Structural health monitoring (SHM) is being widely evaluated by the aerospace industry as a method to improve the safety and reliability of aircraft structures and also reduce operational cost. Built-in sensor networks on an aircraft structure can provide crucial information regarding the condition, damage state and/or service environment of the structure. Among the various types of transducers used for SHM, piezoelectric materials are widely used because they can be employed as either actuators or sensors due to their piezoelectric effect and vice versa. This paper provides a brief overview of piezoelectric transducer-based SHM system technology developed for aircraft applications in the past two decades. The requirements for practical implementation and use of structural health monitoring systems in aircraft application are then introduced. State-of-the-art techniques for solving some practical issues, such as sensor network integration, scalability to large structures, reliability and effect of environmental conditions, robust damage detection and quantification are discussed. Development trend of SHM technology is also discussed. For metallic or composite aircraft structures, in-service conditions and failure modes are generally complex and may not be accurately predicted. Because of this, the aerospace industry typically uses conservative time-based or usage-based scheduled maintenance practices that are overly time-consuming, labor-intensive, and very expensive. Furthermore, as structures age, maintenance service frequency and costs increase while performance and availability decrease. One possible method of ensuring structural safety is to inspect structures frequently and keep abreast of their structural condition. The use of condition-based maintenance coupled with continuous on-line structural integrity monitoring could significantly reduce the cost of inspection.Structural Health Monitoring (SHM) is perceived as a significant method for determining the integrity of structures involving the use of multidisciplinary fields including sensors, materials, signal processing, system integration and signal interpretation ,2,3. TheDuring the past two decades, different SHM system technologies, including both passive and active monitoring, have been developed through the use of built-in distributed sensor network integrated with metallic and composite aircraft structures ,7,8,9. T(1)Optimized sensor layout: Most SHM systems require an expert with years of experience to know where and how to install sensors for optimal damage detection for every new application.(2)Self-diagnostics: Most SHM systems do not have built-in self-diagnostics and thus cannot distinguish between damage to the structure and damage to the sensors themselves.(3)Environmental compensation: Most SHM systems are overly sensitive to environmental changes and do not have effective compensation techniques to allow practical field use.(4)Probability of detection: Most SHM systems cannot easily provide quantifiable specifications for resolution or probability of detection (POD) for every new application or sensor configuration.(5)Damage quantification: Most SHM systems do not output quantitative damage sizes with associated uncertainty values.(6)Airworthiness compliance: the SHM technology needs to meet not only the intended functions for a specific application, but also the requirements of airworthiness complianceWhile the SHM technology has shown great promise in the laboratory, the transition into field applications have been pretty slow. While most of major civil aircraft OEMs, such as Airbus, Boeing and COMAC, have been paying close attention to it, and evaluating it in the real world, none fully succeeded to incorporate it within their civil aircrafts yet. This is due to a number of factors including :(1)OptimA complete, robust SHM system is one that combines the essential elements needed for implementation including state-of-the-art techniques to optimize sensor placement, perform self-diagnostics, compensate for the influence of environmental variation, generate POD curves and output quantitative damage sizes.This paper first provides a brief overview of piezoelectric transducer-based SHM technology developed in the past two decades. The piezoelectric transducer-based SHM systems including sensor network and diagnostic hardware with principles of monitoring, identification algorithms of damage and impact are summarized. The requirements for practical implementation and use of SHM systems in aircraft application including the design principles of SHM systems and some key issues are then introduced. State-of-the-art techniques for solving some practical issues, such as sensor network integration, scalability to large structures, reliability and effect of environmental conditions, robust damage detection and quantification are discussed. Development trends of SHM technology are also discussed in this paper.A typical piezoelectric transducer based SHM system consists of a sensor network mounted on or embedded into a host structure, portable diagnostic hardware, and data analysis software, as shown in Sensors that transform the parameters of structure state, such as damage, load and temperature, into the corresponding sensing signals are the basis of SHM. They are usually mounted on the surface of a structure or embedded into the structure in the form of a network. The sensor network plays an important role in the performance of the SHM system. The ability of sensors and actuators in the network to communicate with each other defines the intelligence of the system. The type, location, and number of sensors and actuators critically affect the sensitivity and performance of the SHM system. As the number of sensors increases, the integration of such a sensor network with a structure can be very challenging or become impractical. The SMART Layer technology originally developed at Stanford University offers a simple and efficient way to integrate a large PZT sensor network onto a structure with high reliability ,16. OnceThe layer is made of a thin dielectric film with an embedded network of distributed PZTs that can be used as either actuators or sensors, as shown in The characteristic features of the layer include: (1) ease of installation, (2) adaptability to any structure with complex geometry, (3) use of an area sensing network, (4) actuation and sensing capabilities, (5) signal consistency and sensor reliability, and (6) shielding to reduce EM noise. The layer can either be surface-mounted on existing structures or embedded inside composite structures during fabrication thereby providing a built-in nondestructive assessment of the structure states. Similar to SMART Layer, another novel lightweight diagnostic film with sensors/actuators and a multiple-path wiring option using inkjet printing was recently developed. The diagnostic film allows for systematic, accurate, and repeatable sensor placement. Furthermore, the film is highly flexible and adaptable for placement on complex configurations. The diagnostic film offers significant weight reduction compared to other available technologies .Piezoelectric sensor networks integrated with a structure can be used for both active and passive sensing. In the active-sensing mode, both wave propagation and electromechanical impedance (EMI)-based SHM methods have been developed for damage detection. The passive SHM mode is used in real time to monitor impact events including both impact location and energy.A typical active diagnostic hardware for wave propagation-based SHM has the built-in capability to generate a specific waveform for structural diagnostics, collect sensor data at a high sampling rate and resolution, and multi-channel capability to accommodate a network of piezoelectric elements. As shown in In the wave propagation-based SHM, guided waves, such as Lamb and Rayleigh waves, are most widely used for damage detection in metallic and composite structures. Guided waves used for damage detection are introduced into a structure at one point by a piezoelectric actuator and sensed by another piezoelectric sensor at a different position (pitch-catch), or the same piezoelectric element (pulse-echo), as shown in A key advantage of using piezoelectric elements is that a larger area of the structure can be monitored with fewer transducers, which is vitally important for the monitoring of large-scale structures. Other sensors, like optical fiber-based types, can only scrutinize smaller, specific areas, thus leaving larger areas of a structure unmonitored.The electromechanical impedance (EMI) technique is another active SHM method promoted by piezoelectric transducers. In the EMI-based SHM, the electrical parameters of a piezoelectric transducer are measured to identify damage. The damage near the transducer causes a stiffness change and affects the structure’s resonant characteristics which will accordingly change the electrical impedance of transducer due to the electromechanical coupling. The diagnostic process of active SHM is usually based on the comparison of the current sensor responses with previously recorded sensor responses (baselines) from the undamaged structure. The differences between the two sets of signals are what contain the information about any existing damage or other anomalies. Since the EMI method was first proposed in 1993 by Liang , it has Based on the previous study ,32, the The passive diagnostic hardware consisting of sensor amplifier and data acquisition unit is simpler than the active diagnostic hardware. The function of impact monitoring system is to collect stress wave signals generated by impact loads through sensor network on the structure, estimate impact location and reconstruct impact energy or force history, so as to monitor external impact on aircraft structure. The principle of impact monitoring is shown in Purely passive systems exist only for detecting impacts and other load changes, with any diagnosis of damage or further analysis being left to other systems. In contrast, purely active systems used to detect damage also suffer from their own drawbacks. For instance, active systems must constantly query the structure, resulting in wasted energy and expense when they are not needed. Therefore, it is desirable to combine active and passive systems in order to bring forth the advantages of both systems. With the active-passive diagnostic hardware integrated within a single sensor layer, upon detection of impact event, the transducers are engaged in an active mode to actively scan the impact area to determine the location and size of any resulting damage region. Details of the active-passive diagnostic system can be found in the literatures ,34.Algorithms for damage detection using guided wave were extensively investigated during the past two decades. Comprehensive reviews on the state of the art of Lamb wave-based damage identification approaches can be found in literatures ,36. The In recent years, damage diagnosis imaging methods based on ultrasonic guided waves have been widely concerned and developed to depict the position and area of damage. The main damage imaging methods include phased array imaging, delay and sum, tomography, migration imaging, and so on. The comparisons of several damage detection techniques are listed in In phased array imaging, several PZTs in the sensor network are used to form a dense sensor array. It is the same as the principle of a radar that the phase of excitation signal of each PZT can be adjusted independently so that Lamb wave propagation can be focused on the specific direction for the far field or on the specific location for the near field because of the sum of Lamb wave field excited by all PZTs. If the guided wave beam is controlled to focus to every corner, omnidirectional scanning and rapid detection of a plate-type structure with limited access can be realized. Giurgiutiu et al.  realizedIn the Delay-and-Sum (DAS) method, according to the damage scattering signal received by multiple pitch-catch paths, the damage is located according to the group velocity and the propagation time of guided wave. The scattering signal is processed by short-time Fourier transform, wavelet transform or Hilbert transform. The signal energy after signal processing is mapped to the potential damage point, which results in that the signal energy summed at the location of real damage reaches a maximum. DAS imaging formulation was firstly proposed and verified by the experiment by Wang et al. . MichaelUltrasound guided wave tomography (GWT) originates from the X-ray computer tomography (CT) technology commonly used in medicine. In this method, sensors are usually arranged around the monitored area and form a great number of transmitting-receiving paths. The monitoring principle is that the signal changes of guided wave varying with the damage are fusing to contour the damage. Common methods of guided wave tomography include standard parallel projection, fan-beam projection and double-cross projection . Khare eGao et al.  proposedMost of wave propagation-based damage detection methodologies rely on the use of baseline data collected from the structure in the undamaged state. It is well known that some environmental effects, such as temperature, load, humidity, radiation, will also cause changes in the sensor signals, and will thus interfere with most damage detection schemes and cause the false alert. There are some research reports about temperature compensation to reduce the effect of the change of amplitude, phase and wave shape, such as baseline signal stretch (BSS) , OptimalTo overcome the limitations of environmental effects, baseline free damage detection technique was proposed. Baseline free time reversal method has been extensively studied by the researchers ,92,93,94In addition, a novel methodology for optimal sensor placement based on maximum area coverage for damage detection using Lamb waves was also developed. A feature of the optimization approach lies in the fact that it is independent of characteristics of the damage detection algorithm making it readily up-scalable to large complex composite structures such as aircraft stiffened composite panel .The comprehensive reviews on EMI methods can be found in the literature ,96,97,98The EMI methods have been demonstrated to effectively detect various damages, such as crack and load damage ,100, debIt should be noted that the affectivity of DIs depends on the selection of EMI signature components  and the way of EMI signature measurement and conversation. For example, Budoya and Baptista  used twoTo quantitatively identify the type and size of damage, lots of work were further investigated. Since the EMI signals are usually obtained in the frequency domain, it is not easy or intuitive to make implementation. Therefore, some researchers were making attempts to analyze the EMI signals in a view of time domain ,115,116.Similar to guided wave signals, the EMI of piezoelectric transducer can be significantly affected by environmental and operational conditions, such as loads, vibration and temperature ,118, as The piezoelectric transducer is required to be small and