Leptin and resistin are cytokines whose plasma levels correlate with adiposity. Leptin is a hormone synthesised and released from adipocytes and can be transported into the brain. Resistin is produced in adipocytes in rodents and in macrophages in humans, particularly macrophages that have infiltrated adipose tissue. Both hormones can act within the brain to influence sympathetic nerve activity. Leptin appears to have a generalised sympatho-excitatory actions whilst resistin appears to increase sympathetic nerve activity affecting the cardiovascular system but inhibits sympathetic nerve activity to brown adipose tissue, which contrasts with leptin. Since both hormones can be elevated in conditions of metabolic dysfunction, interactions/crosstalk between these two hormones in the brain is a real possibility. This review describes the current knowledge regarding such crosstalk within the central nervous system. The evidence suggests that with respect to sympathetic nerve activity, crosstalk between leptin and resistin can elicit enhanced sympatho-excitatory responses to the kidneys. In contrast, with respect to food intake, resistin has weaker effects, but in regard to insulin secretion and thermogenesis, leptin and resistin have opposing actions. Thus, in conditions in which there is increased resistin and leptin levels, the result of crosstalk in the central nervous system could contribute to worse cardiovascular and metabolic complications. In conditions like metabolic syndrome, overweight/obesity and Type 2 diabetes, there is increased body weight, insulin resistance, hyperglycemia, hypertension, and metabolic dysfunction including elevated levels of leptin and resistin. Both these hormones have central and peripheral actions. This review will focus on the central actions of these hormones on metabolic function and cardiovascular regulation and discusses what is known of interactions/crosstalk between leptin and resistin, particularly in relation to cardiovascular and metabolic regulation.ob/ob, db.db and lean control mice in the mid-60s led investigators to hypothesise a circulating factor that was a key regulator of metabolism. In 1994, leptin was first identified by Friedman as a circulating factor originating from fat tissue that was able to regulate energy balance  and Brandt’s vole (Lasiopodomys brandtii) that acclimate to a cold environment by increasing body mass and energy expenditure. In such cases plasma leptin levels are low in contrast to conditions in which rewarming takes place where body mass and energy expenditure drop and plasma leptin levels increase /cocaine- and amphetamine-regulated transcript (CART) and reduces the activity of agouti-related peptide (AgRP)/neuropeptide Y (NPY)/GABA-containing neurons located more medially in the ARC . Note thDirect projections from the ARC to other hypothalamic nuclei play key roles in mediating the metabolic effects of Leptin. The ARC projects directly to the paraventricular nucleus, a key neuroendocrine and integrative nucleus, and ARC neurons also project directly to the dorsomedial hypothalamus (DMH). As indicated earlier, leptin receptors are present on POMC/CART and on NPY/AgRP/GABA-containing neurons within the ARC which would suggest that the pathways involved in mediating leptin’s anorectic effects involve activation of the POMC/CART neuronal population that projects to the hypothalamic paravemtricular nucleus (PVN) and the DMH which results in activation of melanocortin receptors by the neurotransmitter, alpha-melanocyte stimulating hormone (MSH) released from the POMC/CART neurons, and inhibition of the NPY/AgRP/GABA neurons. However, this is clearly not the full story since knockdown of leptin receptor-positive POMC neurons only leads to mild obesity . SimilarEating a meal can provide gustatory pleasure, thus reward and desire can play major roles in dietary intake. Leptin may influence these pathways to modify dietary intake. The reward pathways in the brain involve the mesolimbic dopaminergic pathways from the ventral tegmental area (VTA). Leptin receptors are present in the VTA but knockdown of the leptin receptor-positive dopaminergic neurons in the VTA did not appear to influence motivational food-seeking behaviour but did increase anxiety . Howeverob/ob mice  has significantly reduced innervation of brown adipose tissue and correlates with their reduced thermogenic responsiveness to cold  and corticotrophin (CRH). TSH acts upon the thyroid gland to enhance thyroid hormone production which ultimately can lead to increased basal metabolic rate and increased energy expenditure . The PVN to cold .Leptin, acting within the brain, can increase blood pressure in pre-clinical studies and it can contribute to the hypertension associated with obesity , 2017. IIt is also noteworthy that peripheral sites of action can also contribute to leptin’s cardiovascular actions, particularly recent work which describes a key role for circulating leptin acting within the carotid body . In thatThe intracellular transduction pathways involved in the metabolic and sympatho-excitatory responses induced by leptin involve PI 3-Kinase, ERK1/2, and MTORC1 . HoweverResistin is a member of the resistin-like molecule hormone family which is characterised by 10 conserved cysteine residues and a conserved cysteine residue located near the amino terminal end . ResistiIt has been almost 20 years since resistin was first discovered, yet its specific receptors have not been identified. Several candidates have been suggested and these include a metabolite of of a proteoglycan known as decorin. This may be involved in growth of white adipose tissue . Adenyl Resistin was originally identified due to its association with insulin resistance (hence its name) . Of partResistin has been reported to elicit acute reductions in dietary food intake  but thisSeveral clinical studies have reported correlations between plasma resistin levels and high blood pressure . AdditioThe mechanisms that could contribute to elevations in blood pressure are changes in sympathetic nerve efferent outflow. Acute intracerebroventricular administration of resistin elicits significant increases in sympathetic nerve activity including that to the skeletal muscle vasculature and kidneys , 2012. IResistin does not induce a generalised increase in sympathetic nerve activity, rather it appears that sympathetic nerve efferent outputs to organs/tissues that are important in cardiovascular regulation are increased but sympathetic nerve activity to metabolically important tissue  is reduced. Thus, the sympathetic efferent pathways influencing cardiovascular outputs are increased by both resistin and leptin. In contrast, the metabolic sympathetic efferent outputs elicited by resistin opposes the effects of leptin.The intracellular mechanisms involved in the transduction of the renal sympathetic nerve responses elicited by centrally administered resistin is mediated by PI 3-kinase . The cenThe intracellular transduction mechanisms mediating the central responses to resistin on sympathetic nerve activity to brown adipose tissue involves ERK 1/2 dependent pathways. However, the inhibition of PI 3-kinase did not influence the sympatho-inhibitory action of resistin . Thus, tAs we have seen earlier, leptin and resistin act centrally to increase sympathetic nerve activity to the kidneys and skeletal muscle vasculature , 2012. IThe potential central crosstalk between leptin and resistin in the regulation of blood pressure, heart rate and sympathetic nerve activity to the kidney was recently investigated. There was no evidence of crosstalk in blood pressure or heart rate effects. However, when resistin was administered intracerebroventricularly 15 min after leptin the increase in renal sympathetic nerve activity was at least double that observed with each hormone alone  suggestiThe sympatho-excitatory action of leptin on renal sympathetic nerve activity is not affected by high fat diets. However, diets high in fat lead to a reduction in the sensitivity to the anorexigenic actions of leptin . This seWhether a high fat diet could influence the cardiovascular effects of resistin and the crosstalk between leptin and resistin has been investigated. A moderate high fat diet (22% fat) which induced a 40% increase in whole body fat mass did not influence the blood pressure or heart rate responses to leptin and resistin combined . The senThere is a very limited number of studies that have investigated central crosstalk between leptin and resistin with respect to metabolic function. The potential for such an interaction is high. For example, and as indicated earlier in this review, leptin administered centrally elicits increases in sympathetic nerve activity to brown adipose tissue . In contNot only is there crosstalk on energy expenditure, leptin and resistin may interact centrally to affect dietary intake and thus body mass. Leptin is a potent anorectic whilst resistin may also reduce food intake acutely. Chronically, however, the decrease in food intake is not sustained. Furthermore, the chronic administration of leptin together with resistin intracerebroventricularly resulted in attenuation of the effects of chronic central administration of leptin on food consumption  Table 1Table 1. Glucose homeostasis can also be affected by chronically administered leptin and resistin acting within the brain. Leptin can increase peripheral insulin sensitivity but reduces glucose-stimulated insulin secretion by acting within the hypothalamus. Resistin, also acting in the hypothalamus increases insulin sensitivity (but to a much lesser extent than leptin) but reduces glucose-stimulated insulin secretion, in contrast to leptin. Thus, here too, is the potential for central crosstalk . Indeed,According to the World Health Organisation, there are approximately 1.9 billion adults over 18 years of age, 38 million children under 5 years of age and over 340 million children between 5-18 years of age who are overweight or obese . Of greaA characteristic of obesity, in both humans and in animal models, is an increase in sympathetic nerve activity to the kidneys . This efThe recent hypothesis that hyperleptinemia is the cause of leptin resistance in metabolic function (see below) has raised the possibility that partially reducing leptin levels increases sensitivity to leptin and this would influence the cardiovascular effects of leptin in high fat fed conditions. This has not been investigated to date, but if it were found to occur, it would suggest that the “selective leptin resistance” related to cardiovascular regulation in high fat fed conditions discussed earlier in this review, may need review and that partial reduction of leptin levels in the high fat fed conditions should see even greater cardiovascular responses to leptin. This is an intriguing suggestion and the implications for crosstalk with resistin may be even further cardiovascular complications. Clearly, this is an area for further investigation.in vivo studies following chronic administration of leptin and resistin.Although there are limited studies on the central crosstalk of leptin and resistin on metabolic function, the evidence indicates that the presence of resistin reduces the actions of leptin on food intake and it has opposing actions on insulin secretion and thermogenesis. In regard to food intake and insulin secretion, the crosstalk has been found in preclinical In recent work, evidence is suggesting that partially reducing the high leptin levels (20–80%) in rodents fed a high fat diet