A bidirectional in vitro brain–computer interface (BCI) directly connects isolated brain cells with the surrounding environment, reads neural signals and inputs modulatory instructions. As a noninvasive BCI, it has clear advantages in understanding and exploiting advanced brain function due to the simplified structure and high controllability of ex vivo neural networks. However, the core of ex vivo BCIs, microelectrode arrays (MEAs), urgently need improvements in the strength of signal detection, precision of neural modulation and biocompatibility. Notably, nanomaterial-based MEAs cater to all the requirements by converging the multilevel neural signals and simultaneously applying stimuli at an excellent spatiotemporal resolution, as well as supporting long-term cultivation of neurons. This is enabled by the advantageous electrochemical characteristics of nanomaterials, such as their active atomic reactivity and outstanding charge conduction efficiency, improving the performance of MEAs. Here, we review the fabrication of nanomaterial-based MEAs applied to bidirectional in vitro BCIs from an interdisciplinary perspective. We also consider the decoding and coding of neural activity through the interface and highlight the various usages of MEAs coupled with the dissociated neural cultures to benefit future developments of BCIs.  A brain–computer interface (BCI) builds a direct bridge between the brain and the surroundings to understand, recover and boost neurologic functions, which is also promising for utilizing human intelligence, essentially a subtle computing power2. BCIs have been in the limelight for years, and the emerging “metaverse”, where everyone is immersed in virtual reality, is another primary usage scenario of the technology5. In particular, an in vitro bidirectional BCI, which can both monitor and modulate a brain on a chip, is distinguished by its variety of potential applications8. Such devices help overcome the obstacles of in vivo neuroprostheses by decreasing complications and resulting in the mutual benefit of both technologies9. Additionally, the ex vivo BCI also reveals important biological mechanisms and widens the applications of micro biosensors, contributing to biological computers and neuromorphological computing10. This review aims to facilitate the fabrication of in vitro bidirectional BCIs and popularize the technology by explaining the underlying mechanism of bidirectional communication.Based on the actuations of billions of neurons, the brain ensures individual survival in the rapidly changing environment and is responsible for behavior, cognition and emotionμm, roughly corresponding to the scale of neurites or neuron somas. Thus, the changes in the ion concentration of the microenvironment arising from individual neuronal activity can be registered and sorted as spikes or local field potentials (LFP)11. MEAs achieve multisite readouts of neuronal ensembles noninvasively and at cellular resolution, which is low-cost and label-free. And Thomas et al.12 demonstrated the first in vitro test using the device on brain tissue in 1972 even before Vidal et al.13 proposed the innovative BCI in 1973. In summary, MEAs are superior for in vitro brain research.A microelectrode array (MEA), a tool for detecting cellular electrical activities, is the key to the ex vivo biosensing system. It is composed of ten to several thousand electrodes arranged on a rigid or flexible base, forming a customized pattern, which is also known as multielectrode array. The diameters of the published microelectrodes range from 5 to 50 14. Bioelectrical interfaces with nanostructures generally possess improved mechanical compliance and biocompatibility15. Additionally, the nanocoating on electrodes increases the electrochemical reaction area and therefore improves the probing and stimulating performances16.Methods for improving MEA performance abound, a ubiquitous and effective one of which is the introduction of nanomaterialsThis review is generally divided into four sections, covering device manufacturing, data analysis, application prospects and development direction of the in vitro bidirectional BCI Fig. . To appeThis article aims to provide (1) a general overview of the design for high-performance MEAs facilitating brain–computer interaction; (2) a step-by-step guide to analyzing extracellular neural signals; (3) examples of the stimulation paradigm for modulating brain activity in vitro; and (4) case studies illustrating applications of ex vivo bidirectional BCI.The past decades have witnessed a surge in the applications of BCIs encompassing recovering and enhancing brain functions. Although the current use of BCIs is limited to patients, the ultimate goal is to also have healthy people take advantage of the benefits of BCIs. Sorting vast types of BCIs into different categories is necessary , has been explored as a therapeutic avenue in artificially generating sensory input and restoring motor function, already utilized by technologies such as deep brain stimulation, cochlear implants, and visual simulation18.Current BCIs embrace neuromodulation to achieve bidirectional communication, not only focusing on data acquisition as in the past. The two-way interactions between the brain and computer have provided insight into brain-to-brain connections, maximizing the efficiency of information exchange. At the same time, novel bidirectional BCIs integrate real-time feedback to modulate physiological and pathological neural activities, which is urgently needed to assist precise and personalized treatments in neurodegenerative diseases such as Parkinson’s and epilepsy19. Therefore, simplified in vitro models have become great assets for clinical applications to identify the functions and mechanisms of various electrical stimuli, avoiding vain efforts based on “trial and error” and taking safety and efficacy into consideration20.In addition, noninvasive in vitro BCIs using cultured brain cells not only eliminates the need to penetrate the brain but also provides authenticity, accuracy and a high signal-to-noise ratio. Important factors are the reproducibility and controllability of the in vitro neural cultureCombining these advantages, in vitro bidirectional BCIs are worthy of attention, particularly the popular MEA-based BCI. At the forefront of bioelectronics, MEA-related neuroscience studies are committed to introducing novel materials, constructing elaborate structures and integrating new technologies for better interplay between neurons and electronics. And the utilization of nanomaterials improves the overall properties of the MEA and is highlighted here.In vitro neuronal culture comes from brain slices or organoids derived from dissociated neurons or stem cells Fig. . Ex vivo22, which is why MEAs are widely applied to detect electrical information from ex vivo brain tissues. Using MEAs, the electrical signals generated following routine neuronal activities are easy to read and quantify without fluorescent dyes or transgenes23. Different cultures have distinctive properties: acute slices retain sophisticated structures of natural neural networks, while the cell types and the tissue density of slices might cause the tissue section to undergo functional degradation or early death; and dissociated neural cultures generally can be preserved much longer than slices, and the density and complexity of the formed neural network can be easily controlled by the implanted cell types and ratio, culture density, incubation time and cultivation conditions24, yet the formation of mature networks takes time. Regardless of the cell or tissue source, a stable and nontoxic MEA interface is required.First, the electrophysiological records of in vitro neural culture appear consistent with those in vivo27, due to natural evolutionary optimization28. Suitable technologies, such as MEAs, are needed to reveal and utilize the underlying mechanisms. MEAs, which can achieve multichannel parallel recording, are outstanding research tools to investigate individual neurons functioning in isolation and the information processing by multiple neurons interacting with each other through specific synaptic motifs29 , a phenomenon detailed by brain connectomics30.Neuronal circuits intrinsically hold outstanding characteristics for encoding temporal and spatial relationships, as well as performing functions in memory and learning32. So, adding countable functional connections can revolutionize inhomogeneous neuronal networks33. Applying ES to a neural network, imitating endogenous electrical signals, can alter the connection structure and thus the network34. Therefore, realizing the overall control of the network with ES is feasible, which is why it is common to use MEAs for the nondestructive and reversible neuromodulation via ES36.In addition, there is tangible evidence that neural networks inherently have the characteristics of small-world networks: almost every neuron is linked with each other through only a few connectionsConsidering the attributes of isolated neural networks, MEAs are preferable for their bidirectional communication compared with other available technologies. This review mainly focuses on monitoring and modulating neural networks on MEAs. Special attention is given to the analysis of multisite neuronal firing activities because valuable multichannel data are difficult to acquire by other recording methods.Here, the design principles and fabrication processes of the MEA are introduced, and emerging materials and structures used in fabrication are reviewed specifically. Then, nanomaterial-modified electrodes are classified into four categories; in vitro bidirectional BCIs can be improved by nanomaterial-based modification because it comprehensively enhances the performance of the electrodes. The involvement of nanomaterials in 3D-printed MEAs is briefly introduced.11.MEAs and the neurons adhered to them constitute the typical biohybrid model for interrogating and modulating neural pathways. The key component is the neural microelectrodes, which are capable of electrophysiological and electrochemical detection. Distinguished from other tissue-level recording methods , MEA records cell-scale charge transfer and actions at the same level, providing neuroscientists with an unprecedented perspective inside the spatiotemporal dynamics of the brain. Often, multisite spikes and LFPs are sampled from an MEA after amplification, filtering and sorting. Spikes or action potentials (APs), recognized as a detected voltage potential above a threshold, are the most typical and fundamental activity of neurons. LFPs originate from synchronized synaptic currents, which are equal to the average signals from clustered neurons37. That is, MEAs that possess many channels and high spatiotemporal resolution are preferred, especially when the MEAs can perform long-term continuous probing on live organisms with abiotic stability.There are three significant criteria for evaluating MEAs coupled with dissociated neurons: scale, resolution and biocompatibilityThe traditional structure of MEAs based on MEMS technology comprises (i) a base layer where ubiquitous conductive materials such as gold, titanium, and platinum can be attached and deposited, (ii) a patterned metal layer as electrodes for transduction from bioelectrical signals to machine-processable electrical signals , and (iii) an insulation layer protecting internal interconnection responsible for the size of the electrode. Devices Fig.  and corr38. To quantify the performance of the electrodes, typically, the impedance, charge storage capacity (CSC), charge injection capacity (CIC) or charge injection limits (CIL), water reduction potential and oxidation potential (water window) and stability should be determined41.Comprehensive and feasible characterizations of MEA are necessary, ensuring that the devices meet the demands for recording and stimulation16. High CSC, CIC or CIL are the determinants of effective neuromodulation since the regulation involves the transition from electron flow to ion flow at the