lightweight enough when EMI method is used in aerospace application. Current transducers are often designed using piezoceramics, so-gel thin-film, composite piezoelectric films. The design and manufacture aspects of piezoelectric film deposition on different substrates are an interesting hotspot. For example, Hoshyarmanesh and Abbasi  depositeMany studies have been conducted for impact identification, including estimation of impact location and reconstruction of impact force history or energy ,153,154.Gaul et al. presented a method that uses piezoelectric films to detect the flexural waves in plates . The arrChoi et al. proposed an optimal smoothing and filtering algorithm based on distributed built-in piezoelectric sensors to reconstruct the force history of impact on composite beams . This imPark et al. further developed the impact identification algorithm using a built-in diagnostic technique. This method does not rely on the full-scale mathematical model of structure, nor does it require large scale acquisition of training data to establish neural network, but obtains the transfer function which can reproduce the physical behavior of system through experimental testing . MuellerIt should be pointed out that the model-based method is quite intractable for complex structures. Even if it can simulate the response well, it is difficult to reconstruct the excitation formula of input force from the output sensor signals simply.Worden and Staszewski developed an algorithm for impact location and quantification on a composite panel using neural networks ,137,138.Recently, Fu et al.  applied It is well known that wireless sensor networks (WSNs) offer numerous advantages over conventional wired systems, such as low weight and cost, scalability, flexibility, and ease of deployment. Some studies using a piezoelectric transducer based WSN to detect impact for aerospace and other industries have been conducted ,152,153.To design an integrated SHM system that can be practically used for aircraft applications in real world, some main issues shall be considered carefully: the definition of system function, composition of the system, and airworthiness compliance, and so on.The objectives to be monitored for aircraft application, especially for civil aircraft, can be classified to two categories: one is monitoring of structure state parameters, such as impact energy, strain, temperature; the other is detection of structural damages, such as cracks, delaminations, corrosion, and so on. Before an SHM system is designed, the functions of the SHM system should be well defined for the specific application based on what is required to be monitored and the readiness of the technology used to monitor it. In general, impact damage over large areas in composite structures, cracks at hot spot areas and strain distributions at some critical areas must be monitored.As described above, there are many types of sensors that can be used for SHM, including piezoelectric, fiber-optic, ICM, CVM, MEMS, strain gauges, etc. Besides piezoelectric transducers, it is often necessary to integrate various types of sensors in an integrated SHM system to complete the multidimensional measurements of structure since each individual sensor has its own advantages and limitations. For the giving conditions of usage and structural application, the sensor technology selected for the integrated SHM system shall meet three criteria: (1) the function to complete a specific measurement, (2) the capability to be integrated with the host structure, and with other hardware systems to constitute integrated SHM system, and (3) the maturity and network capability. (1)As an electric device on the aircraft, the SHM system must meet the electronic and electrical regulations for airworthiness, which include requirements about power supply, anti-static protection, fireproofing, high-intensity radiated fields (HIRF) protection, lightning protection, and anti-burst of oil tank, etc.(2)The integrated SHM system shall meet electrical wiring interconnection system (EWIS) regulations. The design and installation of wires, cables, connectors and switching devices shall satisfy with the requirements of electrical grounding, physical isolation, line shield and protection, and material flame-retardant, etc.(3)As an installed structure on the aircraft, the sensor network of SHM system shall meet the installation regulations for airworthiness, which include the requirements concerning weight, load distribution, material compatibility of sensor and adhesive layer with the aircraft structure, applicability and durability of sensor network, accessibility and detectability of the overall system, etc.The SHM system used for aircraft structures must meet the requirements of airworthiness compliance. According to the characterization of SHM system, its airworthiness compliance needs to be considered in three aspects:More specifically, the following requirements shall be addressed for designing an SHM system in an aircraft application: (1) function and performance requirements, including the capabilities of detecting and assessing structural damage, the minimum damage size to be detected, probability of detection (POD); (2) operation requirements, including ease for setup, a simple GUI; (3) safety requirements, including no adverse affect on the structural integrity of aircraft and personnel using it; (4) reliability & environmental requirements; (5) installation requirements; (6) maintainability requirements; (7) weight requirements.Besides the airworthiness compliance described above, there are several key issues that need to be addressed before the SHM technology can be widely used for aircraft. These key issues include: capability of large sensor network, self-diagnosis, environmental adaptability and quantitative monitoring results.Aircraft structures to be monitored are typically large in size, and need a large sensor network for sufficient damage detection and structural state sensing. Building such a multifunctional large sensor network for aircraft structure is very difficult. There are many challenges and issues that need to be solved, including: (1) how to build a large sensor network and integrate a variety of sensors together, (2) how to establish the communication link between the sensor networks and processing center, (3) how to process all the information from a variety of sensors. SMART Layer technology provides an effective way to integrate a certain number of sensors and actuators with metal/composite structures . HoweverThe concept of multi-modal sensing capabilities with built-in sensor network was proposed. An expandable lightweight polymer-based flexible sensor network that can host a high-density array of multiple types of sensors was developed ,157. As The SHM system designed for aircraft applications in the real world needs to provide the defined functions under different operation environments of aircraft. Survivability of the SHM system and reliable results under these environments are two important issues of environmental adaptability.The durability of the PZT sensors must be entirely guaranteed through the life of the host structure. However, the strength of the PZT is often much lower than that of the host structure, which is a critical problem to be solved urgently. It was demonstrated that the PZT bonded the structure can be used without any degradation even if the applied strain to the host structure exceeds the failure strain of PZT by introducing compressive pre-stress via a specific cure cycle . AnotherThe reliability of piezoelectric transducer based SHM system under harsh environments has been investigated ,160,161.If one or more sensors are degraded, damaged, or missing, the SHM system may not function properly and can give false indications of structural damage. Measuring the impedance of each channel can be used to find an open or short circuit. This can indicate a missing sensor or damaged connection/wiring, but a degraded or damaged sensor may go undetected using the impedance method. To resolve this problem, a reasoning process using the active sensor signals has been developed and implemented to detect degraded or damaged sensors that the impedance method may miss .An integrated three-step method shown in One of the elements that are essential to the practical usage and implementation of any structural health monitoring system for aerospace applications is to provide well-defined resolution including a quantifiable probability of detection (POD) and damage size/severity along with measure of uncertainty when a damage is detected. Although many SHM systems have demonstrated that they can detect and locate damage on a structure, providing quantified damage sizes still remains a challenging task, even in a laboratory environment.For the detection of impact damage over large area in composite structures, probability of detection (POD) was introduced as a standard measurement for quantifying the reliability and robustness of built-in structural health monitoring systems. However, traditional POD curves are generated through extensive testing which is not practical for every new structure and sensor configuration. To overcome this difficulty, Acellent has developed a method to compute the POD ,162. AftQing and Beard et al.  proposedThere are also a lot of studies devoted to making use of statistical and machine learning methods to solve the uncertainty problem caused by environmental variation, dispersive effect, measurement error, and so on. The use of an artificial neural network (ANN) was proposed for Lamb wave-based damage detection in laminates by Su et al. . DworakoSupport vector machine (SVM) with robust anti-noise characteristics was employed for damage classification with more detection rate than ANN by Agarwal et al. . Some clAfter more than 20 years of development, SHM technology is being used more and more in the aerospace industry. The objects of SHM application have gradually expanded from simple metallic structures to more complex composite structures, while the monitored physical parameters have expanded from strain and temperature to various kinds of damage, and the diagnostic results have gradually developed from qualitative to quantitative. Focusing on the form diversity, feature complexity, damage diversity and concealment of aircraft structures, exploring the development trend of health monitoring of aircraft structures from monitoring process, innovative sensor design, new monitoring methods or technologies is an important way to improve its technical maturity. In the field of aerospace industry, the development trends of SHM can be summarized as the following ,180:(1)The sensing technology is developing to the multi-field coupling sensing technology.Sensors have been widely applied on aircrafts to monitor the strain, temperature, etc., of the aircraft structure, but the damage detection SHM technology can bring direct benefit to the maintenance of aircraft, which is still in the laboratory and flight testing stage, and also only focuses on small local areas. From relatively simple monitoring of stress/strain, temperature to direct monitoring of damage is an obvious development trend of SHM . In the (2)Sensors are developing towards miniaturization, intellectualization and in-situ integration, and then can be seamlessly integrated with composite structures.With the rapid development of materials science, manufacturing technology, microelectronics and information science, many multi-functional materials have emerged, which can integrate sensing, actuating, communicating and computing. They provide a technical basis for the miniaturization, integration and intelligent development of sensors . It is a(3)The monitoring process is developing to the life cycle of structure design, manufacture, service and maintenance of aircraft structure, especially aircraft composite structure.Composite materials have been and continue to be widely used in aerospace because of their high strength-to-weight and stiffness-to-weight ratios. Due to the complexity of aircraft composite structures and the harsh service environment, the damage and failure mechanisms of composite structures are diverse. It is difficult to fully characterize the service state of composite structure by a single physical parameter. The development of multi-field coupled hybrid sensor networks and combinatorial characterization methods are necessary. The construction of the multi-mode holographic sensing capability, the realization of health monitoring of composite structure from design, manufacture, service to maintenance of life cycle, and the formation of innovative concepts and design methods of smart composites are the important development direction of SHM.(4)Monitoring methods are developing from linearity to nonlinearity, from low frequency to high frequency, while diagnosis results are developing from qualitative to quantitative.With the development of solid mechanics, computational mechanics and non-linear theory, non-linear monitoring methods The performance of SHM systems is developing from detection of event, location and size of damage to the detection of effects.In order to quantify the performance of SHM systems, we can classify them by their output with four sequential levels called Technology Classification Levels (TCL): Detection of event (TCL1), Detection of location (TCL2), Detection of magnitude/size (TCL3), Detection of the effect (TCL 4) . At presStructural health monitoring will have a great impact on structure design, improving safety and reliability, and reducing maintenance and operation costs for aircraft. Among the various types of transducers, piezoelectric materials are being widely used for SHM because they can be used as either actuators or sensors.An overview of piezoelectric transducer-based SHM technology developed in the past two decades was first provided in the paper. SMART Layer-based sensor networks, the principles of active and passive monitoring, and diagnostic algorithms were briefly summarized.The requirements for practical implementation and use of SHM systems in aircraft application, and the challenges needed to be solved in system integration, damage quantification, environmental compensation approach, and airworthiness compliance approach in order that the SHM technology can be widely used in aircraft, were then addressed.Development trends of SHM technology were also discussed in this paper. The object of SHM applications has gradually expanded from simple metallic structures to more complex composite structures, while the the monitoring of physical parameters has expanded from strain and temperature to various kinds of damage, and the diagnostic results have gradually developed from qualitative to quantitative.Although extensive studies are still needed in the areas of damage quantification, environmental compensation, system integration, airworthiness compliance and verification method to fully implement an SHM system in aircraft applications, it is clear that the SHM technology will provide a cost-effective, in-service, cradle-to-grave diagnostic system for monitoring the structural integrity of aircraft.


