental and the few clinical studies conducted on direct intracranial drug delivery in epilepsies. Epilepsies are serious chronic neurological disorders that affect more than 50 million people worldwide. Apart from the occurrence of spontaneous recurrent seizures, epilepsies can be accompanied by comorbidities such as depression, anxiety, and cognitive deficits . The maiEven more challenging, about 30% of epilepsy patients do not become long-term seizure-free with currently available ASDs , with evDespite the development of many new ASDs, the percentage of patients suffering from DRE has not been considerably reduced over the last decades ,8. In paDrug-resistant epilepsies may, in fact, not necessarily be drug-resistant in all cases because the suggested mechanisms for medically refractory epilepsies include (among many others) alterations of drug uptake into the brain as well as pharmacokinetic alterations in the periphery ,20. We tFurthermore, targeted intracranial administration of ASDs or other appropriate drugs may not only offer an alternative to systemic drug treatment but also to focus resection. Intracranial drug delivery approaches may be advantageous in patients in which the seizure focus is located in an eloquent brain area. The risk of irreversible functional loss, for example, of movement, speech, or sensation induced by focus resection , is likeThe brain networks involved in seizure generation from a limbic or neocortical focus and its propagation and modulation pathways are summarized in The clear advantages of intracranial drug delivery over systemic drug delivery include (a) the possibility to reach higher drug concentrations in key target brain areas of the epileptic network compared to systemic administration, (b) the possibility to use otherwise toxic substances not suitable for systemic administration, (c) the reduced risk of causing systemic and/or neurological adverse effects, (d) the possibility to use substances for which the BBB is not permeable, and (e) to overcome BBB-associated drug resistance mechanisms. Disadvantages include (a) the invasive procedure necessary for intracranial drug delivery, (b) the difficulties associated with the need for long-term administration of an appropriate drug, and (c) the difficulties associated with the fact that the distribution of an appropriate drug within the brain parenchyma cannot be reliably set to the desired range. Nevertheless, intracranial pharmacotherapy is a network-specific treatment approach, which aims to specifically target the epileptic network underlying epilepsy.Intracranial drug infusion has been studied by targeting different compartments of the central nervous system (CNS), i.e., via the intraparenchymal, ventricular, or transmeningeal route. Apart from the seizure focus, seizure propagation pathways or circuits belong to the epileptic network and have been shown to offer suitable anatomical targets for therapeutic interventions , which wThe present review is about intracranial drug delivery as a strategy to overcome drug-resistant focal epilepsies with or without secondary generalization, because the majority of patients with DRE suffer from focal onset epilepsy. The generation of other epilepsy types such as absence epilepsies involves different cortical and subcortical brain circuitries and mechanisms . In addiDifferent technical strategies aim to achieve network-specific pharmacological seizure-control in contrast to systemic drug treatment. Concerning direct intracranial drug delivery, several techniques  to adminMost proof-of-concept animal studies utilize acute on-site injections (bolus deposition) via stereotaxically implanted cannulas into the target region, while chronic (continuous or discontinuous) drug release is required for further development of this treatment strategy aiming to achieve long-term seizure control. An approach to prolong the duration of antiseizure effects by intracranial drug delivery is the use of drug-loaded biodegradable or nonbiodegradable polymer-based implants or bioceramics slowly releasing ASDs or other appropriate compounds, which then passively diffuse through the surrounding tissue. These drug carriers are experimentally implanted at seizure-modulating brain targets in order to achieve a gradual, more or less continuous drug release directly into the target region of the epileptic network ,44. AlthHowever, three main disadvantages are associated with the use of polymer-based implants to suppress epileptic seizures. First, the drug diffuses passively through the brain parenchyma, resulting in uneven drug distribution within the target area and a limited extent of delivery . Drug peAnother strategy for continuous intracranial drug delivery is the use of external or (subcutaneously) implantable drug pumps connected to a  catheter/cannula system located in the ventricular system or specific targets within the brain parenchyma. Osmotic pumps connected to a catheter system also have the disadvantage of releasing the drug by passive diffusion ,59,60,61The intention of CED is to distribute the drug at higher concentrations, more evenly, and over a larger range than when administered by diffusion alone ,49,50,51Risk factors or complications associated with the direct delivery of drugs into the brain must be thoroughly investigated for each infusate to be tested. While toxic effects by a mild increase in intracranial pressure during CED are thought to be minimal, neurotoxicity from direct effects of drugs on neurons and glial cells are more relevant and may exclude some drugs or dictate dose and infusion rate . FurtherA receptor agonist muscimol for 12 to 24 h into the epileptic focus of patients with DRE did not damage adjacent brain parenchyma or adversely affect seizure surgery outcome. As mentioned above, continuous intracranial drug delivery can be associated with the development of pharmacological tolerance [Extensive preclinical and clinical experience exists with CED of drugs to treat brain tumors and neurological diseases such as Parkinson’s disease  and haveolerance ,57, thusolerance , such asFurthermore, the development of intracranial drug administration only when needed, i.e., timed delivery in relation to seizure activity  will prAlthough invasive techniques are associated with several risks of complications such as mechanical device failure, catheter obstruction, catheter misplacement, infection, rejection, CSF leak, and the need for refill ,62, highIn most preclinical studies on epilepsies, different types of rodent models are used. A general overview of seizure and epilepsy models is given, for example, by ,72,73. AA receptor antagonists pentylenetetrazole . PTZ can be injected subcutaneously (sc), intraperitoneally (ip), or intravenously (iv). Further chemoconvulsants used are, for example, the GABAA receptor antagonists bicuculline  and the volatile flurothyl. Chronic seizures or epilepsies are also induced by electrical or chemical means. Chronic seizure models of TLE can be induced, for example, by electrical kindling  of limbic structures such as the amygdala or hippocampus [Briefly, acute seizures can be induced electrically, for example, in the maximal electroshock seizure (MES) or the 6 Hz 44 mA test, or chemically, for example, by the administration of the GABApocampus . Chronicpocampus .Numerous different seizure/epilepsy models have been used during the past decades to investigate intracranial drug delivery approaches. However, a careful choice of the animal model may accelerate the further development of this treatment strategy for sysDRE.First, concerning acute and chronic seizure models, those which allow reliable and repeated seizure threshold determination are advantageous because they permit easy quantification of seizure susceptibility over time in individual animals, i.e., before, during, and after (long-term) drug delivery . For example, the determination of electrographic or motor seizure thresholds in kindled rats as a chronic seizure model of TLE ,76,77 asA receptor agonist muscimol [Second, models of DRE such as TLE models or models of refractory neocortical epilepsies may better predict the seizure-controlling outcome of intracranial drug delivery approaches in humans, although this is not yet known. The kindling model of TLE is well-known to be highly predictive of at least systemic drug efficacy against focal limbic seizures in humans . Especiamuscimol  by usingDirectly targeting the seizure focus or areas close to the seizure focus with intracranial drug delivery is an obvious approach. Accordingly, numerous studies have addressed this strategy, aiming to re-establish a balance between excitation and inhibition within the abnormally excitable seizure focus and associated epileptic network. The seizure focus can be targeted directly by intraparenchymal drug delivery, indirectly by transmeningeal diffusion of an appropriate compound administered to the epidural or subdural (subarachnoid) space, or indirectly by drug delivery into the ventricular system .Injections or infusions of drugs into the CSF via icv or intrathecal delivery in order to bypass the BBB is clinically established and has been used for several decades to treat different CNS disorders such as brain tumors, infectious meningitis, and intractable pain . The icvAdvantages of drug infusions into the ventricular system include the relatively easy access and the lack of need to clearly localize the epileptic focus . HoweverDespite the disadvantages mentioned above, animal studies utilizing acute icv injection of different types of compounds, including ASDs in different seizure and epilepsy models, suggested that the delivery of drugs into the CSF may be an alternative route in the treatment of sysDRE. In these acute icv injection studies, a wide range of drugs with GABAergic and non-GABAergic mechanisms of action have been shown to have antiseizure potential in different preclinical models, e.g., GABA uptake inhibitors in the acute scPTZ seizure test in mice . Mixed aHowever, for long-term symptomatic seizure suppression, chronic infusion of appropriate substances into the ventricle system is inevitable. Furthermore, depending on physicochemical properties of the drug, such as liposolubility and ionization at local pH, not every drug penetrates deeply into the brain parenchyma after acute icv injection. This is the reason why high doses are often necessary to reach antiseizure efficacy with bolus icv injections. Thus, an acute bolus may be more toxic and less effective than small icv doses delivered repeatedly or continuously . The occIn a comprehensive study, acute ip, acute icv, and chronic icv (7 days via osmotic pump) administration of valproate was investigated using the amygdala-kindling rat model of TLE to directly compare efficacy, toxicity, and drug levels between the three different administration routes . At doseA pathological and behavioral study in pigs (ventricular ependyma comparable between humans and pigs) indicated behavioral adverse effects at doses higher than three mg/day of the ASD phenytoin and higher than 1.5 mg/day of valproate chronically infused icv . HoweverOommen et al.  infused A first-in-man clinical study recently investigated chronic , continuous unilateral icv infusion of valproate in patients suffering