Transcranial (electro)magnetic stimulation (TMS) is currently the method of choice to non-invasively induce neural activity in the human brain. A single transcranial stimulus induces a time-varying electric field in the brain that may evoke action potentials in cortical neurons. The spatial relationship between the locally induced electric field and the stimulated neurons determines axonal depolarization. The induced electric field is influenced by the conductive properties of the tissue compartments and is strongest in the superficial parts of the targeted cortical gyri and underlying white matter. TMS likely targets axons of both excitatory and inhibitory neurons. The propensity of individual axons to fire an action potential in response to TMS depends on their geometry, myelination and spatial relation to the imposed electric field and the physiological state of the neuron. The latter is determined by its transsynaptic dendritic and somatic inputs, intrinsic membrane potential and firing rate. Modeling work suggests that the primary target of TMS is axonal terminals in the crown top and lip regions of cortical gyri. The induced electric field may additionally excite bends of myelinated axons in the juxtacortical white matter below the gyral crown. Neuronal excitation spreads ortho- and antidromically along the stimulated axons and causes secondary excitation of connected neuronal populations within local intracortical microcircuits in the target area. Axonal and transsynaptic spread of excitation also occurs along cortico-cortical and cortico-subcortical connections, impacting on neuronal activity in the targeted network. Both local and remote neural excitation depend critically on the functional state of the stimulated target area and network. TMS also causes substantial direct co-stimulation of the peripheral nervous system. Peripheral co-excitation propagates centrally in auditory and somatosensory networks, but also produces brain responses in other networks subserving multisensory integration, orienting or arousal. The complexity of the response to TMS warrants cautious interpretation of its physiological and behavioural consequences, and a deeper understanding of the mechanistic underpinnings of TMS will be critical for advancing it as a scientific and therapeutic tool. Basic questions such as which parts of the gyrus, which cell types, and which neuronal compartments are preferentially excited by TMS are still subject to research. The relationship between the biophysical properties of cortical neuronal populations and the efficacy to produce action potentials with TMS in these populations is also still incompletely understood. The same is true for the mechanisms that determine the propagation of the direct TMS-induced neuronal excitation within local cortical circuits and along neural projections to interconnected brain regions.This consensus paper gives an account on what we currently know and what we still do not know about how TMS “engages” its target, the brain. We summarize the current level of understanding of how TMS targets neural structures in the stimulated area and how the regional induction of action potentials impacts on the cortical target area at the cellular and microcircuit level. We also discuss how regional neural excitation of the stimulated cortex influences neural function of remote brain areas. Plasticity-inducing effects of TMS are not covered. We first synthesize some general considerations about TMS and its underlying mechanisms in 2.2.1.The direct neural response to the TMS-induced electric field is complex, involving a mixture of neuronal populations . InvasivBy systematically varying the intensity and duration of the TMS pulse, one can derive a curve, which delineates a threshold function for evoking a motor evoked potential (MEP). This curve reflects the membrane excitability of the neural structures that are stimulated with TMS and can be mathematically described by its strength–duration (S-D) properties and the closely related neurophysiological concepts of chronaxie and rheobase . Rheobasin vitro peripheral nerves, which found that long, straight axons are activated at the site of peak electric field gradient. Introducing bends or terminations (e.g. cut ends) shifted excitation to these locations, where the threshold was inversely proportional to the electric field magnitude , and bends  . Within Fig. 2B1) by TMS is relatively insensitive to voluntary contraction, GABA-agonists, or the inhibitory paired-pulse paradigm, short interval intracortical inhibition (SICI)  . Since fTMS at low intensities for most coil configurations is known to directly activate axons in cortical grey matter , but juxIn summary, there are several accounts of where the TMS-induced electric field primarily excites the intracortical axons. The notion that TMS primarily causes intracortical excitation of myelinated axons at their terminals is biophysically the most plausible account. Regardless of which of the various accounts is correct, they all predict that the efficacy of TMS to depolarize the axonal elements critically depends on the orientation of the axonal element relative to the direction of the induced electric field and the magnitude of the electric field. It is also possible that multiple mechanisms play a role in TMS excitation depending on the stimulation parameters, including intensity, direction, and pulse waveform.1-wave generated by monosynaptic inputs to corticospinal neurons, whereas A-P oriented currents in M1-HAND preferentially evoke later I-waves  is used for TMS. Direction-specific effects are clearly evident when stimulating the M1-HAND in the precentral gyrus with a figure-of-eight coil and a monophasic pulse configuration see . A TMS p I-waves . Biophys I-waves   and retrograde (antidromic) direction, inducing forward and backward information flow within the stimulated pathway . An anteBoth local transsynaptic excitation of neural structures in the targeted cortex as well as the transsynaptic propagation of excitation to remote brain regions are modulated by state-dependent factors, including ongoing neuronal firing rate and intrinsic excitability. The motor response to TMS of M1 is influenced by the “motor state” at the time of stimulation: Using identical stimulation settings, the MEP amplitude obtained at rest can be facilitated or suppressed by motor imagery involving the target muscle . DependiElectroencephalographic (EEG) recordings of the TMS-evoked EEG potentials (TEPs) during non-rapid eye movement (NREM) sleep have also convincingly demonstrated that the brain state at the time of stimulation determines the brain’s response to TMS . NREM slThe induced electric field does not only excite neuronal structures in the central nervous system. Peripheral co-stimulation of sensory and motor axons in the face or neck region and intracranial sensory and motor axons at the base of the skull may also be effectively excited by TMS . TMS indThere are several other sources of somatosensory co-stimulation, including the excitation of mechanoreceptors due to TMS-induced vibration, and reafferent somatosensory stimulation caused by TMS-evoked muscle twitches . The durIn addition to co-stimulation of peripheral somatosensory and motor fibers, TMS also produces a loud click in the coil case, which leads to auditory stimulation via air and bone conduction . The vibThe multiple peripheral effects of TMS result in substantial “off-target” brain stimulation. Multisensory peripheral co-stimulation is inherent to TMS and needs to be taken into consideration when designing a TMS study because it may conflate or obscure the true transcranial effects of TMS on functional brain activity and render the causal interpretation of neurophysiological, behavioral or therapeutic effects ambiguous . Of notePeripheral co-stimulation effects should be assessed and reported in detail. The magnitude of peripheral co-stimulation should be minimized, and its impact should be masked or matched by experimental control condition as much as possible . There a2.2.When targeting a cortical area located on the lateral surface of the hemisphere, cortical patches located in the gyral crown will always be exposed to a stronger electric field than cortical patches located in the wall of the gyrus or at the fundus of a cortical sulcus . This isThe limited depth penetration has important practical implications for the use of TMS. When using a stimulation intensity that is sufficient to effectively target the fundus region of a cortical gyrus, TMS will always result in a stronger concurrent stimulation of more superficial parts of the gyrus, such as the top and lip regions in the crown. A depth gradient in terms of effective stimulation also exists when stimulating cortical areas in the mesial wall. When targeting mesial cortical areas such as SMA or primary motor leg area, the superficial cortex close to the midline of the hemispheric convexity will be exposed to a stronger electrical field than the mesial cortical areas located in the interhemispheric fissure. Another implication is that TMS is unable to directly target deep structures of the cerebral hemispheres such as cingulate cortex, medial parts of the temporal lobes, cortex around the insular fissure, thalamus and basal ganglia. However, these deep brain structures may be effectively stimulated by a spread of excitation from effectively stimulated superficial cortical areas to the cingulate cortex via white-matter connections. Regarding depth penetration, a notable exception are local electric field maxima caused by non-uniformities in the heterogeneous tissues . These lThe prevailing view is that TMS activates primarily myelinated cortical axons at their bends, bifurcations or terminations , with loin vitro studies combining inductive magnetic stimulation with electrophysiological recordings from nerve preparations, cell cultures or rodent brain slices  may be preferentially activated by TMS . The impThe ability of TMS to excite distal elements of the axonal tree broadens the range of potential intracortical target sites: TMS may effectively stimulate medium or long-range cortico-cortical axons that project into the targeted area or short-range connections formed by local axon collaterals. Cortico-cortical axons are myelinated in cerebral white matter, but they become less myelinated and thinner as they branch out in grey matter and ultimately form axon terminals. They also tend to bend as they leave the white matter before ramifying within the cortex . Similar considerations apply for intracortical axon collaterals of the local, outward-projecting pyramidal axons . Axonal The action potentials initiated directly by the TMS pulse propagate orthodromically to cause synaptic transmission at all downstream connections, including both outward projections as well as local connections via the dense intracortical axon collaterals. Theoretical considerations suggest action potentials may fail to propagate through axonal branch points in an activity-dependent manner if there is a change in electrotonic load; however, orthodromic action potentials have been shown to propagate reliably throughout intracortical axon arbors for physiologically relevant firing rates (<200 Hz) in numerous experimental preparations and brain regions . PropagaAs pointed out in At very low TMS intensities, inhibitory neurons may primarily be excited transsynaptically via low-threshold excitatory inputs. This notion is also supported by the results of a triple-pulse TMS study, in which two sub-threshold conditioning pulses increased the inhibition of the motor output evoked by a supra-threshold test stimulus . AlthougTMS may also directly activate the axons of inhibitory interneurons, which show considerable spatial dispersion and arborization in grey matter . The larPyramidal cells have a bipolar structure formed by the dendritic tree, soma and axon, being oriented perpendicular to the cortical surface. Tranchina and Nicholson used analytical analysis of basic cable theory and predicted peak polarization at the soma by uniform E-field due to impedance mismatch between the axon and dendrite . Some mocortical column cosine theory also predicts strongest activation by the E-field orientation parallel to cortical columns, i.e. parallel to the main somato-dendritic axis of