Neural vasopressin is a potent modulator of behaviour in vertebrates. It acts at both sensory processing regions and within larger regulatory networks to mediate changes in social recognition, affiliation, aggression, communication and other social behaviours. There are multiple populations of vasopressin neurons within the brain, including groups in olfactory and visual processing regions. Some of these vasopressin neurons, such as those in the main and accessory olfactory bulbs, anterior olfactory nucleus, piriform cortex and retina, were recently identified using an enhanced green fluorescent protein-vasopressin (eGFP-VP) transgenic rat. Based on the interconnectivity of vasopressin-producing and sensitive brain areas and in consideration of autocrine, paracrine and neurohormone-like actions associated with somato-dendritic release, we discuss how these different neuronal populations may interact to impact behaviour. In addition to its well characterized role as a neurohypophyseal hormone, vasopressin  acts within the brain to modulate circadian rhythmicity, social recognition, aggression, affiliation and other social behaviours in vertebrates  and accessory olfactory bulbs (AOBs) respectively, where olfactory information undergoes complex processing and integration by multiple intrinsic and extrinsic cell types  and accessory olfactory bulbs (AOBs) of male and female rats and V1aR and V1bR expression and/or binding has been described in the olfactory bulbs of a variety of rodents , suggesting potential para/autoregulation  is a cortical region that integrates olfactory information sent from the MOB with input from the PIR, multiple regions of the amygdala and other brain regions , a component of fox urine, increases Egr-1 in both lateral and dorsal subdivisions of the AON of male and female rats, suggesting a role for this cortical region in the processing of both conspecific and heterospecific signals  is a three-layered cortex that lies caudal to the AON. It is divided into anterior and posterior regions, with the anterior region receiving widespread input from the AON and the posterior region being involved in integrating information from the anterior PIR and other brain regions to construct a perception of odour quality , closely associated with the PIR, sends and receives information from the MeA and COA and represents an area of potential integration between the main and accessory olfactory processing pathways Pitkanen . It projThe olfactory tubercle (OT), an amalgam of multiple functional regions, including the islands of Calleja (IC), is located medial to the PIR Ikemoto . It compThe OT, especially medial aspects, is sensitive to vasopressin. Vasopressin-ir fibres have been described in medial aspects of the OT in male rats and male and female mice , a nucleus in the pons of the hindbrain, sends noradrenergic inputs to the olfactory bulbs, AON, PIR and OT  are multi-faceted interconnected structures with a number of reciprocal connections with olfactory processing areas and other brain regions  and MeA (accessory olfactory system) are interconnected  is the location of the mammalian master clock, which coordinates peripheral clocks and facilitates circadian physiology, morphology and behaviour  by activating V1aR, V1bR and oxytocin receptors to mediate changes in olfactory and light/visual processing and social behaviour. Vasopressin signalling systems sometimes show differences associated with the natural history of the species examined, developmental stress, social condition and the ebb and flow of gonadal steroids across annually repeating life history stages. Some vasopressin signalling pools are sexually dimorphic, while others are not. All of these factors must be considered to achieve a more complete understanding of vasopressin’s role in sensory processing and behavioural modulation.


Angiogenesis—the sprouting and growth of new blood vessels from the existing vasculature—is an important contributor to tumor development, since it facilitates the supply of oxygen and nutrients to cancer cells. Endothelial cells are critically affected during the angiogenic process as their proliferation, motility, and morphology are modulated by pro-angiogenic and environmental factors associated with tumor tissues and cancer cells. Recent in vivo and in vitro studies have revealed that the gap junctions of endothelial cells also participate in the promotion of angiogenesis. Pro-angiogenic factors modulate gap junction function and connexin expression in endothelial cells, whereas endothelial connexins are involved in angiogenic tube formation and in the cell migration of endothelial cells. Several mechanisms, including gap junction function-dependent or -independent pathways, have been proposed. In particular, connexins might have the potential to regulate cell mechanics such as cell morphology, cell migration, and cellular stiffness that are dynamically changed during the angiogenic processes. Here, we review the implication for endothelial gap junctions and cellular mechanics in vascular angiogenesis. Vasculogenesis leads to the creation of the heart and the first primitive vascular plexus inside the embryo and its surrounding membranes, as can be observed in yolk sac circulation. In contrast, angiogenesis is the physiological process of neovascular formation from pre-existing blood vessels during adult tissue homeostasis and tumorigenesis , and is The vascular network, which supplies oxygen and nutrition, is necessary for tumor growth and cancer cell proliferation. Anti-tumor angiogenic therapy has shown some promise in the treatment of several cancers . AlthougGap junctions consist of connexin (Cx) family protein, which has four transmembrane domains and two extracellular loop domains ,20. The Post-translational modification of Cxs carboxyl terminal residues play an important role in the regulation of the Cx protein life cycle to include oligomerization, trafficking, gap junction formation, gating function, and internalization ,29,30. CEndothelial cells play a critical role in regulating vascular inflammation , blood cCx expression pattern in endothelial cells is dependent upon vessel type, be it arteries, veins, or lymphatic vessels. Cx37 and Cx40 are co-expressed in the arterial endothelial cells of healthy vessels ,54, wherSeveral studies have shown that aberrant gap junction function and Cx expression in endothelial cells contributes to the promotion of endothelial dysfunction and vascular inflammatory diseases such as atherosclerosis. For example, Cx37 and Cx40 are found at lower levels during the early stage of atherosclerosis , while dPro-angiogenic factors that are released from tumor tissues and cancer cells ,74, are In addition to VEGF, basic fibroblast growth factor (bFGF) and hypoxia are well known as a pro-angiogenic factor and a conducive environment. It has been reported that microvascular endothelial cells facilitate gap junction function and Cx43 expression in response to bFGF stimulation . StimulaEndothelial gap junction function and Cx expression are assuredly regulated by pro-angiogenic and environmental factors. It has also been reported that Cx expression and gap junction function in tumor cells , myocadiSeveral groups have investigated the impact of Cxs on the development of the cardiovascular system, which is closely related to angiogenesis. Mutations in the gene for Cx43 (GJA1) were found to cause a hypoplastic left-heart syndrome . Cx43-deSome reports have shown the relevance of endothelial Cxs expression and vascular angiogenic potential in endothelial cells using in vitro angiogenesis assay. Knockdown of Cx43 using specific siRNAs reduces tube formation and cell proliferation of human aortic endothelial cells . The dowWhile these studies have provided extensive evidence that endothelial Cx expression modulates angiogenesis, the specific impact of each Cx on angiogenesis remains unclear. Notably it is believed that any endothelial Cx expression may modify the expression of other Cxs ,105,107.Endothelial cells dynamically alter cell mechanics such as cell morphology, cell proliferation, and cell migration during angiogenesis ,109. EndThe intracellular domain of Cx protein interacts with other proteins to aid the structural stability of cell-cell junctions sustained by cytoskeletal scaffolds . Due to The interaction between Cx and cytoskeletal proteins contributes to the regulation of cellular stiffness which is defined as the physical property of a cell to resist deformation in the response to any applied force. A contraction force generated by the actomyosin cytoskeleton and F-actin has been inseparably linked to the regulation of cellular stiffness ,126. ActIt is thought that endothelial cellular stiffness may be a determinant factor of leukocyte adhesion to endothelium . In geneIn addition to leukocytes, endothelial cells themselves have been shown to modulate their own migration, proliferation, and morphological changes in response to extracellular substrate stiffness ,137. In We are beginning to understand that gap junction and Cx in endothelial cells might serve as a connection center between biological function and cell mechanics in the context of angiogenesis. In this review, we provided an overview of the endothelial gap junction function and Cxs expression found under pro-angiogenic conditions and the functional role of endothelial gap junctions and Cxs in cell mechanics during the angiogenic process. Although several studies have demonstrated gap junction-/Cx-dependent regulation of angiogenesis, these mechanisms are still speculative and controversial. Notably, analysis of alterations to gap junction functionality and Cx expression patterns in pro-angiogenic stimuli in neovasculature are needed in order to properly characterize these de novo blood vessels involved in tumor angiogenesis. The involvement of gap junction and Cx in the regulation of cellular stiffness and cellular mechanics via their interaction with intracellular molecules has been shown; however, the dual roles of Cx as a mechanosensor and mechonotransducer remain unclear. Cellular stiffness- and mechanics-based mechanisms hold promise of helping us better understand the physiological and pathological components of angiogenesis and endothelial cell functionality. Additionally, gap junction- and Cx-mediated cell-cell interactions in a number of other cell types such as vascular smooth muscle cells, pericytes, fibroblasts, macrophages, and tumor cells also contribute to tumor angiogenesis through the expression of pro-angiogenic factors. Thus, further studies on the basic biology of gap junctions and Cxs in these type cells are needed for further elucidation, with a particular emphasis on the interplay of angiogenesis and tumor development. We speculate that gap junction and Cx targeting approaches may be relevant to the development of new treatments for cancer patients.


