Anaphylaxis is a systemic hypersensitivity reaction that can be life threatening. Mechanistically, it results from the immune activation and release of a variety of mediators that give rise to the signs and symptoms of this pathological event. For years, most of the research in anaphylaxis has focused on the contribution of the immune component. However, approaches that shed light on the participation of other cellular and molecular agents are necessary. Among them, the vascular niche receives the various signals  that elicit the range of anaphylactic events. Cardiovascular manifestations such as increased vascular permeability, vasodilation, hypotension, vasoconstriction, and cardiac alterations are crucial in the pathophysiology of anaphylaxis and are highly involved to the development of the most severe cases. Specifically, the endothelium, vascular smooth muscle cells, and their molecular signaling outcomes play an essential role downstream of the immune reaction. Therefore, in this review, we synthesized the vascular changes observed during anaphylaxis as well as its cellular and molecular components. As the risk of anaphylaxis exists both in clinical procedures and in routine life, increasing our knowledge of the vascular physiology and their molecular mechanism will enable us to improve the clinical management and how to treat or prevent anaphylaxis.Anaphylaxis, the most severe allergic reaction, involves a variety of immune and non-immune molecular signals that give rise to its pathophysiological manifestations. Importantly, the vascular system is engaged in processes relevant to anaphylactic events such as increased vascular permeability, vasodilation, hypotension, vasoconstriction, and decreased cardiac output. The novelty of this review focuses on the fact that new studies will greatly improve the understanding of anaphylaxis when viewed from a vascular molecular angle and specifically from the endothelium. This knowledge will improve therapeutic options to treat or prevent anaphylaxis. Anaphylaxis is a systemic reaction with a range of clinical manifestations due to the varying involvement of several organs and systems. At the cellular and molecular levels, most existing research focuses exclusively on the immune component. However, interactions between vascular and immunological microenvironments are responsible for most manifestations of anaphylaxis, including the most severe. Therefore, precise understanding of the cellular communication between immune and resident cells within the complex pathophysiology of this pathological event is necessary. This review provides an overview of the human vascular system in anaphylaxis and seeks further understanding of the dysregulation of its underlying processes.Anaphylaxis is considered the most serious manifestation of allergic disorders. The World Health Organization defines it as “a severe, life-threatening systemic hypersensitivity reaction characterized by being rapid in onset with potentially life-threatening airway, breathing, or circulatory problems and is usually, although not always, associated with skin and mucosal changes” . In the Several triggers can induce anaphylaxis, the most common being foods, drugs, and Hymenoptera venoms, although in some cases the cause of the reaction is unknown (idiopathic) –14. In pThe major molecular mechanism underlying anaphylaxis is the classic allergic IgE-mediated reaction involving mast cells and basophils . Mast ceInterestingly, after experiencing an anaphylactic reaction, some patients have no detectable levels of allergen-specific IgE , 31, 32.Specifically, the implication of both monocytes and macrophages has been demonstrated in passive and active systemic anaphylaxis , 89, 90.Other reactions triggered by drugs  are also capable of activating mast cells and basophils as well as degranulation led by the Mas-related G-protein coupled receptor member X2 (MRGPRX2) –95. ThisConcerning the participation of other immune cells in these events, accumulation of eosinophils was detected in passive cutaneous anaphylactic reactions in guinea pigs, as well as in spleens and coronary arteries from anaphylactic human cadavers , 100. SpOverall, a large number of anaphylactic mediators coming from different cellular sources are released both into the bloodstream and in local microenvironments Table 1.Classically, the best-characterized mediators have been classified into preformed and newly synthesized. Those stored in cytoplasmic granules contain highly sulfated polysaccharides (heparin or other proteoglycans), tryptase, chymase, histamine, and PAF, being mainly released from mast cells and basophils , 24. HowIn relation, multitudes of soluble products from both the contact (bradykinin) and the complement system  are also released in the pool of mediators , 110. ThIn addition, other molecular players such as extracellular vesicles (EVs), microRNAs, and metabolites are just starting to gain relevance as surrogate biomarkers in anaphylaxis but also participating in their underlying molecular pathways , 111–113Altogether, this large variety of molecular signaling pathways contributes to amplify the number and heterogeneity of processes occurring in these pathological events.The cellular