pyramidal  axonal hillocks of pyramidal cells. This heterogeneity in part explains the multitude of physiological effects that can be elicited with TMS see . Dependi2.3.Pharmacological manipulations have been instrumental in three ways to enhance our knowledge about what is being stimulated by TMS. These different lines of research combined TMS with MEP recordings, EEG or positron emission tomography (PET), respectively.A) receptor, such as benzodiazepines and barbiturates, decrease MEP amplitude at stimulus intensities clearly above corticomotor threshold  blocker such as carbamazepine, lamotrigine or phenytoin increase the corticomotor threshold . Since Vhreshold . This sthreshold . Benzodihreshold , 1996b.  neurons , or onto neurons . Finally neurons . This bo neurons .A receptor agonists enhance the N45 response evoked by single-pulse TMS of left M1 in the non-stimulated right hemisphere  When using small animals like rodents, the conventional TMS coils are relatively large with respect to the size of the brain, exciting relatively large tissue volumes compared to studies in humans, while also reducing the peak induced electric field strength due to the small head size . It veryIn vitro studies of magnetic stimulation of acute brain slices, organotypic cultures or cell cultures allow for a better controlled geometry of induced electric fields and the measurement of acute and discrete changes in cellular processes. However, the translational value of such studies is limited because of the different physiological conditions of in vitro preparations compared to the state of an awake human brain controlled by numerous modulatory systems. Despite of these inherent limitations, animal research is critical to resolve what is being stimulated with TMS at the single-cell and cell-circuit level.In vitro and in vivo recordings have not only been used to study the biophysical response properties of single neurons to inductive magnetic stimulation  orientation of the induced electric field favored the activation of callosal axons and emphasized the involvement of inhibitory interneurons  excitation of neurons by the TMS pulse was evident, nor an activation of thalamocortical inputs.In addition to invasive electrophysiological measurements, a range of novel optical imaging technologies have become available that offer high functional specificity as well as spatial and temporal resolution while avoiding contamination by electromagnetic artifacts . Opticalrneurons . Two-phoA seminal study by Using a TMS-setup suitable for inducing callosal activity with a mediolaterally oriented electric field, it has been shown that rTMS induces changes in neuronal activity markers primarily within cortical layers II/III . Using aThe question of what is stimulated by TMS also includes the functional impact of TMS-induced electric fields that are subthreshold to elicit action potentials. In principle, any “hot spot” of suprathreshold TMS is surrounded by brain regions of subthreshold stimulation level, but even in a “hot spot” only a portion of neurons might be effectively discharged while the local electric field may remain subthreshold for other neurons. Subthreshold de- or hyperpolarization of the neuronal cell membrane can affect synaptic responses and the orthodromic and antidromic propagation of action potentials, even if not directly eliciting action potentials. Although primarily demonstrated with repetitive stimulation , magnetic fields of 1 mT, or even less, are able to change intracellular calcium levels and the activity of downstream molecular pathways .2.5.Online neuroimaging can detect acute effects produced by TMS in any cortical and subcortical region throughout the brain and with high spatial and temporal resolution . This isPET and functional magnetic resonance imaging (fMRI) provide whole-brain coverage at good spatial resolution . A critiParametric PET studies of rCBF and rCMRglc showed that rTMS trains not only lead to dose-dependent activity changes at the cortical stimulation site, but also in remote cortical regions known to form a functional network . This woIt is worth noting that a local increase in the BOLD response measured from structures situated directly under the stimulating coil is not always seen in TMS-fMRI studies . This maPerhaps the most relevant contribution of TMS-neuroimaging studies has been the confirmation of the idea that TMS to a cortical site can also influence activity in subcortical brain regions , as wellTMS may also be combined with EEG recordings. EEG captures the cortical response to single TMS pulses with high temporal resolution. Online TMS-EEG studies have consistently shown that a single TMS pulse gives rise to a rapid propagation of activity across putatively interconnected regions, including areas in the hemisphere contralateral to TMS . The corIn addition to its transcranial mode of action, TMS excites the brain through afferent neuronal channels activated by concurrent auditory and somatosensory stimulation . TMS cauDue to its low temporal and spatial resolution, proton magnetic resonance spectroscopy (proton-MRS) has preferentially been used to capture regional metabolic changes after prolonged TMS  . Yet, prAs pointed out at the end of 2.6.Dual-site TMS paradigms generally combine the delivery of a conditioning stimulus over a cortical area to activate putative pathways projecting onto M1-HAND with a test stimulus over M1 . The tesThis may, however, not always be the case. When considering that TMS of M1-HAND induces corticospinal descending volleys that leave the cortex several milliseconds after the TMS pulse has been applied, a conditioning pulse given several milliseconds before the test pulse has enough time to modulate the TMS effects in M1 via an indirect route that uses additional subcortical or cortical areas as relays. For instance, a polysynaptic pathway underlies cerebellar-cortical dual-site effects, because there is no monosynaptic connection between any cerebellar structure and M1-HAND. Tractography derived from diffusion weighted MRI scans may provide some hints on the white matter pathways that are engaged by a dual-site TMS paradigm. If microstructural properties of a certain white matter tract scales with the measures of effective connectivity, as obtained by dual-site TMS, this may increase the confidence that the physiological interactions are mediated through these cortico-cortical or cortico-subcortical tracts .The cortico-cortical interaction may vary when tested in the context of a task-free state  or during a specific motor or cognitive task . These tThe conditioning stimulus was applied through the implanted electrodes of the DBS device and triggered the TMS pulse . In patiLong-term potentiation (LTP)-like or long-term depression (LTD)-like effects in M1-HAND have been observed after dual-site paired associative stimulation (PAS) targeting M1-HAND bilaterally , PMv andConventional dual-site approaches have primarily targeted pathways that project onto M1, using the MEP evoked with a test TMS pulse over M1-HAND as a convenient read-out. In principle, dual-site TMS can also be applied to areas outside M1. This requires different physiological read-outs to probe the functional impact of the conditioning pulse. The combination of dual-site TMS with EEG has been proposed for this purpose using the TEP as read-out . Yet inh3.The motor cortex rostral to the central sulcus, especially M1-HAND, has been the most popular cortical target for studies exploring the mechanisms of action of TMS . Using tWhen Barker and colleagues introduced TMS in 1985, they also targeted the M1-HAND. In their letter to Lancet, they included a figure featuring a MEP recorded from a hand muscle to illustrate the ability of TMS to probe the function of corticospinal projections . Indeed,Another important neurophysiological property of the MEP is that a slight pre-activation of the target muscle is sufficient to produce a consistent facilitation of the MEP compared to TMS with the target muscle at rest. This MEP facilitation is mediated by physiological changes at both spinal and cortical levels which renders the spread of local precentral excitation along the corticomotor pathways to the target muscle more efficient in a pre-activated relative to a resting state . This isAlthough it is easy to record the MEP with surface electrodes, its underlying neurophysiology is complex. The MEP results from synchronized corticomotor excitation of fast conducting corticomotor neurons and propagation to the motor units of the target muscle . Synchro3.1.Some neurophysiological characteristics of TMS-evoked MEPs and their relevance in terms of the mechanism of action of TMS have shortly been mentioned in 3.1.1.When a slightly suprathreshold TMS pulse is given to the M1-HAND, multiple descending volleys can be recorded at short intervals from the corticospinal tract  and multInvasive recordings from electrodes implanted into the epidural space of the spinal cord also revealed that TMS evokes corticospinal descending volleys mainly via a transsynaptic mechanism . At inteTMS may also give rise to an earlier volley, the so-called direct wave (or “D-wave”) which is produced by direct activation of corticospinal axons in the subcortical white matter . HoweverIn humans, the cortical circuits implicated in the generation of I-waves can be probed non-invasively with paired-pulse TMS targeting the motor hot spot in the precentral gyrus . A firstBiphasic TMS pairs, triplets, or quadruplets separated by an ISI that corresponds to the individual trough between the first and second SICF peak facilitates MEP amplitude across a wide range of TMS intensities . Short-l3.1.2.When using a figure-of-eight coil and a monophasic pulse configuration to stimulate the M1-HAND, MEP latency and amplitude depend on the current direction induced in the cortex . When moThe orientation-dependent differences in latency can be attributed to differences in preferential I-wave activation as revealed by epidural recordings of the descending corticospinal activity and single motor unit recordings . The P-ABased on this work, it can be concluded that different neuronal populations or circuits are targeted in the precentral hand knob by TMS when using different coil orientations that produce differently oriented currents with respect to the local axis of the precentral gyrus . This noIt should be noted that biphasic (full-sine wave) pulses also display orientation dependent effects when targeting the precentral motor hand knob, though to a lesser extent than monophasic pulses . This is3.1.3.TMS of M1 also produces inhibitory effects that have a lower threshold than the one necessary to evoke a MEP . The TMSA modeling study using morphologically realistic models of inhibitory basket cells in lamina 4 of precentral cortex estimated that their axon terminals may have a relatively low threshold for TMS activation . They alAlternatively, it may be that most interneurons are activated transsynaptically via excitation of axon terminals that belong to the axonal arbor of excitatory pyramidal neurons, which were predicted to have the lowest activation thresholds . It woulAlthough MEPs are sensitive to current direction, However, the situation may be more subtle . For ins3.2.3.2.1.The part of the M1 hosting the motor representation of hand muscles (M1-HAND) has a convex shape with an outwards curvature towards the parietal cortex. As pointed out in the previous sections, the characteristic curvature of the central sulcus renders the M1-HAND easily recognizable as knob-like structure on the cortical surface or axial slices or of structural MRI scans. Therefore, Yousry et al. coined the term precentral motor hand knob . TMS wil3.2.2.The human M1 is located in the anterior bank of the central sulcus, covering the caudal wall of the precentral gyrus . In non-The transition zone in which the rostral M1-HAND is gradually transformed into PMd may vary across healthy individuals and thus influence how the M1-HAND is stimulated by TMS . This infast monosynaptic corticospinal projections to the cervical spinal motoneurons  and caudal (new) part based on the absence (old) or presence (new) of cortico-motoneuronal cells . The rosoneurons . Howeveroneurons . Fig. 5 The homotopic representations of the rostral and caudal M1 are strongly