from drug-resistant focal seizures with or without secondary generalization . ValproaIn summary, icv delivery is a promising approach to achieve anticonvulsant effects while reducing systemic toxicity but does not necessarily improve neurological toxicity due to unwanted distribution of the drug into areas not involved in the epileptic network in addition to the wanted diffusion to epileptogenic areas.Apart from icv delivery of drugs, compounds with antiseizure activity can be applied to the brain surface via implantation of drug-releasing catheter systems or cups/carriers into the epidural or subdural (subarachnoid) space overlying a neocortical seizure focus. The concept of drug delivery through spinal meninges is clinically established for the administration of local anesthetics and analgesics, for example, during obstetric pain relief or lower extremity surgery. Concerning intracranial drug delivery, the transmeningeal approach has the advantage of being less invasive than direct intraparenchymal or icv drug delivery, but it requires seizure-suppressing molecules that can penetrate the meninges and diffuse into the underlying neocortex in sufficient amounts. Epileptic foci located deeper in the brain parenchyma are not sufficiently reached by this approach . TherefoA receptors upon continuous exposure to GABA, thereby not only inducing tolerance but also increasing the risk of GABA withdrawal seizures, similar to what is described for intraparenchymal GABA delivery [In a series of early proof-of-principle studies, acute epidural or subdural administrations of different compounds were shown to suppress chemically induced neocortical epileptiform activity in a concentration-dependent manner. Muscimol applied topically to the cortex in rats suppressed neocortical epileptiform activity induced by penicillin, bicuculline, and picrotoxin, albeit not strychnine . Acute edelivery ,111. In delivery . Furtherdelivery .Again, chronic continuous or discontinuous drug delivery is necessary for long-term seizure suppression. In a chronic transmeningeal drug delivery study, Tang et al.  showed tDrug-loaded polymers also have been investigated using the transmeningeal route. Halliday et al.  subduralBecause epidurally or subdurally located drug-releasing polymer systems are highly limited in drug load capacity, a frequent refill or exchange would be necessary for long-term transmeningeal drug delivery. The success of transmeningeal pharmacotherapy in epilepsies thus argues for drug delivery on demand in closed-loop systems and/or the use of subcutaneously implanted microinfusion pumps with larger drug reservoirs connected to appropriate catheter systems. Nevertheless, in a first-in-man study on intracranial drug delivery in epilepsies, a lidocaine-soaked gel foam was placed for several minutes onto the pia mater above the seizure focus of three patients prior to tissue resection, and within minutes a clear reduction in interictal EEG spike activity was detected . BecauseThe most direct and obvious approach to deliver a drug to its target in the brain is to administer it directly into the parenchyma. Compensating local deficits in inhibition or reducing hyperexcitation can be considered as a restorative network modulation. Mainly the epileptogenic zone of the brain will be modulated, thereby minimizing neurological toxicity in addition to the lowered risk of systemic adverse effects. Depending on the used technique, the compound diffuses passively  or convection-enhanced (microinfusion pumps connected to catheter system) through the brain interstitium. In general, preclinical studies indicate that intrafocal drug delivery approaches are well tolerated, although behavioral toxicity was not investigated in many of the studies (see below). The occurrence of slight adverse effects may be related to the used drug and can be partially explained by drug diffusion into neighboring brain areas. Likewise, observed anticonvulsant effects are probably not always induced solely by modulation of the seizure focus, but also by infiltration of adjacent seizure-modulating areas.Kelso et al.  directlyIndeed, there is a considerable amount of preclinical studies providing evidence for anticonvulsant activity by direct delivery of many different drugs with a variety of mechanisms of action into or close to the seizure focus . Many ofAcute microinjection of lidocaine into a bicuculline-induced seizure focus in the piriform cortex suppressed electrographic and behavioral seizures in rats . The intIn addition, acute bolus delivery of arsono analogs of GABA and aspartate, but not the arsono analog of glutamate, into the amygdala exerted anticonvulsant effects by elevating afterdischarge thresholds in amygdala-kindled rats ,136. TheA receptor binding in response to frequent seizures have been found near the frontal piriform cortex ipsilateral to the presumed cortical focus, irrespective of where interictal or ictal activity occurs in the cortex [Systemic pilocarpine-induced seizures were prevented by microinjection of the NMDA receptor antagonists 2-amino-7-phosphonoheptanoate (APH) into the piriform cortex . Local ie cortex . This are cortex .Again, acute intracerebral drug applications have a limited effect duration. To prolong the effect of a drug on the seizure focus, delivery from implantable osmotic pumps, polymer carriers, and chronic CED has been investigated.Kohane et al.  directlyWhile GABA itself is not appropriate for systemic administration because of its low ability to pass the BBB, this property is of interest for intracerebral infusion. Continuous (7 days) intracortical  and intrPolymeric microspheres loaded with phenytoin and injected into the hippocampus caused anticonvulsant effects in the rat tetanus toxin model of epilepsy . Likewis(Neuro) peptides only show limited capacity to pass physiological barriers after oral or iv injection. The neuropeptide thyrotropin-releasing hormone (TRH), known to exert anticonvulsant effects, showed prolonged anticonvulsant activity in response to sustained release from polymeric microdisks implanted into the amygdala in the rat amygdala-kindling model . The nucContinuous CED of high-molecular-weight ω-conotoxins, acting as irreversible presynaptic N-type calcium channel antagonists, into the amygdala of rats via an external pump over 20 min induced long-lasting (about 1 week) anticonvulsant effects on amygdala-kindled electrographic seizures and thresholds, whereas intra-amygdalar carbamazepine induced only short-term anticonvulsant effects . NoteworStein et al.  were theHeiss and colleagues published a series of studies in which they investigated CED of muscimol into the brain parenchyma of nonhuman primates ,165,166,Based on the above described preclinical feasibility experiments in nonhuman primates ,165,166,As mentioned in the introduction, resection of the epileptogenic zone as a surgical epilepsy treatment may be effective for selected patients with sysDRE. Achieving seizure freedom by focus resection is more likely in patients with electro-clinically concordant structural lesions in the temporal lobe, while the expected outcome is less successful for patients with extra-temporal lobe foci, focal-to-bilateral tonic-clonic seizures, inconspicuous imaging findings, and occurrence of psychiatric co-morbidities . HoweverAs illustrated in Pathophysiological models of basal ganglia in epilepsies do not only include changes in firing rates and excitation/inhibition but also changes in firing patterns, including circuit-wide oscillations and burst discharges. Accordingly, in vivo, single-unit recording studies in amygdala-kindled rats modeling TLE showed seizure-outlasting plastic network changes in all basal ganglia regions investigated, including striatum , SNr 17,178, STNInvestigations on human patients showed that the basal ganglia are hyperactive during focal to bilateral tonic-clonic seizures . In a reAbout 40 years ago, Gale and Iadarola ,190 wereMost studies on intraparenchymal drug delivery targeting basal ganglia structures again were performed by acute microinjections. The SNr receives direct inhibitory (GABAergic) input from the striatum and GPe and excitatory (glutamatergic) input from the STN. Accordingly, numerous studies showed that acute bilateral microinjection of GABA-mimetic drugs such as vigabatrin and muscimol or NMDA receptor antagonists into the SNr suppresses seizures in rat models of TLE. Acute bilateral microinjection of the irreversible GABA degradation inhibitor vigabatrin  or the NBilateral microinjection of vigabatrin into the SNr of rats suppressed amygdala-kindled seizures ,199. BilBilateral microinjection of muscimol or the NMDA receptor antagonist 2-amino-7-phosphono-heptanoic acid (AP7) into the SNr protected against motor seizures evoked by unilateral bicuculline injections into the anterior piriform cortex . BilaterA receptors) protected against convulsive seizures evoked by focal injection of bicuculline into the anterior piriform cortex [Neurons in the SNr receive excitatory substance P input from the striatum. Accordingly, substance P antagonists microinjected bilaterally into the SNr also attenuated motor seizures induced by maximal electroshock or iv-injected bicuculline . Furtherm cortex .Apart from vigabatrin, further ASDs have been tested regarding their ability to control seizures upon intranigral delivery. Midazolam protected against pilocarpine-induced secondarily generalized seizures after acute bilateral microinjection into the SNr, NAcc, and caudate-putamen . PhenytoA receptor blockade by picrotoxin abolished the anticonvulsant effect of topiramate in the SNr, showing that the anticonvulsant effect of topiramate was mediated via nigral GABAA receptors.Meurs et al.  comparedNoteworthy, several ASDs administered systemically are known to reduce the firing rate of SNr neurons in naïve rats ,211. It A receptor subunit composition, glutamate receptor distribution, and anatomical connections differ between anterior and posterior SNr , with higher GABA immunoreactivity levels in the aSNr compared to pSNr in both sexes, but with higher levels in female compared to male rats [Many studies showed that the seizure-controlling properties of the SNr depend on subregion-, age-, and sex-specific features, and on rat strain ,214,215.ale rats . Kindlinale rats . Muscimoale rats , but notale rats , emphasiale rats , althougale rats . Subregiale rats ,218. FurThese and other reports initiated further acute intracerebral drug delivery studies investigating putative anatomical basal ganglia sites of anticonvulsant actions. Indeed, all investigated basal ganglia neurons are able to modulate different seizure types depending on the drug delivered. In line with the basal ganglia direct inhibitory pathway , acute bApart from the modulation of GABAergic and glutamatergic neurotransmission, modulation of dopaminergic transmission mainly within the striatum has been studied with regard to its ability to modulate seizures. Acute microinjections of dopamine D2 receptor agonists into the dorsal striatum have been shown to control different experimental seizure types, including pilocarpine-induced limbic motor seizures in rats, whereas injections of D2 antagonists in the same region caused proconvulsant effects ,223. NotB