is sufficient to attenuate the leptin resistance and improve leptin sensitivity such that weight loss and insulin sensitivity and glucose homeostasis are improved from the hyperleptinemic condition , 2020. IThe opposing actions on thermogenesis, mediated by opposing changes in sympathetic nerve activity to brown adipose tissue have been observed following acute administration of leptin or resistin alone . If a siThe adipokines, resistin and leptin are produced in fat tissue and each can act in the brain to affect metabolic functions, like food intake, insulin sensitivity and release and energy homeostasis. They also influence cardiovascular regulation. Leptin and resistin alone increase sympathetic nerve activity to the vasculature and kidneys and their actions are enhanced, as shown by the larger excitatory response observed in renal sympathetic nerve activity, when both are present compared to their individual responses. These effects are mediated by actions within the hypothalamus indicating crosstalk, probably involving similar intracellular transductions pathways.By contrast, metabolic actions of leptin and resistin alone can oppose each other. The central actions of leptin include decreases in food intake, decreases in insulin release and elevated thermogenesis mediated via increased sympathetic nerve activity to brown adipose tissue. The central actions of resistin elicits decreased food intake but increased insulin secretion and reduced thermogenesis. When resistin was combined with leptin, the magnitude of the decrease in food intake induced by leptin was reduced and the decrease in insulin secretion induced by leptin was reduced, suggesting the centrally mediated crosstalk between these two hormones may be detrimental to the metabolic regulation in conditions of hyperleptinemia and hyperresistinemia. The potential for crosstalk on the responses of leptin and resistin on sympathetic nerve activity innervating brown adipose tissue has not been investigated directly. However, given the clear opposing actions on this sympathetic outflow following acute administration of each hormone, we suspect that crosstalk between leptin and resistin on thermogenesis would also negatively impact energy homeostasis.The evidence, therefore, suggests that in conditions of hyperleptinemia and hyperresistinemia, such as overweight, obesity or metabolic syndrome, the potential for central crosstalk between leptin and resistin is high and the result could contribute to worsened metabolic function and cardiovascular dysfunction . We suspEB wrote the review and designed the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Significant progress has been made toward model-based prediction of neral tissue activation in response to extracellular electrical stimulation, but challenges remain in the accurate and efficient estimation of distributed local field potentials (LFP). Analytical methods of estimating electric fields are a first-order approximation that may be suitable for model validation, but they are computationally expensive and cannot accurately capture boundary conditions in heterogeneous tissue. While there are many appropriate numerical methods of solving electric fields in neural tissue models, there isn't an established standard for mesh geometry nor a well-known rule for handling any mismatch in spatial resolution. Moreover, the challenge of misalignment between current sources and mesh nodes in a finite-element or resistor-network method volume conduction model needs to be further investigated. Therefore, using a previously published and validated multi-scale model of the hippocampus, the authors have formulated an algorithm for LFP estimation, and by extension, bidirectional communication between discretized and numerically solved volume conduction models and biologically detailed neural circuit models constructed in NEURON. Development of this algorithm required that we assess meshes of (i) unstructured tetrahedral and grid-based hexahedral geometries as well as (ii) differing approaches for managing the spatial misalignment of current sources and mesh nodes. The resulting algorithm is validated through the comparison of Admittance Method predicted evoked potentials with analytically estimated LFPs. Establishing this method is a critical step toward closed-loop integration of volume conductor and NEURON models that could lead to substantial improvement of the predictive power of multi-scale stimulation models of cortical tissue. These models may be used to deepen our understanding of hippocampal pathologies and the identification of efficacious electroceutical treatments. Encouraged by advances in computation, new multi-scale neural network models have demonstrated the ability to predict spatiotemporal patterns of activity in complex neural tissue systems when activated by detailed models of stimulating electrodes  has been established as a valid and intuitive numerical approach to estimating electric fields induced in animal tissue systems through extracellular electrical stimulation Cela, . The AM The algorithms proposed herein were developed through utilization of data previously published and generated from a multi-scale model of a rat dentate gyrus. The following sections briefly summarize the construction, feedforward simulation, and validation of a complex multi-scale AM-NEURON model of the hippocampus . Once nodal voltages in the AM model are solved, voltage at neuronal compartments are found by tri-linear interpolation from the 8 nearest nodes and applying this voltage as an extracellular battery via the extracellular mechanism in NEURON—this is done for each compartment in every neuron –this causes currents within each cell that are appropriate given their location within the field estimated by the AM model.Because both neuronal and volume conductor models are electrical circuits, interfacing the two intuitively involves passing node voltages or currents back and forth  were then used to calibrate the AM for LFP estimation. This case was selected for LFP estimation analyses that follow because stimulating in the infrapyramidal blade allowed stimulation artifacts to be as far from the boundaries of the volume conductor model as possible, somewhat reducing the required complexity. This proved important owing to the dozens of variations of meshed models (complexity and geometry) that were used to analyze numerical vs. analytical performance.The model was simulated on a 4,040-processor high-performance computing cluster owned by the authors and supported through the University of Southern California Center for High-Performance Computing.in vitro or in vivo electrophysiology. It is on this basis that we justify establishing a homogeneous analytical estimate of LFPs as a proxy ground-truth to validate the numerical methods developed in the sections that follow.Previous studies, by these authors and others in the community, used simple analytical estimations of local field potentials to validate their neuronal models . While tThe analytical method used in this study is the point-source equation, as follows:n current sources, I. σ is the inverse of tissue resistivity  and r is the path length from source to recording location . A spatial scaling factor was then applied to expand this volume, ensuring that current sources near the edges of the model would be less affected by boundary-driven current shunting. The bounding volume scalar factor used in this study was 142%, which was set after determining that the relative field amplitude of edge-most currents are >95% relaxed by the time they reach the nearest boundary. This volume was then meshed with either grid-based hexahedral or unstructured tetrahedral  meshes with resolution (maximum edge-length) ranging from 40 to 180 μm edge-lengths. Tetrahedral meshes were constructed through Python scripts that make API calls to Gmsh 4.5 and TetGen 1.5.1 using Delaunay triangulation  . Therefoon Chew, .L, and edge cross-sectional area, A, to be the only scalars of importance in converting local tissue resistivity, ρ, to resistance, R, according to:Tetrahedralizations or hexahedralizations  are unbrA was a constant 0.25 μm2 for all meshes in this study. Because unstructured meshes are not orthonormal, it stands to reason that there should be different rules for assigning or scaling distance-derived resistances based on the angle and number of adjoining connections. Following analysis done by Duffin in the late 1950's, fractional conductance within and across tetrahedra can be approximated as proportional to the cotangent of internal angles  going to 0 as ϑ approaches 0 and infinity as ϑ approaches π/2. Therefore, very narrow elements are effectively short-circuited and meshes of a regular grid that yield tri-rectangular tetrahedra are electrically equivalent to hexahedralizations of the same grid. Whether this rule remains useful for obtuse tetrahedra was not studied, as none were generated in these models.This overall approach of applying material properties to tetrahedral meshes (Equations 4–7) has the nice feature of Following construction of a primary mesh and conversion to a circuit, new resistors were added between the hull nodes and a virtual ground. Each new wire connected to ground from a hull node had a resistance equal to the average of all other wires connected to the hull node being considered.The fundamental paradigm of the AM was then extended to incorporate incident and dynamic current sources from NEURON compartments into an already existing meshed model . For theThe steps for current splitting include, first, relative distance-weighted division of currents into eight or four parts, for hexahedral and tetrahedral elements, according to each of the nodes in the voxel that contains the source in the AM mesh. Secondly, these divided currents are then applied as a new static current source at each node.This relatively complex algorithm was also compared with a simpler current shifting algorithm which applies all current sources at the nearest respective node in the mesh according to LFP estimations via these two algorithms were compared to each other and a point-source estimated LFP for both computational performance and estimate accuracy. Comparison was performed through calculation of a time-averaged absolute residual, or error, with respect to the analytical solution at recording locations evenly distributed throughout the transverse plane of the tissue model.Once a resistor network was constructed and properly grounded, either the splitting or shifting algorithms were used to add a set of currents to the model. While the geometry of the model remained the same for every time step, a new netlist was written for each time step to account for changing current source amplitudes. Current source-node assignments remained the same for every time step for a given mesh geometry and resolution. These netlists were then loaded in series and nodal voltages were solved using the matrix solvers described at length by Cela . Figure https://github.com/bingsome/Neurospice). There remain, however, significant hurdles for adaptation of this method to different tissue systems, including but not limited to: tissue geometry, heterogeneous dielectric properties, and the complexities of constructing a biologically realistic NEURON model capable of generating meaningful virtual extracellular currents. Further, the value of such a tool may be somewhat reduced should we not focus future efforts on solving possible computational bottlenecks to be raised in the discussion portion of