and reciprocally interconnected , but theThere is convincing evidence that this rostro-caudal segregation also exists in the human M1-HAND and has been labeled Brodmann area BA4a and BA4p . The posThe rostro-caudal segregation of M1-HAND and its variable and smooth rostral border have important implications for the ability of TMS to stimulate M1-HAND. One may intuitively assume that focal TMS targeting the precentral gyrus causes a homogenous stimulation of the entire M1-HAND, but this is not the case. Since the strength of the induced electric field attenuates with the distance from the coil, the rostral M1-HAND (BA4a) in the upper wall of the precentral gyrus will always receive a stronger current than the caudal M1-HAND (BA4p) in the lower wall of the precentral gyrus. In other words, the rostral M1-HAND (BA4a) is more susceptible to TMS-induced neural excitation because it is closer to the stimulation coil. The relative magnitude of direct neural excitation of caudal versus rostral portions of M1-HAND will depend on the temporal properties of the stimulus, as well as properties of the different neuron populations.This poses a problem: Although stimulation in rostral M1-HAND might activate corticospinal neurons, their slow conduction velocities would produce MEPs later than we observe. The shortest latency MEPs are evoked by a latero-medial (L-M) oriented TMS pulse or a single high-voltage transcranial electrical pulse (TES) . The cenThis raises the question of what is being stimulated. The MEP threshold for L-M oriented TMS is higher than for P–A, which may mean that the induced current can spread far enough into the anterior bank of the central sulcus to activate the large fast-conducting corticospinal neurons in area BA4p. The situation is not as clear for conventional P-A oriented TMS. One possibility is that despite its lower threshold compared with L-M oriented TMS, the induced current still manages to spread deep enough to activate synaptic contacts directly on the corticospinal neurons of area BA4p. This seems unlikely in view of all the evidence that activation occurs in the gyrus and tip of the anterior bank. A second possibility is that P-A TMS activates intracortical axons in the gyrus that monosynaptically excite fast-conducting corticospinal neurons in area BA4p . The addThe uncertainty about site of activation has one more implication. If TMS activates neural elements in the precentral crown, but recruits corticospinal neurons in the sulcal wall, where are I-waves generated? Although numerous models for I-wave generation exist , at pres3.2.3.The superficial parts of the PMd located in the crown of the precentral gyrus represent a spatial hot spot for TMS-induced neurostimulation. This superficial part of PMd is closest to the coil and therefore is exposed to the strongest electric fields in the crown and lip regions of the precentral gyrus see . This paResearch in non-human primates provided converging evidence for a strong functional interaction between PMd and M1, demonstrating dense reciprocal monosynaptic cortico-cortical connections between the two areas . IntraceThese biophysical and neuroanatomical considerations strongly support the idea that concurrent excitation of neuronal elements in PMd  results in relevant transsynaptic excitation of neural elements in M1-HAND via short-range cortico–cortical premotor–motor connections . This inAs pointed out previously, TMS induces stronger electric fields in the caudal PMd, located in the precentral crown-lip region, than in M1-HAND, located mostly in the sulcal wall. This implies that intracortical inhibitory interneurons in PMd might be excited more strongly than their counterparts in M1-HAND located in the central sulcus  and 5. DIn summary, the anatomy of the precentral hand knob has several important implications for TMS. First, the curvature of the hand knob needs to be considered when mapping corticomotor representations of M1-HAND or examining effects of direction specificity regarding the induced electric field in the precentral gyrus. Second, the precentral gyrus is not a homogenous area which is equidistant to the TMS coil, but it hosts the caudal PMd (BA6) in the crown-lip region and the rostral and caudal M1-HAND (BA4a/p) in the sulcal part of the gyrus. These regions differ in their sensitivity to be excited by TMS because of differences in coil-cortex distance and the spatial orientation of cortical axonal structures and the induced electric field. This implies that PMd and M1-HAND are concurrently stimulated by TMS, but also in an inherently different fashion. Importantly, the strongest “local dose” in terms of the induced electric field is achieved in the crown-lip region covered mainly by the caudal PMd and to a variable degree by the rostral M1-HAND . However, direct activation of M1-HAND  in the anterior wall may partly contribute to MEP generation when using higher intensities of TMS or when inducing a L-M directed current.3.3.Experiments examining descending I-waves in the corticospinal tract have revealed important insights into how TMS activates the corticomotor system. Although other sections do refer to I-waves, we included a dedicated section to provide comprehensive coverage of this important topic. A single TMS pulse given to the human precentral motor cortex produces repetitive descending volleys in the fast-conducting axons of the corticospinal tract. In animals, several descending corticospinal volleys have also been identified after M1 stimulation: a short latency “direct” volley that is believed to originate from the direct activation of corticospinal axons , followed by a series of later “indirect” volleys  numbered according to their temporal order . These mIn humans, the short-latency D-wave and the transsynaptically generated corticospinal I-waves have been recorded directly in conscious subjects who have had electrodes implanted surgically in the epidural space of the cervical cord for control of pain . These lThe possibility that different cortical circuits could be activated using transcranial stimulation in humans was first suggested by the pattern of discharge of single motor units in the muscle evoked by TMS pulses over the motor cortex. A single pulse produced several peaks of increased firing probability that were presumed to result from arrival of excitatory postsynaptic potentials at spinal motoneurons from the D- and I-wave volleys. The data showed that later peaks could be evoked in isolation by changing the direction of the current induced in the brain . The posThe contribution of different cortical circuits in I-wave generation is also supported by These observations indicate that the physiology of I-waves is complex. Several theories about I-wave production have been proposed. Initial theories postulated the existence of a chain of intra-cortical interneurons projecting upon the corticospinal cells and hypotesized that an intensity dependent activation of the different interneuron circuits produces early and late I-waves. More recently, it has been hypothesized that the I-waves might be produced by cortical networks with specific oscillatory properties activated by transcranial stimulation. Based on a canonical cortical circuit model, Other models are based on physiological properties of the pyramidal cells. Very fast oscillations with a frequency comparable to the I-waves (~600 Hz) have been observed in neocortex of rats and cats  and humaElectrophysiological recordings of descending corticospinal volleys probe the orthodromic conduction of action potentials along large fast-conducting axons. Small slow-conducting axons are heavily underrepresented when measuring corticospinal orthodromic and antidromic conduction, although they outnumber by far the larger fast-conducting axons . This in3.4.Field calculations and measurements help to understand how coil geometry, its position and the head anatomy affect the induced electric field evoked in the precentral motor cortex. Even simplified head models, such as spherical models , alreadyRegional spots of high electric field strength may also occur in the white matter underlying the crown of the gyrus see . This grInstead, corticofugal axons may be activated in the white matter at their bends by electric fields directed outward at the bend. Given that the main axons projecting from the tip of the crown into the white matter are mostly perpendicular to the field, which is tangential to the scalp, it is unlikely that these axons are stimulated by this mechanism. However, axons projecting from the transition zone between crown and wall of the precentral gyrus will bend downwards into the white matter at angles approaching 90°. These axonal bends might constitute a low threshold site of excitation .While the field calculations proved useful to generate hypotheses on the likely stimulation positions in the brain, more validation studies are still required. For instance, the validity of field calculations could be demonstrated by showing that they can predict electrophysiological properties of TMS. The issue of validation concerns both the predicted field pattern (as it might be imprecise due to model uncertainties) and the sometimes implicitly used assumption that mostly positions of high field strength are stimulated. Indirect support comes from a range of motor mapping studies which used TMS with figure-8 coils at the optimal orientation and consistently demonstrated that the center of gravity is situated above the crown of the precentral gyrus , in lineMost TMS studies using MEPs as primary read-out make the implicit assumption that TMS caused a homogenous stimulation of the entire M1-HAND. As discussed in more detail in cortical column cosine theory of TMS efficacy  , it was suggested that it rather occurs where the field component causing an inward current flow perpendicular to the cortical layers is strongest , 2009. Tefficacy . Common l cells) , 2015. Gositions . The mosositions . This reositions .cortical column cosine theory . The lack of activation in the remaining four participants was attributed to juxtacortical axonal excitation, but such activation should also lead to transsynaptic activation of cortical patches created by ortho- and antidromic propagation of action potentials. Other It has been argued that the site of peak activation in the precentral cortex as revealed by e theory . Using Hicipants . In thesicipants . It shouand knob : Applyinand knob . Subtracand knob . In contand knob .A note of caution is warranted, when using functional brain mapping of regional neuronal activity to corroborate biophysical models of direct neuronal excitation by TMS. If rk level . For insctive MT . A clustctive MT . While tctive MT .cortical column cosine theory provides a phenomenological explanation of the directionality effects of TMS. Since large parts of M1-HAND are positioned in the posterior wall of the precentral gyrus  prior to a suprathreshold test stimulus inhibits the test response. This form of inhibition is termed SICI and is dependent on both the intensity of the conditioning and test stimuli . SICI caeceptors  or, as deceptors .3.5.2.B receptors  describes the relative electromyographic silence seen following the MEP evoked by single-pulse TMS applied during a voluntary contraction. The duration of the CSP increases with stimulus intensity and typically has a maximum duration of 200–300 ms, while -in contrast- the level of voluntary contraction has little influence on the duration . Spinal eceptors . Howeverensities .3.5.3.B dependent networks contribute to LICI  is studied by using suprathreshold conditioning and test stimuli applied at ISIs of 50–200 ms. Epidural spinal recordings have shown that the later part of the LICI is associated with suppression of the late I-waves . GABAB d to LICI . These f to LICI . LICI an to LICI . Evidenc to LICI  and may eceptors . The int3.6.All intracortical neuronal elements in M1 that contribute to TMS-induced transsynaptic excitation of fast-conducting cortical output neurons are strongly modulated by afferent activity. Sensory input from the contralateral limb reaches the M1 either through the somatosensory cortex or more directly via thalamo-cortical afferents. Electrical or mechanical stimulation of afferent activity may either depress or enhance the amplitude of MEPs evoked