receptor agonist baclofen completely suppressed the seizures, and this effect was largely abolished by co-injection of a GABAB receptor antagonist, suggesting that in this basal ganglia region, GABAB receptors are more important at least for modulation of tonic seizures [Acute bilateral microinjection of the GABA uptake inhibitor tiagabine into the GPe of rats exerted anticonvulsant effects on tonic seizures induced by PTZ . Howeverseizures .A remarkable nonselective seizure modulation can also be achieved through the STN, which is composed of glutamatergic projection neurons. The STN is part of the indirect basal ganglia pathway and receives strong GABAergic input from the GPe. In addition, the STN receives fast cortical glutamatergic input via the hyperdirect pathway . It monoA receptors by muscimol, while vigabatrin probably acts more physiologically depending on the activity of GABAergic connections. Apart from drug-dependent differences in inducing adverse effects upon targeted inhibition of basal ganglia, site-specific effects are known. Muscimol injected into the SNr of rats provoked locomotor activation in addition to the contralaterally directed postural asymmetry observed from STN injection [Acute microinjection of muscimol bilaterally into the STN of rats suppressed absence seizures , preventnjection . We recenjection . Furthernjection .Nevertheless, most animal studies investigating seizure modulation through the basal ganglia traditionally concentrated on the SNr, although it was not clear, which structure (SNr or STN) is more promising regarding seizure control. We were the first who provided a direct comparison between systemic administration of vigabatrin and its targeted acute microinjection into different brain regions, including aSNr, pSNr, and the STN, regarding anticonvulsant efficacy and occurrence of adverse effects . TargetiAlthough not directly compared, a cell transplantation study also indicated that bilateral grafting of an inhibitory cell line into the STN might be more effective in raising seizure thresholds than placing the same cell line bilaterally into the SNr .Again, most studies were designed as acute injections, while few studies investigated chronic drug delivery into basal ganglia regions in epilepsy models. The first study investigating long-term drug delivery into the basal ganglia in a model of TLE was performed by Kokaia et al. . GABA-reThe promising results obtained from acute intrasubthalamic vigabatrin delivery  describeThe exact mechanisms underlying the antiseizure effects of basal ganglia manipulations are not fully understood. Early activation of the cortex and SNr emphasizes the important role of these structures in generalized amygdala-kindled seizures . It is aIn summary, the basal ganglia, especially the SNr and the STN, were profoundly investigated as a common seizure gating and control mechanism, being relatively nonselective as to the type of seizure or seizure origin they can influence, which is a highly attractive feature with regard to translation into clinical application. Clinical studies support the concept of an inhibitory role of the basal ganglia during TLE and other types of epileptic seizures ,168. MosThe thalamus is a complex structure containing multiple nuclei with diverse functional roles, including relaying information between limbic structures, basal ganglia, cortical regions, and brain stem areas ,238. In The DM, which is strongly connected to the amygdala, piriform cortex, and other limbic structures, is known to have an important role in the propagation and modulation of focal to bilateral tonic-clonic seizures ,241,242.Further thalamic regions were investigated for their ability to modulate limbic seizures, for example, the ventromedial (VM) and the posterior nucleus (PO) of the thalamus . MuscimoConsidering the ample evidence available on the role of basal ganglia in limbic seizure control, it was obvious to investigate basal ganglia target structures apart from thalamic regions for their role in pharmacological seizure modulation. Especially the SC , but also the PPN , were investigated by intracerebral drug delivery approaches to define their role and verify their efficacy in limbic seizure control. The SC is reciprocally connected with basal ganglia regions and sends efferents to thalamic and brain stem regions .As mentioned in chapter 5.1, the SNr provides tonic inhibitory influence onto the deep/intermediate layers of the SC and inhibition of these GABAergic nigral neurons is well-known to suppress numerous seizure types with different origins. Accordingly, disinhibition of the deep layers of the SC exerts an anticonvulsant action, as was proved by the observation that bilateral, but not unilateral, application of bicuculline to the SC attenuates limbic motor seizures induced by unilateral bicuculline injection into the anterior piriform cortex in rats . The mecThe importance of the SC was confirmed and extended by recent optogenetic studies in rats, showing that activation of deep/intermediate layers of the SC neurons suppressed seizures originating in diverse brain networks, including the limbic system . UnilateScattered data are also available for targeting further subcortical structures, including lateral habenula, which is considered an important relay between basal ganglia and limbic structures such as the hippocampus . For exaBilateral microinjections of bicuculline into the zona incerta exerted an anticonvulsant effect on pilocarpine-induced seizures, while muscimol was proconvulsant .Although bypassing the BBB by intracranial drug delivery is a highly attractive approach associated with a number of advantages over systemic drug administrations (see Introduction Section), several challenges must be addressed before translation into clinical use is realistic.Apart from defining the most promising target site for each subset of sysDRE patients, choosing a drug feasible for long-term intracranial delivery is inevitable. For clinical relevance, several compound requirements should be met, including stability at body temperature, efficacy at concentrations not significantly exceeding physiological osmolarity in brain extracellular compartment, efficacy at physiological pH, water solubility, and lack of local neurotoxicity at therapeutic doses ,108. SomPharmacoresistant epilepsy is a multifactorial and variable phenomenon, for which numerous further mechanisms are under investigation apart from the BBB alterations and peripheral pharmacokinetic mechanisms discussed in the introduction. Some of these further mechanisms will probably also interfere with intracranial drug delivery. According to the target hypothesis , epilepsOther hypotheses include disease etiology, the intrinsic severity model, genetic and epigenetic factors, inflammatory processes, and neural network alterations  expressed cytochrome P450 metabolic enzymes , therebyTaken together, although peripheral mechanisms for ASD resistance can be circumvented, it is nevertheless likely that intracranial drug delivery induces individually distinct responses definable as responders and nonresponders. Animal experiments performed by our group support this concept by revealing responders and nonresponders after intracranial microinfusions of vigabatrin  and muscAs mentioned in chapter 3, numerous animal models not only exist for seizure/epilepsy but also for DRE. Most of them are laborious and therefore not suitable for fast screening. Nevertheless, future research directions should involve investigations of intracranial drug delivery approaches in well-defined animal models of DRE.A receptors. However, GAD activity was reduced in the STN of vigabatrin responders as well as nonresponders with no intergroup differences, although low sample sizes limit the validity of this finding [Similar to systemic ASD administrations , the dev finding . Likewis finding . Discont finding ,65.3 in rats and about 240 mm3 in humans [Most preclinical studies on intracranial drug delivery in seizure/epilepsy models were conducted in rodents. Because of the much smaller volume of brain structures in rodents compared to humans, it is much more difficult to restrict drug distribution to the boundaries of the target region. This entails challenges for safety investigations in rodent models because few mm drug distribution distances in rats or mice may cause neurotoxic adverse effects by infiltrating brain areas surrounding the intended target zone. For example, the STN has a volume of about 0.8 mmn humans . Acute in humans , as welln humans  into STNn humans . BecauseConsidering the size of human brains, the limited diffusion range rather could be a problem depending on the technical approach used. For example, limited penetration distances (mm range) were described by passive diffusion from polymer implants . HoweverApart from size differences, discrepancies in connectivity between rodent and primate brains could influence the outcome of intracranial drug delivery approaches. For example, the STN differs between rats and humans concerning connectivity with other brain areas . Beyond There is broad experimental evidence that the modulation of part of the epileptic network by targeted intracranial drug delivery is sufficient to control focal seizure initiation, propagation, or generalization. Promising target regions include the seizure focus itself and seizure-controlling structures of the epileptic network remote to the epileptogenic zone. Among the remote regions, especially basal ganglia structures such as the STN were defined preclinically as key nodes exerting a widespread influence on seizure circuitry when pharmacologically modulated. These remote key structures are known to be relatively nonselective as to the type of seizure or seizure origin they can influence. Therefore, they are highly attractive targets for the development of therapeutic intracranial drug delivery approaches for sysDRE patients not eligible for focus resection. However, while most preclinical studies so far utilized acute intracranial bolus injections, long-term targeted delivery is required for suppression of spontaneous recurrent seizures. Future preclinical studies should take this requirement into account. In addition, even though remarkable antiseizure effects can be obtained preclinically by intracranial drug delivery approaches, there are challenges to be solved before this strategy is clinically applicable. These challenges include the risk of development of pharmacological tolerance in response to intracranial drug delivery and pharmacoresistance mechanisms apart from epilepsy-induced BBB alterations.Although invasive surgical procedures are inevitable, bypassing the BBB by targeted intracranial drug delivery is a promising strategy worth of further development to circumvent BBB-associated drug resistance mechanisms and to lower the risk of systemic and neurologic adverse effects. Until now, very few clinical trials confirming the feasibility of targeted intracranial drug delivery in medically intractable epilepsy patients have been performed. Overall, the existing data show that targeted modulation of neuronal activity is a reasonable research strategy for the investigation and treatment of epilepsy patients resistant to systemic pharmacotherapy.