electrode–tissue interface via Faradaic reactions and/or the charging and discharging behaviors on the electrochemical double layer around the microelectrodes. The CSC refers to the maximum charge the electrode-electrolyte interface can hold within the water window determined via cyclic voltammetry16. This parameter is closely related to the electrochemical surface area, taking the microstructure into consideration. Thus, an increased CSC indicates that the electrochemical reactions on the improved electrode can be more efficient. The CIC or CIL, defined as the maximum injected charge per unit area from the electrodes, is the limit where the delivered waveform polarizes the electrode without irreversible reactions measured by the biphasic current pulsing. For stimulation, the CIC or CIL is crucial to prevent damage to the electrical–biological interface by avoiding irreversible Faradaic reactions. Generally, the higher the CSC is, the higher the CIC or CIL.In particular, the impedance is the most important: when the densely arranged microelectrodes reach the equivalent size of the cell to achieve large-scale detection, the noise and signal attenuation depend on impedance. Because of the frequency-dependent nature of neural electrophysiological signals, commonly, the impedance magnitude is taken at 1 kHz (center frequency of spike activity) as a representative figure from Bode plots displaying impedance against frequency using electrochemical impedance spectroscopyFor cellular recording and stimulation, high-performance MEAs must meet a number of requirements at the same time. Basically, good electrical characterization of an MEA is necessary to realize probing with a high signal-to-noise ratio. Since the cells are grown directly on the electrodes and the distance between neurons and the device is related to signal strength, the morphology of the neuron–electrode interface also plays a decisive role. Meanwhile, high biocompatibility and weak biological toxicity are prerequisites for cell adhesion as well as neural signal readout. To obtain superior MEA characteristics, innovations in materials and structures have received special attention.42. For conducting layers, nonmetallic materials such as graphene have become intriguing electrode materials due to their excellent electrochemical properties, high flexibility and necessary biocompatibility43. Specially treated metal materials also have unexpected gains; for example, holey gold has extraordinary transparency44. The transparency makes it possible to observe morphological changes during measurements, and thus the system can facilitate simultaneous optical and electrical regulation. Other transparent materials, such as indium tin oxide (ITO), carbon nanotubes (CNTs)45, and silver nanowires (AgNWs)46, have attracted considerable attention. ITO is both transparent and highly biocompatible, and the other two materials are ideal candidates for building flexible and stretchable devices. More information about the modification of the conducting layer will be discussed later. However, the insulation layer to protect the conducting wires, which is critical for defining the electrode area and acquiring cell signals, remains the most neglected and awaits innovation47.Various materials have been recently introduced in the base layer, conducting layer and insulation layer. For the base layer, common rigid substrates are still popular, while flexible polyimide-based probes, involving parylene, polyimide, polydimethylsiloxane (PDMS) and hydrogels, are gaining recognition for their ability to noninvasively interrogate a 3D culture of neurons with bidirectional functionality25. A recent study found that neural assemblies on MEAs with poly-dl-ornithine coating resulted in better survival and improved electric characteristics compared to PEI coating48. Additionally, novel hydrogels enhance biocompatibility and thus are used as the interface between electrodes and cells7.Surface treatment is the last step before seeding cells. Usually, the MEA is plated with poly-d-lysine or poly ethylene imine (PEI). Then, laminin, found in the extracellular matrix, is placed directly over the electrodes to provide a focal substrate for cell adhesion, thus increasing the quality of the seal49. In addition, a facile height-controllable fabrication method for gold pillar-shaped electroplated microstructures was developed50. Vertical nanowire MEAs grown mainly by focused ion beam deposition or other common techniques also have high aspect ratios51. The most promising 3D vertical electrode is the mushroom-shaped microelectrode, whose unique morphology promotes engulfment by cells without triggering cell repair mechanisms52. Traditional planar electrodes only record neural activity from the outside of samples, while 3D electrodes can collect and mediate signals from the inside, offering alternatives to monitor activity with higher fidelity in developed neural tissues such as brain slices. For macrostructure, flexible MEMS technology allows planar MEAs to have customized shapes. Advances in materials sciences facilitate the stacking and bonding of 2D MEAs to build a discrete 3D MEA. An MEA specific to the shape of a hippocampus slice was fabricated :poly(styrene sulfonate) (PEDOT:PSS) is the most popular conducting polymer. Devices fabricated from PEDOT:PSS exhibit outstanding CSC and CIL59. The soft consistency, high porosity and high water content perfectly mimic living tissues, which makes hydrogels attractive for minimally invasive neural prostheses to reduce mechanical mismatch. The soft coating significantly alleviates the problems of hard devices, such as high impedance and strong foreign body reaction. Moreover, hydrogel coatings can wrap a variety of nanoparticles, forming nanocomposites59.It is also worth noting that conducting polymer hydrogels made of PEDOT, PEG, poly, and poly(acrylic acid), among others, have a three-dimensional network structure of polymer chains for retaining waterMetal nanomaterials with high dielectric constants have always been the first choice for electrode modification. Sputter deposition, laser roughening, and electrodeposition are valid fabrication methods. Compared to conventional metal conductors, the nanometer-sized features of most metals, such as platinum, iridium oxide or titanium nitride, lead to enhanced charge injection limits for neural stimulation. However, the lack of biocompatibility and corrosion resistance prevent the devices from being stably used for monitoring over time.57, and a Au hierarchical nanostructure was deposited to improve the electrochemical surface area in a more effective manner58. In particular, quasi-1D metal nanowires with substantial length and ductility facilitate a decrease in resistance and an increase in the complexity of the spatial structure62.The optimization of the nano metal materials is achieved through rare materials and novel structures. Precious metals are promising candidates for stimulating electrodes due to their high charge injection limit and corrosion resistance, and liquid metals and shape memory alloys may adapt to the complex and soft environment of organisms. For traditional metal nanoparticles such as platinum (Pt) and gold (Au), novel microstructures rejuvenate them: laser-roughened Pt and microporous Pt have been used to fabricate stable electrodes with low threshold potential and low impedance17. In particular, there is potential for electrochemical usage. Carbon-based electrodes have been accepted as the gold standard for fast scan cyclic voltammetry to detect dopamine63.Inorganic nonmetallic materials for neural electrode modification are mainly carbon-based materials, such as carbon nanotubes (CNTs), multiwall carbon nanotubes (MWCNTs), carbon nanofibers (CNFs), graphene, and porous graphene. They offer intriguing properties for neural recording, such as reduced impedance, reliable electrochemical stability, fast electron transfer kinetics and biocompatibility. These materials are also known to feature a wider electrochemical potential window and a high CIC, which is beneficial for cellular stimulation purposes44. Porous graphene reduces the stacking between graphene layers and gains the advantages of porous materials. CNTs are the most frequently used carbon materials for neural applications, and MWCNTs provide easier preparation, lower cost and increased porosity. CNFs are gaining attention because of their extraordinary strength, large surface area, biosafety and stability. Carbon atoms with different binding modes can form other microstructures, such as large grains in single-layer graphene (LG-SLG). LG-SLG via chemical vapor deposition was put forward and applied successfully to MEAs, where large synchronous neural activities were recorded in an earlier development stage64.Specifically, graphene substrates can enhance the viability of neurons and the length of neurites but with a high error rate and poor uniformity in handling and fabrication39. For a positive example, CNFs mixed with PEDOT generated a desired synergetic effect, resulting in a microelectrode interface with lower impedance55. The good analytical performances of PEDOT:CNF composites come from the effective electronic exchange and the available reactive surface, which help catalyze the electron transfer reaction. The deficiency of the nanocomposites is the performance degradation following delamination, largely due to free-standing nanoscale components. For stability, strong mechanical characteristics and high electrical performance, ‘dead’ volumes or surfaces and the inhomogeneous distributions of phases should be avoided65. Nonconducting elastomeric biomaterials and conducting hydrogels are promising candidates to serve as matrices for conducting fillers to produce composite coatings with favorable electrochemical characteristics and biocompatibility62.Nanocomposites are combinations of nanoparticles. Some associations can yield synergistic effects, while others cannotUnlike conventional manufacturing, 3D printing with nanomaterials exhibits excellent capabilities in fabricating exquisite mini stereostructures without clean rooms in a programmable manner, which further advances the design of MEAs.66. Additionally, an MEA utilizing conductive nanoporous carbon ink was manufactured, whose structural resolution was comparable to those fabricated in cleanroom. The nanoporous carbon microelectrodes exhibited high specific capacitance and recorded the spontaneous activity of HL-1 cells extracellularly67. Moreover, highly controlled directional printing of quasi-1D metal nanowires may promote the development of 3D MEAs68. Composites with superior properties cannot be ignored. The function and biocompatibility of 3D needle-like electrodes ; height: 33 ± 4 μm) using nanocomposite ink combining PEDOT:PSS and MWCNTs were verified through proof-of-principle biological experiments )nts Fig. 69.70. The joint application of multiple technologies to the design of MEA is also attractive. Multifunctional electrodes combined with optogenetic technology, microfluidic technology, flexible electronics and other emerging technologies can meet the high-level needs of comprehensive experiments involving multiple conditions and factors.The potential design, fabrication and characterization of nanomaterial-based MEAs for in vitro bidirectional BCIs are presented here. The renewal of MEAs depends on the material and structure, and a more affordable and more useful device is a consistent pursuit. There is a higher desire for transformable MEAs that can adapt to the morphological changes that come with the growth and development of brain tissue in vitro during long-term culture71 and thin film transistor (TFT) technology72, are hopefully to be improved by nanomaterials.Nanomaterial-modified MEAs using MEMS technology have widely served as in vitro bidirectional BCIs to collect neural activities and realize dynamic feedback control. Nanomaterial-based 3D printing of MEAs is a promising method. Other fabrication methods, such as complementary metal-oxide-semiconductor (CMOS) technologyNoticeably, the best tests for comparing electrodes are usually in 3-electrode cells with phosphate buffered or physiological saline. However, the electrolyte solution is too simple to compare to a multicomponent mixture in a natural biological environment. Therefore, the values measured are merely approximations of the microelectrode behavior wrapped in a neural network. Moreover, protocols indicating the variation in these parameters over time after implantation remain to be explored.73. However, to date, the interactions between the input electric pulse sequence and collected electric signals have been difficult to interpret.Classical experiments involving communication with neuronal cells date from the early 1790s, when Luigi Galvani first stimulated frog legsES depolarizes the membranes in close proximity as a controllable and reversible switching, which is clearly beneficial to work in the interactions with neurons. Simultaneous multisite recording of cellular neural signals, which is difficult to obtain without an MEA, is an important prerequisite for interpretation of neuronal behaviors, which is a more complex task than judging whether animals or humans behave as expected. Thus, the data processing of electrical activity captured by MEAs and the corresponding stimulation paradigms deserve careful study.74. Remarkably, the network dynamic patterns represent a specific operation mode, revealing memory traces, information flow and so on75. This section provides a step-by-step guide for those who want to decode the characteristic activity of in vitro neural cultures, including the analysis of the following: cellular activity, population activity, network dynamics and other phenomena.Decoding neural signals is fascinating but complicated. Ex vivo brain tissue from various species has the capability to process multichannel data and exhibit the universal fundamental property of plasticity. Inside in vitro neural networks, not only can individual neurons run as sophisticated information-processing units, but their connection patterns through synapses also enable the execution of computational tasks77. A spike is the action potential (AP) detected by the MEA when neurons fire, and a group of temporally overlapping spikes (a series of APs) firing at a frequency much higher than the mean firing rate (MFR) is termed a burst. Both activities are intrinsic properties of neural networks79.The basic electrophysiological features describing neuronal activity extracted from MEA recordings are spikes and bursts80. Periodicity, the repetition of spike patterns, can be evaluated using autocorrelation or Welch’s periodogram. A poststimulus time histogram of spikes can also be computed to investigate the effects of stimuli on neurons81. More electrophysiological features can be taken into consideration78. Burst analysis is similar.Analysis usually starts with the basic quantitative parameters of spikes. Normally, MFR is paramount due to the intrinsic variability of neural activities. The interspike interval is used to characterize the firing pattern and discern regular and irregular firing. For mature neurons, spontaneous neuronal firing occurs in a periodic fashion, patterning with intervals82 or the Fano factor (the ratio between spike-count variance and mean)80. Recently, multifactor cluster analysis was introduced in the classification of spikes83, and unsupervised machine learning methods have been applied to cluster spike patterns84 and burst patterns85.Further analysis includes the classification of signals, unveiling the inner heterogeneity for comprehensive understanding. For example, spikes from putative inhibitory and excitatory neurons are routinely classified based on the half-peak waveform width of the largest waveform and the polarity for each unit86. The average electrical activities of neurons around the electrodes are represented as LFPs or the overall spike characteristics. LFP is the dominant signal acquired by diversified electrophysiological recording techniques, accompanied by many mathematical analysis methods87. Therefore, attention is given to collective activities represented by multichannel spike trains, which are difficult to record without an MEA.Brain functions come from the cooperative activities of multiple neural clusters, which is why population activity is important. Additionally, decoding large-scale neuronal dynamics provides robustness and reliability for revealing normal physiological activities and abnormal pathological phenomena88. The hallmarks of neuronal spontaneous activity are intermittent SBs, separated by periods of silent activity, and the intervals between SBs are distributed approximately lognormally89. To assess changes in network bursts, the following parameters were analyzed: SB duration, interSB interval and SB percentage80. Interestingly, some neurons always fire ahead of the SB, as if they trigger the network activity, which are called ‘burst leaders’. SBs are believed to play an essential role in many neural processes, such as generating multifunctional patterns, learning, connecting sensory circuits, and encoding and maintaining memory, and are associated with various neurodegenerative diseases, including epilepsy, Parkinson’s disease, Alzheimer’s disease and schizophrenia74.To characterize the complex behavior of population-wide activities, spontaneous events and synchronization must be taken into consideration. When the majority of the neurons in the biological network fire simultaneously, the dramatic reverberating neural event is called a ‘network burst’, ‘spontaneous burst’, ‘super burst’ or ‘population burst’, abbreviated ‘SB’ in this article. The bursts of elevated population activity, correlated in space and time, are supposed to be tied to neuronal avalanches and synchronized oscillation90. The cross-correlation between neurons is used to measure the degree of synchronization80. The Pearson correlation coefficient is a widespread canonical measure used to verify spike train synchrony. A novel measure of correlation, the spike time tiling coefficient, is also valuable and independent of the object-specific firing rate. Notably, bursts, well-organized neuronal events, appear when large-scale neurons become synchronic80. These activities, similar to those observed in vivo, are related to the plasticity and dynamics of the neural network.Different from instantaneous synchronization, synchronous firing refers to when two or more neurons fire in a similar pattern, i.e., sequential synchronization80. The measure of modularity, representing the community structure employed in assemblies of functionally distinct clusters, is usually identified in graph-theoretic methods91. Neurons or active electrodes can be regarded as a subset of nodes, and the links correspond to the relation between the nodes  is calculated to exhibit network structure95. Similarly, compared with a relatively sparsely connected single neural network, when one neural network is interlaced with another, the synaptic latency representing the time required for signal transmission is direct evidence of connectivity53. To facilitate the study of network dynamics, handy software toolboxes have been developed to verify the functional network97.To reveal functional associations, conditional firing probabilities and cross-correlation coefficients provide usable descriptions of relationships between firing activities80. Based on graph theory, these electrophysiological activities can be mapped to an appropriate coordinate system as points, and the change across periods can be quantified using, for example, the Euclidean distance or Mahalanobis distance98. The matrix constructed by the relationship of multichannel data before, during, and after a disturbance can be compared using the similarity index99.After building functional networks, the dynamic changes of the network need to be determined to investigate whether functional connections appear, disappear or reappear. Comparing a quantified parameter characterizing population activity is feasible100. More attention should be given to the interconnections between events of multilayers. Machine learning algorithms are adopted in the statistical analysis of neural networks in vitro to integrate cellular electrophysiological features, synchronized population events, network connectivity and others to create a high-fidelity multidimensional profile78, which is a valuable guideline.Some factors are not independent from each other; for instance, a phase shift appears between the spike and LFP101. Therefore, ES with different settings applied to various types of cells may have diverse effects. Here, representative paradigms for ES and their functions are listed with key parameters , and the differentiation effect was assessed106. Instead, we focus on direct electrical coupling, which uses electrodes to inject charge through neural electrical interfaces. As indicated earlier, a lot has been achieved through ES. Nevertheless, developing new paradigms for modulation is meaningful, especially those that can be adaptively personalized based on the fluctuations in the characteristics of isolated neural networks107. Other breakthroughs lie in increasing the precision of stimulation by limiting the changing electrical field to a certain area108.Concerning the information input, we ignore indirect electrical coupling, such as implementing an electromagnetic field although parallel plates, field-effect transistors, induction coils or solenoids, generating ultrasound acting on ion channels and using optogenetic tools to manipulate neurons109. On MEAs, the in vitro neuronal network model converts complex or vague mechanical and chemical factors into readable electrical signals through interactions with the microenvironment, which is advantageous. Therefore, MEAs coupled with in vitro brain samples have routinely served as biosensors to evaluate the effects of, for example, chemical drugs110, biomaterials, and physical forces on brain cells111. Moreover, the information extracted through MEAs facilitates biological regulation, such as controlling the differentiation of NSCs80 and suppressing epileptiform activity in rat hippocampal slices112.The development of in vitro BCIs brings infinite possibilities in four fields in particular: (a) physiological research, (b) biosensing, (c) biological regulation and (d) neurocomputing Fig. . Basical113. A neuronal network is similar to an “organic computer” with intelligence. A series of ESs can encode customized information, and the computation results can be decoded from the detected electric signals. Bidirectional in vitro BCIs have already exerted excellent control capability in robot movements114 and demonstrated superior learning ability in playing games107. Hopefully, quick and effective encoding and decoding of neural activity can facilitate the connection of several brains from different sources in vitro to manage parallel operation with higher precision.Neurocomputation is the most striking usage because of the exquisite structure and dynamic adaptability of natural neural networks based on synaptic plasticityMEAs are designed with affordability and flexibility and they can be customized tools to modulate neural networks. In particular, the nanoscale roughness and porosity of nanomaterial modification improve cell adhesion, electrochemical reaction area and charge storage capacity, and tissue sealing between nanoparticles and cells yields a high signal-to-noise ratio. In summary, nanomaterial-based MEAs have outstanding strengths for bidirectional BCIs in vitro: (1) they possess inherent and instant bidirectional communication ability through electric signals with millisecond temporal resolution, as well as a gradually growing encoding dictionary and an increasingly standardized decoding path; (2) they acquire information directly from neuronal activity, not indications; (3) they provide a unique global perspective from individual cell to neuronal network; and (4) they offer continuously optimized devices due to the rapid development of related disciplines.In conclusion, this is a distinctive review centering on in vitro bidirectional BCIs with a broad overview of nanomaterial-based MEAs and neural signal–electric signal translation on MEAs. Interdisciplinary information is collected through bidirectional in vitro BCIs for the application of MEAs affiliated with living organisms in more situations. The unlimited possibilities of the devices are only partly displayed, aiming to inspire interest in the promising field.To advance the development of bidirectional in vitro BCIs, the problems mainly exist in exploring new and effective stimulation paradigms mimicking sensory input and decoding neural activities in a more comprehensive manner, such as combining electrophysiological signals and electrochemical signals with high-performance BCI Fig. . Neverth