by a single supra-motor threshold TMS pulse . The modWhile the effects of homotopic afferent inputs on singe-pulse MEP amplitude are mostly inhibitory (SAI and LAI), afferent inputs induce a disinhibition of local inhibitory circuits  , 2014. TSuprathreshold TMS at rest as used in SAI paradigms induces BOLD activations in the anterior and posterior subregions of M1-HAND , correspIn summary, the synchronized stimulation of afferent volleys can be used to study rapid and slow effects on corticomotor excitability as well as the excitability of cortical interneuron circuits in the M1. These effects have mainly been studied in the M1-HAND.4.4.1.Focal TMS with a figure-of-eight coil can be used to selectively target areas in M1 which represent a specific body part because of the somatotopic arrangement in the pericentral sensorimotor cortex. Most TMS studies of M1 targeted M1-HAND, but TMS can also be used to study the cortical representations of other body parts, in particular the representations of the leg or face . The pri4.2.The M1 is a unique area, generating direct corticospinal motor output via large fast-conducting pyramidal neurons (giant Betz cells) . As poinIt is common practice to adjust the intensity of TMS to the individual MT, even when targeting other brain regions. However, it is unlikely that the cortical MT obtained with TMS over the M1 is a reliable indicator for the efficacy of TMS in areas outside M1. Both cortical MTs and phosphene thresholds  allow standardizing intensities in controlled studies to a certain extent. Most studies, however, do not find a correlation when individual PTs are compared with individual resting or active MTs , but see4.3.TMS has contributed to understanding the temporal dynamics of parietal involvement in wide range of tasks probing attention, spatial and sensory-motor processing . Yet, a While it has been shown that studies benefit from individualized coil placement based on structural or functional MRI , a conseThe effects of parietal stimulation on a network level can be explored independently of behavioural read-outs with dual-site TMS by applying a conditioning TMS pulse at a parietal site and a test pulse at M1 see . These pMultimodal stimulation studies combining TMS with functional MRI or EEG have further helped to understand network effects outside of M1: TMS-fMRI demonstrated that stimulating IPS produced effects on the BOLD signal in visual area V5/MT+ in a task context and stimulation intensity specific manner . EEG rec4.4.Visual cortex comprises posterior cortical regions V1–V6 and has been targeted by TMS to clarify the working mechanisms of TMS or to investigate the functional specialization of specific visual areas. The latter category of studies used focal TMS to temporarily disrupt functional processing in the cortical target region. This interference approach proved to be useful to demonstrate and characterize causal links between functional specialization of visual cortical areas and visual perception. Its temporal resolution enabled to assess the temporal flow of information between functionally or anatomically connected visual areas. Seminal studies in the field confirmed that TMS over early visual areas V1/V2 in the occipital lobe can impair visual perception of shortly presented three-letter stimuli at a specific time window, peaking 70–100 ms after the presentation of the visual stimulus . Later iTMS over the visual cortex can not only disrupt perception but can also induce a transient perception of a flash of light, referred to as phosphenes. Phosphenes elicited by TMS of V1/V2 are small and static, whereas those induced by TMS over V5 are large, reflecting the cortical magnification factor, and are moving . Early sComplementing the MT derived by TMS targeting the precentral cortex, the phosphene threshold (PT) has been used to probe visual cortical excitability alone or in combination with other methods, e.g., with EEG or fMRI e.g., . ApplyinStudies in animals have revealed important insights into the neurophysiological mechanisms of action of TMS by targeting visual cortical areas see . In the Many of these findings in animals have implications for the application of TMS to study the visual system in humans. For instance, measurements of spiking activity and local field potentials in the cat visual cortex suggested that TMS has state-dependent effects: higher pre-TMS activity predicted larger post-TMS responses . In humaDevelopments in the field during the last decade imply that the application of rhythmic TMS is a promising tool to entrain cortical activity . It was 4.5.TMS to the prefrontal cortex (PFC) has helped to establish the role of the PFC in cognitive functions like memory , languagThe effects of prefrontal TMS are not only mediated by TMS-induced changes in the PFC region that is directly stimulated by TMS. Given the complex interactions of the PFC with other areas, TMS affects the functional interaction of the stimulated area with connected brain areas, which may contribute substantially to the effects of prefrontal TMS at the behavioral level. These connectivity-based network effects should not be viewed as a limitation. Methodological control conditions achieved by stimulation of cortical areas or by looking at time course of interactions between nodes will reveal distinct specializations. For example, it might be possible to establish functional connectivity and a hierarchy of control  by means of multi-site TMS or TMS in combination with neuroimaging procedures . For insPrefrontal TMS has also been used to characterize the differential involvement of the same cortical area in different cognitive tasks. Here, the same protocol may induce opposite effects based on the specific cortical engagement and capacities related to the cognitive function that are engaged by the experimental task. For example, the same TMS protocol applied to the same PFC region may facilitate a cognitive function, e.g., action naming , yet impPrefrontal TMS is often personalized by adjusting the intensity to individual resting motor threshold as determined at M1-HAND, but, as discussed above, this dosing procedure relies on the assumption that prefrontal cortex and precentral gyrus are equally responsive to TMS. 4.6.Premotor areas are located rostral to M1 and thus can be targeted using the precentral motor hot spots as reference points. The premotor cortex has traditionally been functionally divided into dorsal (PMd) and ventral (PMv) premotor areas. Recent studies however suggest a more complex organization, e.g. that PMd itself is composed of several functional subdivisions . The PMdMost TMS studies have typically investigated the role of PMd and PMv in the context of skilled hand movements. It has been shown that PMd contributes to the anticipatory control of grasping movements when they are conditioned to external cues, such as arbitrary associations between color and object weight , but alsTMS applied over PMv was shown to disrupt hand shaping and alter the pattern of hand muscle recruitment when grasping an object . Interes4.7.One major challenge for quantifying the effects of cerebellar stimulation is the lack of any direct, non-invasively measurable output. The effects can only be inferred from secondary measurements, either physiological or behavioural. Physiological measurements allow for evaluation of both instantaneous (online effects) or delayed consequences (after-effects) of cerebellar TMS, whereas behavioural read-outs are only suited (with few exceptions) to evaluate after-effects of cerebellar TMS. Two physiological approaches to probe the impact of cerebellar TMS stand out so far, as they offer excellent temporal resolution: one approach measures modulatory effects of cerebellar TMS on corticomotor excitability in M1 , while tDespite their ideal temporal resolution, there is one major limiting factor: the scalp-to-cerebellar cortex distance is more than three times longer and more variable than the distance from scalp to M1-HAND. This leads to poor focality and neuronal population targeting . Indeed,At the same time, the critical role of the distance between the coil and the cerebellar target also represents an advantage for applying an ideal, active sham stimulation. The active sham condition is represented by a real stimulation delivered with the exact same intensity as in the real TMS condition, but with the coil placed 5 cm below the cerebellar target on the back of the neck . This reA better understanding of what structures within the cerebellum are responding to TMS could also help us adjust the stimulation intensity and orientation appropriately. A few studies have addressed this problem by modelling the effective electric fields that are induced by TMS in the neuronal structures of the posterior fossa . They coTwo experimental approaches can currently provide clues regarding the structures that are stimulated within the cerebellum. One approach employs dual-site conditioning-test TMS targeting the cerebellar hemisphere and the contralateral M1-HAND. The second approach involves single TMS pulses delivered over the ocular motor vermis  during saccade adaptation. For dual-site cerebellar-cortical TMS, the first coil is placed over the cerebellum and applies a conditioning stimulus to the posterior cerebellum. This conditioning cerebellar stimulation can reduce the amplitude of MEPs evoked by a test pulse delivered over M1-HAND 5–7 ms after the cerebellar conditioning stimulus . The effThe conditioning-test approach is most frequently referred to as cerebellar-brain inhibition (CBI) or less commonly, but more specifically, cerebello-motorcortical inhibition . This ceAnother approach employs single TMS to target the ocular motor vermis  during saccade adaptation. TMS of these structures can induce hypometric contralateral and hypermetric ipsilateral saccades , accelerEvidence for the influence of cerebellar TMS on non-motor cortical areas in humans is still indirect, coming mostly from fMRI studies. Both intermittent and cTBS of the cerebellum appear to modulate the functional connectivity between the cerebellum and the default mode network, dorsal attention system, and frontal and parietal cognitive regions . More in5.More than three decades have passed since TMS was introduced as a non-invasive method to stimulate the human cortex. Although it has undoubtedly pushed the frontiers of human neuroscience and interventional neurophysiology with a potential for therapeutic applications, we still lack a comprehensive understanding of which neural elements in the cortex are primarily targeted with TMS. It is fair to conclude that TMS induces action potentials by depolarizing myelinated axonal structures in the stimulated cortex and that this regional excitation may spread via cortico-cortical and cortico-subcortical connections to connected brain regions. Both the regional as well as remote effects due to spread of excitation via neural fibers are highly state-dependent and involve excitatory (glutamatergic) and inhibitory (GABA-ergic) neurons. More detailed statements regarding the preferential site of excitation  or the sensitivity of distinct classes of intracortical neurons are still debated, although some progress has been made in recent years to clarify these questions. More basic research on the biophysics and neurobiology of TMS is needed to get further basic insight into its mechanisms of action. Preclinical research in animals, particularly recordings from single cells and the use of advanced methods such as optical imaging and optogenetics in combination with neuroimaging and biophysical modeling need to work hand in hand to clarify how TMS interacts with the human brain. The complexity of the brain’s response to TMS provides a challenge for the interpretation of its (neuro)physiological and behavioural effects , and cau