Free radicals, generally composed of reactive oxygen species (ROS) and reactive nitrogen species (RNS), are generated in the body by various endogenous and exogenous systems. The overproduction of free radicals is known to cause several chronic diseases including cancer. However, increased production of free radicals by chemotherapeutic drugs is also associated with apoptosis in cancer cells, indicating the dual nature of free radicals. Among various natural compounds, curcumin manifests as an antioxidant in normal cells that helps in the prevention of carcinogenesis. It also acts as a prooxidant in cancer cells and is associated with inducing apoptosis. Curcumin quenches free radicals, induces antioxidant enzymes , and upregulates antioxidative protein markers–Nrf2 and HO-1 that lead to the suppression of cellular oxidative stress. In cancer cells, curcumin aggressively increases ROS that results in DNA damage and subsequently cancer cell death. It also sensitizes drug-resistant cancer cells and increases the anticancer effects of chemotherapeutic drugs. Thus, curcumin shows beneficial effects in prevention, treatment and chemosensitization of cancer cells. In this review, we will discuss the dual role of free radicals as well as the chemopreventive and chemotherapeutic effects of curcumin and its analogues against cancer. Radical species are comparatively highly reactive, due to the presence of an unpaired electron that imparts a high degree of electrophilicity. These free radicals act as oxidants or reductants by either donating an electron to or accepting an electron from other reactive molecules [Free radicals are generally reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are capable of independent existence. ROS and RNS can broadly be categorized into two groups—radicals and non-radicals. Superoxide  or endogenous (produced in the body), solubility in water  or lipids , or the size and nature of the antioxidant molecule, i.e., enzymatic  or non-enzymatic . Studies have shown that antioxidants reduce the occurrence of carcinogenesis. It has been observed that the consumption of fruits and vegetables, rich in antioxidants, in daily diet causes a reduction in the risk for cancer incidence . In contReactive oxygen species (ROS), produced either exogenously  or endogenously , are related to a wide variety of human disorders. Excessive ROS causes damage to proteins, DNA, and RNA leading to genetic alterations in cells. On the other hand, low levels of ROS are essential for various biological functions, including cell survival, cell growth, proliferation and differentiation, and immune response . Lethal 2−•), hydrogen peroxide (H2O2), and hydroxyl radical (HO•) as well as secondary metabolites including lipid peroxides, peroxynitrite (ONOO−), and hypochlorous acid (HOCl). Because of their high reactivity, they cause damage to the macromolecules and disrupt biochemical function [The high accumulation of ROS and/or low level of antioxidants in cells causes an imbalance in redox status, which is known as oxidative stress. Oxidative stress is implicated in a variety of pathologies including cancer. ROS are short-lived and highly electrophilic molecules generated by the partial reduction of oxygen to form superoxide , transcription factors , components of ubiquitin/proteasome system and autophagy/lysosomal protein degradation systems, molecular chaperones, and cytoskeletal proteins are susceptible to altered physiological function by ROS . These mCancer cells have a hypermetabolism that produces a large amount of ROS compared to normal cells. At the same time, cancer cells have marked antioxidant capacity that help in maintaining the redox balance. Recently, anticancer therapies that induce oxidative stress by increasing ROS and/or inhibiting antioxidant level have received great attention . The incSince cancer cells possess comparatively high ROS levels, these cells have a higher sensitivity towards increased prooxidant and decreased antioxidant levels ,26. ThisGarcinia species), also caused synergistic reduction of cell viability in SKOV-3 cells with doxorubicin and this effect was found to be correlated with increased cellular ROS accumulation [Besides these, several natural compounds also exhibit anticancer effects by inducing ROS. Resveratrol, a dietary product present in grapes, vegetables and berries, has been shown to exert an anti-cancer effects on melanoma cells through the induction of ROS. It has also been shown to induce ROS-p38-p53 pathway by increasing the gene expression of phosphorylated p38 MAPK and activating the p53 and ER stress pathway . A combimulation . Similarmulation .Other types of anticancer drugs cause cancer cell death by depleting antioxidant levels. For instance, buthionine sulfoximine synergistically affects the chlorin e6 (Ce6)-based photodynamic therapy (PDT) of colorectal cancer cells  and seveAs described above, ROS plays a key role in carcinogenesis. Therefore, reducing ROS levels can reserve the initiation of tumorigenesis. Curcumin has shown to be a well-established antioxidant compound by innumerable researchers. Various studies have also shown that curcumin blocks the tumorigenesis process in many rodent carcinogenesis models including skin, duodenal, forestomach, and colon carcinogenesis and therefore works as a chemopreventive agent ,47,48. IAs curcumin has antioxidant activity, it exhibits a chemopreventive effect by reducing ROS levels and by inducing antioxidant enzymes . In a stCurcumin also exhibits potential benefit against arsenic-induced oxidative stress in humans. A field trial was conducted in volunteers consuming arsenic contaminated ground water. Among 286 volunteers, half were treated with curcumin (500 mg twice daily) and remaining half were assigned with placebo. After three months, DNA damage and oxidative stress were analyzed in blood. The blood samples from endemic regions showed severe DNA damage with increased levels of ROS and lipid peroxidation. The antioxidant enzymes showed depleted activity in the placebo treated group. However, curcumin intervention reduced the DNA damage, retarded ROS generation and lipid peroxidation and raised the level of antioxidant activity . Thus, cS-transferase and quinone reductase. This suggests that curcumin may prevent liver and kidney carcinogenesis by inducing antioxidant enzymes [Curcumin also exerts chemopreventive efficacy by increasing the activity of antioxidant enzymes and phase II-metabolic enzymes in the liver and kidneys. Curcumin (2%) when fed to mice for 30 days showed a significant enhancement in the activities of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and catalase. In addition, curcumin enhances the activities of glutathione  enzymes . Moreove enzymes , while i enzymes . Such obThe transcription factor Nrf2 is believed to be one of the crucial players in regulating the various antioxidant genes including hemeoxygenase (HO-1). Activation of the Nrf2 antioxidant pathway signaling is reported to play an important role in cancer prevention . InteresCurcumin is a known potent anti-inflammatory agent that prevents tumor progression and exerts a chemopreventive effect on carcinogenesis via suppressing inflammation. Increasing evidences suggest the involvement of ROS as a mechanism by which chronic inflammation drives cancer initiation and progression. Chronic inflammation stimulates ROS generation which leads to tissue injury and promotes carcinogenesis ,71,72. TConsidering the poor absorption and bioavailability of curcumin, several analogues of curcumin have been synthesized and optimized for better antioxidant properties . DimethoThe chemopreventive efficacy of curcumin has been evaluated in combination with several other natural and synthetic compounds. Interestingly, the chemopreventive potential of curcumin in combination with resveratrol has been shown in multiple studies. Curcumin in combination with resveratrol prevented lung carcinogenesis in rats . In a stThe cancer therapeutic benefits of curcumin have been demonstrated in multiple types of cancers including, breast, prostate, skin, lung, liver, brain, stomach, cervical, ovary, multiple myeloma, leukemia and several other types of cancer . Prior e2O2 [Along with chemopreventive properties, curcumin has also chemotherapeutic effects against different types of cancers through multiple mechanisms including oxidative stress pathway . Curcumi2O2 ,90. Inde2O2 . Further2O2 .It has also been observed that the treatment of leukemic cells with curcumin increases intracellular ROS levels over a threshold which was found to be linked with the induction of anti-tumorigenic activity . In melaAlong with ROS production, depletion of antioxidant enzymes enhances the anticancer property of curcumin. In a study, curcumin suppressed the growth of human leukemic cells via ROS-independent glutathione depletion, which leads to caspase activation and further apoptosis in leukemic cells. Curcumin treatment to leukemic cells also downregulates the expression of the inhibitor of apoptosis proteins (IAPs), phospho-Akt, c-Myc, and cyclin D1, probably through the suppression of glutathione . In anotBesides curcumin, its analogues  also exhA2 without the β-diketone moiety, displayed antiangiogenic activity. It has been demonstrated that A2 exerts its antiangiogenic activity mainly through inducing endothelial cell death via elevating NADH/NADPH oxidase-derived ROS [PGV-1) has been studied for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 -benzylidine-cyclopentanone) is a monocarbonyl analogue of curcumin and has stronger potency than the lead compound, curcumin. It has been found that PGV-1 exhibits 60-fold higher efficacy compared to that of curcumin in inhibiting K562 cell growth. PGV-1 also inhibited the proliferation of leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer cells. Interestingly, PGV-1-induced cell death is mediated by an increase in intracellular ROS levels through inhibition of ROS-metabolic enzymes [Another monocarbonyl analogue of curcumin, ived ROS . Further enzymes . Nakamae enzymes . Further enzymes .Accumulated preclinical evidences suggest that the effectiveness of chemotherapy is being limited due to drug resistance, therapeutic selectivity, and undesirable side effects. In this regard, curcumin has proved its ability to sensitize drug-resistant cancer cells and enhance therapeutic efficacy of drugs. As ROS production by curcumin plays a pivotal role in cancer cell death, it can also mediate chemosensitization. Studies showed that curcumin improves the sensitivity of cancer cells to chemotherapy drugs by regulating a variety of signaling pathways . An in vThe cancer therapeutic response of curcumin has been shown to be improved by tolfenamic acid . Tolfenamic acid has been reported to inhibit the growth of human cancer cells in vitro and in vivo. However, the combination of tolfenamic acid and curcumin showed higher growth inhibition when compared to either single agent. Further, another study showed that the combination treatment upregulated the ROS level that led to an increase in apoptosis in colorectal cancer cells . SubtoxiCombinations of drugs may enhance the therapeutic effect of individual drug treatment. Combinations of natural products are broadly explored in cancer therapy. In a study, curcumin was combined with arabinogalactan (found in Larch trees) and treated to the human breast cancer cells. This combination promoted cell growth inhibition and apoptosis induction in human breast cancer cells by increasing ROS level as well as loss of mitochondrial membrane potential and reduction of glutathione. In addition, this combination inhibited the progression of breast tumors in mouse model . It has Oxidative stress—caused by an imbalance between free radical generation and the antioxidant defense system of the body—potentially results in the initiation and progression of cancer. However, free radicals, specially ROS, play an important role in therapeutic drug action. Thus, ROS has both pathological and pharmacological importance in the body. These findings suggest the use of strategies to either suppress or induce ROS. One of the strategies is to decrease oxidative stress by quenching ROS and/or increasing antioxidant enzymes that can subsequently inhibit carcinogenesis. Another strategy is to use ROS inducing agents in cancer patients that can induce apoptosis in cancer cells. Use of antioxidants in cancer patients have shown unfavorable results for the treatment efficacy of therapeutic drugs. Studies have shown that antioxidants cause increased cancer incidences, probably due to the inhibition of ROS mediated apoptosis of cancer cells. However, high consumption of fruits and vegetables rich in antioxidants are associated with decreased risk of cancer incidence. In view of this, curcumin is considered as an antioxidant as well as a prooxidant, since it reduces oxidative stress in normal cells and induces ROS production in cancer cells. Although several studies have investigated the beneficial role of curcumin, this review will help to better understand the ROS associated chemopreventive and anticancer mechanism of curcumin.