In general, reproductive performance exhibits nonlinear changes with age. Specifically, reproductive performance increases early in life, reaches a peak, and then declines later in life. Reproductive ageing patterns can also differ among individuals if they are influenced by individual-specific strategies of resource allocation between early-life reproduction and maintenance. In addition, the social environment, such as the number of available mates, can influence individual-specific resource allocation strategies and consequently alter the extent of individual differences in reproductive ageing patterns. That is, females that interact with more partners are expected to vary their copulation frequency, adopt a more flexible reproductive strategy and exhibit greater individual differences in reproductive ageing patterns.Riptortus pedestris by ensuring that females experienced monogamous  or polyandrous conditions .In this study, we evaluated the effect of mating with multiple males on both group- and individual-level reproductive ageing patterns in females of the bean bug We found that group-level reproductive ageing patterns did not differ between monogamy-treatment and polyandry-treatment females. However, polyandry-treatment females exhibited among-individual variation in reproductive ageing patterns, while monogamy-treatment females did not.Our findings provide the first empirical evidence regarding the influence of the social environment on individual variation in reproductive ageing patterns. We further suggest that the number of potential mates influences group- and individual-level reproductive ageing patterns, depending on which sex controls mating. We encourage future studies to consider interactions between species-specific mating systems and the social environment when evaluating group- and individual-level reproductive ageing patterns. A senescent decline in reproductive performance has been documented across many animal taxa –3. In inIn particular, the number of available male mating partners can be a potential determinant of nonlinear reproductive ageing patterns in females. Reproductive ageing patterns are expected to differ between females allowed to mate with multiple males  and females that mate with only one male throughout their lifetime . Females can increase longevity and egg production rates by mating with multiple partners if they receive direct benefits from males during mating, such as nuptial gifts or nutritious ejaculates –19, and In addition, differences in nonlinear reproductive ageing patterns between monogamous and polyandrous females in insect species are expected to depend on which sex controls mating. In insect species where females are coerced into copulation and males control mating, females are more likely to concede into superfluous copulations as the number of males increases –24. ThesMoreover, female individuals are expected to vary in nonlinear reproductive ageing patterns, and the extent of individual differences in nonlinear reproductive ageing patterns within monogamous females may differ from that within polyandrous females; such differences are also expected to depend on which sex controls mating. Individuals are expected to differ in life-history strategies according to resource allocation to early-life reproduction or body maintenance –27, poteRiptortus pedestris) at the group and individual levels. The bean bug R. pedestris (Hemiptera: Alydidae), a major pest of leguminous crops in East Asia, is a multivoltine species that produces two to three generations per year ). Females were randomly assigned to one of three mating treatments .We used Mann–Whitney We first analysed the effect of mating treatment on group-level reproductive ageing patterns  using a univariate mixed-effects model, where weekly egg production was fitted as the response variable. In the model, we included female identity as a random effect and fitted mating treatment , linear and quadratic ages (weeks since eclosion as adults), the age (in weeks) at the last observed reproduction and the interaction between mating treatment and other covariates  as fixed effects. In the model, we fitted age (in weeks) as age-1 (in weeks) to ensure that the intercepts of our models represented the number of eggs produced during the first week after eclosion as an adult. The age at the last observed reproduction was used to control selective disappearance . In addi2 effect, Model 4) to assess among-individual variation in reproductive ageing patterns. In all the models above, residual variances were assumed to be age-specific  and not correlated across ages.We also constructed treatment-specific random intercept and slope models by adding random effects sequentially to assess among-individual variation in the linear/quadratic patterns of reproductive ageing in each treatment. In all the treatment-specific models below, we fitted weekly egg production as the response variable and included linear and quadratic ages (weeks since eclosion as adults) and the age (in weeks) at the last observed reproduction as fixed effects. We did not use within-individual mean-centring but rather chronological ages (in weeks) because within-individual mean-centring is sometimes problematic when calculating individual differences in ageing slopes , 45. We 2, df = 0) and P [20/1” in the results.All models were implemented in ASReml  and solved using the restricted maximum likelihood method. We assessed the significance of fixed effects using conditional Wald F tests. The significance of variance attributable to random effects was determined using likelihood-ratio tests (LRTs) and calculated as twice the difference in log likelihood between the full model and a reduced model where the random effect of interest was removed; the P value was calculated assuming an equal mixture of P(χ df = 1) , 47. ThiIn addition to comparing models that assessed individual differences in reproductive ageing patterns, we conducted an analysis of variance (ANOVA) to compare total lifetime egg production among the mating treatments. ANOVAs were performed using Statistica software .P = 0.22; total copulation duration: U = 117.5, Z=− 0.21, P = 0.83) , possibly due to the production of fertilised eggs. However, there was no difference in female body weight between the monogamy and polyandry treatments .At the group level, the weekly egg production of females showed negative quadratic changes with age in all mating treatments; however, the slopes differed by treatment  did not vary in their nonlinear reproductive ageing patterns. However, when the number of potential mates increased , females exhibited variation in their nonlinear reproductive ageing patterns. This result was in line with our prediction because females control copulation frequency and duration in R. pedestris. Thus, we suggest that species-specific mating systems  and mate availability  can shape individual variation in reproductive ageing patterns in insects.Our results show that the social environment, such as the number of potential mates, influences individual- as well as group-level patterns of reproductive ageing in females of the bean bug R. pedestris, females control mating and copulate only when males mount females and display courtship behaviours, including up and down movements of their antennae and forelegs [R. pedestris males cannot coerce females to mate. In our observation, when females are reluctant to mate, they actively kick males with their hind legs, preventing males from mounting them and displaying courtship signals. Additionally, we found that there were no differences in copulation frequency or duration between monogamous and polyandrous females and that males in the polyandry treatment experienced copulation frequencies and durations that were half those of males in the monogamy treatment. This result indicates that R. pedestris females that live with multiple potential mates refuse to engage in prolonged mating with a single male. Thus, as R. pedestris females control mating decisions, female copulation frequency is not a function of the number of potential mates, resulting in the lack of differences in group-level nonlinear reproductive ageing patterns between females in the monogamy and polyandry treatments.Mated females showed faster reproductive ageing than virgin females that laid unfertilised eggs, implying the presence of costs related to mating and the production of fertilised eggs. However, we did not find differences in lifetime egg production between monogamous and polyandrous females, indicating that their lifetime reproductive costs were similar. This idea was also supported by the lack of differences in group-level nonlinear reproductive ageing patterns between monogamous and polyandrous females. In forelegs . That isR. pedestris females prefer males that have longer hind legs and produce more rapid courtship tapping [R. pedestris females will vary in their copulation frequency and reproductive strategies when multiple mates are available, thus resulting in greater individual differences in reproductive ageing patterns. For example, in the polyandry treatment that provides the opportunity to mate with multiple available males, some females may increase copulation frequency with multiple males early in life  and experience rapid reproductive ageing, whereas other females may be choosy, maintaining moderate copulation frequency throughout their lifetime and experiencing slow reproductive ageing. In contrast, in the monogamy treatment where only a single male is available, females could not be choosy. Although there was an extra male in the monogamy treatment, he was unavailable for copulation. Thus, females were not in direct contact with him, were unable to precisely assess his quality and could not be choosy. We also found that female copulation frequency did not differ between the monogamy and polyandry treatments, suggesting that monogamy-treatment females were less choosy (or not choosy) than polyandry-treatment females. Therefore, all the monogamy-treatment females indiscriminately allowed male mating attempts and showed similar negative quadratic reproductive ageing patterns among individuals.Despite the lack of differences in group-level reproductive ageing patterns between treatments, polyandry-treatment females exhibited significant among-individual variation in their nonlinear reproductive ageing patterns, while monogamy-treatment females did not. One explanation for this difference is that the increase in the number of potential mates did not affect female copulation frequency at the group-level but increased the among-individual variation in female copulation frequency. That is, females might vary in reproductive strategies  in response to increased mate availability , 49; thi tapping , indicatTherefore, in insect species in which females control mating decisions, increased mate availability facilitates variation in female reproductive strategies, leading to an increase in the extent of individual differences in nonlinear reproductive ageing patterns. However, the opposite is expected in insect species in which males obtain copulations through coercion. In such species, more females are predicted to experience multiple