Background: Hepatocellular carcinoma (HCC) is a prevalent malignancy with a high mortality rate. Cellular senescence, an irreversible state of cell cycle arrest, plays a paradoxical role in cancer progression. Here, we aimed to identify Hepatocellular carcinoma subtypes by cellular senescence-related genes (CSGs) and to construct a cellular senescence-related gene subtype predictor as well as a novel prognostic scoring system, which was expected to predict clinical outcomes and therapeutic response of Hepatocellular carcinoma.Methods: RNA-seq data and clinical information of Hepatocellular carcinoma patients were derived from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC). The “multi-split” selection was used to screen the robust prognostic cellular senescence-related genes. Unsupervised clustering was performed to identify CSGs-related subtypes and a discriminant model was obtained through multiple statistical approaches. A CSGs-based prognostic model-CSGscore, was constructed by LASSO-Cox regression and stepwise regression. Immunophenoscore (IPS) and Tumor Immune Dysfunction and Exclusion (TIDE) were utilized to evaluate the immunotherapy response. Tumor stemness indices mRNAsi and mDNAsi were used to analyze the relationship between CSGscore and stemness.Results: 238 robust prognostic differentially expressed cellular senescence-related genes (DECSGs) were used to categorize all 336 hepatocellular carcinoma patients of the TCGA-LIHC cohort into two groups with different survival. Two hub genes, TOP2A and KIF11 were confirmed as key indicators and were used to form a precise and concise cellular senescence-related gene subtype predictor. Five genes  were selected from the TCGA training dataset to construct the prognostic CSGscore signature, which could precisely predict the prognosis of hepatocellular carcinoma patients both in the training and validation datasets. Multivariate analysis verified it as an independent prognostic factor. Besides, CSGscore was also a valuable predictor of therapeutic responses in hepatocellular carcinoma. More downstream analysis revealed the signature genes were significantly associated with stemness and tumor progression.Conclusion: Two subtypes with divergent outcomes were identified by prognostic cellular senescence-related genes and based on that, a subtype indicator was established. Moreover, a prognostic CSGscore system was constructed to predict the survival outcomes and sensitivity of therapeutic responses in hepatocellular carcinoma, providing novel insight into hepatocellular carcinoma biomarkers investigation and design of tailored treatments depending on the molecular characteristics of individual patients. Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer, the fourth leading cause of death among all cancers, and its incidence is rising rapidly in recent years . In the Cell senescence is a process in which cells stop dividing or lose their proliferative capacity, can be induced by a couple of stresses such as the activation of oncogenes and DNA damage caused by conventional chemotherapies/radiotherapies . TherefoTriggered by the p53 tumor suppressor, cellular senescence has been recognized as a suppressive factor of HCC by inducing the cell-autonomous program of cell-cycle arrest or apoptosis . In linehttps://portal.gdc.cancer.gov/) using the “TCGAbiolinks” package (. 114). The transcriptome profiles and survival data of 238 HCC patients from the International Cancer Genome Consortium (ICGC) database (ICGC-LIRI-JP) were acquired to serve as the external validation set. All data were normalized and processed as previously reported  between HCC and adjacent normal tissues were screened by using the “limma” package , with thhttps://david.ncifcrf.gov/tools.jsp) was used to perform the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and a network showing the most significant enriched pathways was drawn by the package “ggraph.”The package “limma” was utilized to screen the DEGs between two CSG subtypes of HCC, followed by the enrichment analysis of biological functions and pathways. The “clusterProfiler” R package was applied to conduct the Gene Ontology (GO) enrichment and the most significant terms were visualized by using the “ggplot2” package. Meanwhile, the online DAVID tool  and the validation set (n = 114) at a ratio of 2:1, and the ICGC-LIRI-JP dataset (n = 238) was selected as an external validation set. In the training set, two feature selection algorithms suitable for high dimensionality were applied to screen the most subtype-relevant features: LASSO regression and the Random Forest. The expression of the top 208 DEGs between two CSG subtypes with the cutoff of |logFC| > 1.5 and adj.P.Val<0.01 was used as the input variable and the CSG subtype status was used as the binary outcome (0 or 1). For the LASSO selection operator, the “multi-split” strategy was adopted to minimize the effect of arbitrary choice in the random sample split process , where β indicates each selected prognostic DECSG. Next, the CSGscores for the HCC patients in the training, validation, and external dataset were computed, and all the patients were categorized into the high- and low-risk groups depending on the median CSGscores in the training set. Kaplan-Meier survival curves and log-rank tests were used to compare the divergent survival outcomes in both groups. Time-dependent receiver operating characteristic (tROC) curves were drawn to evaluate the predictive power of CSGscore. Besides, stratified survival analysis was used to explore its additional prognostic value in subgroups divided by clinicopathologic variables.We examined the utility of the CSGs for the construction of a prognostic CSGs scoring system with the aforementioned prognostic DECSGs. Based on the TCGA training dataset, all the 238 robust prognostic DECSGs were subjected to LASSO-COX and stepwise regression algorithms to generate the best subset of prognostic genes, which were subsequently used to build the prognostic CSGs signature: CSGscore = Σ(coef (β)*EXPUnivariate and multivariate analyses were conducted to verify the independent prognostic capacity of CSGscore. Based on the results of Univariate analysis, a CSGscore-integrated nomogram was established with the “rms” package. Calibration curves were plotted to estimate the consistency between predicted probabilities of 3- and 5-year survival and actual ones. The decision curve analysis (DCA) curves at 1-, 3-, and 5- years were depicted to compare the net benefits of the nomogram and that of the pathologic stage or tumor burden status. Kaplan-Meier analysis was further applied to test the clinical relevance of the nomogram for OS, DFS, PFS, and DSS.https://tcia.at/home), and a lower IPS stands for worse sensitivities to immunotherapy (50) values of 138 chemo/targeted drugs  between distinct CSGscore groups. Immunophenoscore (IPS) representing the tumor immunogenicity of each sample was acquired from The Cancer Immunome Atlas (TCIA) . Finally, the correlation of CSGscore genes’ expression and clinicopathologic parameters including tumor grade, pathologic stage, tumor burden status, and CSG subtype was also investigated via boxplots.The innovative OCLR machine-learning algorithm-derived stemness indices including mRNAsi and mDNAsi have correlated with multiple clinical observations in malignant tumors such as poor survival, tumor metastasis, and therapeutic resistance . Thus, whttps://www.rstudio.com/).Kaplan-Meier and log-rank tests were used to compare the survival outcomes for OS, DFS, PFS, and DSS in different groups. Wilcoxon test or Kruskal-Wallis test was used for the comparisons of continuous variables in two or three groups. Correlation coefficients and statistical significance were calculated by Spearman correlation analysis. Pearson Chi-square test was utilized to examine the distribution differences of categorical variables. The R package “glmnet” and “randomForest” were used to perform the LASSO and random forest algorithms for dimensionality reduction. The “pROC” package was used to assess the predictive accuracies for the CSG subtype predictor and the “timeROC” package was used to determine the prognostic reliability of the CSGs scoring system. All data visualization was completed in RStudio  of DNA replication, cellular component (CC) of chromosomal region, and molecular function (MF) of catalytic activity acting on DNA . For thePreliminary work inspired us to investigate potential biomarkers with indicative roles in the classification of CSGs-related HCC subtypes to achieve a precise and concise model for CSG subtype categorization of HCC. We first picked out the top 208 of 784 DEGs between the two HCC subtypes with the cutoff of |logFC| > 1.5 . Next, wTo evaluate the capability of CSGs in the prognosis prediction of HCC, the abovementioned 238 robust prognostic DECSGs were applied as the input variables for the LASSO-COX regression analysis to construct a prognostic model, which resulted in 10 potential genes for the next incorporation into the stepwise regression . The finTo validate the stability and robustness of CSGscore for OS prediction, we adopted the TCGA-validation dataset and the whole TCGA cohort as internal validation sets and chose the ICGC-LIRI-JP dataset as an external set. Using the same cutoff value that was derived from the training set (0.9824545), all patients in each validation set were categorized into high- and low-risk groups. As The correlation between CSGscore and clinicopathologic features was subsequently explored. As presented in The accurate preoperative prediction of OS might help surgeons to make better clinical decisions and comprehensive nomograms can be used for the combined diagnosis or disease status prediction with multiple indicators. In this situation, accuracy was of the essence, so we were prompted to construct a novel nomogram combing the CSGscore model and multiple clinicopathologic traits to constitute a quantitative tool for predicting the clinical outcomes of HCC patients. While univariate analysis identified the CSGscore classifier, stage, CSG subtype, and tumor burden were prognostic variables, in multivariate analysis, only CSGscore and tumor stage were proved to be independent prognostic indicators for HCC . AfterwaOngoing efforts are being made to decipher the interplay between cellular senescence and immune response in the tumor microenvironment to determine whether senescence changes the role of the immune system from anti-tumor to pro-tumor response. In this context, we eagerly mined the potential correlation between CSGscore and the tumor immune microenvironment of HCC. We found most of the immune checkpoint genes (ICGs) were highly expressed in the CSGscore-defined high-risk group compared to the low-risk group . Moreove50 values of 138 chemo-/targeted drugs were calculated by “pRRophetic” algorithm for HCC patients and 10 common chemo/targeted drugs were selected to show the different IC50 values of them in low- and high-CSGscore groups  from 238 robust prognostic DECSGs, which were subsequently employed to construct the CSGscore risk signature as a scoring system for HCC prognostic prediction. The following validation of the internal and external validation datasets revealed that patients in the high-risk group defined by CSGscore were proved to have a worse prognosis. Among these CSGscore genes, PSRC1, TMEM45A, CCT5, and STC2 were high-risk factors, while SOCS2 was a protective factor. A few studies reported that PSRC1 was a key gene in cancer. For instance, Wei et al. identified PSRC1 as a poor prognostic gene in HCC , and ManIn advanced HCC, immunotherapies have rapidly evolved in recent years, and most of them focused on monoclonal antibodies against CTLA-4 and PD-1 that block immune checkpoints pathways to reactivate the tumor-killing activity of immune cells in the TME . AlthougIt is well known that cancer stem cells (CSCs) contribute to a high rate of cancer recurrence, as well as resistance to radiotherapy or chemotherapy . To dig Taken together, we comprehensively evaluated the cellular senescence patterns in HCC and proposed a novel CSG subtype classification system with clinical significance. A CSG subtype predictor was successfully established with high accuracy. In addition, we constructed a novel CSGs-based prognostic signature - CSGscore, which was proved to be a robust and precise indicator for the prediction of OS and therapeutic responses. The correlations of CSGscore genes and immune cell infiltration might advance the dynamic interplay between TIME and cellular senescence in different subgroups of HCC. Finally, the integrative analysis of the CSGscore genes also suggests they are of great importance in the process of tumor progression and represents a promising direction for novel biomarker development of HCC.