Mycobacterium tuberculosis infection in humans. Severe inflammation and extensive tissue damage drive the morbidity and mortality of this manifestation of tuberculosis (TB). Antibiotic treatment is ineffective at curing TBM due to variable and incomplete drug penetration across the blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barriers. Adjunctive corticosteroid therapy, used to dampen the inflammation, and the pathologic manifestation of TBM, improves overall survival but does not entirely prevent the morbidity of the disease and has significant toxicities, including immune-suppression. The rabbit has served as a fit for purpose experimental model of human TBM since the early 1900s due to the similarity in the developmental processes of the brain, including neuronal development, myelination, and microglial functions between humans and rabbits. Consistent with the observations made in humans, proinflammatory cytokines, including TNF-α, play a critical role in the pathogenesis of TBM in rabbits focusing the attention on the utility of TNF-α inhibitors in treating the disease. Thalidomide, an inhibitor of monocyte-derived TNF-α, was evaluated in the rabbit model of TBM and shown to improve survival and reduce inflammation of the brain and the meninges. Clinical studies in humans have also shown a beneficial response to thalidomide. However, the teratogenicity and T-cell activation function of the drug limit the use of thalidomide in the clinic. Thus, new drugs with more selective anti-inflammatory properties and a better safety profile are being developed. Some of these candidate drugs, such as phosphodiesterase-4 inhibitors, have been shown to reduce the morbidity and increase the survival of rabbits with TBM. Future studies are needed to assess the beneficial effects of these drugs for their potential to improve the current treatment strategy for TBM in humans.Tuberculous meningitis (TBM) is the most devastating form of extrapulmonary  Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is transmitted via aerosols exhaled by patients with active disease and acquired by new contacts via inhalation of infectious droplets, resulting in the establishment of infection in the lungs. However, the bacteria can be transported by infected macrophages/dendritic cells from the lungs to other organs, such as lymph nodes, the spine, and the brain, resulting in disseminated extrapulmonary disease  treatment modalities for this disease. Thus, aspirin and thalidomide, both known to have anti-inflammatory properties, have been tested for their ability to control pathogen-mediated inflammation in TBM . This non-barbiturate sedative, shown to be a potent antiemetic drug was widely prescribed for the treatment of morning sickness among pregnant women during the early 1960s. Although it became commonly used in Europe and Asia, the US Food and Drug Administration did not approve the sale of thalidomide in the US, due to concerns about the safety of the drug. This concern was later compounded by the finding that administration of thalidomide to pregnant women resulted in teratogenic effects, such as limb malformation in the newborns  and confirmed with the presence of Iba-1, a marker of microglia. A PET-imaging study of pediatric rabbits with TBM revealed localization of the tracer around TB lesions in the brain and the site of injection at the 21st-day post-infection. In these animals, enlargement of the TB lesion was noted with time. The bacterial burden in brains, with superficial medial tuberculoma, increased until 14 days of infection after which it remained stable. Gradual dissemination of Mtb into the lungs was observed until 21 days that became stable with no significant increase until 35 days post-infection. At this time, microscopic examination revealed deep tuberculomas with increased Mtb CFU.The developmental stages of the brain in children makes them highly vulnerable to TBM. To understand the pathogenesis of TB during early brain development, a pediatric rabbit model of TBM was created (Tucker et al., 11C rifampin, isoniazid, and rifampicin for 6 weeks, followed by PET imaging. Results from this study revealed limited penetration of rifampin that diminished as early as 2 weeks post-treatment. PET scans also unveiled absence of antibiotics at the site of TB lesions (Tucker et al., The penetration of antibiotics across the BBB during a multi-drug treatment regimen was evaluated in the pediatric rabbit TBM model (Tucker et al., Taken together, the rabbit model of TBM recapitulates the pathologic manifestations seen in human disease. This model is useful to test therapeutic interventions after the development of clinical signs and to evaluate the effect of different regimens of anti-TB and immunomodulatory treatments on the course of the disease (Sampaio et al., Although the rabbit model recapitulates several clinical and pathologic manifestations of TBM in humans, it has some limitations. In the rabbit model, Mtb is inoculated directly into the intra-cisternal region. This may not reflect the natural dissemination that occur in patients with TBM. Similarly, disease manifestations of TBM in rabbits are associated with the nature of the mycobacterial strains and the infectious inoculum used. However such associations are not well-established in human patients. Therefore, future studies should fine-tune the rabbit and potentially other animal models of TBM, to mimic the natural route of Mtb transmission, without compromising the pathology of human disease. Nonetheless, experimental TBM in outbred rabbits displays the heterogeneity in host response, as seen in human patients (Tsenova et al., During the last decade, several structural analogs of thalidomide have been synthesized and evaluated for their potency as selective TNF-α inhibitors (Corral et al., M. bovis, was shown to recapitulate the pathophysiology of human TBM (Tsenova et al., A rabbit study of sub-acute CNS mycobacterial infection, established by intra-cisternal injection of low-dose pathogenic In contrast to IMiDs, SelCIDs selectively inhibit TNF-α production and do not stimulate lymphocyte proliferation; they have minimal effect on the production of other pro-inflammatory monocyte cytokines (Gordon and Goggin, Since TBM continues to have an unacceptably high morbidity and mortality rate, particularly in young children, there is an urgent need for new, efficient adjunctive therapies for this devastating manifestation of TB. Results from the published literature suggest that the rabbit model of TBM can provide a useful tool for expanding our understanding of the pathologic process of CNS infection and disease. This preclinical model can facilitate the selection of optimal candidate adjunctive therapies to control the disease and improve the outcome of Mtb infection in the CNS of humans. The safety profile of PDE4i, described in this review, and their selective inhibition of monocyte-derived TNF-α, identify them as potantially efficacious adjunctive drugs to be used with antibiotics for the successful treatment of human TBM.CV and SS conceived the concept. GK and SS designed the framework. RK, PS, AK, CV, GK, and SS wrote the manuscript. CV, GK, and SS edited the manuscript. All authors have read and agreed to publish the manuscript.GK was a member of the board of directors of Celgene corporation from 1998-2018. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The autonomic nervous system (ANS) plays a major role in maintaining homeostasis through key adaptive responses to stress, including severe infections and sepsis. The ANS-mediated processes most relevant during sepsis include regulation of cardiac output and vascular tone, control of breathing and airway resistance, inflammation and immune modulation, gastrointestinal motility and digestion, and regulation of body temperature. ANS dysfunction (ANSD) represents an imbalanced or maladaptive response to injury and is prevalent in pediatric sepsis. Most of the evidence on ANSD comes from studies of heart rate variability, which is a marker of ANS function and is inversely correlated with organ dysfunction and mortality. In addition, there is evidence that other measures of ANSD, such as respiratory rate variability, skin thermoregulation, and baroreflex and chemoreflex sensitivity, are associated with outcomes in critical illness. The relevance of understanding ANSD in the context of pediatric sepsis stems from the fact that it might play an important role in the pathophysiology of sepsis, is associated with outcomes, and can be measured continuously and noninvasively. Here we review the physiology and dysfunction of the ANS during critical illness, discuss methods for measuring ANS function in the intensive care unit, and review the diagnostic, prognostic, and therapeutic value of understanding ANSD in pediatric sepsis. The autonomic nervous system (ANS) is a unique system as it regulates functions in nearly all organ systems . Along wIn response to severe infections and other injuries, the central nervous system (CNS) activates the sympathetic branch of the ANS in order to make the necessary physiological and metabolic adjustments to overcome the acute physiologic stress . ANS dysThere are several reasons why understanding ANSD in the context of sepsis is crucial to the care of the critically ill septic child. First, sepsis is defined as a “life-threatening organ dysfunction caused by a dysregulated host response to infection” . AlthougIn this paper we will review the physiology and dysfunction of the ANS during critical illness, discuss methods for measuring ANS function in the pediatric intensive care unit, and review the diagnostic and prognostic value of ANSD in pediatric sepsis.The ANS is responsible for regulating all innervated organs , a remarkable task often overlooked by clinicians , 25, 26.sympathetic nervous system (SNS) is often characterized as the system in charge of the “fight or flight” response; however, it also carries important roles in day-to-day, non-danger situations  is often referred to as the “rest and digest” system, and is in charge of energy conservation, digestion, and waste removal. The vagus nerve, which innervates most organs and tissues between the larynx and the small intestine, controls the vast majority of parasympathetic functions in the body . Loss of function is best exemplified by spinal cord injuries, where the lack of