components of the vascular compartment are both target and effector resident cells in anaphylaxis. Thus, the following section expands on the morphological and functional particularities of the cardiovascular system in order to broaden its knowledge in anaphylactic reactions. The circulatory system is composed of a set of interconnected tubular organs that form a closed circuit in the human body. It is divided into the blood vascular system, which carries blood, and the lymphoid vascular system, which transports lymph . HoweverThe functions of the blood vascular system are as follows: transportation of substances entering the body from the external environment (mainly nutrients and oxygen), transfer of molecules such as hormones, hydraulic force generation, regulation of heat, ultrafiltration in the kidney, and defense through the transport of immune cells and mediators . This ciBox 1The heart is an organ located in the central part of the thoracic cavity and made up of two thin-walled atria and two thick-walled ventricles. The right and left parts of the heart are separated by a septum, so blood from one side does not mix with the other  arise from the heart and transport blood to the muscular arteries. In each ventricular systole, the heart pumps a very large volume of blood, causing distension of the elastic walls, which accumulate the potential energy released during the diastole. Therefore, they push the blood when the heart is relaxed, acting as a secondary propulsion pump complementing the heart and resulting in a continuous flow  are the most abundant arteries in the human body. They usually appear near the organs, and their function is to distribute the blood through the different regions of the body since, by contracting their muscular component, they can regulate the size of their lumen . Due to their thinness, they do not present tunics and are the organ of the circulatory system with the highest level of gas and nutrient exchange. There are three types of capillaries: continuous, fenestrated, and sinusoids according to the specialized subpopulations of EC , but morphological and functional differences exist between them. The ti consists of a single and extensive layer of endothelial cells (ECs) that forms a physical barrier between blood and tissues, allowing the selective transport of molecules through it  and/or elastin fibers supported mainly by a collagen matrix. VSMCs are fundamental homeostatic players in different diseases such as hypertension and atherosclerosis (2+ (iCa2+) levels. Indirectly and with the main purpose of maintaining homeostasis, ECs also contribute to vascular tone modulation via synthesis and release of vasoactive substances  that contributes to the disruption of junctional protein complexes. In addition, iCa2+/calmodulin (CaM) activates myosin light chain kinase (MLCK) leading to the phosphorylation of myosin light chain 2 (MLC-2) and resulting in increased acto-myosin contractility, stress fibers, and the disruption of the endothelial barrier. In turn, myosin light chain phosphatase (MLCP) dephosphorylates MLC-2 producing cellular stability. MLCP inhibition is achieved by Rho kinase (ROCK) activation downstream of the guanine nucleotide exchange factor, Trio, which initiates the activation of RhoA  and adherent junctions (AJ). TJs are mainly composed of occludins and claudins that bind to the actin cytoskeleton and α-catenin. In AJs, VE-cadherin is the major structural protein, and they contribute to barrier stabilization by providing mechanical cohesive strength between ECs , 179. Di of RhoA , 181. Inociation , 183. Moociation , 183–185ociation .In contrast, other mediators stabilize the endothelial barrier, mostly to maintain homeostasis. These barrier-stabilizing agents, through adenylate cyclase (AC), increase cyclic adenosine monophosphate (cAMP) levels in ECs. It activates the protein kinase A (PKA) and guanine nucleotide exchange factors such as Rac1 which lead to inhibition of the small GTPase RhoA and the strengthening of the actin cytoskeleton stabilizing the endothelial barrier , 178. SpAdequate control of peripheral vascular pressure is essential, as it provides the driving force to pump blood to the organs . Vasodil2+ concentration, ensuring that MLCK can no longer phosphorylate MLC-2 and leading to relaxation of the VSMCs and vasodilation. Furthermore, NO activates MLCP and, by dephosphorylating MLC-2, causes additional relaxing effects  and the underlying production of NO have been probably characterized as the most harmful vasodilator and hypotensive factor , 202–204 effects , 204. An effects . In addi effects –154. The effects , 207. Tr effects –210. Bra effects .The regulation of the vascular system presents a dilemma because a third block of important circulatory disturbances may also appear in anaphylaxis. Severe reactions seem to present vasoconstriction of the vessels in the thoracic cavity leading to cardiac arrest . The cor2+ which activates MLCK leading to the phosphorylation of MLC-2 and resulting in increased acto-myosin contractility  alter blood flow, resulting in impaired vascular homeostasis, which may lead to anaphylactic shock , 197.The exacerbated fluid extravasation, in combination with the