copulations as the number of males increases, potentially decreasing individual differences in copulation frequency as well as in reproductive ageing patterns in females. Thus, future studies should determine how species-specific mating systems  interact with mate availability to influence the extent of individual differences in reproductive ageing patterns as well as group-level reproductive ageing patterns.G. campestris, male calling efforts declined with age in groups with a balanced sex ratio but did not decline in groups with a female-biased sex ratio [Pieris napi), males cannot coerce females to mate; thus, females exhibit among-individual variation in mating frequency even with a male-biased sex ratio [Our results also suggest that social environment factors, such as the sex ratio, affect not only group-level female reproductive ageing patterns but also the extent of among-individual variation in female reproductive ageing patterns in insects. As the sex ratio is known to affect the cost of early-life reproduction as well as age-specific mortality , it may ex ratio . Moreoveex ratio , 49 and ex ratio . This vaGasterosteus aculeatus), the throat colour of males  changes with age; no significant genetic variation in colouration-indicated ageing was found in males who experienced warm winters  in their juvenile stage, whereas males who experienced temperature regimes equivalent to those in the wild over the winter  exhibited genetic variation in colouration-indicated ageing [P. napi) that we mentioned above, individual differences in female mating frequency were suggested to stem from unpredictable weather conditions [In addition to the social environment, (early or current) environmental quality is expected to influence the extent of variation in individual- and group-level patterns of reproductive ageing in insects. Environmental effects on individual or genetic variation in ageing have rarely been studied in vertebrates (but see ), and, td ageing . In the nditions . These fnditions ; thus, tR. pedestris females did not change but that females exhibited individual differences in their patterns of reproductive ageing with increases in the number of potential mates. Previous studies have assessed the relationship between ageing and the social environment at the group level . Here, iewed in , we furtiewed in , 61. Our


Anesthetic management is a great challenge during the surgical resection of pheochromocytomas and paragangliomas (PPGLs) due to potential hemodynamic fluctuations and/or postoperative complications. Although combined epidural-general anesthesia is commonly used in PPGLs surgeries, there is still no consensus whether combined epidural-general anesthesia was superior than general anesthesia alone for these populations. For the first time, we conducted this systematic review and meta-analysis to summarize the effects of combined epidural-general anesthesia versus general anesthesia alone on hemodynamic fluctuations as well as postoperative complications in patients undergone PPGLs surgeries.This systematic review and meta-analysis was performed according to the preferred reporting items for systematic reviews and meta-analyses statement. The primary outcome were hemodynamic fluctuations, including intraoperative hypotension, postoperative hypotension, and hypertensive crisis. Secondary outcome was the incidence of postoperative complications during hospital stay.Finally, three retrospective cohort studies involving 347 patients met the inclusion criteria. A meta-analysis was not performed since outcomes from included studies were not available to be pooled. On the basis of the findings of non-randomized controlled trials (RCTs) literature, 2 studies suggested that combined epidural-general anesthesia was associated with intraoperative and postoperative hypotension, although one study reported that epidural anesthesia use reduced the incidence of postoperative complications in patients undergone surgical resection of PPGLs.Currently, no published RCTs have yet assessed clinically relevant outcomes with respect to the application of epidural anesthesia during PPGLs surgeries. Well-designed RCTs should nonetheless be encouraged to properly assess the efficacy and safety of epidural anesthesia for PPGLs surgeries. The diagnosis of PPGLs requires both evidence of excessive release of catecholamines and the anatomical localization of catecholamines-secreting tumor. The tumor-node-metastasis (TNM) system for PPGLs, based on the extent of the primary tumor (T stage), the extent of lymph node metastases (N stage), and the presence of distant metastases (M stage), has been introduced in the 8th Edition of the AJCC Cancer Staging Manual. Surgical resection, including open and laparoscopic approach, is the mainstay for treatment of PPGLs. Although laparoscopic resection is superior in terms of many aspects, open resection is more recommended for large or locally invasive pheochromocytomas, or multifocal tumors including metastases. Previous study indicated that surgical resection of the primary tumor could improve overall survival in patients with metastatic PPGLs. In addition to surgical resection, chemotherapy and radiopharmaceutical treatments also showed potential benefits for patients with PPGLs, such as the combined chemotherapy protocol of cyclophosphamide, vincristine, and dacarbazine, and radiopharmaceutical treatments using 131I-MIBG and 131I-Ultratrace Iobenguane.Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors deriving from the chromaffin cells of the adrenal medulla and extra-adrenal neural crest tissues.,11 Generally, general anesthesia is the standard anesthetic technique for the surgical resection of PPGLs.,13 In recent years, epidural anesthesia gains superiority when used as a supplementary technique of general anesthesia in many aspects, including postoperative analgesia, reduction of opioids consumption, less cardiovascular and respiratory complications.,15 Therefore, the combined epidural-general anesthesia has been frequently used in PPGLs surgeries.–18 Increasing numbers of studies reported that epidural anesthesia may prevent the fluctuations in hormone levels and facilitate hemodynamic stability for patients undergoing PPGLs surgeries.–23 However, epidural anesthesia was also implicated in intraoperative and/or postoperative hypotension due to decreased peripheral vascular tone,,24 which might be worsen for PPGLs surgeries because of alpha-adrenoceptor blockade and abrupt reduction of circulating catecholamine levels.,25 Epidural anesthesia was even reported to exacerbate intraoperative hypotension but not prevent hypertensive crisis during PPGLs surgery, which may be result from the fact that epidural block was not effective to suppress the catecholamine surge during tumor manipulation but exaggerate the sympathectomy and vasodilatory effects after tumor ligation. Several studies have compared the effects of combined epidural-general anesthesia with general anesthesia alone on hemodynamic instability and postoperative complications in patients undergone PPGLs surgeries,–18 but the results remain controversial. It is thus worthwhile to conduct a systematic review and meta-analysis to summarize the efficacy and safety of epidural anesthesia in the surgical resection of PPGLs.During PPGLs surgeries, anesthetic management is a great challenge due to dramatic hemodynamic fluctuations, such as hypotension, hypertensive crisis, and high occurrence risk of postoperative complications.No ethical approval is required because this is a literature-based study. This systematic review and meta-analysis was conducted in agreement with the preferred reporting items for systematic reviews and meta-analyses guideline.PubMed, EMBASE, Cochrane Central Register of Controlled Trials (CENTRAL), and Web of science were systematically searched from the inception to June 3, 2021 with the following terms: “pheochromocytoma or paraganglioma,” “general anesthesia or general anesthesia” and “epidural.” The language was restricted to English. Additional studies were obtained by reviewing the references of relevant articles.Inclusion criteria: Study design: Randomized controlled trials (RCTs) or retrospective cohort studies; Population: adult patients undergoing surgical resection of PPGLs; Intervention: combined epidural-general anesthesia; Control: general anesthesia alone; and Primary outcomes: hemodynamic fluctuations, including intraoperative hypotension, postoperative hypotension, and hypertensive crisis; Secondary outcome: the incidence of postoperative complications during hospital stay. Reviews, conference abstracts, comments, and case reports were all excluded.Two authors independently screened the titles and abstracts of all the studies obtained from the search for the potentially relative studies. Then, the full-text of potentially eligible studies were retrieved and reviewed for study inclusion. Any disagreements were discussed with the third author.Two authors independently extracted the following data from included studies: authors, publication year, study design, sample number, participants’ age, types of surgery, anesthetics of epidural anesthesia, intraoperative vasoactive medications, and primary outcomes. We contacted corresponding authors of included studies for missing or unreported data. Discrepancies were resolved by discussion with the third author. The assessment contains the following items: confounding, selection of participants, departure from intended interventions, missing data, measurement of outcomes, and selective reporting. The estimated risk of bias for each item was rated as “low,” “moderate,” “serious,” or “critical.” Any disagreements were discussed with the third author.Two authors independently assessed the risk of bias for each included study by the Robins-I tool.–18 involving 347 patients were included  baseline after ligation of the adrenal vein. Hypertensive crisis was defined as a SBP > 200 mm Hg or an increase > 30% of SBP baseline during the operation.The characteristics of included studies were shown in Table All included studies were rated as having a moderate risk of bias with the ROBINS-I tool. The details of risk of bias for each included study was present in Figure  reported the incidence of postoperative complications in patients undergone open surgery of pheochromocytoma. They found that the incidence of postoperative complications was lower in patients with combined epidural-general anesthesia than in those with general anesthesia alone . Furthermore, the severity of postoperative complications was lower in patients with combined epidural-general anesthesia compared with general anesthesia alone (P = .017). Additionally, combined epidural-general anesthesia was identified as one independent factor  that associated with lower occurrence of postoperative complications using multivariate Logistic regression.Data in these included studies were not available to be combined for meta-analysis as each predefined outcome was reported in only 1 study. Li et al compared the effects of combined epidural-general anesthesia with general anesthesia alone on the incidence of postoperative hypotension in patients undergone open resection of PPGLs. They showed that patients with epidural anesthesia had a higher incidence of postoperative hypotensive compared to those without epidural anesthesia . Moreover, epidural anesthesia was demonstrated as an independent factor associated with postoperative hypotension .Wiseman et al compared the effects of combined epidural-general anesthesia with general anesthesia alone on hemodynamic instability in patients during PPGLs surgeries. The incidence of intraoperative hypotension was higher in patients with combined epidural-general anesthesia than in those with general anesthesia alone . No difference was found in the incidence of hypertensive crisis . The combined epidural-general anesthesia was suggested to be an independent predictor of intraoperative hypotension , but had no association with hypertensive crisis .Jeon et al,30 Epidural