sympathetic tone leads to orthostatic hypotension, low resting blood pressure, loss of circadian blood pressure fluctuation, and bradycardia . Sympath2 receptors. Sympathetic input increases SA node-mediated heart rate (chronotropy), AV node and ventricular conductivity (dromotropy), and atrial and ventricular contractility (inotropy). Parasympathetic input through the vagus nerve has a negative effect on all of these cardiac functions, except ventricular contraction, which is unaffected . Whe. Whe7). The best-described ANS-immune system interaction occurs through the cholinergic anti-inflammatory pathway, which modulates the innate immune system response. Initially the CNS senses inflammation through both the humoral route (mediated by cytokines) and the neural route ] , 5. This1-adrenergic receptors in reticuloendothelial macrophages can lead to pro-inflammatory cytokine release, although in prolonged septic states, epinephrine down-regulates IL-6 and TNF-α production . In . In 1). Activation of the HPA axis results in cortisol release from the adrenal cortex, while activation of the SNS leads to a release of catecholamines from the adrenal medulla. The net effect of these pathways leads to increased glucose production and availability, which is an adaptive response to stress designed to enhance energy availability to vital organs . CortisoThe enteric nervous system branch of the ANS plays a key role in the modulation of gastrointestinal motility and digestion . The vagThe ANS appears to also have a direct effect on the gut microbiome. For example, microbes are capable of responding to neuroendocrine hormones. Specifically, catecholamines may induce microbial change to more pathogenic phenotypes, and these microbes may have exponential growth in response to host stress within hours of sensing higher catecholamine levels , 36. In Core body temperature is tightly regulated by the CNS via behavioral and autonomic responses. The behavioral responses  are by far the most powerful strategies, but many critically ill patients lack the ability to implement behavioral responses and thus depend heavily on autonomic defenses such as sweating and pre-capillary vasodilation in response to heat, and arteriovenous (AV) shunt constriction and shivering in response to cold . The AV When hyperthermic, skin vasodilation can have significant impact on hemodynamics given that skin blood flow can increase up to the equivalent of 60% of the cardiac output . In homePotential confounders that could affect the measurement of ANS function in the pediatric intensive care unit include age, mechanical ventilation, neuromuscular blockade, the administration of catecholamines and sedatives, and pre-existing autonomic dysregulation. Some studies in critically ill adults suggest that mechanical ventilation and catecholamines do not affect HRV or baroreflex and chemoreflex sensitivity , 39. SedCardiovascular homeostasis requires continuously varying levels of sympathetic and parasympathetic inputs to the heart, resulting in a continuously changing heart rate, even during normal resting conditions . HRV is There are several examples of successful evaluations of real-time analysis of HRV in the intensive care unit , 43–45. Similar to HRV, the ANS regulates variability in the respiratory rate with input from chemoreceptors and baroreceptors . DysreguThe baroreflex sensitivity measures the ability of the ANS to modulate the vagal and sympathetic tone with changes in blood pressure . InvasivStimulation of the peripheral and central chemoreceptors by decreases in arterial oxygen levels and/or pH or increases in arterial carbon dioxide produce an increase in respiratory rate and sympathetic tone. In the ICU, chemoreflex sensitivity can be calculated as the regression slope of arterial oxygen tension to heart rate interval duration . DecreasAs a key component of the host response to infection, dysregulation of the ANS response is likely an important contributing factor to organ dysfunction in patients with sepsis . ANSD, t2 agonists like dexmedetomidine and ß1-blockade with esmolol has been associated with improvement in vasoplegia in shock states (Advanced understanding of ANSD pathophysiology in pediatric sepsis could also help identify potential therapeutic targets. Sympathetic over-activity, which is common in sepsis and other critical illnesses, can lead to tachyarrhythmias, diastolic dysfunction, inflammation, immune suppression, platelet aggregation, and gastrointestinal dysmotility , 27, 33.k states . Statinsk states , 58. Andk states .The ANS plays a major role in maintaining homeostasis in response to infections and other types of stress. ANSD represents an imbalanced or maladaptive response and is prevalent in pediatric sepsis. Better understanding of ANSD in pediatric sepsis could have significant diagnostic, prognostic, and therapeutic implications.LS-P and CB designed the review. All four authors made substantial contributions to drafting and final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AI and handling Editor declared their shared affiliation.


To define a profile of the muscle groups affected by patellofemoral pain syndrome (PFPS) to determine a pattern of functional weaknesses around the knee. Sixty-three female patients were randomly selected, and 17 included in this study, receiving a clinical evaluation with pre-established protocol which evaluated the quadriceps, abductors, range of motion in the internal rotators and ankle dorsiflexors, pelvic tilt, and dynamic valgus. Losses were seen in abductor strength and ankle dorsiflexor range of motion in comparison with the contralateral limb . There was a loss of quadriceps strength and decreased amplitude of internal hip rotation when compared to the Kujala score . Dynamic valgus and pelvic fall were greater in comparison with the Kujala score . Clinical evaluation showed deficits in the quadriceps and abductor muscle groups, as well as decreased range of motion in the internal hip rotators and ankle dorsiflexors and increased dynamic valgus and pelvic drop. Level of Evidence IV; Case series. Consequently, many authors have emphasized exercises to strengthen the vastus medialis obliquus (VMO).The objective of this study is to define a profile of muscular involvement and mechanical changes involved in PFPS in order to determine a pattern of involvement for functional weaknesses around the knee, optimizing future training protocols to treat PFPS.We randomly selected 63 female patients; of these, 17 with knee pain were effectively assessed after the exclusion criteria were carefully applied. All patients underwent magnetic resonance imaging (MRI) and were evaluated by the same team composed of three orthopedic physicians and two physiotherapists.The inclusion criteria were: female patients between eighteen and forty-five years with anterior knee pain. The exclusion criteria were: any degenerative changes of the knee or hip, history of patellar dislocation, clinical evidence of knee instability, meniscal or other intra-articular injuries, evidence of edema, Osgood-Schlatter disease or Sinding-Larsen-Johanssen syndrome, patellar tendinopathy, chondral injury, osteoarthritis, neurological involvement affecting the gait, joint or muscle injuries in the hip, lumbar pain, sacroiliac pain, patients who previously received surgical treatment in the knee or hip joints, and bilateral complaint of anterior knee pain, since all patients were assessed in comparison with the healthy knee.Within the sample group, muscular strength was assessed in the abductors (SA), in the external rotators of the hip (SEHR), and the quadriceps (QS). A calibrated Hand Held MicroFET 2 manual dynamometer  was used to assess these parameters. Range of motion was assessed via ankle dorsiflexion (RAD) and internal rotation of the hip (RIHR). A calibrated AM-2 angle meter  was also used.The EVA and Kujala scores were calculated, in addition to the degree of dynamic valgus and pelvic drop.Each calculation was repeated three times and the average of these values was used.The results were statistically evaluated by the most appropriate method, using SPSS software . All tests were performed at a 5% significance level.This study was approved by the Brasil Platform for ethics in human research (CAAE: 60238116.9.0000.0023) and an informed consent form was signed by all patients.Clinical evaluation of the groupings in which muscular strength was measured showed only a loss of strength in comparison with the contralateral knee, with a statistically significant result (0.031) for the abductors. When range of movement was assessed, loss was seen in RAD (0.040), as demonstrated in Comparison of dynamic valgus and pelvic drop correlated to the Kujala score  showed statistically significant results .  AccordinThere was a loss in QS and decrease in RIHR when these variables were correlated to the Kujala score .  Accordin,-PFPS is known to involve anterior knee pain without any chondral changes in the femoropatellar joint, according to Petersen et al.-Recent studies have shown that PFPS does not appear in the knee joint, but rather in the decreased amplitude of internal rotation of the femur, due to weakness of the hip abductors (gluteus medius and minimus muscles).Weakness of the gluteus medius and minimus muscles causes pelvic instability, and consequently the patient cannot support the pelvis for one minute while standing on the affected leg, thus determining pelvic drop, as reported by Petersen et al.,,,Besides pelvic instability, weakness of the hip muscles causes a leg alignment known as dynamic valgus.To the best of our knowledge, no single assessment combining results related to the muscular forces around the knee and the biomechanical changes that corroborate with the etiology of PFPS can be found in the recent literature. Although we effectively evaluated 17 patients, a number that could represent bias in our results, this bias was minimized by the regularity and expressive statistical results of the evaluated parameters, and our study was strengthened by the selective exclusion method applied.Understanding PFPS is absolutely essential in order to be able to treat this condition, and our results can guide future training efforts for treatment.Patients with PFPS in this study demonstrated a reduction in SA, decrease in RIHR and reduction of RAD, and increase in pelvic tilt and dynamic valgus.