Ultrasound imaging is widely used to evaluate the neuromusculoskeletal system, and recently, a particular interest is mounting in assessing the bone tissue and fractures. Ultrasound can be considered a valuable diagnostic tool to perform a first-line evaluation of bone tissue, especially in particular settings without direct access to X-ray imaging and/or in emergency conditions. Moreover, different healing phases of bone fractures can be accurately assessed by combining the B-mode modality and (high-sensitive) color/power Doppler optimizing the management of patients—e.g., planning of progressive loads and rehabilitation procedures. In this review, we summarized the role of ultrasound imaging in the management of bone fractures and described the most common sonographic signs encountered in the daily practice by assessing different types of bone fractures and the progressive phases of the healing process.The online version contains supplementary material available at 10.1186/s13244-022-01335-z. US represents an alternative to XR for the occult bone fractures diagnosis.Knowledge of sonographic patterns is crucial for the detection of challenging fractures.US allows an early assessment of the callus formation and bone healing.The online version contains supplementary material available at 10.1186/s13244-022-01335-z. Ultrasound (US) imaging is a diagnostic technique characterized by several advantages; indeed, it is a cost-effective, non-invasive, reproducible examination and, unlike X-ray (XR), does not use ionizing radiation, resulting in a safe technique, particularly in the pediatric population , 2. In cRecently, particular interest has growth in the diagnostic potentialities of US in bone tissue pathologies . Some auIn the emergency setting, US imaging can be performed to assess in real-time the correct reduction in distal radius fractures , 21 and In this pictorial review, we summarize the main imaging features on US assessment of bone fractures. Starting from the normal sonographic appearance of the bone and superficial soft tissues, here we describe pathological findings of different types of bone lesions and the sonographic patterns of different healing phases.Imaging of the bone is traditionally performed with standard XR, CT, MRI, or scintigraphy. The first-line imaging tool in fracture diagnosis is represented by standard X-ray, while US is traditionally used for the evaluation of surrounding soft tissue , 24. LikSonographic assessment of the superficial bones must be performed with high-frequency transducers—usually more than 8 MHz—that guarantee high spatial resolution but limited penetration in depth . For deeComprehensive knowledge of the sonographic patterns of different anatomical structures of the neuromusculoskeletal system in physiological conditions is paramount to correctly interpreting the ultrasound findings in pathological conditions. The bone surface presents as a hyperechoic line —with theDue to the high density and compact structure of cortical bone, US waves are reflected determining a hyperechoic (bright) line which represents the cortical bone surface . Bone frImpact fractures can occur in individuals of all ages, and their shape and anatomical location are highly dependent on several factors—e.g., the individual bone quality and the dynamics of trauma. For instance, in elderly people, impact fracture may also occur after a minor trauma due to pathological conditions affecting the resistance of bone tissue such as osteoporosis and vitamin D deficiency . UltrasoSeveral authors have demonstrated that the diagnostic accuracy of ultrasound imaging in identifying impact fractures is higher at the level of diaphysis of long bones, and lesser at the ends-of-bones and near the joints . LikewisLongitudinal  a large amount of gel should be used to minimize the involuntary compression of the small vessels with the ultrasound probe—i.e., the “suspension technique,” (2) the probe should be kept extremely firm during Doppler evaluation to avoid/minimize motion artefacts with fake vascular signals, (3) pulse repetition frequency should be kept as low as possible to depict small-size vascular elements and slow blood flows, (4) we suggest to increase the gain of Doppler until it reaches background noise, and then (by slowly decreasing it) is possible to remove the unreal signals, preserving the real microvascular network, (5) the size of Doppler box should be accurately evaluated considering a significant reduction in Doppler sensitivity for a too much large region of interest. Likewise, modern ultrasound equipment can also present dedicated software to optimize the visibility of small vessels with slow flows usually known as high-sensitive Doppler modalities—e.g., the superb microvascular imaging (SMI) or the microvascular flow imaging (MVFI).Interestingly, it has been widely demonstrated that vascular signals within the fractured segment of the bone progressively reduce as the callus develops . LikewisIn order to correctly interpret the sonographic findings, a comprehensive knowledge of the multiple and progressive stages of the bone fracture healing process is essential Table  50]:Tab:Tab50]:T*7 days: during the first week, a hematoma can be observed surrounding the fracture site and presenting as a hypoechoic or anechoic irregular area . At this stage, many vascular elements originating from the periosteum  stage is usually visible on conventional X-rays. Each and every aforementioned phase can be assessed by combining the grayscale modality and color/power Doppler. Indeed, in the early phases, the presence of vascular signals can be considered a positive sonographic finding suggesting an active phase of healing  with a dome shape hematoma (red) (A) or can show a post-traumatic disruption of the periosteal layer (yellow) with a diffusion of the blood (red) within the surrounding soft tissues (B). The subperiosteal hematoma (red) is often related to a mechanical disruption of the Sharpey’s fibers (black lines) with dissociation of the anatomical interface between the cortical bone and periosteum (yellow) (C).Additional file 2: Video 1. High-sensitive power Doppler clearly shows feeding vessels penetrating the bone cortex and traveling within nutritional foramina.Additional file 3: Video 2. Cartilaginous plate in between the metaphysis and epiphysis of the distal femur in young volunteer.Additional file 4: Video 3. Long-axis view of a post-traumatic rib fracture with bulging of the periosteum and subperiosteal hematoma.Additional file 5: Video 4. Sonographic tracking of a post-traumatic rib fracture. Of note, the transverse acoustic window easily shows the focal interruption of the cortical bone.Additional file 6: Video 5. Hill–Sachs defect presents as a depression of the cortical bone in the posterolateral side of the humeral head.Additional file 7: Video 6. A longitudinal scan shows the avulsion fracture of the base of the 5th metatarsal bone at the attachment site of the lateral cord of plantar fascia after an ankle sprain.Additional file 8: Video 7. A transverse scan shows the hematoma surrounding the bony fragment detached from the base of the 5th metatarsal bone after an ankle sprain.Additional file 9: Video 8. Local hematoma can be nicely observed surrounding the cortical irregularity due to a post-traumatic fracture of the distal radius.Additional file 10: Video 9. High-sensitive power Doppler allows to observe the microvasculature within the hematoma surrounding the bone fracture.Additional file 11: Video 10. High-sensitive power Doppler shows the vascular invasion—originating from the thickened periosteum—of the fibrous callus during the healing phase.Additional file 12: Video 11. The color Doppler clearly shows neovessels located within the superficial portion of the (hypoechoic) fibrous callus in a patient with a post-traumatic fracture of the humeral neck.Additional file 13: Video 12. High-sensitive power Doppler shows the microvasculature within the thickened periosteum and around the partially calcified callus.