anesthesia has been reported beneficial to the hemodynamic fluctuations and the postoperative complications associated with PPGLs surgeries.,22 Although epidural anesthesia has been commonly used as a supplementary technique of general anesthesia in PPGLs surgeries, it is unclear whether combined epidural-general anesthesia was better than general anesthesia alone for these patients. Therefore, we performed a systematic review and meta-analysis, and finally identified 3 retrospective cohort studies–18 that compared the effects of combined epidural-general anesthesia with general anesthesia alone on the hemodynamic fluctuations and postoperative outcomes in patients undergone PPGLs resections. Unexpectedly, the combined epidural-general anesthesia was suggested as an independent predictor of both intraoperative hypotension and postoperative hypotension, although it could reduce the incidence of postoperative complications compared with general anesthesia alone.During surgical resection of catecholamine-secreting tumors, mainly including pheochromocytomas and paragangliomas, hemodynamic instability can occur and be life-threatening.,32 which is likely due to a decreased peripheral vascular tone. For PPGLs surgeries, the incidence of intraoperative hypotension was as high as 41% to 81%.,34 One recent study demonstrated that, compared with general anesthesia alone, combined epidural-general anesthesia was associated with a higher incidence of intraoperative hypotension in video-assisted thoracoscopic lung cancer resections. However, there is no consensus whether epidural anesthesia leads to the intraoperative and/or postoperative hypotension in patients with PPGLs surgeries. The included studies in our review showed that epidural anesthesia was an independent risk factor for both intraoperative and postoperative hypotension of patients undergone resection of PPGLs, and the incidence of hypotension in patients with epidural anesthesia was 88.7% and 58.8%, respectively. This high incidence was also suggested to be partly caused by the abrupt reduction in circulating catecholamines after resection of PPGLs as well as by preoperative alpha/beta-blockade use. Kang et al compared the effects of combined epidural-general anesthesia with general anesthesia alone on hemodynamic instability during resection of PPGLs. The primary outcome was the occurrence rate of post-resection hypotension. Consistent with the results of Wiseman et al, their published conference abstract showed that combined epidural-general anesthesia was an independent risk factor of post-resection hypotension after PPGLs surgeries.Previous studies reported that the incidence of hypotension associated with epidural block for open abdominal surgeries was about 15% to 33%,,39 It is necessary to control patients’ blood pressure at a desirable level prior to PPGLs surgeries. Uncontrolled preoperative hypertension may increase the risk of perioperative complications in patients undergoing PPGLs surgeries, and usually cause intraoperative hypotension due to the impaired regulation ability of blood pressure.,42 One of the included studies found that a high baseline mean blood pressure (MBP) was associated with the occurrence of intraoperative hypotension during PPGLs surgeries. In their study, the cutoff value of mean blood pressure to predict intraoperative hypotension was 93.7 mm Hg. Similarly, 1 published abstract of Kang et al showed that increased MBP baseline was an independent risk factor of post-resection hypotension for PPGLs surgeries. Therefore, the baseline level of MBP may serve as a predictor for intraoperative and/or post-resection hypotension for PPGLs surgeries.Patients with PPGLs usually suffered from hypertension. Beta-blockers are usually used to control tachyarrhythmias resulting from the use of alpha-blockers in the preoperative management of patients with PPGLs.,43 Generally, alpha-blockers but not beta-blockers are thought to be the major medication factor that leading to postoperative hypotension after PPGLs resection. Surprisingly, Wiseman et al reported that alpha-adrenoceptor blockers were not a predictor of postoperative hypotension. Interestingly, 1 abstract also reported that alpha-blocker premedication was not associated with post-resection hypotension in PPGLs surgeries. Similarly, a meta-analysis showed that preoperative alpha-blockade use had no effects on hemodynamic fluctuations compared to no alpha-blockade use in phaeochromocytoma surgery. Wiseman et al first identified beta-blockers as an independent risk factor of postoperative hypotension for resection of PPGLs. Notably, beta-blocker itself can also reduce blood pressure or exacerbate the hypotension induced by alpha-blockers.,45 The effects on blood pressure of beta-blockers may be prominent in patients with alpha-blockers premedication for PPGLs surgeries, particularly for those received epidural anesthesia. Therefore, it should be cautious when performing epidural anesthesia on the patients with alpha-blockers and beta-blockers premedication for resection of PPGLs.In addition of epidural anesthesia, the use of beta-blockers was suggested another independent factor that associated with postoperative hypotension for resection of PPGLs.,47 Tumor manipulation is the most important factor to cause hypertensive crisis by inducing a catecholamine surge during PPGLs surgery.,48 The combined epidural-general anesthesia technique has been reported to facilitate hemodynamic stability before tumor isolation and resection.,23 However, one of our included studies showed that combined epidural-general anesthesia had no interaction with hypertensive crisis during PPGLs surgeries but was associated with hypotension. This may be explained by that the epidural block was not sufficient to suppress the catecholamine surge during tumor manipulation but exaggerated the sympathectomy and vasodilatory effects after tumor ligation.,50The hypertensive crisis is another common feature of hemodynamic instability with an occurrence rate of 51% to 85% in PPGL surgeries, which is usually caused by excessive release of catecholamine.,52 Interestingly, an increased attenuation number on unenhanced computed tomography was demonstrated as the only independent predictor of hypertensive crisis. Meanwhile, an increased attenuation number on unenhanced computed tomography was also an independent predictor for intraoperative hypotension. The underlying mechanism of this correlation remains unclear, further studies are needed to determine this.Mean attenuation (Hounsfield unit: HU) value on unenhanced computed tomography was suggested as a diagnostic tool for PPGLs.,54 A meta-analysis showed that epidural analgesia was associated with a lower incidence of pulmonary complications after abdominal and thoracic surgeries, probably due to reduced opioid consumption, earlier mobilization, and improved cough. Epidural anesthesia may also contribute to improve the balance between myocardial oxygen consumption and supply as well as to relieve the activation of postoperative neuroendocrine, metabolic and inflammatory response. One of the included studies first reported that combined epidural-general anesthesia was associated with a lower incidence of postoperative complications in patients undergone open surgery of pheochromocytoma. However, 1 major limitation of their study was that patients’ data were collected until hospital discharge. Therefore, their results might have underestimated the incidence of postoperative complications because a prospective study found that about 1 to 3rd of complications occurred between discharge and postoperative 30 days. The effects of combined epidural-general anesthesia on the occurrence of overall, including both short-term and long-term postoperative complications for PPGLs surgeries need to be further assessed.Mounting evidence has demonstrated that epidural blockade could improve the postoperative complications of various type of surgeries. However, no difference was found in short- or long-term postoperative outcomes between these two operative approaches in PPGLs surgeries, although open approach was associated with longer duration of hospital stay. Likewise, the operative approach may also influence the effects of epidural block on the outcomes of patients underwent PPGLs surgeries. Among the included studies in this review, 2 studies enrolled patients receiving open surgery,,18 while 1 enrolled patients receiving laparoscopic or open surgery. However, no published studies have reported the effects of different operation approach on the effects of epidural block for PPGLs surgeries. Further prospective RCTs are needed to determine this.The operative approach for PPGLs resection may influence the outcomes of patients. Currently, the operation approach for PPGLs resection mainly includes laparoscopic and open resection. Patients with larger malignant PPGLs were more likely to receive open approach, while laparoscopic approach appears to be safe for tumor size < 6 cm.,60 Moreover, LA is associated with a lower perioperative morbidity and shorter length of stay than OA.,62 In recent years, robotic-assisted adrenalectomy is used increasingly, and a meta-analysis showed that robotic-assisted adrenalectomy is a superior technique to conventional LA in treating adrenal tumors, even in pheochromocytoma. Therefore, it will also be interesting to evaluate the effects of epidural anesthesia for patients with robotic-assisted resection of PPGLs.For adrenalectomy, several studies found that laparoscopic adrenalectomy (LA) has greater advantages than open adrenalectomy (OA) in terms of postoperative pain, patient satisfaction, and the length of hospital stay and recovery times. the use of beta-blockers, the baseline of mean blood pressure, and the attenuation number on unenhanced computed tomography. Other factors, such us the surgical approach may also be involved in the effects of epidural anesthesia during PPGLs. These factors should be noted and validated in future studies when investigating the effects of epidural anesthesia in PPGLs.From this systematic review and meta-analysis, we summarized several factors that may influence the outcomes regarding the use of epidural anesthesia in PPGLs, including patients gender, perioperative blood transfusion, 97, and 146, respectively. Secondly, the results between included studies were not available to combined for meta-analysis due to each outcome was only reported in an individual study. Thirdly, the quality of included studies were relatively low, as the overall risk of bias were serious to critical. Therefore, large samples, multicenter, randomized controlled trials are required to determine the efficacy and safety of combined epidural-general anesthesia in surgical resection of PPGLs.There were several limitations in this study. Firstly, all included researches were retrospective cohort studies, and the number of included studies and sample were small. Only 3 studies were included in this study, the sample was 119,In summary, no published RCTs have yet assessed clinically relevant outcomes with respect to the application of epidural anesthesia during PPGLs surgeries. On the basis of the findings of non-RCT literature, the outcomes from three retrospective studies suggests that combined epidural-general anesthesia was associated with both intraoperative and postoperative hypotension, although it reduced the incidence of postoperative complications in patients undergone surgical resection of PPGLs. Therefore, in choosing between combined epidural-anesthesia and general anesthesia alone for PPGLs surgeries, potential benefits should be balanced against the risk of hypotension. Well-designed RCTs should nonetheless be encouraged to properly assess the efficacy and safety of epidural anesthesia for PPGLs surgeries.Conceptualization: Shuai Zhu.Data curation: Min Yang, Chao Kang.Formal analysis: Min Yang, Chao Kang.Methodology: Min Yang, Chao Kang.Validation: Min Yang, Shuai Zhu.Writing – original draft: Min Yang.Writing – review & editing: Shuai Zhu.