this manuscript.It is our eventual goal to release a software tool that will enable other investigators to optimize and later estimate LFPs within tissue models of arbitrary geometry and conductivity. Therefore, subsequent to acceptance of this manuscript, code to generate a meshed volume, integrate current sources, solve nodal voltages, and visualize results will be distributed via a public Github repository  and current source handling algorithms (splitting vs. shifting). Models were cross-compared for both computational performance and accuracy with error calculated with respect to point-source LFP estimates as a proxy ground truth. These estimates were made at recording locations evenly distributed throughout the transverse plane of the tissue model.The point-source equation was used to estimate LFPs in 1222 evenly distributed locations on a 2d-grid within a transverse plane at 50 μm under the stimulating electrodes and centered over the NEURON model. At very coarse resolutions current splitting had the advantage of 11–30% better accuracy at a computational penalty of 220–350% longer solve times , 10. TheBecause the number and spatial distribution of current sources remained the same for every meshed model, the preprocessing step of assigning sources to voxels or nodes was particularly impactful for very coarse meshed models. While this step involves pairwise relational distance calculation for millions of points and, therefore, increases in complexity with increasingly complex meshes, preprocessing time did not increase nearly as fast as did the time required to solve the circuits.Neither tetrahedral nor hexahedral meshes were universally superior in the analyses performed. For very coarse meshes, tetrahedral models were markedly faster to solve and could be more accurate than hexahedral but not without substantial cost in both dynamic memory utilization and model complexity –12. As mshifted) numerical estimates corresponded well with the analytical estimates  volume conduction model design is complicated by geometric and anatomic variety of electrodes and tissue and (ii) prediction of neural tissue system behavior is complicated by the nuances of tuning biologically detailed models of neurons and synapses. Through synthesis of the results presented in this paper and those that provided the feedforward AM-NEURON model described previously, we propose a bidirectional algorithm that deepens the cross-scale links between models of bulk-tissue dielectric behavior and cellular and network behavior of complex neural tissue systems  electric fields generated in neural tissue by stimulating electrodes, (ii) evoked behavior of neural tissue that lies within that volume, and (iii) the electric fields that are generated by that behavior completes the critical components of an algorithm that couples feedforward and feedback interactions between virtual electrical stimulating, recording devices, and computational models of neural tissue. An algorithm for bidirectional communication between AM and NEURON domains could prove a powerful approach to understanding electrode-based brain-machine-interfaces and electroceutical devices. While the accuracy of the numerical method with respect to analytical solutions could have been performed with randomized non-biological current sources, previous work enabled the use of a highly detailed  systems .A stable and efficient approach to solving fields arising in tissue from endogenous current sources allows us to begin inquiry into several new areas of work. Some of these include the following: (i) providing clarity regarding the extent to which volume conduction of transmembrane currents reinforces population synchrony, (ii) use of the model framework to validate and optimize spike-sorting algorithms for engineering applications, and (iii) investigation of more fundamental questions about the biology of specific neural circuits by determining with greater accuracy which components of a neuron or neurons in a network actually cause the features that emerge in evoked potentials  and those from the artifact phase (+0–2.0 ms). While beyond the scope of this present report, these differences warrant further analysis and may become the focus of subsequent studies. Because recording locations are often very far from the stimulation location, artifacts in LFPs far from the stimulation can be estimated accurately with very coarse meshes without adequately capturing the activity nearest to the virtual recording electrode  is likely to be negligible with respect to other sources of error in computational neural systems and modeling .Mesh design challenges represent one specific limitation for most numerical approaches to solving electromagnetic fields, the AM included. For every new problem and new tissue geometry, new meshes must be designed as there is not likely to be a universally optimal mesh volume, fundamental geometry, or resolution. Technically speaking, each model must find a new optimum for the following three variables: geometry, resolution, and mesh material properties . For LFP estimation, these calculations must also consider the spatiotemporal distribution of endogenous current sources.Computational demands and model complexity represent additional critical challenges that must be faced. Approaches to mitigating these demands are being developed by the authors and others who have a need for mature electromagnetics numerical solvers. Potential approaches include mesh reduction or refinement and parallel solution of the AM. Alternative solvers may be considered which have already implemented massively parallel solution of RC circuits, such as the Xyce environment distributed by Sandia National Labs  cluster. While the authors had access to HPC resources, the implementation to be distributed only requires a single-machine and will be multi-thread enabled. This means that investigators must be especially aware of the potential performance impact of increased quantities and spatial distribution of current sources relative to their meshed models. Future implementations may potentially be capable of performing preprocessing and/or solution via GPU, as many of the respective operations are reduceable to matrix algebra first principles.Ultimately, relative compute-time performance of analytical vs. numerical methods is dependent upon compute-hardware, the specific software implementation, and the spatial resolution of both current source inputs and recording locations to be solved. Thus, while there remain significant advantages for numerical over analytical methods in solution of complex and heterogeneous problems, it is difficult to conclude that numerical methods are always faster or more efficient than analytical for homogeneous volume conduction problems. However, for inhomogeneous problems, there is significant advantage afforded by application of a numerical method over analytical.The present study demonstrates an intuitive numerical approach to estimating local field potentials generated by a detailed computational model of cortical tissue. AM-NEURON LFP estimation, the analysis presented herein, and its synthesis with earlier work makes three recognizable contributions to model-based analysis of complex neural systems and electroceutical devices: (i) validation of a fundamental approach to incorporating incident and dynamic current sources into a pre-existing meshed model, (ii) suggestion of an equivalent-circuit algorithm that unifies intracellular and extracellular neuronal models, and (iii) clarification of the impact of mesh geometry on computational performance and model accuracy. Together, these contributions point the way forward in the development of more sophisticated models that seek to understand the bi-directional interaction between brains and brain-computer interfaces or electroceutical devices.https://github.com/bingsome/Neurospice).Project code to generate and solve fields arising from endogenous current sources can be found in a Python library (Neurospice) that is publicly distributed on Github (Rapid advances in computing and neural interfacing technologies have encouraged the development of model-based approaches to predicting brain behavior. Combining meshed models with discrete solutions to the partial differential equations which govern the spread of currents forms the foundation for powerful models of electrical stimulation of neural tissue. But, as these models increase in complexity, the need becomes more severe for the development of sophisticated computational approaches to understanding the fields generated by not only active electrodes but also active neurons. Due to the spatial, temporal, and magnitudinal differences in electrode vs. neuron generated fields, different numerical algorithms may be required to accurately predict voltage gradients throughout affected volumes when generated by numerous neuronal sources. To address this concern, we have conducted a study which explores the impact on field estimation of mesh geometry and algorithms for handling spatial mismatches between current sources and meshed nodes. Establishment of a robust algorithm for numerical estimation of field potentials arising from neural activation could provide a path toward more predictive modeling of large-scale recording systems, neural network behavior, and extracellular neuron-neuron interactions.CB constructed the models and conducted the analysis. JP and CG contributed to mathematical formulations. J-MB, JP, and CG contributed to project and manuscript design. J-MB, DS, GL, and TB oversaw development of models and analysis. GL and TB were involved in project conception and procurement of funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The physical architecture of materials plays an integral role in determining material properties and functionality. While many processing techniques now exist for fabricating parts of any shape or size, a couple of techniques have emerged as facile and effective methods for creating unique structures: dealloying and additive manufacturing. This review discusses progress and challenges in the integration of dealloying techniques with the additive manufacturing (AM) platform to take advantage of the material processing capabilities established by each field. These methods are uniquely complementary: not only can we use AM to make nanoporous metals of complex, customized shapes—for instance, with applications in biomedical implants and microfluidics—but dealloying can occur simultaneously during AM to produce unique composite materials with nanoscale features of two interpenetrating phases. We discuss the experimental challenges of implementing these processing methods and how future efforts could be directed to address these difficulties. Our premise is that combining these synergistic techniques offers both new avenues for creating 3D functional materials and new functional materials that cannot be synthesized any other way. Dealloying and AM will continue to grow both independently and together as the materials community realizes the potential of this compelling combination. The past two decades have seen significant progress in two fields of metallurgy, the dealloying and additive manufacturing of metals. Dealloying is a selective corrosion process that has been extensively developed into a highly effective and facile method for producing self-organized, bicontinuous nanoporous and nanocomposite metals. Additive manufacturing (AM) has revolutionized the design and production of customized metal parts by employing layer-by-layer