Oxidative Stress in Myocardial and Neural Remodeling” evaluates the damage of oxidative stress specifically to the cardiovascular and nervous systems, defines underlying mechanisms, and proposes novel therapeutic targets.Endogenous oxidative stress arises as an attempted reparative response to injuries, including hypoxia, myocardial infarction, stroke, and radiation-induced phototoxicity. More clinically relevant than ever, oxidative stress has recently been implicated as the key player in the induction of severe acute inflammatory critical illness by the coronavirus SARS-CoV-2 . Indeed, in pulmonary hypertension, right ventricular function is the major determinant of survival. Mikhael et al. review evidence that in chronic pulmonary hypertension, oxidative stress induces structural remodeling of not only the pulmonary vasculature but also the right ventricle, eventually causing right heart failure. They discuss why the preclinical success of antioxidant therapies fails to translate clinically .Starting with the heart, two reviews shed light on the cardiac-chamber specificity in oxidative stress response. Li et al. delineate a molecular pathway linking oxidative stress, diet, and inflammation with myocardial fibrosis and identify a novel potential target for antifibrotic therapy. Using mouse models of doxorubicin-induced cardiomyopathy, they demonstrate how trimethylamine N-oxide (TMAO), a gut microbiota-dependent metabolite of specific dietary nutrients, can aggravate myocardial fibrosis through activation of the Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome .Delving deeper into the mechanisms of structural remodeling by oxidative stress, Zhao et al., on the other hand, investigated the mechanistic role of oxidative stress in electrical remodeling, particularly during synergy with existing structural and electrical remodeling by noncardiomyocytes, including myofibroblasts . Using a novel in-vitro two-dimensional model of interface between cardiomyocytes and noncardiomyocytes that they developed, the authors demonstrated a functional proof-of-concept for the contribution of oxidative stress to the dual protective and proarrhythmic roles of myofibroblasts and other noncardiomyocytes. Not only connexin43-rich noncardiomyocytes can enable passive, decremental, anisotropic impulse propagation, but they can also, independently or synergistically with oxidative stress, induce new arrhythmia triggers, dynamic functional conduction block, non-reentrant and reentrant arrhythmias.Carbone et al. emphasize the critical role of neutrophils in cardiac and neural remodeling mediated by the oxidative stress of ischemia-reperfusion injury as neutrophils are activated early for massive production of reactive oxygen species. The authors discuss how anatomical and functional differences between these two organs shape their responses to oxidative stress and suggest that attention to these differences may improve translational success of preclinical trials . Supporting Carbone et al.'s conclusion, Vaillancourt et al. demonstrate that the activities of reactive oxygen and nitrogen species may extend beyond local tissues. They discover how oxidative stress in pulmonary hypertensive rats induces neuroinflammation remotely in the thoracic spinal cord . Denver et al., on the other hand, explore the mechanistic synergism between coexisting oxidative stress in heart and brain and the resultant clinical manifestations. Based on the insight that the central nervous system vasculature is particularly vulnerable to oxidative stress .The next three articles examine how oxidative stress causes adverse structural remodeling in both the cardiac and central nervous systems concomitantly. Ishiyama et al. discover that in Ménière's disease, endogenous oxidative stress primarily targets the vascular endothelial cells of the blood labyrinthine barrier for structural remodeling. The inflammation triggered by oxidative stress causes pericytes to degenerate and migrate, thereby compromising the structural integrity of the blood labyrinthine barrier and its critical function in ionic and fluid homeostasis .Exploring yet another vasculature system, Deres et al. conduct a test of the efficacy and safety of a novel anti-inflammatory drug, bradykinin B1 receptor antagonist FGY-1153 in spontaneously hypertensive rats. The authors conclude that this novel agent provides moderate protection against the development of hypertensive cardiomyopathy and has no cardiovascular toxicities . However, additional randomized preclinical and clinical trials are necessary to confirm these promising findings regarding the efficacy and safety profile of FGY-1153.Moving from pathophysiology to therapeutics, Taken together, the collection of original research and review articles presented in this Research Topic provides an overview and update of insights into the critical role of oxidative stress on myocardial and neural remodeling. We hope that this collective knowledge would inspire and drive further research for innovative and effective anti-oxidative therapeutic strategies to reduce the burden of morbidity and disability due to oxidative stress.TN conceptualized this Research Topic, recruited co-editors, and wrote the original draft. All authors contributed to manuscript revision, approved the submitted version, and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Obesity is a complex disease attributable to many factors including genetics and environmental influences. Growing evidence suggests that gut microbiota is a major contributing factor to the pathogenesis of obesity and other metabolic disorders. This article reviews the current understanding of the role of gut microbiota in the regulation of energy balance and the development of obesity, and how the microbiota communicates with host tissues, in particular adipose tissue. We discuss several external factors that interfere with the interplay between gut microbiota and host tissue metabolism, including cold exposure, diet regimens, and genetic manipulations. We also review the role of diet-derived metabolites that regulate thermogenesis and thus energy homeostasis. Among the gut microbial metabolites, we emphasize short-chain fatty acids, which could be utilized by the host as a direct energy source while regulating the appetite of the host through the gut-brain axis. Obesity is a worldwide epidemic, with sedentary lifestyles and increased food intake likely the main causes . HoweverRecent studies suggest that the composition and activities of gut microbiota not only associate with obesity but cause it. The gut microbiota not only contributes metabolites and energy to the host but also controls the absorption of nutrients in the intestine, thereby influencing human energetics . Consideob/ob mice  are fatter than germ-free mice  even though they consume fewer calories . Then, a/ob mice , or diet/ob mice , into ge/ob mice . Similar/ob mice .To address the causal role of the microbiome in obesity, the microbiomes of human twin pairs discordant for obesity were transplanted into C57BL/6 germ-free mice . These hSpecific bacteria strains have been associated with obesity and type 2 diabetes. Firmicutes and Bacteroidetes are the two dominant gut microphyla , and theAkkermansia muciniphila of class Verrucomicrobia has been most extensively studied , a secreted protein that inhibits lipoprotein lipase activity  and Type II Iodothyronine Deiodinase (Dio2) in their interscapular BAT compared to mice that received microbiota from donors housed at thermoneutrality (29°C)  . The rolA direct link has been established between gut microbiota and diet-induced obesity . For insUcp1 expression.Diet-induced weight-loss intervention improves low gut microbial gene richness, thus mitigating the dysmetabolism of obese patients . Considein vivo and in vitro indicates that gut microbiota plays a role in shaping host adaptability to dietary lipid uptake, possibly as early as in the digestive phase.In addition, germ-free mice are resistant to high-fat diet-induced obesity, accompanied by malabsorption of dietary lipids, while HFD-fed SPF mice develop obesity and exhibit an increased abundance of the family Clostridiaceae in their intestines . The facA. muciniphila and Lactobacillus bacteria, which are capable of producing small-chain fatty acids (SCFAs) that may provide the host with beneficial health outcomes  regimen in diet-induced obese mice results in weight loss and an improved metabolic profile associated with increased beige fat development . Notably the gut . Consist the gut .However, short-term dietary interventions appear to have no effect. A randomized 1-week-long dietary intervention found no effects of different types of bread on clinical parameters nor on gut microbial composition . AnotherN-acylethanolamines (NAEs) are the best characterized endocannabinoids, and increased NAE levels are associated with obesity and metabolic comorbidities , the NAE endocannabinoid synthesizing enzyme, is more susceptible to diet-induced obesity and metabolic dysregulation  acetate (C2), propionate (C3), and butyrate (C4), which are the major end products of microbial fermentation of dietary fiber, serve as the primary energy source for colonic epithelium . A randoSeveral studies suggest that SCFAs activate G protein–coupled receptors (GPCR) to affect the target organ of the host. For example, Gpr43, which has a strong affinity to acetate and propionate, plays an important role in mediating the microbial input in WAT metabolism . Gpr43-KA recent study proposed that butyrate mediates the thermogenic stimulation of BAT, as administering butyrate sodium to microbiota-depleted mice partially rescues impaired thermogenesis and promotes fat oxidation . Other sA. muciniphila modulates histone deacetylase level and the expression of genes involved in satiety and host lipid metabolism  ratio compared with the high BFR obese group. This finding indicates that the expression levels of genes implicated in glucose and energy homeostasis in adipose tissue could be epigenetically regulated by gut bacterial populations. In support of this, Faecalibacterium prausnitzii. Their results disclosed the influence of different composition of gut microbiota in obesity and type 2 diabetes on the epigenetic regulation of genes. On the other hand, some reports have demonstrated that fecal micro-RNAs (miRNAs) can shape the composition of the gut microbiome  is strongly associated with cardiovascular risks and host inflammation . Cholinee tissue . Since TCyp7b1), a hepatic enzyme that mediates alternative bile acid synthesis. The elevated bile acid secretion changed the microbiota composition and increased adipocyte thermogenesis (Clostridium species, which is classically described as being responsible for the conversion of primary bile acids into secondary bile acids, reversed the metabolic phenotypes in the KO mice (In addition to SCFAs, bile acids appear to play an important role in mediating the interactions between microbiota and host tissues. Bile acids are synthesized from cholesterol by a process orchestrated by multiple liver enzymes . Afterwaogenesis . Confirmogenesis . In lineogenesis . Notablyogenesis . Further KO mice . This reAnother nutrient whose gut microbial metabolites have been associated with adipocyte function is resveratrol. Resveratrol is a non-flavonoid polyphenol compound that is naturally found in a wide variety of plants, such as grapes and peanut skin . BesidesThe evidence