Acute renal injury (AKI) is a complex clinical syndrome, involving a series of pathophysiological processes, in which inflammation plays a key role. Identification and verification of gene signatures associated with inflammatory onset and progression are imperative for understanding the molecular mechanisms involved in AKI pathogenesis. Non-coding RNAs (ncRNAs), involved in epigenetic modifications of inflammatory responses, are associated with the aberrant expression of inflammation-related genes in AKI. However, its regulatory role in gene expression involves precise transcriptional regulation mechanisms which have not been fully elucidated in the complex and volatile inflammatory response of AKI. In this study, we systematically review current research on the intrinsic molecular mechanisms of ncRNAs that regulate the inflammatory response in AKI. We aim to provide potential research directions and strategies for developing ncRNA-targeted gene therapies as an intervention for the inflammatory damage in AKI. According to the guidelines issued by the KDIGO  organization, acute kidney injury (AKI) is a complex clinical syndrome with systemic effects characterized by rapid impairment of glomerular filtration function . The conThe etiology of AKI is extraordinarily complex, but it is essentially related to irreversible damage to the renal parenchyma . In the Pathologically, AKI is related to a variety of biological processes and usually described as an injury to the renal tubular epithelium and vascular system which manifests by multiple forms of programmed cell death, a delayed proliferation of renal resident cells, and an intense intrarenal inflammatory response . In addiNon-coding RNAs (ncRNAs) account for ∼90% of non-coding sequences transcribed in the human genome, and less than 2% of transcribed coding genes translate into functional proteins . NeverthAKI is considered an inflammatory disease with systemic effects . InflammThe complex interaction between the renal parenchyma and the circulatory system is closely related to the pathogenesis of AKI . The rapvia the DAMPs  recognition receptors of the tubular epithelial cells and innate immune cells residing in the kidney . The moshe DAMPs . Cell neAt different stages of AKI injury, quiescent macrophages undergo phenotypic polarization influenced by the microenvironment to differentiate into two functional states with specific roles . In the Chemokines and their receptors are crucial in the inflammatory response and can promote acute neutrophil- and monocyte–macrophage-dependent inflammatory responses, which are closely associated with the inflammatory damage in AKI . Early iMonocytes recruited from peripheral blood can differentiate into tissue-specific macrophages , which cThe balance between pro- and anti-inflammatory mediators affects the tissue repair process after acute inflammatory injury, whereas an imbalanced inflammatory response can impede normal tissue repair as well as lead to abnormal remodeling and dysfunction. Ideally, the balance between pro- and anti-inflammatory mediators ensures the later repair of the renal tissue . HoweverAs a result of the potentially devastating nature of uncontrolled inflammatory responses, the expression of inflammation-associated genes is tightly regulated at several different levels . RegulatNF-κB is a critical nuclear transcription factor that regulates the inflammatory response and expression of various inflammation-related genes, including inflammatory cytokines, chemokines, and adhesion factors . NF-κB iNotably, TNF-α and IL-1β both form a positive feedback loop by activating the NF-κB signaling pathway. The TNF receptor (TNFR) and IL-1 receptor (IL-1R) mediate the activation of the NF-κB pathway, leading to the transcription of downstream inflammatory cytokines . Both ofThe detrimental effects of AKI cause upregulation of cytokine and chemokine expression through TLR activation on the plasma membrane of renal tubular epithelial cells. In turn, activation of these cells recruits inflammatory macrophages and natural killer cells to the site of damage, further amplifying the inflammatory response and leading to apoptosis of renal tubular epithelial cells . The besSilent information regulator transcript 1  is a histone deacetylase with various biological functions . It has via inhibition of NF-κB activity in LPS-induced renal tubular epithelial cells -dependent protein kinase (AMPK), the master metabolic regulator (energy sensor) in eukaryotic cells , has beeal cells . MAPK, aal cells .Inflammatory vesicles are the primary signaling receivers of the classical pathway of cell pyroptosis and are composed of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 precursor . The twoLess than 2% of the human genome is transcribed into mRNA with protein-encoding functions . For thevia the classical pathway. In this pathway, primary miRNAs (pri-miRNAs) transcribed from DNA sequences are cleaved into precursor miRNAs (pre-miRNAs) at their stem-loop structure by the RNase III enzyme, drosha  that lack the complete open reading frame, thus lacking protein-coding potential . They plLncRNAs play a central role in epigenetic processes and chromatin regulation. LncRNAs can interact with DNA, other RNAs, and proteins through nucleotide base pairing or a domain generated by RNA folding, which endows lncRNAs with extensive ability to regulate gene transcription and perform biological functions . SpecifiCircRNAs are a type of endogenous ncRNA that possess the structural characteristics of a covalently closed continuous loop . This loThe function of most circRNAs remains largely elusive, but its most apparent mechanism of action is acting as a “sponge” of miRNAs to regulate gene transcription and expression . CircRNAThere is growing evidence that ncRNAs play an important role in AKI. However, the specific mechanisms by which ncRNAs regulate the expression of inflammation-related genes in AKI have not been fully elucidated. Clarifying the functional roles of ncRNAs and their intrinsic molecular mechanisms in regulating inflammatory responses will provide potential research strategies for developing targeted ncRNA gene therapies as an intervention for the inflammation-induced damage during AKI .The most widely studied type of ncRNAs in AKI is the miRNAs. Many studies have shown that miRNAs play an essential role in kidney-related physiological and pathological processes by regulating the expression of post-transcriptional genes . The resvia NF-κB signaling. Yang et al. found that miRNA-125a-5p indirectly inhibited the TLR4/NF-κB signal transduction by targeting TRAF6, thus inhibiting the expression of pro-inflammatory cytokines and reducing LPS-induced kidney injury  . Unlike Exosomes are vesicles of 30–120 nm in size released into the extracellular space by the fusion of multivesicular bodies with the cell membrane . Their pThe abnormal expression of lncRNAs in AKI during inflammation is closely related to the inhibition or activation of inflammatory-related genes and signaling pathways. Enhancing or inhibiting gene transcription by interacting with transcription factors is the primary way lncRNAs participate in regulating inflammatory responses . Cytoplavia inhibition of the TLR4/NF-κB signaling pathway by targeting miRNA-374a-3p (via miRNA-22-3p (Nuclear transcription regulation is an essential function of lncRNAs, and its influence on the NF-κB pathway is most important. LncRNAs can affect the expression of inflammatory factors by acting on the activation of NF-κB and related signaling pathways. A study on the up-regulation of SNHG14 in LPS-induced AKI indicated that the SNHG14/miRNA-495-3p axis promoted the expression of inflammatory cytokines by activating NF-κB signaling . The act-374a-3p . Feng etNA-22-3p .Some lncRNAs, such as CCAT1 and HOXA-AS2, have protective effects, as they can reduce the inflammatory response in AKI by inhibiting the NF-κB signaling pathway. A study in LPS-induced AKI showed that up-regulation of CCAT1 could restore the inhibitory effect of SIRT1 on NF-κB by targeting miRNA-155. This, in turn, reduced the LPS-induced inflammatory response and apoptosis of renal tubular epithelial cells . AnotherWhen cell pyroptosis occurs, inflammation can be caused by the release of pro-inflammatory cytokines IL-1β and IL-18. The active N-terminal of GSDMD is the “molecular switch” needed to open the cell membrane pores and release inflammatory factors in the classical cell pyroptosis pathway . LncRNAsvia the miRNA-424-5p/TXNIP axis (via the miRNA-577/GOLPH3 axis (he ceRNA network consisting of lncRNAs, miRNAs, and mRNAs, is involved in the pathogenesis of AKI by altering the expression pattern of inflammation-related genes . For insNIP axis . The latPH3 axis .Based on high-throughput sequencing technology and bioinformatics analyses, many studies have revealed differential expression profiles of circRNAs in AKI . Similarvia the miRNA-338/FOXA1 axis (A study found that the circRNA BNIP3L can promote LPS-induced apoptosis and the inflammatory response by the miRNA-370-3p/MyD88 axis in HK-2 cells . AnotherXA1 axis . CircRNAXA1 axis . The latXA1 axis .Two different studies have shown that the circRNA VMA21 can simultaneously serve as a sponge for miRNA-9-3p and miRNA-7-5p. Furthermore, up-regulation of circRNA VMA21 can alleviate sepsis-related AKI by inhibiting the expression of these two miRNAs . AnotherOur current understanding of the function and role of circRNAs is still in its infancy, and further research is needed to clarify the specific regulatory mechanism of circRNAs in the inflammation of AKI. However, the study of circRNAs as a therapeutic target for AKI will undoubtedly be a hot topic in the future.Understanding the role of ncRNAs in AKI inflammation can provide potential research directions and strategies for the development of ncRNA-targeted gene therapy as an intervention for AKI inflammation injury. However, studies into the regulation of inflammation by ncRNAs have mainly been conducted in animal experiments, with a huge gap to fill before clinical trials. Therefore, extensive research is needed to further explore the clinical value of ncRNAs. In addition, identifying homologous ncRNAs between experimental animals and humans is a tremendous work in itself . Most ncin vivo represents a fascinating new dimension in therapeutic regulation of the inflammatory response in AKI. Technically, in vivo anti-sense oligonucleotide therapy for targeted regulation of ncRNAs is feasible. However, many challenges must be overcome from an experimental research perspective before clinical translation, including careful evaluation of potential off-target effects caused by low ncRNA specificity. Future work is needed to address the shortcomings of the current research and increase our understanding of the regulatory mechanism of ncRNAs in AKI. A better understanding of the mechanisms behind ncRNAs will potentially result in its safe and effective use as a precise treatment for patients with AKI.Inflammation is an extremely important part of the development of AKI, and ncRNAs involved in the regulation of gene expression play an important role in this process. At present, research into the role of ncRNAs in regulating the inflammatory response in AKI has mainly focused on miRNAs and lncRNAs, though elucidating the mechanism of circRNAs has been in full swing. The mechanistic role of ncRNAs in the inflammatory response of AKI is gradually being uncovered, with multi-target inhibition or overexpression shown to be effective at reducing AKI-related inflammation in animal or cell models. Inhibition or rescue of these dysregulated ncRNAs


A nine-year-old girl with no history of abdominal surgery was presented to the emergency department with a seven-day-lasting abdominal pain, yellow vomiting and constipation; the stool was brownish mixed with small stones. There was no history of fever or similar episodes. Her development history revealed multiple episodes of earth and stone-eating since her 3 years. The physical examination revealed a well-developed child with stable vital signs, the abdomen was slightly distended and tense in its lower part. There was a palpable lump in the hypogastric area and in left iliac fossa. Rectal examination demonstrated a rectal ampulla full of small stones. Abdominal X-ray showed gravel inside the large bowel and rectum with a distended transverse and left colic angle. Manual evacuation and colonic lavage were done and repeated twice daily for three days. On the fourth day, an abdominal X-ray showed clearance of all stones from the colon and no signs of colonic obstruction or perforation.