construction, and the development of AM continues to advance rapidly as commercial industries adopt this emerging technology.The development of dealloying and that of additive manufacturing have paralleled one another. While aspects of both technologies emerged much earlier, both dealloying and additive manufacturing techniques did not begin to see widespread popularity until the early-to-mid 2000s. In the early 2000s, a series of publications identified the fundamental kinetic processes leading to porosity evolution in dealloying, leading to the identification of many new materials systems that could be processed in this way. In turn, these inspired studies on the mechanical properties and catalytic properties of nanoporous materials. That same decade also saw explosive growth in the additive manufacturing sector owing to the production of low-cost AM units and the development of more user-friendly 3D modeling software. These innovations enabled adoption by not only the manufacturing industry but also the general public . Since tWe argue here that AM and dealloying are inherently and uniquely complementary: not only can we use AM to make nanoporous metals of complex, customized shapes, but dealloying can occur simultaneously during AM to produce unique composite materials with nanoscale features of two interpenetrating phases. Such complementarity in materials processing will undoubtedly lead to many fascinating and impactful advances.Dealloying was once only considered a detrimental corrosion process, most commonly as the dezincification of brass alloys, but the field has now grown to encompass an array of processing techniques that take advantage of this corrosion process to make self-organized nanoporous metal networks with extremely high surface areas ,3. TheseThe variety of dealloying methods has grown as researchers have identified new dealloying media beyond aqueous acidic solutions. Because selective dissolution is driven by some difference in how the elements of the precursor alloy interact with the dealloying medium, changing the dealloying medium changes the driving force behind the dealloying. With more dealloying pathways comes a greater number of compatible material systems, resulting in new nanoporous metals that were previously inaccessible via dealloying. While many variations of dealloying now exist today, electrochemical dealloying was the earliest form of dealloying that attracted attention from the community for the synthesis of nanostructured materials ,10. As tLiquid metal dealloying, unlike electrochemical dealloying, utilizes the difference in enthalpies of mixing between each element of the precursor alloy with a molten third element, which will act as a liquid metal solvent. The third element is chosen such that, at the dealloying temperature, the sacrificial element will dissolve out when the precursor alloy is immersed in a molten bath of the third element, due to a positive enthalpy of mixing between the two elements. As the dissolving species is removed from the precursor, the remaining element reorganizes into a bicontinuous porous structure. This phenomenon was first discovered by Harrison and Wagner in 1959  and was Alternatively referred to as solid metal dealloying or solid-state interfacial dealloying, solid-state dealloying is the selective removal of one element from the precursor alloy via solid-state diffusional mechanisms into a metal solvent, forming a bicontinuous microstructure, again resulting from the relative rates of dissolution and interface reorganization. Like for liquid metal dealloying, the driving force behind solid-state dealloying is the difference in enthalpies of mixing between the elements of the precursor alloy and the dealloying medium, leading to selective dissolution. The transport kinetics in solid-state dealloying are generally slower by a few orders of magnitude compared to those in both electrochemical and liquid metal dealloying; however, for the same reason, the ligament size of the dealloyed material tends to be much finer. As such, solid-state dealloying has, to date, been limited to thin film geometries or as an interfacial effect. Wada et al. first reported this technique in 2016, using solid-state dealloying to create a dealloyed interfacial region between the two layers of a diffusion couple . They prIn the latest variation of dealloying to be carefully elucidated—vapor phase dealloying—the difference between the vapor pressures of the two precursor alloy elements is used to drive the sublimation of one element at an elevated temperature, while the remaining element forms a porous metal structure . The earThe recent introduction of these new techniques has increased the compositional diversity of dealloyed nanoporous metals and nanocomposites. All of these dealloying methods, each with their own advantages and limitations, can be used together with additive manufacturing (AM) to gain further control over the final shape and microstructure of materials in AM parts, which then, in turn, affect material properties and potential use. Because of their high surface area and relative structural stability, dealloyed materials by themselves have already proven to be incredibly versatile across several industries. Here, we discuss three categories of dealloyed materials’ applications that could benefit from the integration of AM into their processing: (1) electrochemically active materials such as electrocatalysts for fuel cells, battery electrodes or electrochemical actuators; (2) structural materials; and (3) substrates for biomedical applications such as implant surfaces.Some of the earliest work on dealloyed nanoporous metals evaluated their electrocatalytic properties ,40. LikeThe introduction of liquid metal dealloying and solid-state dealloying techniques opened the door to a greater variety of applications for nanoporous metals by increasing the compositional range of dealloyed materials to include non-noble metals, such as refractory metals, which are defined by their resistance to high temperatures and mechanical wear. Because the dealloying solvent is metal, these two dealloying methods can be used to create high-strength metal–metal nanocomposites. Liquid metal dealloying has already been demonstrated as an effective and facile method for making nanocomposites with remarkable strength and toughness . HoweverDealloyed materials have also demonstrated potential for use in next-generation biomedical implants. The design criteria for biomedical implants are often rigorous and require tailored material properties, which can be provided through dealloying. For example, orthopedic implant materials must exhibit a mechanical response similar to that of bone. A mismatch in stiffness results a disproportional load distribution between the bone and the adjoining implant, which can lead to bone regeneration and implant failure ,47,48. TThe term “additive manufacturing” was formally established in 2009 by the ASTM  F42 Technical Committee to define a new class of manufacturing processes that employed layer-by-layer deposition to print components from 3D model data . This deHowever, 3D printed metal components, unlike like their conventionally machined counterparts, commonly exhibit micro-porosity and anisotropic fracture behavior . UsuallySeveral methods of AM currently exist, the majority of which fall into one of the seven categories of AM processes defined by the ASTM F42 Technical Committee ,66. HereDirect ink writing (DIW) describes a class of droplet-based 3D printing processes. An ink deposition nozzle generates a pattern onto the substrate, which sits on a computer-controlled translational stage . Figure The most promising metal powder bed fusion technique currently is selective laser melting (SLM), in which a high-power laser is used to fuse metal powders together into a solid part . Inside Some progress has already been made in using dealloying and AM methods together for the controlled formation of hierarchical porous structures, but this work can be expanded upon. There are also new pathways that can be opened with the combination of these two types of material processing; while the existing literature has focused on employing dealloying techniques as a post-processing treatment for 3D printed structures, here, we introduce the possibility of using laser-based AM to induce porosity formation during the printing process such that the material is already dealloyed before it leaves the build chamber. While rapid solidification during SLM introduces new complications to the dealloying process, it also provides a vision for a one-step method to produce metal nanocomposites with the material property advantages of a dealloyed microstructure coupled with the manufacturing flexibility of 3D printing. We will then discuss how further processing can produce nano/micro-porous powders from AM nanocomposites.Interest in hierarchical porous structures stems from their potential applications in microfluidics and catalysis, where a large surface-area-to-volume ratio and fast mass exchange are both necessary for optimal performance ,79. Exam2/g in nanoporous graphite, for example [A number of traditional methods exist for fabricating hierarchical porous metallic materials—templating , electro example . Bulk naThis approach was examined in Song et al.’s review on hierarchical porous metal structures , but at More recently, Fujita et al. also reported the use of an alternative AM method, selective laser melting (SLM), to make hierarchical porous Cu structures . They weBoth of the previously mentioned studies used chemically dealloying, which is the dealloying method with the longest history and largest body of research, but we anticipate that complementing chemical dealloying with alternative dealloying methods can grow the applications of this technique to include metals beyond the typical noble metals used in chemical dealloying to include metals from nearly every section of the periodic table. Additive manufacturing has been used to construct components across a wide spectrum of compositions, which means dealloying is the limiting factor in the compositional variety of alloys that can be used in this process to create hierarchical porous metals. Electrochemical dealloying itself is fairly versatile, but its composition range is limited by the requirement that the surface mobility of the remaining metal’s atoms fall within a narrow “Goldilocks” range . If the In addition to hierarchical porous structures, additive manufacturing and dealloying together can also enable the fabrication of new nanocomposite materials with improved microstructure and shorter processing times. Here, we discuss the combination of laser-based additive manufacturing—specifically, selective laser sintering/melting (SLS/M)—and dealloying to create metal–metal nanocomposites formed in situ during the melting process. The motivation here is the general observation that materials with small microstructural features generally exhibit higher strength than their bulk counterparts. While selective laser melting itself has been used to fabricate metal composites ,76, incoWhat if a two-component powder mixture was fed into an additive manufacturing system such that dealloying occurred during SLS/M? To our knowledge, this has not been studied in a systematic way. Localized laser interactions with an Ag-Au thin film immersed in nitric acid have been reported as an effective method for patterning the film by selectively dealloying areas irradiated by the laser , but intAs proof of principle of