From the global perspective, stroke refers to a highly common cause of disability and death. Ischemic stroke (IS), attributed to blood vessel blockage, preventing the flow of blood to brain, acts as the most common form of stroke. Thus far, thrombolytic therapy is the only clinical treatment for IS with the approval from the FDA. Moreover, the physiology barrier complicates therapeutically and diagnostically related intervention development of IS. Accordingly, developing efficient and powerful curative approaches for IS diagnosis and treatment is urgently required. The advent of nanotechnology has brought dawn and hope to better curative and imaging forms for the management of IS. This work reviews the recent advances and challenges correlated with the nano drug delivery system for IS therapy and diagnosis. The overview of the current knowledge of the important molecular pathological mechanisms in cerebral ischemia and how the drugs cross the blood brain barrier will also be briefly summarized. Stroke is a brain disorder that is attributed to a dramatic blood vessel rupturing or blocking within the brain, thereby preventing the flow of blood to the brain. As impacted by a significant effect exerted by disease worldwide, stroke continues to be a main health issue in the globe, representing the 5th top reason for death within the USA as well as the third primary reason for potential life lost worldwide. Nearly a population of 15 million encounter stroke each year in the globe, with 33% caused to be permanently disable, and 40% caused to die . It is eTo date, though several pre-clinical therapies have reported that neuroprotectants (including apoptosis suppressors and antioxidant) exert good effects for treating IS, none of them are efficacious in humans . The mosThe central nervous system (CNS) has developed several barriers for protecting its invasion in circulating blood cell, neurotoxic molecule and pathogen. Among the mentioned, BBB is the most extensive and exclusive, under the formation of brain microvasular endothelial cells (BMECs) lining the micro-scale brain vessels, followed by tightly correlated astrocyte and pericyte. It was previously shown that BBB is a dynamic and functional neurovascular unit well separating CNS from circulating mechanism and protecting CNS against possibly adverse toxin, chemical and infection within blood , 14. In As a primary disease for mortality and morbidity globally, IS has aroused surprisingly less attention of the nanobiotechnology research compared with tumor or diabetes , 23. TheIS, attributed to the flow of blood blocked to the brain, is the most common type of stroke . Brain i2+-dependent enzyme excessively  [Oxidative stress refers to part of downstream consequences of excitotoxicity induced by the production of ROS and is generally indicated to activate the channels of apoptosis, necrosis and autophagy, thereby facilitating the final infarct size in brain . The intuclease) . In cereuclease) , 34.+/K+-ATPase, thereby depolarizing the neuronal membranes with Na+ abnormal influx and K+ efflux [+/Ca2+ exchanger orientation and introducing Ca2+ to cells, which causes excitotoxic amino acids release, including glutamate. The uncontrolled accumulation of glutamate can overstimulate the metabotropic glutamate receptor and broadly stimulate the receptors of N-methyl-D-aspartic acid (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid (AMPA), thereby disrupting calcium homeostasis of CNS [2+ content in cells [It is generally acquired that the first change after cerebral ischemia is a down-regulation within cerebral blood flow, thereby causing inadequate delivery of oxygen and glucose. As impacted by oxidative phosphorylation acts as the major energy production source, the decreased glucose and oxygen supplies will decouple oxidative phosphorylation, followed by the lessened ATP production as well as ion pump function injury . The dec+ efflux . The inss of CNS . Then thin cells . Calciumin cells .Membrane integrity loss and organelle failure refer to the critical systems of cell apoptosis within ischemia. Yet existing studies have obviously implicated systems following apoptotic-like channels and cascade, in particular in the penumbra. Both caspase-dependent and caspase-independent systems are presented. The cell apoptosis channels show correlations with an apoptotic-like system within cerebral ischemia. For instance, Cytochrome c released from mitochondria receives the modulation to be anti-apoptotic Bcl2 family member. Cytochrome c release can activate downstream caspases through apoptosis body formation and caspase stimulation is modulated indirectly by caspase secondary catalyst obtained from mitochondria through hindering inhibitors of apoptotic protein (IAP). Effector caspases (Caspase 3 and 7) target substrate, dismantling cells through the process to cleave metabolic, repair, cytoskeletal, homeostatic and cell signaling proteins. Caspase-independent cell apoptosis is likely to be vital. According to a system, poly-(ADP ribose) polymerase (PARP) stimulation boosts the release of apoptosis-inducing factor (AIF), thereby being translocated to the nucleus, while the system of which binding to DNA and boosts cell apoptosis based on a system awaiting elucidation.Inflammation starts few hours when cerebral ischemia occurs. The IS can trigger the release of a wide range of inflammation-related markers including inducible nitric oxide synthase (iNOS) and eicosanoids, cell adhesion molecules , cytokines  . Leukocyvia intravascular therapy or intravenous thrombolysis. The vital element determining the traditional therapy’s efficacy is rapid thrombolysis in a small window of time (3–4 h) for ensuring minimal brain impairment. Surgical intervention including mechanical endovascular thrombectomy has also been developed to restore blood flow in the occlusion of an intracranial vessel. Nevertheless, the mentioned intervention has not had the ability to prevent death or disability in primary cases. Table At present, the routine therapies to manage IS and correlated complications largely concentrated on drugs to remove the thrombus, as well as to minimize deleterious ischemic injuries to neurons Until recently, intravenous administration of rt-PA in terms of thrombolysis continues to be the only commercially-available treatment approved by the FDA. This therapy is largely determined by the narrow time for being effective (less than 30% of stroke patients) and also increases the risk of intracerebral hemorrhages . RecentlSecondary prevention of stroke can also be done by antiplatelet therapy, anticoagulants accompanied with or without surgical treatment. The provision of anticoagulants like aspirin and statins for the patients refers to the sole cheap option, whereas the mentioned antiplatelet drugs are almost adopted to be precaution for subsequent or probable episodes, and capable of repairing or shielding the influenced region from more injury in no circumstances . TherefoAs a promising curative approach, the neuroprotective agents aim at improving survival of neurons after ischemic stroke to increase the curative window, as well as inducing neurological repair to improve functional outcomes. For this reason, they are commonly used in combination with drugs of other categories. Some classes of neuroprotectants consist of free radical scavengers, γ-aminobutyric acid (GABA) agonists , calciumIt is obvious from clinical trials or reports that a single drug is ineffective, but a mixture of multiple therapeutic drugs with different targets are required for better prevention and management of IS. The major toxicity of systemic administration of conventional drugs  is the activation of plasminogen in regions with normal circulation leading to hemorrhagic complications . BesidesAnother major challenge of stroke treatment is to bypass the physiological barriers ) raised by the brain. Moreover, the presence of efflux transporters (i.e. P-glycoproteins) in BBB and CBB also provides additional obstacles for drug delivery to the brain. Hence the next generation therapeutics for stroke therapy is expected to cross the BBB effectively. In brief, the clinical trials and reports obviously show an urgent need to apply the methods for swiftly crossing the physiological barriers and targeting site-specific administration of multiple curative agents at a wide range of time points to the management of IS.The BBB is one of the most vital component between the nervous system and rest part of the body, which protects the brain from harmful stimulation. BBB permeability is controlled by tight junction protein complexes , which limits the paracellular diffusion between brain capillary endothelial cells (BCECs). Although stroke can destroy this barrier and lead to increased vascular permeability, the BBB maintains brain homeostasis . It is rAlthough passive delivery may facilitate the transport of nanocarriers across compromised BBB to some extent, lower dose and poor distribution of nanocarriers into the brain still remain great challenges . AnotherA major requirement of IS treatment refers to visualizing the diseased site under great resolution and sensitive property for achieving precise diagnosis and continuously curative result monitoring. Numerous molecular imaging methods were employed to image the brain including MRI, positron emission spectroscopy (PET), computed tomography (CT), digital subtraction angiography (DSA), single photon-emission computed tomography (SPECT), ultrasound and optical imaging , 62. Benvβ3-specific Fe3O4 nano-scale probe with surface modified by RGD tripeptide was prepared. The RGD peptide could be a particular recognition motif of integrin receptors, facilitating Fe3O4 nano-scale probe localization on collateral vessels and achieving MRI visualizing. Likewise, the PEGylated ultrasmall paramagnetic Gd2O3 nanoparticles with diameter of 5 nm were adopted to achieve cell labeling and tracking in the brain with MRI  were introduced as contrast enhancers. For instance, an investigation conducted by Ito A er al. reported that MNPs conjugated with antibodies were capable of exhibiting multiple-functionality as a selective biology-based recognizer of target molecules, as well as a MRI contrast enhancement agent . It was 64Cu and then coated it with PEGylated phospholipids. Biodistribution and in vivo PET/CT imaging researches on the probes indicated high initial blood retention with moderate liver uptake, thereby enabling them to be attractive contrast agents for disease researches [18 F-modified polyglucose nanoparticles that exhibited high affinity for macrophages in MRI and PET imaging of ischemic animals have been reported. The mentioned data suggest that macroflor PET/MRI could be a clinical device for the non-invasive monitoring of macrophage biology [PET refers to a method to analyze brain functions with the use of a radioactive tracer, thereby allowing for metabolic marker mapping and physiology-related factor within the brain region . As one searches . Likewis biology .5 nanoprobes with a mean diameter of 7 nm were also developed and employed as a contrast agent for CT angiography (CT-A) and CT perfusion (CT-P) imaging. This nanoprobe has unique advantages over traditional iodinated CT agents, such as much lower dosage needed, metabolism primarily through liver and higher imaging efficiency at different voltages [CT refers to a highly powerful non-invasive diagnostic imaging method in modern medicine, to be integrated and processed to generate tomographic images of the brain. Compared with MRI, CT is not sensitive to the differentiation between ischemic penumbral and infarct core region . Accordivoltages . In addivoltages , 80. Apavoltages . AccordiUltrasound images are achieved by adopting a small probe as a transducer and gel placed directly on the skin to image the blockage or defects in blood vessels. It creates images of the inside of the body with the use of sound waves known as echogenicity. Blood vessels are known to exhibit poor echogenicity and hardly reflect the ultrasound waves even under a high intensity. As a result, ultrasound contrast enhancers are introduced into the system, which are usually aerated microbubbles exhibiting robust echogenic properties made of albumin, lipids or synthetic polymers , 83. OveSo far, several clinical trials have been conducted for imaging of IS while very few of them involve nanocarriers. The benefits of nano-based interventions in the context of IS imaging have been clearly demonstrated in many in vitro and pre-clinical studies. Considering that many inorganic materials do not naturally exist in the body, fully evaluating the biodistribution and toxicity after systemic administration is urgent needed. This is expected to initiate more clinical trials involving nanoparticle-mediated imaging strategies for IS in the future Table .Apoptosis or cell injury within ischemic core occurs fast, causing the difficult protection of such a region by conventional approach. Common drawbacks that limit traditional drug treatment consist of the physiological barriers, fast metabolism or degrading process, as well as unsatisfactory distributing profile. The nano drug delivery systems are capable of deriving from material exhibiting organic backgrounds or artificial features. As a drug-delivery system with high prospect in treating brain disorder, the nanocarriers exhibit benefits in preventing