strongly suggests that alterations in gut microbiota diversity and composition contribute to the pathogenesis of obesity and obesity-related metabolic disorders. However, the limitations and pitfalls of the studies should be noted. Many of these studies are observational. In addition, differences in the lab environment could contribute to differences in the microbiome and its effects. Also, many studies used mice, which have gut anatomies that are different from humans. Future studies are needed to gain a more accurate understanding of host-microbiome communications. Based on the existing preliminary data identifying epigenetic mechanisms as a regulator of gut microbiota composition, a dietary approach targeted to favor a more beneficial bacterial population and thus epigenetic changes might be effective in the prevention of obesity. For this, the accurate analysis of microbiome diversity and composition will be key. Large-scale sequencing studies have already identified organisms and their relative abundance in purified DNA by sequencing specific regions of the 16S or 18S ribosomal genes . In paraAccumulating studies have started to examine the effect of gut microbiota transplantation on insulin sensitivity and microbiota composition in humans. SK and HX co-drafted manuscript. HX crafted art work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Breast cancer is the second most commonly diagnosed cancer in American women following skin cancer. Despite overall decrease in breast cancer mortality due to advances in treatment and earlier screening, black patients continue to have 40% higher risk of breast cancer related death compared to white patients. This disparity in outcome persists even when controlled for access to care and stage at presentation and has been attributed to differences in tumor subtypes or gene expression profiles. There is emerging evidence that the tumor microenvironment (TME) may contribute to the racial disparities in outcome as well. Here, we provide a comprehensive review of current literature available regarding race-dependent differences in the TME. Notably, black patients tend to have a higher density of pro-tumorigenic immune cells  and microvasculature. Although immune cells are classically thought to be anti-tumorigenic, increase in M2 macrophages and angiogenesis may lead to a paradoxical increase in metastasis by forming doorways of tumor cell intravasation called tumor microenvironment of metastasis (TMEM). Furthermore, black patients also have higher serum levels of inflammatory cytokines, which provide a positive feedback loop in creating a pro-metastatic TME. Lastly, we propose that the higher density of immune cells and angiogenesis observed in the TME of black patients may be a result of evolutionary selection for a more robust immune response in patients of African geographic ancestry. Better understanding of race-dependent differences in the TME will aid in overcoming the racial disparity in breast cancer mortality. Breast cancer is the second most common cancer in women in the U.S. following skin cancer, and is the second leading cause of cancer death , 2. In bA widening racial gap in survival has also been observed for women in the US Department of Defense healthcare system , as wellIndeed, several studies have indicated that racial disparity in breast cancer outcome between patients of African compared to those of Caucasian ancestry are due to biological factors including differences in gene expression patterns of tumor cells as well as differences in the local milieu (or context) in which cancer cells reside, typically referred to as the tumor microenvironment (TME) . TME encBreast cancer is an extremely heterogeneous disease at multiple levels, including histologic subtype, grade, hormone and growth factor receptor status, as well as gene expression pattern . MoleculA number of immune cells reside within the TME and contribute to cancer progression. Among the well-studied ones are tumor-associated lymphocytes (TILs), regulatory T cells (T-regs), neutrophils, tumor-associated macrophages (TAMs), and myeloid-deprived suppressor cells (MDSCs).TILs, the most abundant immune cells within breast TME, convey a good prognosis especially in patients with triple negative (TN) disease –28. In pUnlike TILs, increased number of T-regs in breast TME has been associated with decreased relapse-free and overall survival , 35. ThiNeutrophils have been typically involved in the pathophysiology of acute infection and elimination of bacteria. However, about a decade ago, studies in pre-clinical models of cancer demonstrated that depending on the levels of chemokines in TME, tumor-associated neutrophils (TANs) may develop an either pro- or anti-tumor phenotype . More reThe role of TAMs in the progression of breast cancer has been extensively studied –47. ThisMyeloid-derived suppressor cells (MDSCs) represent well-established mediators of the immunosuppressive tumor microenvironment, and also serve as critical regulators of angiogenesis, cancer cell invasion and migration, as well as pre-metastatic niche formation –64. MDSCMicrovascular density has been consistently associated with tumor progression and outcome in breast cancer , becausein vivo and in vitro  are the most common stromal cell type of mesenchymal origin in the tumor microenvironment . Howeverin vitro . This stAccording to several studies it seems that single nucleotide polymorphisms (SNPs) in the FGF family of genes may influence the risk for breast cancer in patients of various racial backgrounds. In particular, SNP variants in the fibroblast growth factor receptor-2 (FGFR-2) gene and/ or the FGFR-2 promoter are associated with an increased risk of breast cancer in Chinese women , 80, Norin vivo studies in the future.Likewise, CAFs and ECM may affect breast tissue density, which has important clinical implication not only for cancer progression but also for mammographic screening. Indeed, in a large multiethnic study, it was shown that women of Hispanic ancestry had the highest mammographic breast density, followed by AA and EA women . To whatin vitro studies  and breast cancer risk. . InteresSince cytokines operate in integrated networks, a more complete understanding will be gained with the exploration and accurate measurements of multiple cytokines simultaneously (known as cytokine patterns or signatures), using advanced proteomic technologies . A wide Several groups have previously compared breast cancer gene expression patterns between AA and EA patients using high throughput approaches such as RNA sequencing and found differences in signaling pathways primarily related to angiogenesis, chemotaxis and immunity , 55. HowA similar prediction could be made using five genes most differentially expressed in the breast cancer stroma, PSPHL, CXCL10, CXCL11, ISG20, and GMDS. Importantly, this separation was independent of estrogen receptor expression status. Interestingly, CXCL10, CXCL11, and ISG20 are IFN-γ-regulated genes, which is consistent with the presence of interferon signature found in breast cancer from AA patients . There aEchinococcus granulosus  vs. adjuvant (AC) chemotherapy have no difference in survival , 117, a Other promising therapies targeting the TME are immunotherapies . AlthougDisparities encountered in the TME are a part of the dynamic interplay between local and systemic factors. As discussed above, the most pronounced differences in TME are associated with inflammation and angiogenesis. African ancestry is associated with higher inflammatory gene expression and enhanced bacterial clearance likely due to pathogen-rich geographic ancestry. While aggressive immune response is beneficial in defeating pathogens prevalent in certain geographical regions, it may also be promoting pro-tumorigenic properties in the TME as an unintended consequence. These protective innate immune variants are both disproportionately distributed among racial populations and are linked with racial disparities in cancer . TherefoThe TME is rapidly emerging as a key contributor to cancer progression, and patient outcome. The complex interplay between tumor cells and surrounding immune, vascular, and stromal components continue to be studied extensively. In this review, we highlight the racial differences in TME on cellular, molecular, and genetic levels. Furthermore, we explore systemic immune and cytokine signatures as contributors to the racial disparity in TME. The awareness of these differences and further research will lead to development of race-specific biomarkers and therapeutic targets and ultimately improved personalized cancer treatment.GKa and MO contributed conception and design of the review. GKi, JP, GKa, and MO wrote sections of the manuscript. JS, JC, and PF provided critcial revisions to the content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cancer Research, Xia and colleagues reported that, in cancer, constituents in serine–glycine-one-carbon (SGOC) metabolism exhibit enhanced transcriptional activation and are increasingly utilised, which results in more glucose-derived carbon to serine–glycine biosynthesis. The current work identifies an MYCN-dependent metabolic vulnerability and shows a variety of associations between metabolic reprogramming and enhanced sensitivity to metabolic stress, which may lead the way to unlocking new anticancer therapies. Here, we summarised new insights into the role of SGOC metabolism in the progression of neuroblastoma (NB) with highly activated SGOC metabolism.In a recent study published in  Neuroblastoma (NB) is a highly heterogeneous disease in children that ranges from low-risk tumours that spontaneously differentiate and regress with minimal or no therapy to aggressive high-risk tumours that have a poor prognosis, frequently metastasise and are resistant to therapy. Child survivors often suffer a lifetime of severe adverse effects elicited by chemotherapeutic drug toxicity. Xia and colleagues reported a MYCN-dependent metabolic vulnerability that could suppress the serine–glycine-one-carbon (SGOC) pathway, which offers potential targets for preventing therapeutic resistance in NB. This mechanism may result in fewer required drugs for treatment, thereby reducing the incidence of deleterious adverse effects in the paediatric patients.MYCN is an important member of the nuclear proto-oncogene MYC family, which supports the development of many different tumours, including retinoblastoma, medulloblastoma, prostate cancer, lung cancer and NB. The MYCN oncogene is frequently amplified in malignant NB, and this amplification is considered the single most relevant genetic alteration that promotes cell proliferation, metabolic changes and aggressiveness in NB. Many malignancies would benefit from improved MYCN-targeting therapeutic approaches. Several indirect strategies to target MYCN-driven tumorigenesis exist as follows: (a) MYCN transcription targeting; (b) MYCN protein stability targeting; (c) MYCN dimerisation and transcriptional activity targeting; (d) MYCN-based immunotherapy; and (e) indirect MYCN targeting1. Xia and colleagues2 uncovered a mechanism of aberrant SGOC pathway activation in NB with MYCN amplification, and their findings may provide potential therapeutic targets using three of the abovementioned strategies (excluding immunotherapy).The one-carbon metabolic pathway can generate various molecules, e.g., nucleotides, proteins, and lipids and substrates for methylation reactions, as well as maintain the