this concept, The use of multi-component powders also increases processing options from a thermodynamic and phase stability vantage point. For instance, the single element binder can be replaced with powders of a eutectic alloy with a relatively lower melting point, thus lowering the dealloying temperature. Recent work by Song et al. reported dealloying Ta-Ti alloys in molten Cu-based eutectic alloys, yielding a material with a decreased characteristic ligament width of ~75 nm, much finer than Ta-Ti dealloyed in pure Cu, which exhibits ligaments ~3 μm in diameter . HoweverRefining our understanding of the integration of dealloying with AM will require a detailed kinetic analysis of the as-formed microstructure in this scenario. We see three kinetic factors that should be characterized: (a) the interface velocity, i.e., how fast the interface is moving between dealloyed and undealloyed material; (b) what the concentration gradient of the dissolving species as it moves through the dissolution medium is; and (c) how fast the microstructure of the dealloyed layer is evolving, i.e., how much coarsening there is behind the dissolution front.1/2t as a result of the diffusion limitations of the sacrificial element in the molten bath. In the powder bed geometry of AM, the binder has limited mass and volume, in contrast to the functionally infinite bath model considered in bulk LMD, and thus will more quickly approach the solubility limit of the dissolving species in the molten bath. This will likely slow the overall velocity of the dealloying front and eventually stop dealloying altogether. We expect the dealloying process in the mixed powder environment to be limited by the solubility limit of the dissolving element in the binder phase, instead of its diffusion limit, as it is in bulk dealloying systems.As the molten binder phase becomes saturated with the dissolving species, the driving force for diffusion away from the interface decreases. It is known from bulk LMD kinetic studies that this leads to an interface velocity that slows as Approaching the solubility limit may introduce another problem. The remaining component in the precursor is miscible with the dissolving element, so the solubility of the remaining component in the binder phase increases as the dissolving component saturates the binder phase. If the concentration in the binder phase rises to near the solubility limit, some amount of solid porous structure may also dissolve away into the binder. This may have the effect of destroying the bicontinuous porous structure; an equally valid possibility is that the presence of the less-soluble element in the melt could also instead refine the porosity of the solid structure and make the pore size more uniform. Residual quantities of the more noble element of the precursor should precipitate in the binder phase upon cooling and may be observable with SEM/TEM systems.Due to the diffusion limitations of the dissolving element diffusing through the liquid medium penetrating the dealloyed layer, the interface velocity decreases, allowing the coarsening of the bicontinuous dealloyed layer to becoming significant. In AM, such secondary coarsening should occur only once a powder particle has been fully dealloyed because the dealloying depth is relatively shallow. Therefore, we can hypothesize that because the dealloying times will be much shorter when dealloying powders, this will lead to smaller ligament sizes. However, if the dealloyed powder remains hot, there is a danger that each dealloyed particle will simply collapse to a ball due to capillary forces, destroying the porosity that was formed. Rapid solidification and fast thermal transport in AM should alleviate this effect. However, another advantage of using powders instead of bulk LMD is that the system can be tuned so that the dissolving species will quickly saturate the molten binder phase, especially if the average binder particle size is small and the molten binder interlayer is thin relative to the powder particle diameter. This should stymie further dissolution from the precursor alloy particles, slowing the dissolution kinetics and extending the working time window.Porosity evolution can also occur without melting the binder phase via selective laser sintering and solid-state dealloying. In AM, the starting material would be the same two-component powder mixture, but the mixture would be sintered, rather than melted, by the laser-based AM process. The selective laser sintering of the powder mixture induces solid-state dealloying to produce a metal–metal nanocomposite. The dealloying temperature can now fall below the melting point of the binder phase. Widening the operating temperature window broadens the utility of dealloying to include more materials systems . For ins2O4 particles [2O4, which they attributed to changes to the solidification behavior due to the presence of MgAl2O4 nanoparticles. In a similar study, Vrancken et al. studied the microstructure of Ti6Al4V powder mixed with pure Mo particles processed via selective laser melting and found local variations in the content of Mo with bands of Mo-rich and Mo-poor compositions [The parameters of the sintering or melting process will be critical for the optimization of simultaneous dealloying and solidification. The scan speed, laser power, and melt pool size and shape are only a few of the factors that can be adjusted to improve the quality of the final sample. Previous studies on the additive manufacturing of metal matrix composites can offer insight into additional issues that may arise when processing powders of two different compositions together. Manfredi et al. reported a case study of manufacturing aluminum matrix composites of AlSiMg alloy with two different types of reinforcements, micro-sized SiC particles and nanosized MgAlarticles . They meositions .3 and 104 K/s [With particle sizes of about 10 microns in diameter and interface velocities on the order of 0.1 microns/s, heating and cooling should be rapid relative to microstructural evolution, but the formation of bicontinuous nanoscale ligaments during dealloying may differ under the extremely fast heating and cooling rates typical in a laser-based additive manufacturing setup, where cooling rates are reported to be between 10 104 K/s . Complex 104 K/s . It is w 104 K/s , but add 104 K/s . Many avThe central question is how refined a microstructure we can expect from an additively manufactured and dealloyed nanocomposite material. We have identified mechanisms that should lead to finer and more homogeneous microstructures: shortened dealloying times, leading to smaller average ligament sizes, and lower operating temperatures with either a eutectic alloy dealloying medium or through solid-state dealloying. With laser-based additive manufacturing, we hypothesize that we can produce metal–metal nanocomposites of nearly any imaginable shape, regardless of complexity, and keep the average length scale of the microstructure uniformly below 100 nm. This would be a huge innovation for materials synthesis via liquid metal dealloying and solid-state dealloying.2O2 and elimination of MO in Wang et al.’s experimental investigation into using nanoporous copper powder as a reagent in wastewater treatment [The materials synthesis approach discussed here can also be used to produce porous powders, which have broad applications in commercial technologies. Nanoporous copper powders demonstrated remarkable catalytic degradation of Hreatment . Dealloyreatment . Figure reatment . By replPorous powders can also be backfilled, which presents a unique opportunity to make powders with two interpenetrating bicontinuous phases. Vacuum impregnation is a simple technique to fill the interconnected void space with another material to increase functionality or improve mechanical strength. Bulk liquid metal dealloyed porous refractory metals have been shown to be mechanically robust enough to resist coarsening at temperatures up to 1900 °C . Their hPorous powders may also serve as feedstock material for AM, enabling them to be “re-formed” into a bulk structure with nanoporosity. This solves a problem with AM, namely, that after dealloying, the porous particles are generally disconnected; see With the recent development of novel dealloying techniques and improvements in additive manufacturing processes, an extraordinary opportunity to design new materials has arrived. A variety of dealloying techniques now allows materials scientists to make nanoporous materials and nanocomposites of nearly any metal on the periodic table and to tailor their shape and microstructure, while the additive manufacturing community has made great strides in printing metal samples that are comparable in mechanical strength and stiffness to their conventionally machined counterparts. Hierarchical metal nanostructures represent one area of interest that can take advantage of progress recently made in both fields. Electrochemical dealloying has already been used to make hierarchical nanoporous materials of additively manufactured bulk components, but liquid metal dealloying or vapor phase dealloying could also be useful techniques for this application. More excitingly, AM plus dealloying promises a one-step synthesis of metal–metal nanocomposites with excellent mechanical properties in any form; the microstructure is formed via liquid metal dealloying or solid-state dealloying, while the overall structure can be nearly any geometry imaginable. These possibilities for new materials come with new processing challenges, and it is clear that further work is necessary to maximize the engineering potential offered by dealloying and additive manufacturing.


Metabolic reprogramming between resistance and tolerance occurs within the immune system in response to sepsis. While metabolic tissues such as the liver are subjected to damage during sepsis, how their metabolic and energy reprogramming ensures survival is unclear. Employing comprehensive metabolomic, lipidomic, and transcriptional profiling in a mouse model of sepsis, we show that hepatocyte lipid metabolism, mitochondrial tricarboxylic acid (TCA) energetics, and redox balance are significantly reprogrammed after cecal ligation and puncture (CLP). We identify increases in TCA cycle metabolites citrate, cis-aconitate, and itaconate with reduced fumarate and triglyceride accumulation in septic hepatocytes. Transcriptomic analysis of liver tissue supports and extends the hepatocyte findings. Strikingly, the administration of the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate reverses dysregulated hepatocyte metabolism and mitochondrial dysfunction. In summary, our data indicate that sepsis promotes hepatic metabolic dysfunction and that targeting the mitochondrial PDC/PDK energy homeostat rebalances transcriptional and metabolic manifestations of sepsis within the liver. Sepsis is a potentially life-threatening condition that occurs due to the body’s overwhelming response to an infection. This reaction leads to aberrant immune responses, and if not diagnosed and treated early after its onset, it may limit survival by inducing coagulopathy, altered microvasculature, and dysregulation of the host’s metabolism and organ function . Sepsis The response to sepsis has classically been characterized as a biphasic phenomenon, where the acute phase is characterized by high energy consumption and hyper-inflammation associated with increased oxidation, followed by cellular reprogramming to a low-energy, anti-inflammatory state of immunometabolic