Treatment with these chemicals produced a five- to sevenfold higher yield of grossly visible hepatic tumors in old rats compared to young animals. The enhanced susceptibility of the aged livers to the carcinogenic effect of PPAR agonists could not be explained by differences in levels of peroxisomal and/or cell proliferation between young and old animals, as neither of these responses was exaggerated with aging. Reported studies have shown that activating PPARa results in the suppression of hepatic apoptosis. This effect is expected to diminish the ability of the liver to purge itself of pre-existing neoplastic cells, allowing them to progress to tumors. New findings from our laboratories show that the aged liver is exceedingly sensitive to the antiapoptotic effect of PPAR agonists. In addition, aged livers showed remarkably higher levels of the antiapoptotic protein Bcl-2 than livers of young, adult, and middle-aged animals. Interestingly, the PPARa agonist Wy-14,643 significantly diminished elements of the proapoptotic machinery  in the aged liver, while remarkably increasing elements of this machinery in younger animals. Taken together, while activation of PPARs appears to inhibit apoptosis in livers of senescent animals, activating these receptors seems to stimulate the apoptotic machinery in young animals. This paradoxical effect may be responsible for the exaggerated sensitivity of the aged liver to the carcinogenic effect of agents that activate PPARs.Exposure to agonists of peroxisome proliferator-activated receptor alpha (PPAR


Challenges in organ transplantation such as high organ demand and biocompatibility issues have led scientists in the field of tissue engineering and regenerative medicine to work on the use of scaffolds as an alternative to transplantation. Among different types of scaffolds, polymeric hydrogel scaffolds have received considerable attention because of their biocompatibility and structural similarity to native tissues. However, hydrogel scaffolds have several limitations, such as weak mechanical property and a lack of bioactive property. On the other hand, noble metal particles, particularly gold (Au) and silver (Ag) nanoparticles (NPs), can be incorporated into the hydrogel matrix to form NP–hydrogel composite scaffolds with enhanced physical and biological properties. This review aims to highlight the potential of these hybrid materials in tissue engineering applications. Additionally, the main approaches that have been used for the synthesis of NP–hydrogel composites and the possible limitations and challenges associated with the application of these materials are discussed.  The application of hydrogel incorporated with metal nanoparticles (NPs) has become a new emerging research area in tissue engineering and regenerative medicine. Disease, injury, and trauma often resulted in tissue damage and degeneration. For repair, replacement, or regeneration, the treatment normally involves the transplantation of tissue from the same patient (autograft) or another individual . However, the treatments are risky, because autografts can lead to donor-site morbidity due to infection and hematoma, whereas allografts might be rejected by the host immune system . FurtherIn order to overcome the challenges of high organ demand and biocompatibility issues, scientists in the field of tissue engineering and regenerative medicine are working on the use of scaffolds as an alternative to transplantation. These scaffolds are developed to mimic the extracellular matrix (ECM), act as structural support, and define the potential space for new tissue development as well as enhance the cell attachment, proliferation, and differentiation . In addiThere are various kinds of materials that have been used to facilitate and develop the tissue engineering scaffolds. The examples are metals, natural and synthetic polymers, and ceramics . Among dAlthough noble metal NPs and hydrogel alone have been well-characterized, the research on the application of noble metal NP–hydrogel composites as tissue engineering scaffolds is still limited. In this review, we will focus on recent studies on the development of noble metal NP–hydrogel composites for tissue engineering purposes. We will also discuss the main approaches for the fabrication of the composite materials. In addition, we will point out some possible limitations and challenges related to the application of these hybrid materials.Over the years, noble metal NPs and hydrogels have been widely studied as potential biomaterials for tissue regeneration. Today, scientific innovations have led to the emergence of the composite material made of noble metal NPs and hydrogel, forming an inorganic–organic framework with improved properties. In the past two decades, nanoparticles have been widely studied for a wide range of applications, and noble metal nanoparticles are the attractive nanomaterials due to their uniqueness such as resistance to corrosion and oxidation, and non-reactiveness . Among tAu NPs are also known as colloidal gold. They can be easily prepared in the diameter range of three to 200 nm, as well as in different shapes such as gold nanocubes , gold naFor Ag NPs, they are typically one to 100 nm in diameter . SimilarOther than Ag and Au, the applications of platinum (Pt) NPs have also been explored. For example, iron-Pt (FePt) magnetic NPs were synthesized and utilized to enhance the cell infiltration and distribution of cells within the poly(lactic-co-glycolic acid) salt-leached scaffolds, with a neodymium magnet placed at the bottom . The effA hydrogel is a three-dimensional (3D) network that is composed of cross-linked synthetic or natural polymers. The common natural polymers that are being used are alginate, chitosan, collagen, and gelatin, and examples of synthetic polymers are polyethylene glycol (PEG), polyacrylamide, and polydimethylsiloxane . HydrogeIn the study reported by Zhao et al., a photocrosslinkable gelatin hydrogel was synthesized for skin tissue engineering . The mec3O4 NPs have been loaded into chitosan/PEG hydrogel. The incorporation of the magnetic NPs has resulted in the higher viability and osteogenic differentiation ability of mesenchymal stem cells − ions, followed by the reduction of [AuCl4]− ions by the collagen hydroxyproline residues to form Au NPs that act as the crosslinkers of collagen chains [Another interesting method that has been applied for the preparation of NP–hydrogel composites is to use the metal NPs as the crosslinker. For example, Skardal et al. have utilized the multivalency of Au NPs and applied them to crosslink a printable semi-synthetic extracellular matrix hydrogel consisted of thiol-modified biomacromonomers derived from hyaluronic acid and gelatin for tissue engineering application . Xing etn chains . Recently, this approach has been adopted to crosslink deoxyribonucleic acid (DNA)-Au NP hydrogel. This created new potential applications of this composite material such as bioimaging, diagnostics, and therapeutics . Other tThe potential of noble metal NPs and hydrogel in tissue regeneration have stimulated the high interest of the researchers to further characterize the property of the hybrid of these materials. To address this, the studies on NP–hydrogel composites for regeneration of tissues such as soft tissues, bone tissues, and cardiac tissues are discussed as below.Escherichia coli and Staphylococcus aureus was also reported. The in vivo results have shown that the composite material was efficient at resisting foreign-body reactions and the formation of collagen capsule, allowed cell migration and infiltration [Biocompatibility is an important criterion for the design of biomaterials for soft tissue transplantation. Xu et al. have tested the cytocompatibility of Ag NPs loaded poly(hydroxyethyl methacrylate) hydrogel with mouse embryo fibroblasts. The strong anti-bacterial response toward ltration . Recentlltration . For theltration . As demoltration . Based oltration . On the other hand, in a long-term in vivo study conducted by Grant et al., the Au NP-containing hydrogel was injected into the swine ears. The presence of Au NPs in the construct has probably improved the longevity of the material, since Au NPs have hindered the binding sites of collagenase. Interestingly, the irritation level of the material was retained at the low level, suggesting the nanocomposite as a potential biocompatible soft tissue filler . In a stBased on the gathered findings, the hydrogels containing Au and Ag NPs showed their potential in different types of soft tissue engineering due to their biocompatibility and multiple bioactivities such as antimicrobial and anti-inflammatory properties. The number of cases of bone fractures is increasing every year due to the high frequency of accidents and diseases. Until today, the repair of the infected bone defect remains challenging, creating a demand for the functional biomaterials with osteogenesis and anti-bacterial properties for infected bone repair . TherefoGonzález-Sánchez et al. have demonstrated the synthesis of Ag NPs-based methacrylate hydrogels as a potential biomaterial for bone graft applications. The composite hydrogel exhibited excellent biocompatibility with osteoblast cells. In addition, anti-bacterial activity was reported when the NPs were incorporated by the post-mineralization absorption method . In anotThe positive effect of metal NP-containing scaffolds was not limited to the osteoblast cells. Tentor et al. have demonstrated the enhanced proliferation and growth of preosteoblastic mouse cells cultured on chitosan/pectin thermosensitive hydrogels containing Au NPs . On the Taken all together, Au and Ag NPs were shown to be beneficial to bone tissue engineering, and they were able to retain their bioactivities after being encapsulated into the 3D structure of the hydrogel.In the human body, the heart is a strong power pump due to the myocardium that is made up of tightly packed uniaxial cytoarchitecture and electrically conductive Purkinje fibers, which supply an electrical conductive signal through the whole heart . The regRecently, a study has been conducted on the collagen hydrogel composites containing peptide-modified nanoAu and nanoAg. The neonatal rat cardiomyocytes seeded on the scaffold showed enhanced proliferation, and increased the level of connexin-43 in the presence of electrical stimulation . These fOther than cardiomyocytes, the effect of Au nanocomposites on stem cells has also been explored. A thermoresponsive hydrogel composite made of chitosan and chitosan-stabilized Au NPs has been synthesized and characterized. The incorporation of Au NPs has promoted the differentiation of mesenchymal stem cells into cardiac lineages. This effect could also be due to the presence of electrical cues in the matrix as contributed by the electroconductive property of the Au NPs . Based oThe potential of Au and Ag-containing hydrogels in applications such as skin, bone, and cardiac regeneration have been particularly highlighted and summarized in 2 cells, which are the kidney epithelial cells from Macaca mulatta [Based on the studies as discussed, the incorporation of noble metal NPs such as Ag and Au NPs have added advantageous functionality to the hydrogel scaffolds. However, there are concerns on the cytotoxic effect of the metal NPs, because the interaction of NPs with cells remains controversial, and the mechanism has not yet been fully understood ,92. Inde mulatta . Other t mulatta . This ceIn NP–hydrogel composites, the NPs were entrapped in the 3D structure of the hydrogel, and massive NPs uptake by cells could be avoided upon implantation . HoweverAs evident in the research studies presented in this review, the incorporation of noble metal NPs into hydrogel formulations appeared to be promising in enhancing the functionality of the materials from physical and biological aspects. However, most of the studies presented only involved short-term cell-based and in vivo studies. There is still a large gap between the knowledge of the short-term and long-term effect of the NP–hydrogel composites. Besides, to date, knowledge on the interaction of the materials with cells has been only limited to a few commonly studied cell types. Hence, further studies are crucial in order to fill in this gap, as the complex process of tissue regeneration in real life takes more time and involves the interaction of multiple cell types.