redox status; this process comprises three critical reactions: the folate cycle, the methionine cycle and the trans-sulphuration pathway. Serine and glycine are synthesised de novo from glycolysis through oxidation of the intermediate 3-PGA, and these products fuel one-carbon metabolism.3. Mehrmohamadi and colleagues first proposed the concept of the SGOC metabolic network4 and identified the expansive and heterogeneous functions of this network in human cancer. They showed the first comprehensive systems-level analysis of the expression pattern, metabolic flux, and the correlation with SGOC metabolism and delineated the potential features of the SGOC pathway in ovarian, lung, colorectal, and breast cancer. Unlike the glycolytic network which may be universally overexpressed in the whole network of cancers, the expressions of SGOC-network constituents differ in more complicated ways. They found that SGOC heterogeneity is strikingly apparent, which may confer its predisposed activation in cancer.The SGOC metabolic unit, which includes serine, glycine and one-carbon metabolism, supplies an integration point in cellular metabolism that permits cells to achieve diverse biological functions by converting serine and glycine into several other metabolic products. Locasale considered that serine, glycine and the one-carbon cycle form a vital full circle in cancer metabolism5, Zhao6 and Liu7 showed that serine–glycine metabolism is essential for tumorigenesis. Importantly, they confirmed that ATF4 is a vital regulator of the transcriptional activation of serine–glycine metabolism and the stimulation of cell proliferation. Ding and Zhao identified a genetic mechanism for activating the serine–glycine biosynthetic pathway. Liu and colleagues used the TH-MYCN mouse, a transgenic model of high-risk NB with MYCN overexpression, to show that the serine–glycine synthesis pathways are essential for tumorigenesis. Importantly, Xia and colleagues reported the potential roles of the SGOC pathway based on their previous research in MYCN-amplified NB; these roles may be the hidden Achilles heel of all MYCN-driven cancers. In this report, Xia and colleagues found that SGOC pathway activation requires both MYCN and ATF4 in MYCN-amplified NB, and these factors form a positive feedback loop. The MYCN protein can activate the expression of ATF4. In return, ATF4 can enhance the stability of MCYN by inhibiting the MYCN ubiquitination mediated by FBXW7 13C mass-isotopomer distribution model to show that NB cells with MCYN amplification utilised more glucose-derived carbon for serine and glycine biosynthesis than those without, which suggests a metabolic basis for their differential sensitivities to PHGDH inhibition. They further found that PHGDH inhibition induces metabolic stress and leads to G1 arrest and autophagy in NB cells with MYCN amplification but not those without MYCN amplification. Because PHGDH is the first rate-limiting key metabolic enzyme in the SGOC pathway, PHGDH inhibition may create a metabolic vulnerability in tumours with a highly activated SGOC metabolism. These findings support a recent publication from Reina-Campos and colleagues revealing that PHGDH inhibition is a vulnerability in neuroendocrine prostate cancer that can be exploited therapeutically by targeting the SGOC pathway11.Furthermore, the SGOC pathway connects glycolytic reactions with one-carbon metabolism, the biosynthesis of serine and glycine. Xia and colleagues used a Taken together, these findings by Xia and colleagues reveal new insight into the features of SGOC metabolism in the progression of NB with MYCN amplification or cancers with highly activated SGOC metabolism. The SGOC metabolic network is important not only for nucleotide, protein, and lipid biosynthesis but also for DNA methylation, histone methylation and redox balance. The outputs of SGOC metabolism are required for cell viability and proliferation and are particularly likely to be co-opted by aggressive cancers. Targeting SGOC metabolism may be an effective and less toxic approach to improve outcomes in MYCN-amplified NB patients. The enzymes within the SGOC pathway may be powerful prognostic tools and perhaps clinically relevant targets in all highly SGOC-activated tumours in the future.


Tumor necrosis factor (TNF) is a pleiotropic cytokine powerfully influencing diverse processes of the central nervous system (CNS) under both physiological and pathological conditions. Here, we analyze current literature describing the molecular processes involved in TNF synthesis and release from microglia, the resident immune cells of the CNS and the main source of this cytokine both in brain development and neurodegenerative diseases. A special attention has been given to the unconventional vesicular pathway of TNF, based on the emerging role of microglia-derived extracellular vesicles (EVs) in the propagation of inflammatory signals and in mediating cell-to-cell communication. Moreover, we describe the contribution of microglial TNF in regulating important CNS functions, including the neuroinflammatory response following brain injury, the neuronal circuit formation and synaptic plasticity, and the processes of myelin damage and repair. Specifically, the available data on the functions mediated by microglial EVs carrying TNF have been scrutinized to gain insights on possible novel therapeutic strategies targeting TNF to foster CNS repair. Microglia represent the resident immune cells of the central nervous system (CNS). In physiological conditions they are involved in sensing the environment and regulating brain homeostasis by performing different functions. Upon brain injury, they transform from immunosurveillant into reactive cells and acquire a variety of immune-phenotypes committed to both detrimental and protective functions ,2. ReactAmong the different factors through which microglia exert their regulatory activity on brain functions, TNF is the one that probably best reflects the dichotomous nature of these immune cells, being able to induce harmful or beneficial effects, depending on the specific isoform and receptor subtypes it activates. Indeed, while TNF has been historically defined as a potent inflammatory mediator driving tissue degeneration in disease conditions, growing evidence provides concrete clues of a parallel role of the cytokine in regenerative processes , thus coHere, we review current literature on the mechanisms regulating TNF production, intercellular trafficking and release from microglial cells, with a special attention to the role of microglia-derived extracellular vesicles (EVs) in these processes. Furthermore, we describe the variety of biological effects exerted by microglia-released TNF on different brain functions, which could be particularly relevant for the development of specific therapeutic strategies aimed at containing the progression of brain damage or promoting tissue repair.TNF is a pleiotropic cytokine produced as a precursor membrane-anchored form of 26 kDa, called transmembrane TNF (tmTNF), by immune cells, including microglia, astrocytes as well as other different cell types . After cUpon TNF binding, TNFR1 recruits the TNFR1-associated death domain protein (TRADD) that induces cell apoptosis by activating Fas-associated death domain protein (FADD) and caspase 8 . HoweverThe intracellular signaling pathways activated by TNFR2 are less characterized compared with those of TNFR1. Given that TNFR2 lacks death domain, the receptor only recruits TRAF2 and activates PI3K-Akt/PKB and NF-κB pathways for mediating pro-survival signals ,21. AccoSeveral studies showed that TNF is upregulated in activated microglia at transcriptional and translational level , in partIncreased release of TNF by microglia can enhance the activation of the entire population of microglia in a positive feedback mechanism ,31,32, sIn line with these findings, a large body of evidence indicated that microglial TNF is involved in brain damage following acute injury or chronic neuroinflammation . High le2+ influx in microglia [Under pathological events, microglia and damaged cells release at the sites of CNS injury a large amount of ATP, a danger signal that activates specific receptors, called P2 purinoceptors, ubiquitously expressed on neurons and glia ,41. ATP icroglia  that evoicroglia . Howevericroglia .MAP kinase cascades  are involved in TNF production downstream of P2X7 activation . SpecifiOnce synthetized, TNF is released via conventional or non-conventional pathways . The conThe non-conventional secretory pathways typically mediate release of proteins lacking leader sequences, through an ER-Golgi transport-independent mechanism. Unconventional secretion allows cells to secrete cytokines without the requirement for post-translational modification, which can influence their functions, and may protect the host when conventional mechanisms are compromised, for example under ER stress . SeveralATP is a major signal inducing unconventional TNF secretion via EVs . In macrAs described above, TNF synthesis in microglia strictly depends on the activation state of these cells and it is finely regulated by signals released in the extracellular milieu in response to brain damage, including ATP . SpecifiEVs are small particles formed by a double layer of phospholipids, which are gaining increasing interest as fundamental mediators of intercellular communications in the brain. EVs signals to adjacent cells but they can travel very long distances and deliver complex messages to distant cells . EVs canEVs can be classified based on site of origin in microvesicles (100 nm to 1 µm in diameter), directly shedding from the cell membrane, and exosomes (30–100 nm in diameter), originating from multivesicular bodies in the endosomal compartment . AnotherGiven all the properties described above, EVs hold great potential for several research and clinical applications, ranging from the basic study of pathophysiological mechanisms , to the Nonetheless, EVs represent ideal carriers for the delivery of transmembrane proteins such as tmTNF. Intriguingly, EV production by microglia is massively induced by the same mechanism driving TNF expression, namely activation of the P2X7 receptor by ATP . This suImportantly, EVs can also carry solTNF among their cargoes and the TNF isoform, soluble versus membrane-bound, carried by EVs greatly impacts the response of recipient cells . TmTNF iBesides TNF itself, other important players of the TNF signaling pathway have been reported in EVs, including TNFR1, associated signaling proteins and TACE, the enzyme responsible for tmTNF cleavage and release of solTNF from the plasma membrane. Indeed, TNFR1-containing small EVs have been detected both in vitro ,81 and iThe presence of enzymatically active TACE within EVs has been reported in blood samples of HIV-infected subjects , whose rAs already mentioned, EVs containing TNF and related signaling molecules have been detected in the CSF  and in tCollectively, the evidence described above suggests that microglia-derived EVs may represent ideal vectors for the delivery of TNF molecules, capable to promote both deleterious and reparative responses in target cells.As already discussed, inflammatory stimuli trigger a complex cellular and molecular response in microglial cells, which includes the release of soluble and transmembrane TNF and other related factors. In the following paragraphs, we will discuss the double-edged role of TNF deriving from microglial cells in