drug degradation, improving pharmacokinetic profiles and neurovascular unit access. Curative agent is typically encapsulated, entrapped, adsorbed or chemically attached on the surface of nanocarriers . The nanThe polymer based nanocarriers have a wide range of applications in the IS delivery due to the good biodegradability and abundant functional groups, which make it easy for targeted functional modification. The most common polymer based nanocarriers include polymeric nanoparticles, polymeric micelles, nanogel, dendrimer. The detailed introduction will be listed below.The polymers include not only the synthetic biodegradable polymers including poly(lactic acid) (PLA), poly(L-lactide-co-glycolide) (PLGA), polycaprolactone and polyethylene glycol (PEG) PLGA, but the natural macromolecular systems consisting of chitosan, polysaccharide, gelatin and starch. In recent years, polymer nanoparticles have been broadly used as the delivery systems for the treatment of IS. PLGA is widely employed among the biodegradable polymers mentioned above. It is a biodegradable polymer under hydrolysable ester links approved by FDA. The gradual hydrolyzing process of PLGA leads to a reduction in molecule weight and formation of voids in polymer chains, thereby facilitating the sustained-release of the drug. Mdzinarishvili et al. conducted the application of glutathione-coated PLGA-b-PEG nanoparticles to load thyroid hormones (T3), which has been proven for protecting the brain from ischemic injury through glutathione transporter on the BBB . FurtherBesides synthetic polymer, natural biodegradable polymers  are also commonly used to prepare nanoparticles. For instance, Vemisci et al. have shown that bFGF or z-DEVD-FMK encapsulation in chitosan nanoparticles excited large quantities of nanoparticles crossing the BBB . AnotherPolymeric micelles refer to newly developed nano-scale systems employed for incorporating charged molecules, acting as attractive options to deliver drugs for spontaneously self-assembling property. This nano-micelles are established under the reaction in which double hydrophilic block copolymers covering ionic and non-ionic blocks are reacting with macro-scale molecules pertaining to opposite charge  , 101. FoThe dendrimers, i.e., a type of highly branched and monodispersed macrocyclic polymer micelles, cover numerous features including a core with attached branch, with branch shell around the core, i.e., a surface of multivalence, the internal shell and the inner core covering terminal function-related group and repeating unit . In partNano-hydrogels are hydrophilic 3D polymer network under broad applications within drug delivery and tissue engineering, capable of maintaining a certain three-dimensional shape and loading a large number of charged molecules . Nano-hyLiposomes refer to vesicular carriers receiving the self-assembly, under the formation of amphipathic lipids through the phospholipids and cholesterol mixing within various ratios , 120, isA critical defect of liposomal systems refers to their narrow circulation time in the body. PEGylation of drug-loaded liposomes exhibits particular effectiveness to improve their longevity within systemic circulation . RecentlDespite the advantages of liposomes, currently there are still no liposome-based nanocarriers available in clinical for the treatment of IS. As a drug delivery, liposomes have been broadly proved to improve pharmacokinetics and biodistribution. Yet no currently marketed liposomal curative agents have exhibited an overall survival benefit when directly compared with the traditional drug for IS, which probably result from the lipid instability, drug leakage and insufficient targeting at the target site , 142.SLNs are also stable lipid-based nanocarriers featuring one core of solid hydrophobic lipid for drug encapsulating process, comprising biocompatible lipid including fatty acid and triglyceride , 143. SL2-MNP) conjugated with tPA, proven to be magnetically guided to increase in a thrombolysis model ex vivo. The SiO2-MNP developed here exited a great future to be the carrier of magnetic targeting drug for improving clinically related thrombolytic therapies [Besides polymeric and lipid-based carriers, the inorganic nano-scale materials, including silica, alumina, metals, and carbon, have shown great potential in the treatment of IS . Inorganherapies . Carbon herapies , 157. Thherapies , 159 as herapies , 161. Foherapies . Some auherapies .As reported from recently conducted researches, several specific inorganic nanocarriers themselves can serve as ROS scavengers in the course of cerebral ischemia, especially after reperfusion. Platinum nanoparticles (nPts) exhibited antioxidant activity through the process to scavenge hydrogen peroxide and superoxide anions when tested in vivo . Cerium In the recent years, the newly developed integrated hybrid nanocarriers have been gaining attention including organic-inorganic hybrid nanocarriers and lipid-polymer hybrid nanoparticles. It is well known that the organic-inorganic hybrid nanocarriers have the properties of reducing initial drug burst and adjustable surface chemistry , 169. FuBiomimetic carriers from cells have presented novel option for drug delivery as impacted by the advantage of distinctive functions and prominent delivery mechanisms of endogenous cells. Camouflage by means of a natural cell or cell membrane has gained increasing attention, because it bypasses the recognition of nanocarriers by the mononeuclear phagocyte mechanism, thereby reducing immunogenicity and prolonging circulation .Exosome refers to a type of vesicular carriers secreted by various cells, which has elicited great interest due to its unique characteristics of low immunogenicity, inherent stability, high delivery efficiency and capability to cross the BBB, to be utilized as a drug and gene carrier to treat IS. For instance, Yang et al. have reported that the modified exosomes fusing rabies virus glycoprotein (RVG) and exosomal protein lysosome-correlated membrane glycoprotein 2b (Lamp2b), could efficiently deliver miR-124 to the infarct site . This RVAnother attractive project in the context of cell-based stroke therapy is employing stem cell to remodel the function-related neurons lost as impacted by the ischemia/reperfusion injuries . Various3O4@nanoerythrocyte-T7, MNET) capable of remodeling stroke microenvironment based on free radical scavenging as well as self-adapted oxygen control  after thrombolysis, suppress oxygen-boost via oxygen storage [Erythrocytes, refer to the largest group of blood cells with significantly greater longer life-span as compared with other blood cells. Erythrocytes and erythrocyte membranes are quite easy to obtain, presenting novel insights into drug delivery for advanced targeting ability and biosafety as compared with artificial nanocarriers. Our group developed a bioengineered ROS-responsive nanocarrier for stroke-specific delivery against ischemic brain injury . This birol Fig. . The bio storage , holding2O3 magnetic nanoparticles (PAMNs) for achieving curative purposes, taking into account the relevant role of natural platelets and thrombosis in targeting damaged vascular adhesion [Platelet, generated from megakaryocyte, refers to discoid, anucleate, small blood cells that are critical to hemostasis. Based on the platelets function in thrombus targeting and immune escape, platelets and platelets-like biomimetic nanocarriers play a role in stroke drug delivery to sites of vascular plaque . In termadhesion . Our groadhesion . To solvadhesion .Leukocytes, designed to accumulate in large numbers in areas of stroke injury and inflammation, can be significantly purified to carrier drugs. Inconsistent with erythrocyte and platelet, the distinguished property of neutrophils is chemotaxis, which endows them with the capability of migrating to an infection or inflammation site within minutes. According to several results, inflammation-related monocytes can be adopted to transport drugs to injury sites, whereas the blood monocyte number (nearly 5% of leukocytes) is insufficient for delivery of drug throughout the entire pathogenesis process of cerebral ischemia . Compare2O2-responsive engineered polymer nanoparticles formulated from copolyoxalate covering vanillyl alcohol (VA) (PVAX) was reported as a newly developed ischemia/reperfusion targeted nanotherapeutic agent [It is a key step for the drug therapy to temporarily control the drug release which can be of wide use in stroke therapy and satisfied the request of in vivo temporal and spatial selectivity of cargo release. Accordingly, the study of stimulus responsive carrier is almost inevitable for stroke treatment. A wide range of nanocarriers of natural and synthetic origin, organic and inorganic properties as well as with various shapes, sizes and surface modifications have been explored for drug delivery. In the context of cerebral stroke therapy, a number of unique responsive studies were applied in microenvironment of the brain. For instance, a Hic agent . The ROSic agent . For theic agent , 194. Apic agent , 195. Gaic agent . Stromalic agent . Besidesic agent . This naSome other nanomaterials with new features or multi-functions for IS treatment are emerging. For instance, Wang et al. have developed a near-infrared light (NIR)-driven nano-photosynthesis biosystem for rescue neurons in ischemic brain areas, achieving the new combined features of cell-biological, biochemical, and animal-level behavioral data . Further2+ homeostasis, oxidative stress attributed to ROS generation and cerebral inflammation, which remains a great challenge for clinicians because of the critical concerns involved in its curative window, presence of BBB as well as long-term effects of impaired brain function. The lack of approaches for efficient drug delivery to the brain is one of the main difficulties to develop effective treatment for IS. Besides, owing to the non-specificity, slow onset of action, narrow therapeutic efficacy, and poor patient compliance, clinical value of these conventional medications  is limited.To conclude, IS is a mental medical emergency which happens due to obstruction of blood supply (blockage of vasculature) to specific regions of the brain. The pathophysiology mechanism of IS involves a complex dynamic process, primarily including glutamate excitotoxicity, disruption of CaNowadays, application of nanocarriers  for delivering curative agents is gaining more and more attention as a possible alternative to traditional therapy due to the following points: (i) reduce recognition and clearance by reticuloendothelial system (RES) and prolong the blood circulation time in vivo; (ii) improve the blood stability and enhance the BBB penetration and lesion site targeting; (iii) reduce the off-target toxicity of therapeutic agents; (iv) co-deliver therapeutic agents with different targets for synergistic therapy; (v) achieve the stimulus-reaction . In addition, this nanotechnology also allows for better high resolution visualization of infarct sites, greatly promoting the diagnosis and treatment of IS.Despite the rapid development and potential clinical prospects of nano-delivery system, several difficulties of nanocarriers need to be emphasized in the near future and their clinical transformation is greatly challenged. Firstly, in view of the complex pathological mechanisms of IS, the nanocarriers usually only target one or two pathological mechanisms and fail to provide comprehensive protection for the CNS. Co-delivering different promising drugs into a single nano-delivery system indeed provides a possibility of multifaceted protection, but it inevitable increases the complexity of the nanocarriers and hampers their clinical translation. Secondly, there are many studies on the anti-stroke of nanocarriers, but most of them just focus on the consideration of drug delivery effectiveness. The mechanism of cerebral metabolism and elimination of nanocarriers as well as the long-term safety for clinical application, are still unclear and the correlated neurotoxicity is yet a matter of concern. The safety data and toxicity profiles of nanocarriers after internalization and retention in brain tissues are not attracted enough attention. Due to the toxicity of nanocarriers is a critical factor affecting its clinical transformation, toxicity investigation of nanocarriers should become an important direction for scientists in this field in the further. Thirdly, although BBB penetration and ischemic penumbra focusing effect of nanocarriers can be enhanced by brain-targeted modification or biomimetic approaches, the BBB still remains a major challenge, and a large proportion of nanocarriers still fail to reach the ischemic area. In particular, the pathogenesis of model animals  is not exactly the same as that of clinical patients, leading to the scarcity of clinical evaluation data of existing nanomedicines in human body, which also explains why is tough to realize clinical translation of nano-drug delivery carriers.Looking forward, more in-depth researches are urgently demanded and further studies should be pursued to reveal the actual efficiency of drug delivery for IS and offer more insight on how to achieve the translation of the drug-loaded nanosystems successfully. Despite the above concerns, it is self-evident that nanotechnology is the way forward in diagnosing, treating and managing IS.