paralysis, with accompanying organ failure . AdditioOf particular interest, during the hyper-inflammatory anabolic phase of sepsis, an increase in the expression and activity of pyruvate dehydrogenase kinase 1 (PDK1) consistently occurs . This enThe liver is an important metabolic and immune organ due to its role in nutrient metabolism and production of acute phase proteins. However, our understanding of transcriptional alterations and subsequent metabolic phenotypes elicited by sepsis still remain limited . FurtherTo test the long-term hepatic transcriptional changes elicited by sepsis, we performed RNA-seq in whole livers 30 hr post-cecal ligation puncture (CLP). At this time point, septic mice exhibit tolerance, in which immunometabolic paralysis and end organ dysfunctions decrease survival. Ingenuity pathway analysis (IPA) of the top physiological pathways subject to alteration in response to sepsis revealed a significant increase in the acute phase response pathway, highlighting a persistent inflammatory state in the liver into the chronic phase of sepsis . Of partACOD1 and elevates itaconate in isolate hepatocyte preparations . In line with altered transcriptional regulation of the TCA cycle, we found that sepsis altered the relative abundance of multiple metabolites involved in the TCA cycle . In partarations .Perturbations in OXPHOS have been shown to promote the production of reactive oxygen species (ROS) . FurtherThus far, our data has revealed that sepsis induces significant impairments in hepatocyte OXPHOS, alterations in the TCA cycle, and induction of oxidative stress, findings that are also metabolic hallmarks of fatty liver disease . BecauseSince transcriptional regulation of hepatic lipid metabolism is significantly altered during sepsis, we further investigated the functional manifestation of the observed gene expression profile. Consistent with our transcriptional profiling studies, our unbiased metabolomic screening in hepatocytes also identified lipid metabolism as the top metabolic pathway altered in sepsis . We obseGiven the severe dysregulation in hepatic lipid metabolism triggered by sepsis, we next sought to characterize the global consequences on hepatic lipid profiles. This was achieved by targeted lipidomic screening. Parallel to our unbiased metabolomic screening, we find sepsis leads to profound accumulation of almost all lipid species surveyed . We nextWe have previously reported that PDK inhibition by the pyruvate analog and pan-PDK inhibitor DCA promotes PDC-dependent immunometabolic adaptations to sepsis and increases survival . Given tGiven our previous results indicating the transcriptional changes associated with sepsis in the liver underlie functional alterations in metabolism, we next sought to determine whether the DCA reversal of transcriptional changes leads to reprogramming of metabolism to a pre-sepsis state. Our RNA-seq data showed a positive enrichment score for production of ROS along with negative enrichment for glutathione conjugation. Strikingly, the depletion of redox components cystathionine, cysteine, glycine, hypotaurine, taurine, and glutathione elicited by sepsis was completely reversed by administration of DCA . More imThe failure of over 50 trials of anti-inflammatory and anti-cytokine agents for treatment of severe sepsis has led to a reevaluation of the concept of sepsis as causing death by unbridedled inflammation . InsteadRemodeling of TCA cycle flux is well appreciated to support immune cell effector function in the face of inflammatory challenges . For exaIn response to lipopolysaccharide (LPS) stimulation, macrophages significantly upregulate IRG1 and accumulate itaconate from the decarboxylation of cis-aconitate . One of Similar to immune cells, liver cells increase ROS production during sepsis . We offeSepsis profoundly alters lipid metabolism, resulting in significant increases in plasma fatty acid and glycerol concentrations, changes reported to predict prognosis in septic patients . FurtherIn addition to accumulation of free fatty acids and triglycerides in the liver, our lipidomic analysis revealed significant increases in PLs. In the present study, we find hepatic levels of the PLs phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin to be heightened during sepsis, indicating abnormal metabolism of PLs. PC and PE are the most abundant PLs that make up cellular membranes and an abnormally high ratio of PC and PE has been reported to influence energy metabolism and linked to fatty liver disease and impairment in liver regeneration after injury . AlteredPDC is a master metabolic regulator controlling the conversion of pyruvate to acetyl-CoA in the mitochondria  and its We reported in a sepsis monocyte model that DCA reduced TCA cycle catabolic effects concurrent with increasing amino acid anaplerotic catabolism of branched-chain amino acids, leading to increased TCA-driven anabolic energetics . In the Notably, our interpretation of results is based on a single 30 hr time point, which was selected based on our previous publication showing reversal of disease tolerance mechanisms and increased survival following DCA intervention at 24 hr . Indeed,In terms of molecular mechanisms underlying the hepatic metabolic manifestations of sepsis, several potential candidates exists. Firstly, the mammallian target of rapamycin (mTOR) could be contributing to sepsis-induced metabolic disturbances in the liver. The foundation for this hypothesis is due to mTOR being demonstrated to be an essential component of the inflammatory response as well as underlying the glycolytic switch observed during acute sepsis . The metWhile we did not directly test here the physiological/pathophysiological contribution of individual metabolic pathways during the course of sepsis, we hypothesize that the overwhelming hepatic metabolic response during prolonged sepsis is pathogenic in nature. Our interpretation is based on our DCA results. We have previously demonstrated that DCA treatment reverses immunoparalysis and promotes host survival . Our resAlternatively, it is possible that some of the metabolic changes associated with sepsis in the liver confer host protection and tissue resolution. For example, we observed a significant decrease in hepatic fumarate levels 30 hr post-sepsis and an even further reduction in the DCA-treated septic mice. We speculate that the decrease in endogenous hepatic fumarate may lend itself to organ protection and sepsis resolution. Fumarate derivatives however have been shown to possess anti-inflammatory, antioxidant, and pro-survival properties in the liver during sepsis . WhetherIn particular, further investigation specifically into the PDK pathway to determine which isoform underlies the hepatic manifestions of sepsis is needed. This is warranted given multiple isoforms of PDK expressed in the liver  and DCA Here, we have confirmed and extended our previous findings that stimulation of mitochondrial OXPHOS metabolism by DCA in murine sepsis may be therapeutic in ameliorating the sepsis-induced immunometabolic paralysis of liver and potentially other vital organs. While encouraging, we recognize the limitations of extrapolating such positive findings in murine models to septic patients. Nevertheless, intravenous DCA has been used safely in critically ill patients , supportMale C57BL/6J mice aged 8–10 weeks were purchased from The Jackson Laboratory . All animals were subjected to a 12:12 hr dark/light cycle with ad libitum access to standard rodent chow and water. CLP model was used to induce sepsis as previously described . Mice weHepatocytes were isolated via portal vein perfusion and collagenase digestion as previously described . FollowiRNA was isolated from whole liver tissues using Trizol and the RNeasy RNA isolation kit  according to manufacturer’s protocol. One microgram of high-quality RNA (RIN > 8) was used as a template for library generation using the Illumina TruSeq RNA Sample Prep Kit v2  according to the manufacturer’s protocol. Generated libraries were then poly(A) enriched for mRNA prior to sequencing. Indexed samples were sequenced at 100 bp paired-end protocol with the NovaSeq 6000 (Illumina), generating approximately 20–30 million reads per sample. Sequenced reads were aligned to the University of California Santa Cruz (UCSC) mm10 reference genome using STAR v2.5 as previously described . The mapHepatocytes were isolated described above for metabolomic screening via ultrahigh-performance liquid chromotography–tandem mass spectroscopy (UPLC–MS/MS) . Briefly, 150–200 µl cell pellets per animal were used as starting material. Samples were prepared using the automated MicroLab STAR system . Proteins were precipitated using methanol under vigorous shaking for 2 min followed by centrifugation. Prior to analysis organic solvents were removed with TurboVap  and overnight storage under nitrogen. Dried samples were reconstituted with solvents compatible with the three following analytical methods: (1) reverse phase (RP)/UPLC–MS/MS methods with positive-ion mode electrospray ionization (ESI), (2) RP/UPLC–MS/MS with negative-ion mode ESI, and (3) HILIC/UPLC–MS/MS with negative-ion mode ESI. Resulting samples were analyzed with the ACQUITY UPLC  and a Q-Exactive high-resolution/accurate mass spectrometer  interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. An acidic positive-ion condition was used on an aliquot optimized to detect more hydrophilic compounds. Another acidic positive-ion condition was ran but chromotographically optimized for hydrophobic compounds. Basic negative-ion optimized conditions were also used on a separate C18 column. Resulting raw data was extracted and peaks identified using Metabolon’s hardware and software. Compounds were identified by comparing to known library entries of purified standards or recurrent unknown entities. A library of authenticated standards contain retention time/index (RI), mass to charge ratio (m/z), and chromotographic data  on all library compounds. Three criteria are used to identify chemicals: (1) RI within a narrow window of proposed identification, (2) accurate mass match to the library ±10 ppm, and (3) MS/MS forward and reverse scores between the experimental data and authentic standards. Peaks were quantified using area under the curve. For complex lipid panel, lipids were extracted with methanol:dichloromethane in the presence of internal standards. Extracts were concentrated under nitrogen and reconstituted in 250 ml of 10 mM ammonium acetate dichloromethane:methanol (50:50). Mass spectrometry analysis was performed in a Shimazdu LC with nano PEEK tubing and the Sciex SlexIon-5500 QTRAP . Both negative and positive mode electrospray were used. Individual lipid species were quantified by taking the peak area ratios of target compounds and their assigned internal standards then multiplying by the concentration of added internal standards. Lipid class concentrations were calculated from the sum of all molecular species within a class, and fatty acid compositions were determined by calculating the proportion of each class comprised by individual fatty acids. Mass spectrometry analysis was performed one time. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:eLife and also a valuable addition to the field of immunometabolism. It provides a valuable dataset detailing hepatic metabolic programs induced by well-established septic model. It also contributes to the growing interest in understanding the complex bi-directional biology of inflammation-induced metabolic changes on an organismal level and whether this understanding can lead to novel therapeutic strategies to diseases of pathologic systemic inflammation.All reviewers found this study interesting and also important. It is potentially of broad interest to the readers of Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jos van der Meer as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Michael Bauer (Reviewer #1); Andrew Wang (Reviewer #2).Thank you for submitting your article " reverses sepsis-induced hepatic metabolic dysfunction" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:Sepsis related organ dysfunction is life threatening condition, which is one of the leading causes of death in hospitalized patients. Despite major achievements in cardiovascular, respiratory and renal support the mortality especially related to septic shock remains high, around 50%. Liver is one of the vital organs but unfortunately at present we can only affect its function indirectly on the one hand, and on the other hand liver support devices are far behind as far as efficacy is concerned when compared to that of used in hemodynamics, mechanical ventilation or renal replacement therapy. Therefore, better understanding of the liver in sepsis could be a major step forward. For these reasons the authors aim to perform this experiment should be congratulated.Their aim was to test the long-term hepatic transcriptional changes, for which they designed an animal experiment using the CLP model compared to sham controls. In addition to performing RNA-seq in whole livers, they also investigated whether transcriptional changes manifest in altered hepatic TCA cycle metabolism.They found that in septic mice, 30 hours after CLP, hepatocyte lipid metabolism, mitochondrial TCA energetics and redox balance were significantly reprogramed.Based on the group's earlier findings that pyruvate dehydrogenase kinase (PDK) inhibition by the pyruvate analogue dyhydroacetate (DCA) promotes immunometabolic adaptations to sepsis and increased survival, they also tested the effects of DCA administration in the current experiment. They found similar results, namely that DCA reversed dysregulated hepatocyte metabolism and mitochondrial dysfunction.Essential revisions:We appreciate the high-quality work of the authors, and after careful evaluation our experts raised several concerns, comments and questions, such as:– The importance of metabolic changes and the signalling process within the observation period of 30 hours is missing from the full picture.– The overall impact of the current findings on the outcome is also unclear.– It is also unclear if the observed changes in liver metabolism are adaptive or maladaptive in sepsis.– The link between hepatic damage and mortality in the experiment is also difficult to prove – although the authors suggest this as primary cause of sepsis related death.– The shift towards the accumulation of free fatty acids in the experiment also requires further explanations.– There are no "magic bullets" in sepsis therapy and DCA is certainly difficult to believe being the one – every trial which tested therapies targeting one molecule or process in sepsis and showed promises in the animal laboratory, failed to show clinical benefit (in fact often caused harm).– Furthermore, the prerequisite of recovery from septic shock is the rapid rebalancing in the DO2/VO2 ratio to avoid or correct tissue hypoperfusion and oxygen debt – hence, pharmacological interventions cannot be separated from the global resuscitation process.– Therefore, the clinical relevance of the findings of the current experiment are questionable and renders the need of revision of the Discussion and the conclusions.For more details, please see the reviewers' comments.Reviewer #1:eLife due to the solidity, comprehensibility and coherency of the data. Metabolic changes in the course of sepsis are currently in the focus of the research field and drugs affecting these crucial metabolic changes during sepsis are promising candidates for successful treatment of this fatal syndrome. Before acceptance of the manuscript a revision along the comments attached is required.Mainali et al. present a comprehensive analysis of metabolic changes in the liver in Response to CLP in mice. The investigations include metabolomics, lipidomics and transcriptomics as well as mitochondrial and specific redox parameters. The study reveals a pronounced sepsis induced shift of hepatocyte metabolism and a salvage effect of the PDK inhibitor DCA on increased mortality of septic mice and concomitant metabolic traits. In spite of the restricted novelty of most of the specific findings the reviewer suggests to consider acceptance of the manuscript in The Discussion part of the manuscript is fixed on the interpretation of metabolic changes happening 30 h after CLP surgery. The authors do not touch metabolic changes until (and after) 30 h. Such considerations might be important for embedding the given data in the current concept on physiological and pathophysiological resistance and tolerance responses of immune cells and parenchyma in sepsis. Hence, to reduce its descriptive character the authors might considerably expand the Discussion.A similar restrictive interpretation is also observable for signaling processes controlling metabolism. How are mTOR and AMPK as key mediators of anabolic and catabolic processes involved in the control of the metabolic events observed after sepsis induction? Key references describing this issue like Cheng et al., 2016, are missing in the given manuscript.Reviewer #2:In this study, Mainali and Zabalawi et al. find that significant hepatic metabolic reprogramming occurs in the CLP model of sepsis. Using -omics approaches in ex vivo hepatocytes, they show CLP-induced reprogramming of the hepatic TCA cycle, lipid metabolism, and redox balance. Finally, in continuation of McCall et al., 2018, they show that PDK inhibition, which they previously showed improved CLP survival outcomes, reverses many of these CLP-induced hepatic changes. This is a very well-done study that supports the growing body of work that immunometabolic reprogramming is critical for organ preservation and survival during systemic inflammation.My major reservation in this study is that the authors do not demonstrate the relevance of the presented observations to the overall impact on sepsis physiology. That is, it is not surprising that hepatocytes, like most cells, undergo a lot of reprogramming during sepsis. Are these observed hepatic changes adaptive and promote organ preservation and survival, or are they maladaptive? It is also not clear that hepatic damage (or damage of "metabolic tissues") is the "primary cause of sepsis death". Along these lines, in Figure 5, the reference to the protective effects of PDK inhibition in CLP-driven mortality is difficult to interpret because, in the cited study, the authors clearly show that PDK inhibition given in the same regimen as the current study improves mortality primarily by reversing immunoparalysis and enhancing pathogen clearance. It remains unclear how hepatic reprogramming contributes to this mechanism.Thus, as presented, their findings do not support their main argument that hepatic reprogramming impacts survival in sepsis (Abstract) in either direction. In my opinion, it also does not add, from a pre-clinical perspective, to the potential utility of DCA in treating sepsis beyond their 2018 study.Reviewer #3:1) The data show a significant shift towards accumulation of free fatty acids and especially 16:0, 18:1 and 18:2 in addition to a significant increase in MUFAs and PUFAs in CLP vs. Sham. I had only one question concerning this result: Can the authors provide an explanation for this result shown in Figure 3D and Figure 4—figure supplement 2A? Are the enzymes, normally responsible for elongation and desaturation e.g. ELOVL and SCD respectively, altered under septic conditions?2) As useful information for the research community, it would be helpful if the authors could discuss the altered lipid metabolism in hepatic parenchymal cells  shown in the manuscript within the Discussion section. Essential revisions:We appreciate the high-quality work of the authors, and after careful evaluation our experts raised several concerns, comments and questions, such as:– The importance of metabolic changes and the signalling process within the observation period of 30 hours is missing from the full picture.This important point was raised by reviewer #1. Please see below for details on how we updated our Discussion to touch on acute changes during sepsis that were not covered by our experimental approach.– The overall impact of the current findings on the outcome is also unclear.This point was raised by reviewer #2. We have updated our Discussion to include our interpretation of our findings, particularly in regard to DCA, in context of sepsis treatment and pathogenesis.– It is also unclear if the observed changes in liver metabolism are adaptive or maladaptive in sepsis.This concern was raised by reviewer #2. Please see below for details on how we have updated our Discussion to discuss our interpretation in terms of whether the changes observed are adaptive or pathogenic.– The link between hepatic damage and mortality in the experiment is also difficult to prove – although the authors suggest this as primary cause of sepsis related death.We agree with this comment and have updated our Discussion to soften our conclusions regarding hepatic damage as causal of sepsis related death.– The shift towards the accumulation of free fatty acids in the experiment also requires further explanations.This point was raised by reviewer #3. We have updated our Results and Discussion to address this. Please see below for details.– There are no "magic bullets" in sepsis therapy and DCA is certainly difficult to believe being the one – every trial which tested therapies targeting one molecule or process in sepsis and showed promises in the animal laboratory, failed to show clinical benefit (in fact often caused harm).We agree with this comment that there is no magic bullet to sepsis therapy. However, we have added references in which DCA treatment has been used to treat lactic acidosis associated with sepsis. See below for details.Reviewer #1:Mainali et al. present a comprehensive analysis of metabolic changes in the liver in Response to CLP in mice. The investigations include metabolomics, lipidomics and transcriptomics as well as mitochondrial and specific redox parameters. The study reveals a pronounced sepsis induced shift of hepatocyte metabolism and a salvage effect of the PDK inhibitor DCA on increased mortality of septic mice and concomitant metabolic traits. In spite of the restricted novelty of most of the specific findings the reviewer suggests to consider acceptance of the manuscript in eLife due to the solidity, comprehensibility and coherency of the data. Metabolic changes in the course of sepsis are currently in the focus of the research field and drugs affecting these crucial metabolic changes during sepsis are promising candidates for successful treatment of this fatal syndrome. Before acceptance of the manuscript a revision along the comments attached is required.The Discussion part of the manuscript is fixed on the interpretation of metabolic changes happening 30 h after CLP surgery. The authors do not touch metabolic changes until (and after) 30 h. Such considerations might be important for embedding the given data in the current concept on physiological and pathophysiological