shaping brain functions, including the neuroinflammatory response to tissue damage, neuronal circuit formation and synaptic plasticity, and myelin degeneration and repair .Neuroinflammation can be considered as the first line of defense to preserve or restore the CNS homeostasis in response to harmful stimuli, degenerative conditions and traumatic injuries. This process mainly involves the synchronized action of different cell types, primarily including microglia and astrocytes . The proMicroglia-derived TNF has been implicated in sustaining the pro-inflammatory activation of microglial cells through a positive feedback mechanism mediated by TNFR1 activation . AccordiIn addition to the aforementioned effects on microglial cells, TNF released by inflamed microglia, together with IL-1α and C1q, has been demonstrated to drive detrimental A1 astrocyte activation, leading to a final toxic effect on neurons and oligodendrocytes underlying the pathological mechanisms of several neurodegenerative disorders . NotablySimilar to microglia, activated astrocytes themselves can produce and release TNF, giving rise to an autocrine/paracrine loop of activation and cytokine release . HoweverIn parallel to pro-inflammatory effects, tmTNF-mediated activation of microglial TNFR2 has been demonstrated to be essential for preserving the protective functions of these cells. Consistently, microglia-specific TNFR2 knockout mice display early onset of EAE and increased infiltration of pro-inflammatory immune cells from the periphery . In thisThe best documented role of microglia in controlling neuronal functions comes from the analysis of TNF effects on synaptic connectivity . HoweverTNF released by microglia has an important role in regulating synaptic plasticity . SpecifiTNF not only controls homeostatic synaptic activity, but also induces neurotoxicity via autocrine/paracrine loops involving other endogenous mediators. First, TNF activates TNFR1 on microglia, amplifying its production and release . Second,Furthermore, TNF also induces neuronal death by caspase activation and apoptosis induction, as demonstrated in cultured cerebellar neurons exposed to microglia conditioned medium, containing both solTNF and TNF-carrying vesicles . The samA receptors [Another mechanism by which TNF promotes neurotoxicity is the inhibition of GABAergic transmission. In vitro studies in cultured rat and mouse hippocampal neurons indicate that TNF treatment induces a rapid and persistent decrease of inhibitory synaptic strength as well as a downregulation of cell surface levels of GABAeceptors . This reeceptors . This deSimilar alterations of synaptic transmission are induced by microglial EVs, which potentiate excitatory transmission while inhibiting release of the inhibitory transmitter GABA in cultures, brain slices and in the visual cortex ,127. HowSo far, only a single study implicated the TNF cargo of microglial EVs in neuronal dysfunction. Specifically, it has been shown that exposure of murine microglial cell line BV2 to α-synuclein, to mimic the neurodegenerative environment associated to PD, increases the secretion of EVs enriched in TNF and major histocompatibility complex II (MHCII) molecules and promotes neuronal apoptosis . In lineBesides PD, important roles of microglial TNF have been described in several other neuropathological contexts, such as AD, stress, ischemia, schizophrenia, and bipolar disorder . Like inElevated levels of TNF were found in hippocampal microglia after acute stress and were shown to correlate with working memory deficits. Interestingly, treatment of stressed mice with the TNF inhibitor etanercept rescued such defects in accordance with a reduction in hippocampal TNF .A dramatic upregulation of microglial TNF has been found in animal models of cerebral ischemia , specifiThe action of TNF can be influenced by environmental factors, such as exercise. Indeed, chronic exercise ameliorates cognitive impairment in wild-type mice by reducing the expression of hippocampal TNF and increasing the expression of TNFR1 and TNFR2. These benefits included reduced errors and improvements in spatial learning and spatial memory deficits in the Morris water maze . HoweverGiven “beneficial” and “deleterious” functions of microglia and TNF in neurodegenerative diseases, the clinical application of anti-TNF therapies remains very difficult. However, an in-depth understanding of the effects of microglial TNF on synaptic functions and of the mechanisms underlying its actions may provide important insights for the development of novel therapeutic strategies aimed to limit neuronal loss and to improve cognitive deficits in patients.A common feature of a diverse group of neurodegenerative diseases, from MS to psychiatric disorders and brain injuries, is demyelination, namely the damage to the myelin sheath. Myelin is the lipidic layer produced by oligodendrocytes which, enwrapping neuronal axons, allows the efficient action potential propagation by saltatory conduction within the CNS and provides mechanical and trophic support to neurons . Myelin Microglia heavily influence the pro-remyelinating attempts promoted by OPCs surrounding myelin lesions . Indeed,A very recent paper showed that TNF released by microglial cells, besides being fundamental for efficient clearance and degradation of myelin debris by phagocytes, it is also required for the generation of new myelinating cells within demyelinated areas, suggesting direct effects of this cytokine on OPCs . This isApparently in contrast with the aforementioned results, genetic ablation of TNF resulted in exacerbation of neurological deficits and augmentation of neuroinflammation and demyelination in the EAE mouse model. Of note, these deleterious effects were rescued by exogenous TNF administration , highligThe understanding of such opposing effects of TNF on oligodendrocytes  has incrOf note, TNF has been proved to be a key component of microglia-derived EVs involved in the astrocyte harmful conversion into oligo-toxic cells both in vitro and following lysolecithin-induced focal demyelination in vivo . The impIntriguingly, TNF levels are dramatically decreased, but still detectable, in EVs derived from inflammatory microglia co-cultured with immunosuppressive mesenchymal stem cells (MSC-EVs), which do not cause harmful astrocyte transformation and instead promote oligodendrocyte maturation . Based oActivated microglial cells can release TNF through conventional ER-Golgi dependent or unconventional EVs-mediated secretory pathways in response to different stimuli. Accumulating evidence highlights the powerful but often controversial effects exerted by this cytokine on CNS cells. The dichotomous role of TNF in orchestrating different CNS processes, ranging from neuroinflammation and neurodegeneration to myelin repair, probably represents the main reason why TNF-targeting drugs have been proved to be largely unsuccessful for the treatment of neurological disorders ,168,169.Importantly, recent findings provide new insights on the mechanisms of TNF release and signaling that may be helpful for the development of novel therapeutic approaches. In this respect, the first thing to consider is the existence of two different isoforms, solTNF and tmTNF, which generally induce opposing effects by activating distinct TNFR1 and TNFR2 receptors . A generBesides directly targeting TNF signaling pathways with specific drugs, the mechanism of cytokine release may also represent an important target for an upstream modulation of TNF levels. Of note, unconventional release of TNF via EVs in response to ATP stimulation appears to be the prevalent mechanism of TNF secretion from activated microglia under disease conditions. Thus, blocking the shedding of microglial EVs by using pharmacological inhibitors  might reFinally, the phenotype of TNF-donor cells seems to pivotally determine TNF levels packaged into EVs, which in turn impact the final effects of EVs on target cells. Accordingly, EVs produced by inflammatory microglia contain high amounts of TNF and induce detrimental astrocyte conversion and demyelination ,76. On t


Nature Communications, Leal et al developed a novel liquid biopsy strategy to distinguish tumor-specific ctDNA alterations from cell-free DNA variants associated with clonal hematopoiesis and evaluated the utility of ctDNA as a biomarker for predicting the prognosis of postoperative GC patients.Gastric cancer (GC) is one of the most diagnosed cancers in the world and is mainly cured by surgical resection. However, a large proportion of patients still presents with microscopic residual disease after surgical resection. Patients with microscopic residual disease can be offered appropriate treatment to improve their long-term survival, but for such, it is imperative to timely identify those with residual disease after surgery. Unfortunately, the traditional detection methods for residual disease have poor sensitivity. Recently, with the development of liquid biopsy technology, circulating tumor-derived deoxyribonucleic acid (ctDNA) detection has shown great promise in bringing new hope for increasing the detection rate of microscopic residual disease. In a study recently published inGastric cancer (GC) is the fifth common cancer and third leading cause of cancer-related death worldwide.Nature Communications, titled as “White blood cell and cell-free DNA analyses for detection of residual disease in gastric cancer,” Leal et alWith the development of liquid biopsy technology, the latest approaches, such as circulating tumor-derived deoxyribonucleic acid (ctDNA) detection via liquid biopsy, may bring new hope for detecting microscopic residual disease after GC surgery. Theoretically, the tumor-specific alterations in the circulation of postoperative cancer patients can dynamically and specifically reflect whether these patients have microscopic residual disease or preclinical metastases.All GC patients enrolled in that study were from the CRITICS trial (NCT00407186).Furthermore, the detection of microscopic residual disease without a white blood cell filter did not predict disease recurrence. In contrast, with a white blood cell filter, a significantly shorter median event-free survival, and a significantly higher risk of disease recurrence for patients with detectable tumor-specific mutations after surgery could be observed. All results mentioned above suggested that microscopic residual disease could be accurately detected by the new detection method developed in the study by Leal et al, and microscopic residual disease could be a predictive indicator for the prognosis of postoperative GC patients.Patients with microscopic residual disease would need adjuvant treatment after surgery. It is important to diagnose them as soon as possible. At present, we still rely on traditional methods, such as pathological staging and microscopic residual disease scoring system, to estimate the risk of GC recurrence after surgery.Overall, the discussed study developed a facile approach for detecting microscopic residual disease in GC by distinguishing ctDNA alterations from other cell-free DNA alterations. However, when using this new approach to detect postoperative microscopic residual disease of GC, there are some limitations, especially in GC patients who have localized diseases. Therefore, in the future, it is necessary to improve this approach by using targeted panels specifically designed for GC and incorporating cell-free DNA fragmentation analyses.