Nanotechnology has a wide range of medical and industrial applications. The impact of metallic nanoparticles (NPs) on the proliferation and differentiation of normal, cancer, and stem cells is well-studied. The preparation of NPs, along with their physicochemical properties, is related to their biological function. Interestingly, various mechanisms are implicated in metallic NP-induced cellular proliferation and differentiation, such as modulation of signaling pathways, generation of reactive oxygen species, and regulation of various transcription factors. In this review, we will shed light on the biomedical application of metallic NPs and the interaction between NPs and the cellular components. The in vitro and in vivo influence of metallic NPs on stem cell differentiation and proliferation, as well as the mechanisms behind potential toxicity, will be explored. A better understanding of the limitations related to the application of metallic NPs on stem cell proliferation and differentiation will afford clues for optimal design and preparation of metallic NPs for the modulation of stem cell functions and for clinical application in regenerative medicine. Stem cells possess unique properties, such as self-renewal and the ability to differentiate into multiple lineages based on the specific lineage inducer, which can be utilized for disease therapy . As showESCs are obtained from the inner cell mass of the growing blastocyst and have the potential to differentiate into ectoderm, endoderm, and mesoderm. At present, ethical issues and immune stimulation limit the application of ESCs in the regenerative medicine . The iPSOn the other hand, ASCs, such as mesenchymal stem cells (MSCs), can be easily isolated from a variety of body organs and tissues, such as fat tissue, bone marrow, skin, brain, and bone. ASCs possess self-renewal properties and the capacity to differentiate into several lineages that emulate the original tissue or organ. In addition, the potency of MSCs to differentiate into bone, fat tissue, chondrocytes, and myocytes is evidenced, whereas their differentiation to other lineages, such as neurons, cardiomyocytes, and hepatocytes, is still debatable . HoweverGenerally, stem cell differentiation is laborious, as it requires formulation of various protocols and the addition of a wide spectrum of factors, undefined components, and chemicals that pose an economic burden, and their mechanism of action is not completely characterized. In this context, nanotechnology represents a powerful tool to overcome the drawbacks of the conventional method for promoting stem cell proliferation and differentiation, while improving the efficacy of stem cell differentiation and its applicability to regenerative medicine ,7.The vast scientific interest in the application of nanomaterials in stem cells is attributed to their easy preparation methods, biocompatibility, unique physical and chemical properties, and their efficient interaction with biomolecules. The applications of nanomaterials in stem cell tracking and therapy have been highlighted by various previous reports ,9,10, an2), cerium oxide NPs (nanoceria), Silicate NPs (SiNPs), and iron oxide NPs (IONPs) are metal oxide NPs, which are well-known with biomedical and pharmaceutical applications. Metallic nanoparticles (NPs), with a dimension of 1–100 nm, were first identified in 1857 by Michael Faraday who reported the presence of metallic NPs in an aqueous solution when he recognized the formation of ruby colored-AuNPs after a reaction of gold salt in aqueous solution . CompareThe presence of high energy on the surface of metallic NPs results in metal-metal aggregation and, therefore, synthesis of a stable colloidal solution of metallic NPs is challenging . AccordiA wide range of nanomaterials that possess various properties and functions have been discovered. The tiny size of nanomaterials allows their efficient interaction with the cell surface and various cellular components . Drug deIn this review, we will present a brief overview of the biomedical applications of metallic NPs, as well as the association between the nanotechnology and stem cells and their downstream applications. In particular, the influence of metallic NPs on stem cell proliferation and differentiation and the possible mechanism behind these effects will be elucidated in detail. In addition, the negative effects and toxicity of metallic NPs on stem cells will also be discussed. The mode of action of metallic NPs in stem cell differentiation and proliferation is still obscure. Thus, better understanding of the link between metallic NPs and stem cells will pave the way for therapeutic applications of metallic NPs in regenerative medicine. A number of published studies have highlighted the application of metallic NPs in medicine and disease therapy via various mechanisms. Here, we will briefly summarize the main biomedical applications and the possible mechanisms involved. The application of nanomaterials in the diagnosis of disease has been shown enhanced the speed, sensitivity, and accuracy of the diagnostic process. NPs are considered efficient labeling materials due to their photochemical stability and the fact that their emission spectrum can be modulated . The SPRMetallic NPs can be suitable tools for the rapid analysis of proteins based on immunoassays. For example, research groups attempted to detect the tumor marker α-fetoprotein via the functionalization of AuNPs with an antibody targeting α-fetoprotein . The binThe surface of metallic NPs could be functionalized with various biological recognition components, such as DNA, protein, peptides, aptamers, and specific receptors to produce nano-biosensors. These nano-biosensors provide an accurate and economically feasible method of analysis . Interes6 cells/well, and after 24 h incubation, cells were washed three times with PBS and treated with two mL/well of a mixture of SPIO and iRGD peptides. The binding to the tumor cells were mediated via αvβ3 integrins. SPIO possessed high biocompatibility with iRGD, allowing for highly sensitive MR cancer imaging . G. G154]. INZEB2, which ultimately promoted the osteogenic differentiation of hBM-MSCs [INZEB2 suppressed the expression of ZEB2, which is a key transcription factor implicated in the inhibition of BMP/Smad-associated activation of the transcription of the osteogenic differentiation. The magnetic field-induced assembly (stripe-like) of magnetic IONPs was exploited for the conversion of primary mouse bone marrow cells into osteoblasts . The inthBM-MSCs . In this3O4/BSA-loaded IONP with an external stimulus, such as a static magnetic field resulted in its high uptake rate into hBM-MSCs and a marked promotion of the osteogenic differentiation [Stimulation of Fentiation . Coatingntiation .Coculture of MSCs with cardiac cells led to the cardiac priming of MSCs and therefore enabled the application of MSCs in cardiac disease therapy. Accordingly, IONPs promoted potential crosstalk between the cardiomyoblasts and MSCs via the upregulation of the expression of a gap junction protein, namely connexin 43 in IONP-exposed cardiomyoblasts . IONP-in2O2 and enhanced the progression of the cell cycle by upregulation of the cell cycle-related proteins, such as cyclin B, cyclin D1, and cyclin-dependent kinase 4 (CDK4) [SPIO NPs promoted the proliferation of hMSCs via abrogating the intracellular H4 (CDK4) . Therefo2SiO4 bioceramic NPs, which was evidenced by calcium mineralization, high ALP activity, and upregulation of the osteogenesis-related markers [Zinc is one of the most plentiful trace metals in the human body and was reported to be essential for the regeneration of bone. In this respect, the differentiation of hMSCs into osteoblasts was enhanced upon their culture with polymeric fibrous polyethersulfone-polyethylene glycol (PES-PEG) electrospun composites coated with willemite or Zn markers . The top markers .The high biocompatibility of silica-containing materials allows them to be favorable for application in bioengineering. SiNPs have been reported to promote the osteogenesis of hMSCs, which was indicated via the activation of ALP and formation of bone nodules . SiNP ofMesoporous SiNPs successfully delivered Nurr1 plasmid DNA and Rex1 siRNA into iPSCs within 6 h, which resulted in their neural differentiation . TherefoThe incorporation of nanoceria into a hydroxyapatite coating led to the promotion of proliferation and osteogenic induction, which was attributed to the activation of BMP signaling . In addiThe modification of the bone scaffold with nanoceria enhanced the proliferation of MSCs, which was mediated through the upregulation of the intracellular calcium level and the consequent increase of the expression level of the angiogenic factor, VEGF, which ultimately enhanced the vascularization of the bone grafts . NanocerBarium titanate NPs coated with glycerol-chitosan enhanced the proliferation and the osteogenic and adipogenic differentiation of rMSCs even at high concentrations . OCT4A and upregulation of stem cell stress responsive genes, OCT4B-164, OCT4B-190, and OCT4B-265. Additionally, high ROS levels were detected upon AgNP treatment; however, polysaccharide-coated AgNPs showed low ROS production, and consequently, low toxicity [As detailed above, metallic NPs showed a wide range of positive effects on stem cell differentiation and have a number of applications in regenerative medicine. However, metallic NPs also have negative effects on stem cells; this should be considered during the application of the metallic NPs in stem cell research. AgNP-exposed human and rat-derived embryonic NSCs showed marked toxicity in a dose-dependent manner . AgNP-intoxicity . Anothertoxicity . In thisRecently, a neurotoxicity study of AgNPs was carried out on hESC-derived neurons and astrocytes and toxic effects and morphological changes were detected in the astrocytes upon exposure to high doses of AgNPs (5.0 µg/mL) . Co-treaA previous study has shown the impact of AuNP and AgNP on the proliferation of human embryonic neural precursor cells . In this2 NPs on the proliferation of MSCs was also reported [2 NPs showed a toxic effect in MSCs in a size-dependent manner, which was highlighted by low cell migration, lack of cell membrane integrity, and suppression of the osteogenic differentiation [2 nanotubes larger than 50 nm showed a drastic decrease in the proliferation and differentiation of MSCs [2–COOH nanorods impeded the osteogenic differentiation of rBM-MSCs that contributed to the induction of the expression level of fibroblast growth factor (FGF-2) and transforming growth factor beta 1 (TGF-β1) [The negative influence of TiOreported . TiO2 NPntiation . TiO2 na of MSCs . TiO2–CO(TGF-β1) .The frequent exposure to copper oxide NPs (CuO NPs), which are found within contraceptive devices, semiconductors, and heat transfer liquids, prompted scientific interest in studying the cytotoxicity of CuO NPs. CuO NPs showed a toxic effect in rBM-MSCs in a dose-dependent manner . MoreoveThe impact of the size and concentration of ZnO NP exposure on the toxicity of NSCs was investigated . ZnO NP-Generally, the special physico-chemical properties of SPIO NPs, such as large surface area and the enhanced reactivity of its surface, allow their transmission across the biological membranes and lead to