resistance and tolerance responses of immune cells and parenchyma in sepsis. Hence, to reduce its descriptive character the authors might considerably expand the Discussion.We appreciate this insightful comment from the reviewer. We agree that by choosing one time point at 30 hours post-sepsis does not convey a complete picture in relation to metabolic reprogramming undertaken by the liver during sepsis. While we agree, we do believe that our comprehensive metabolomic, lipidomic and transcriptomic profiling is a first step to understanding how vital organs like the liver are altered in response to sepsis. In an effort to provide a more comprehensive interpretation of our results in the context of literature we have significantly updated our Discussion. In particular, we have included new discussion points relating to acute changes associated with metabolic reprogramming and the effects on pathogen resistance and tissue tolerance. We have also included additional references incorporating previous literature demonstrating transcriptional changes in metabolic pathways dysregulated in the liver during acute sepsis. Moreover, we updated our Discussion to reflect our itaconate findings in the context of tissue tolerance and immunoparalysis based on previous reports.A similar restrictive interpretation is also observable for signaling processes controlling metabolism. How are mTOR and AMPK as key mediators of anabolic and catabolic processes involved in the control of the metabolic events observed after sepsis induction? Key references describing this issue like Cheng et al., 2016, are missing in the given manuscript.We agree with the reviewer that our interpretation of our results in the context of the previous literature was limited. To address this, we have incorporated an additional Discussion point regarding the potential role of mTOR and AMPK signaling in the regulation of metabolism during sepsis. Furthermore, we have made sure to include seminal publications such as Cheng et al. in our Discussion, particularly regarding tissue tolerance and immunoparalysis.Reviewer #2:[…] My major reservation in this study is that the authors do not demonstrate the relevance of the presented observations to the overall impact on sepsis physiology. That is, it is not surprising that hepatocytes, like most cells, undergo a lot of reprogramming during sepsis. Are these observed hepatic changes adaptive and promote organ preservation and survival, or are they maladaptive?We agree with the reviewer that this is a primary limitation to our study. We believe however, that obtaining molecular insight into whether these changes are maladaptive or protective would require substantial experiments that would be outside the scope of the current manuscript. To address this however, we have updated our Discussion to hypothesize our interpretation of our results in regulating host survival or demise.It is also not clear that hepatic damage (or damage of "metabolic tissues") is the "primary cause of sepsis death". Along these lines, in Figure 5, the reference to the protective effects of PDK inhibition in CLP-driven mortality is difficult to interpret because, in the cited study, the authors clearly show that PDK inhibition given in the same regimen as the current study improves mortality primarily by reversing immunoparalysis and enhancing pathogen clearance. It remains unclear how hepatic reprogramming contributes to this mechanism.This is a very important point raised by the reviewer. We have subsequently softened our interpretation of clinical efficacy and therapeutic potential and have taken out statements indicating that hepatic damage is the primary cause of sepsis-related death. Furthermore, we have updated our Discussion to broaden our interpretation of our DCA effects in the context of our previous work. We pose the hypothesis that the effects of DCA in modulating immunoparalysis could in part be due to its effects on restoring hepatic metabolic function. This opens the possibility that hepatic metabolism could be a direct regulator of immunoparalysis in the context of sepsis and warrants future investigation.Thus, as presented, their findings do not support their main argument that hepatic reprogramming impacts survival in sepsis (Abstract) in either direction. In my opinion, it also does not add, from a pre-clinical perspective, to the potential utility of DCA in treating sepsis beyond their 2018 study.We agree that our main argument that hepatic reprogramming impacts sepsis survival is premature to make. To rectify this, we have removed that concept from our manuscript. We have expanded our DCA findings to indicate the potential utility of targeting the PDK axis may be mediated through hepatic reprogramming. Please refer to the Discussion section.Reviewer #3:1) The data show a significant shift towards accumulation of free fatty acids and especially 16:0, 18:1 and 18:2 in addition to a significant increase in MUFAs and PUFAs in CLP vs. Sham. I had only one question concerning this result: Can the authors provide an explanation for this result shown in Figure 3D and Figure 4—figure supplement 2A? Are the enzymes, normally responsible for elongation and desaturation e.g. ELOVL and SCD respectively, altered under septic conditions?We believe this to be an interesting hepatic phenomenon observed during sepsis. We have subsequently revisited our RNA-seq analysis of sham and CLP animals from 30h post-surgery and evaluated the expression of several enzymes involved in elongation and desaturation. We found repressed expression of all enzymes assayed and have offered our interpretation of this data within the Results. Furthermore, we have added additional data to Figure 3—figure supplement 1 panel B.2) As useful information for the research community, it would be helpful if the authors could discuss the altered lipid metabolism in hepatic parenchymal cells  shown in the manuscript within the Discussion section.We appreciate this comment from the reviewer. We have added Discussion points regarding hepatic lipid metabolism in the context of sepsis. Furthermore, we have added discussion surrounding the major lipid sensor PPARa and what is known about its role during sepsis and its potential contribution to our observed phenotype.


Division of Female Pelvic Medicine and Surgery, Department of Obstetrics and Gynecology, Emam Khomeini Hospital, Tehran University of medical sciences proposed a clinically relevant algorithm to guide appropriate decision making based on underlying risk stratification and resource utilization in order to resume elective surgeries, following COVID-19 pandemic crisis. The consequence of standardized decision-making factors and transparency of the principles will provide more assurance, consistency, and reliability on both sides, care providers and the patient. It also will decrease ethical dilemmas and moral criticism for surgeons. Eventually, this approach is applicable in any other disaster preparedness as a logical stratification of surgical indications for the female pelvic floor surgical procedures. The World has experienced an unprecedented health emergency from the COVID-19 pandemic -5.The rational for this advisory was the concern that elective procedures may lead to the spreading of the coronavirus within services and exhaust medical resources necessary to handle coronavirus epidemic . It is vital to classify various levels of necessary care, so surgical procedures are stratified into three categories based on its urgency state:• Category 1, the most urgent, which should be approached within one month.• Category 2 could be performed within three months.• Category 3 could be postponed up to one year.First category includes surgeries which if significantly delayed could cause major harm. Second and third categories are mainly elective procedures that could be delayed for extended time period. Elective procedures can practically be classified into “essential “, which implies the risk of increased adverse outcomes by postponing surgical care for an undetermined time period, contrasted with “non-essential “, which indicates purely elective surgeries that are not time-sensitive .To guarantee the safety and feasibility of elective surgical procedures during the COVID-19 pandemic crisis, each hospital and surgical field should thoughtfully review all scheduled elective procedures with minimized adverse event. Categorization should be done by taking into account the specific circumstances of the patient including the extent of the pain and quality of life and the impact on daily activities if the surgery is deferred .Pelvic floor disorders (PFD) include disorders of urinary and fecal incontinence, pelvic organ prolapses, and other lower urinary and bowel tract dysfunction . AccordiIt is important to take into consideration, many patients continue to have ongoing healthcare demands, but not essential which are currently being deferred due to the pandemic state. Health care providers including surgical services should be prepared to approach to the restrained surgical needs and prioritize cases in an appropriate time.Relatively low and stable incidence of COVID-19 for at least 14 days should be considered to allow facilities to provide care for patients needing non-emergent procedures , 13. It Division of female pelvic medicine and surgery, Department of Obstetrics and Gynecology, Emam Khomeini Hospital, Tehran University of medical sciences, which is a fellowship/residency training group, designed a set of principles and issues to help plan for resumption of elective surgical procedures. This is a committee consensus of Iranian Female Pelvic Medicine and surgery society and has been approved by health ministry. This protocol has been designed according to the female pelvic medicine and surgery fellowship training curriculum and procedures requiring multidisciplinary services such as vesicovaginal fistula or rectovaginal fistula are not included.Whenever effectiveness of non-surgical trials was not sufficient for PFDs, in order to estimate the necessity of surgical interventions, it is important to consider symptom severity from the patient’s perspective and also from clinician’s viewpoint, and evaluating the multifaceted impact of PFD on a women’s Quality of life , 14.Some elective non-essential surgeries will turn into essential at some points in time, depending on how long this pandemic will prevail and considering the impact of PFD on a women’s Quality of life. In this regard, it is recommended to evaluate appropriate indications on a case-by-case basis. Patients may have numerous questions and concerns regarding the risks threatening their safety. Conclusive patient information and clear communication will be paramount . ConsultIn order to reopening of elective surgery, it is required to consider all aspects of care including adequate facilities for pre, intra and postoperative care, access to various blood products, estimated postoperative hospital stay length, need for postoperative ICU admission and whether personal protective equipment (PPE) and COVID-19 tests are available . ProvideIn consequence, standardized decision-making factors and transparency of the principles will provide more assurance, consistency and reliability on both sides, care providers and the patient. It also will decrease ethical dilemmas and moral criticism for surgeons. Eventually, this approach is applicable in any other disaster preparedness as a logical stratification of surgical indications for the female pelvic floor surgical procedures.