Preeclampsia is one of the most serious pregnancy complications. It may be caused by immunological changes in the early placental microenvironment. The contents of small EVs may serve as biomarkers of pregnancy complications. Evidence suggests that the balance between T helper 17 (Th17) and regulatory T (Treg) cells are critical for preventing preeclampsia. The study recruited 39 pregnant women with preeclampsia and 127 healthy pregnant women. We assessed the levels of both Th17 and Treg cytokines  in their plasma and small EVs. We found significant differences in the levels of all cytokines in the plasma between the two groups during the second trimester. We also observed significant differences between the two groups in the levels of EV-encapsulated cytokines IL-21, IL-22, and TGF-β, as well as in total small EVs, during the second trimester. The ROC analysis showed that the classification efficiency (AUC) of TGF-β in small EVs was 0.81. TGF-β had the best discriminant ability of all the single EV biomarkers tested, the cross-validation of the accuracy was 0.89. Th17 and Treg cytokines in plasma and small EVs may contribute to maternal immune activation and clarify the potential mechanisms of small EVs and cytokines in preeclampsia. Preeclampsia affects 2-8% of pregnancies and is characterized by new-onset hypertension combined with proteinuria after 20 weeks of gestation Small extracellular vesicles (EVs) are a group of heterogeneous cell-derived membrane vesicles, including exosomes, microvesicles, and apoptotic bodies T helper 17 (Th17) and regulatory T (Treg) cells are part of the complex immune machinery The elevated levels of pro-inflammatory cytokines in the maternal circulation system may play a central role in excessive inflammatory response and endothelial dysfunction. It is thought that small EVs may support the exaggerated inflammatory state observed in preeclampsia. Recent studies have demonstrated a shift in the balance of Th17 and Treg cytokines in patients with preeclampsia. We hypothesized that the imbalance in Th17, Treg cytokines and maternal small EVs in the plasma, are involved in immunoregulation and may be potential biomarkers for preeclampsia. Our specific aims were: (1) To investigate the association of plasma Th17 and Treg cytokines in preeclampsia patients and healthy controls; (2) To evaluate the association of total small EVs, placenta-derived small EVs, and EV-encapsulated cytokines in preeclampsia patients and healthy controls; and (3) To assess whether these plasma and EV-encapsulated cytokines could be used as biomarkers for preeclampsia.This study was designed as a prospective cohort study, including pregnant women in their early pregnancy. Then tracked to production and investigated disease  happen or not. Pregnant women were recruited at National Cheng Kung University hospital (NCKUH) from October 2015 to February 2019. We then collected the informed consent, questionnaires and their residual blood samples. The definition of pregnancy trimesters in this study: the first trimester was 0-13 weeks; the second trimester was 14-27 weeks; the third trimester was 28-42 weeks to delivery. Participants' samples in first trimester were collected between 12-13weeks, participants' samples in second trimester were collected between 14-27 weeks and participants' samples in third trimester were collected between 28-39 weeks. The cases were included if they met the criteria of American College of Obstetricians and Gynecologists' clinical guidelines for preeclampsia (new-onset hypertension (blood pressure ≥ 140/90 mmHg on two occasions separated by at least 4 h) and proteinuria (> 300 mg/24 h or protein/creatinine ratio>0.30) after 20 weeks of gestation). We got diagnosis data from medical records. Controls were healthy pregnant individuals without other major obstetric complications. Those pregnancies with preeclampsia onset before collecting blood specimen in second and third trimester and the women with other major obstetric complications were excluded. Finally, we totally included 39 pregnant women with preeclampsia and 127 healthy pregnant women as controls. The study was approved by the Institutional Review Board at NCKUH (IRB numbers: B-ER-103-418 and B-BR-106-003-T).Total small EVs were isolated from the plasma using an qEV size exclusion columns . Purification of small EVs using qEV size exclusion columns was performed according to instructions provided by the manufacturer. Plasma samples (150 μL) were first overlaid on qEV size exclusion columns followed by elution with PBS, finally 200 μL fractions were collected. Small EV was analysed by the tunable resistive pulse sensing (TRPS) using NP200 Nanopore  to get the concentration and size distribution of particles. Carboxylated polystyrene beads (200nm) were used to calibrate the concentration and size. Transmission electron microscopy (TEM) was also used to visualize the morphology of small EV. We added 50-100 μl small EVs for ELISA experiments  according to the instructions of the protocol. The concentrations of total and placental small EVs were quantified using CD63  and placental alkaline phosphatase (PLAP)  enzyme-linked immunosorbent assay (ELISA) kits. CD63 is a common small EVs surface marker and PLAP is a syncytiotrophoblast-specific marker, so small EVs derived from the placenta are positive for PLAP.Plasma samples were prepared by brief centrifugation of whole blood samples and stored at -80 °C until analysis. Small EVs samples were prepared by adding 5ml radioimmunoprecipitation assay (RIPA) buffer  with 50ul protease inhibitor  directly to small EVs suspended in PBS to lyse the small EVs membrane and solubilize the proteins. We added 100 μl small EVs for ELISA experiments  according to the instructions of the protocol. We determined the plasma and levels of EV-encapsulated Th17 , IL-21 , and IL-22 ) and Treg  and TGF-β ) cytokines using the ELISA kits according to the instructions provided by the manufacturer. The plasma and small EVs samples from both groups were assayed using the same ELISA plate to minimize experimental variation.https://www.r-project.org/). We compared maternal demographic characteristics and immunological and EV biomarkers between the two groups. For categorical variables, Pearson's chi-squared test or Fisher's exact test was used to determine significant differences. For continuous variables, Student's t-test or the Wilcoxon rank-sum test was used to determine the significance of differences between the two groups. The values for each biomarker were divided into quartiles and included in the logistic regression model as continuous variables. A P-value of < 0.05 was considered statistically significant.All analyses were performed using SAS version 9.4  and R version 3.6.1  curves were constructed using logistic regressions to produce plots of the percentage of true positives (sensitivity) and false positives (1 - specificity) for the multivariate immunological levels in preeclampsia patients and healthy controls. The area under the ROC curve (AUC) was calculated as the predictive value of using plasma and EV biomarker levels to differentiate between preeclampsia patients and healthy controls. In addition, a type of machine learning algorithm, support vector machine (SVM), was used to assess classification efficiency and establish the prediction models for disease status based on multiple cytokines There were significant differences in body mass index (BMI), blood pressure, and parity between the preeclampsia patients and healthy controls: the preeclampsia patients had a higher mean BMI, they had a higher incidence of chronic hypertension, and they tended to have earlier deliveries Table . Indeed,The plasma levels of all five cytokines were significantly higher in the preeclampsia patients during the second trimester, and also of all except TGF-β during the third trimester . Dot ploThe NTA and TEM data of small EVs in both groups were showed in Figure During the second trimester, plasma IL-22 levels had the highest AUC, which was 0.67, of all the candidate plasma biomarkers when considered individually . When thThe total small EVs concentration (AUC = 0.77) and placental small EVs concentration (AUC = 0.71) during the second trimester demonstrated a good classification efficiency Table . Two EV-Using the SVM, we evaluated the performance of a single plasma cytokine as a predictor, and found, after cross-validation, that IL-22  and TGF-β  displayed relatively high accuracy in discriminating between healthy controls and the patients with preeclampsia . HoweverIn terms of the EV biomarkers, total small EVs concentration  and placental small EVs concentration  both exhibited strong classification ability during the second trimester Table . The samWe also used the second trimester and third trimester data of the pregnant women and merged a small part of the recorded data from the first trimester to create generalized additive mixed models (GAMMs). GAMMs as an extension of generalized linear mixed models makes it possible to account for temporal autocorrelational structure in experimental data and assessed the pattern of TGF-β level, which has the highest accuracy, in EV markers, throughout pregnancy and to examine the differences between our two study groups. The predicted concentration of TGF-β in placental small EVs was significantly higher in the preeclampsia patients throughout pregnancy (P < 0.001) .Our results showed that a pro-inflammatory state was present in the patients with preeclampsia, increasing the levels of pro-inflammatory cytokines and raising the pro- and anti-inflammatory cytokine ratios. The disruption of the balance between the pro- and anti-inflammatory cytokines in the peripheral blood was probably associated with the preeclampsia. These results supported by our findings that Th17 cytokines are dominant and contribute to maternal inflammation in preeclampsia. We established that the concentration of total and placental small EVs in the maternal circulation increased during pregnancy, in both normal and preeclamptic pregnancies. These results suggest that, in early pregnancy, presymptomatic women who are going to develop preeclampsia could be identified from the concentration of small EVs in their plasma.In previous studies, little investigation of cytokines in EVs has been conducted. This study not only measured the level of cytokines in both plasma and placenta-derived small EVs, but also compared the level of cytokines between different trimesters. This study shows the potential role of small EVs in the pathophysiology of preeclampsia, providing biomarkers to predict the preeclampsia in early gestation. In our study, we used two markers to detect EVs for placental organ. The concentrations of total and placental small EVs in maternal circulation were quantified using CD63 and placental alkaline phosphatase (PLAP) enzyme-linked immunosorbent assay (ELISA) kits. In this study, we were to quantify plasma and then extract small EVs to detect cytokines, and there was no re-quantification of small EVs after extraction. Our total EVs included placenta small EVs, we detected EVs markers of placenta in total EVs. But we don't isolate specific placenta small EVs from total EVs in this study.The pattern of cytokine release is not a fixed property of the system; it can be modulated, for example, upon maternal immune activation. However, due to restrictions in methodology or other unelucidated bioprocessing of the small EVs, there were inconsistent results for EV-encapsulated cytokines relative to the levels of plasma cytokines. Some EV-encapsulated pro-/anti-inflammatory cytokine ratios were reciprocal to the plasma ratios . These rDominance of pro-inflammatory cytokines appears to be harmful in pregnancy, and anti-inflammatory cytokines regulate and ameliorate the immune response The Production of Tregs can induce by TGF-β, regulate the balance of Tregs/Th17, and is necessary to maintain the suppressive immune function of Tregs Interestingly, impaired placental function in preeclamptic pregnancies results in placental apoptosis and necrosis, which further increases the release of small EVs The effects of placenta-derived small EVs on both the fetus and the mother have yet to be clearly established. Elucidation of the small EVs pathways during normal and pathological pregnancies will allow the development of early screening tests for women at risk of pregnancy complications, monitoring of responses to interventions, and the development of novel EV-based therapies. There are certainly numerous new therapeutic possibilities involving small EVs, because their properties may be useful for the detection, amelioration, or curing of severe diseases. Furthermore, the bioactivity of placenta-derived small EVs may exacerbate the pro-inflammatory state that was normally associated with pregnancy Our results demonstrate that changes in the levels of both plasma and EV-encapsulated cytokines in pregnant women with preeclampsia differed from those in healthy controls. The pro-inflammatory and anti-inflammatory cytokine profile shifted toward a pro-inflammatory state in the maternal peripheral circulation of the preeclampsia patients. In addition, both the plasma and EV-encapsulated cytokine ratios play an important role in the preeclampsia patients. The greatest differences in these biomarkers, even in the concentration of small EVs, between the two groups existed at the start of the second trimester. In addition, the abnormal concentration of total and placenta-derived small EVs may result in the secretion of numerous immunological effectors and be involved in a potential mechanism of immune regulation. Therefore, the plasma and EV-encapsulated cytokines, as well as their ratios, may be useful as non-invasive measurements for identifying women at high risk of preeclampsia during early gestation. However, a larger trial is required to further validate the utility of this approach for population screening. We look forward to constructing a novel prediction algorithm and improved guidelines for the identification of women at high risk of preeclampsia and the development of a personalized approach in the management of this condition.Supplementary figures and tables.Click here for additional data file.