cytotoxicity via the interaction with the cellular components such as DNA, nucleus, and the mitochondria . The surCollectively, the impact of the metallic NPs on the proliferation and differentiation of various stem cells are summarized in In this review, we attempted to briefly discuss the biomedical application of metallic NPs in cancer therapy, drug delivery, imaging, antimicrobial, and cell tracking. The communication of the NPs with cellular components was also discussed. Nanomaterials possess unique properties that enable them to interact with cell membranes via various mechanisms and to be internalized within the nucleus, leading to the specific activation of crucial transcription factors and signaling pathway-associated molecules. However, further studies of the molecular mechanisms involved in NP-stem cell communication and the cellular retention and release of NPs are needed. In addition, efficient methods for the quantification of NP-stem cell interaction will need to be developed. Recently, extensive research efforts have been directed towards the application of nanotechnology in stem cell research, and several nanomaterials showed high potential for the application of stem cells in disease therapy.Here, we discussed how the metallic NPs had a marked influence on stem cell proliferation and differentiation via various modes of action. In addition, the physical and chemical features of the NPs, as well as their functionalization were implicated in NP-mediated stem cell differentiation and proliferation. Of note, metallic NPs showed differential activity towards stem cell differentiation based on the exposure dose and time, as certain NPs promoted differentiation, while others suppressed differentiation. Accordingly, further research will be needed to determine the optimal NP exposure conditions required to obtain appropriate differentiation of stem cells. Moreover, the mechanisms behind metallic NP-based modulations of stem cell function have not yet been completely characterized, and the contradictory data on the effects of certain metallic NPs on stem cell differentiation will need to be explored further. Furthermore, the potent in vitro effects of metallic NPs on stem cell differentiation will need to be investigated through in vivo studies. There is a dearth of studies related to the impact of metallic NPs on iPSC reprogramming and differentiation, and this research will be of particular interest.We also shed light on the limitations of metallic NPs in stem cell biology, primarily related to NP-mediated toxicity. Unfortunately, several metallic NPs have shown a negative impact on stem cell proliferation and differentiation, which is a concern that should not be overlooked. Therefore, future studies to elucidate the mechanisms behind metallic NP-related stem cell toxicity are necessary.Many controversial studies of the toxicities on certain metallic NPs, IONPs, or SPIO NPs that need further validation. Moreover, the in vitro activities of several metallic NPs need to be confirmed in vivo. The hybrids of the metallic NPs with GO was used to efficiently monitored the neuronal differentiation status of the NSC. In addition the hybrid of SiNPs and GO markedly promoted the neuronal differentiation and the axonal growth of NSCs. However, the mechanism implicated in this effect need further in-depth studies. Moreover, novel hybrids of the metallic NPs with other nanomaterials need to be developed for promoting the high-quality stem cell differentiation and for further application in the clinics. In sum, metallic NPs are capable of modulating stem cell differentiation and proliferation, which highlights their potential for stem cell applications. However, further efforts are needed to find the best design and optimal exposure conditions of metallic NPs in order to obtain good quality differentiated stem cells with minimal toxicity, which will allow the innovation of stem cell therapies for incurable human diseases.


The aim of this study was to examine the impact of the implementation of a new Compassionate Care (CC) curriculum, designed by social workers, on the quality of care provided by Certified Nursing Assistants (CNAs) to residents with Alzheimer’s disease (AD). Additionally, the purpose was to create a collaborative network of CNAs that supported each other. The sample included residents and CNAs from an experimental nursing facility and a control nursing facility. At baseline and 12-weeks, data were collected on AD knowledge, self-efficacy, caregiving satisfaction, and affiliate stigma. CNA changes in terms of their knowledge of AD, self-efficacy, caregiving satisfaction and affiliate stigma were analyzed using a two-way mixed method MANOVA. The stress levels of the residents, specifically agitation and salivary cortisol levels, was examined by testing a hybrid multilevel growth model. The final models were able to show how the changes in the CNAs specifically affected these positive outcomes. CNA knowledge and self-efficacy had the most impact on changing agitation levels, and CNA knowledge and agitation levels had the most impact on salivary cortisol levels. The results of this study showed that integrating a compassionate care curriculum into the work that CNAs perform can lead to positive outcomes on knowledge, self-efficacy, caregiving satisfaction, affiliate stigma and a reduction of agitation and cortisol levels in persons with AD.


Nitrergic enteric neurons are key players of the descending inhibitory reflex of intestinal peristalsis, therefore loss or damage of these neurons can contribute to developing gastrointestinal motility disturbances suffered by patients worldwide. There is accumulating evidence that the vulnerability of nitrergic enteric neurons to neuropathy is strictly region-specific and that the two main enteric plexuses display different nitrergic neuronal damage. Alterations both in the proportion of the nitrergic subpopulation and in the total number of enteric neurons suggest that modification of the neurochemical character or neuronal death occurs in the investigated gut segments. This review aims to summarize the gastrointestinal region and/or plexus-dependent pathological changes in the number of nitric oxide synthase (NOS)-containing neurons, the NO release and the cellular and subcellular expression of different NOS isoforms. Additionally, some of the underlying mechanisms associated with the nitrergic pathway in the background of different diseases, e.g., type 1 diabetes, chronic alcoholism, intestinal inflammation or ischaemia, will be discussed. In the gastrointestinal (GI) tract, as in most other organ systems, nitric oxide (NO) plays a defining role in regulating several functions in both physiological and pathological states. NO contributes to the maintenance of GI mucosal integrity and circulation, regulation of secretion, smooth muscle function or mucosal inflammation ,2. NO, pThe involvement of nNOS and NO pathways in enteric neuropathies was well-described in a variety of disorders, like esophageal or internal anal sphincter achalasia, hypertrophic pyloric stenosis, gastroparesis, Chagas’, or Hirschsprung’s disease . This reEnteric neurons reside in two ganglionated plexuses of the enteric nervous system (ENS). The myenteric plexus is situated between the longitudinal and circular muscle layers of the gut tube and regulates the motility of these smooth muscles. The submucous plexus is embedded in the GI submucosal layer and coordinates the absorption, secretion and circulation of the gut wall . The entIntrinsic primary sensory neurons, interneurons and motoneurons are all present in the ENS, therefore the enteric neurons are able to form local reflex circuits in the intestinal wall and can work autonomously from the central nervous system ,8. At thThe neurochemical phenotype of enteric neurons is critical to their function ,14. The NO can be synthesized by nNOS, endothelial NOS (eNOS) and inducible NOS (iNOS) in the different cell types in the body. In the ENS, the major inhibitory non-adrenergic, non-cholinergic (NANC) neurotransmitter is endogenous NO ,16,17,18The proportion of the nitrergic subpopulation when compared to the total neuronal cell number is shown to be different in the two enteric plexuses. The nitrergic neurons account for only a few percent of all neurons in the submucous plexus , while tIt is well established that NOS-containing myenteric neurons are curiously susceptible to diabetic injuries ,27,28 anIn the submucous plexus, the proportion of nNOS-immunoreactive neurons was doubled in the ileum and tripled in the colon, but not in the duodenum of diabetic rats, while the total neuronal number remained unchanged, suggesting neurochemical adaptation of submucous neurons . These rIn addition, in the microenvironment of enteric neurons, the number of eNOS-labelling gold particles was increased in the capillary endothelium of different gut segments , suggestSex dependency on the diabetic nitrergic dysfunction was also observed ; femalesNOS-containing neurons go through a two-phase degeneration process. The decrease in axonal nNOS expression as the hallmark of the first phase is reversible, however, causing irreversible changes, as apoptotic loss of nitrergic neurons occurs in the second one ,40. In aThe impact of chronic ethanol intake on NOS-immunoreactive myenteric neurons in various gut regions has been investigated in our laboratory for more than a decade. The number of nNOS-immunoreactive neurons was significantly reduced in all investigated intestinal regions after 8 weeks of ethanol consumption . Since tSimilarly, electron microscopic study of the distribution of different NOS enzymes after ethanol treatment revealed opposite changes in the quantity of nNOS- and eNOS-labelling gold particles not only in the myenteric ganglia, but also in their close microenvironment . While tAlthough impairments of enteric neurons in different inflammatory diseases have been widely studied ,53,54,55Extensive damage of myenteric neurons as a distinct early event was demonstrated in the inflamed colonic segment in different rat models despite the sustained inflammatory processes ,58. The Among the different subpopulations of enteric neurons, the vasoactive intestinal polypeptide (VIP)-positive and nitrergic neurons display contrary responses to gut inflammation. In the inflamed colonic regions, the number of NOS neurons increased and the percentage of VIP neurons was unchanged in the myenteric plexus of paediatric patients with Crohn’s disease . HoweverThe activity of NOS and the generation of NO were elevated in colonic mucosal biopsies  as well The deleterious effects of intestinal ischemia/reperfusion (I/R) injury on enteric neurons was also revealed in several studies. Alterations in neuronal structures or even neuronal loss were demonstrated in humans  and animHowever, evidence showed that the NOS-containing enteric neuronal subpopulation is more susceptible to severe enteric neuropathies , like I/Swelling and distortion of nitrergic neuronal dendrites and increased relative cell profile area of NOS neurons was observed following I/R in the ischaemic intestinal region ,70,71. MSeveral studies have suggested that nitrergic myenteric neurons are especially susceptible to the development of neuropathy in diseases of the digestive tract ,27,28,34