loss of peripheral vascular resistance, reduces venous return to the heart. In addition, the blood flow is removed from surface areas, reducing oxygen demands and delivering it to vital organs, thereby decreasing body temperature. However, it is insufficient to satisfy the metabolic demands of the human organism. On the other hand, constriction of pulmonary arteries, together with the effect of tightened airway smooth muscle, leads to reduced oxygen uptake. Therefore, to make up for this lack of oxygen in the body, breathing becomes rapid and deep and the heart rate increases (tachycardia). However, a second phase characterized by bradycardia occurs because this organ cannot remedy the influence of the previous manifestations of this pathological event, causing cardiac collapse and death of the patient , 223.A wide variety of soluble mediators have been proposed as participants of the anaphylaxis pathophysiology , 71. TheIn this sense, most of the studies have mainly been carried out in experimental models . Specifivia its different adrenergic receptors  would determine the magnitude of the anaphylactic events. Furthermore, the role of their receptors (GPCRs) in human anaphylaxis is not yet fully understood, but they result to be essential in preventing or inducing vascular effects. Therefore, GPCRs are the most promising therapeutic targets of study nowadays and the degree of responsiveness of ECs and VSMCs is an important factor to determine in anaphylaxis.in vitro human—models is necessary. This fact is one of the major limitations to study this pathological event. A big piece of the literature around it derives from animal models. For that reason, conclusions must be taken cautelous when translated at human reactions. Strategies based on functional in vitro studies by using human cell cultures combined with sera would provide clues about cellular and molecular happenings occurring in specific anaphylactic microenvironments. Therefore, abundant investigations are aimed at finding vascular targets revealing future alternative strategies to treat or prevent anaphylaxis. Efforts must be focused on alternative therapies called to specifically modulate the whole or parts of the vascular system that benefit the clinical management of these life-threatening events in the near future.Due to the complexity to investigate in human subjects the cellular and molecular mechanisms of anaphylaxis, the improvement and reproducibility of animal—or Conceptualization and—original draft preparation, VE. Writing—review and editing, EN-B, SF-B, AY-M, and VE. Funding acquisition, VE. All authors contributed to the article and approved the submitted version.This research was supported by grants from the Instituto de Salud Carlos III (PI18/00348 and PI21/00158) and FEDER Thematic Networks and Cooperative Research Centers RETICS ARADyAL RD16/0006/0013. This work was also supported by the SEAIC (19_A08) and Alfonso X el Sabio University Foundations. EN-B was granted by funding from the Community of Madrid included in the project FOOD-AL (CM_P2018/BAAA-4574).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Pharmacological, physical and cognitive treatments reduce pain by addressing all pain dimensions. Nonetheless, drugs may be ineffective, and physical activity is not always viable. In contrast, cognitive therapies have usually good outcomes, a wide range of applicability and no side effects. Their efficacy, however, is influenced by cognitive and psychophysiological traits. In this Opinion article hypnotizability is used as a model to support the view that specific psychophysiological traits and cognitive strategies can not only reduce pain, but also modulate the pain-related autonomic and immune activity, induce cortical plasticity relevant to pain control, and assist in the choice of the most appropriate treatment.Hypnotizability, or hypnotic susceptibility, is a multidimensional trait stable through life  characterized by low sensitivity to opiates, high consumption of opioids for post-surgery and cancer pain and low placebo responsiveness more frequently than lows, with mediums displaying intermediate frequencies  C385A polymorphism (rs324420) responsible for endocannabinoids (eCBs) degradation is not significantly different between hypnotizability groups but the polymorphism frequencies indicate a trend to higher degradation efficiency from lows to highs , which modulates the sensory and emotional components of pain  promotes the release of brain dopamine and noradrenaline  the parasympathetic circuit, limited to vagal afferents and efferents; (b) the parasympathetic-neuroendocrine circuit, which is responsible for the release of corticosteroids; (c) the cytokine-vagal-sympathetic circuit, involving noradrenergic pathways and adrenal epinephrine  and electrical direct Transcranial Stimulation (dTCS) are efficaciously used to modulate the activity of pain-related circuits (Klein et al., The pain matrix structure, activity and connectivity (Legrain et al., The authors equally contributed to the paper and agreed on its content.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.


Lymphedema is a condition that affects up to 130 million subjects worldwide. Since it is related to several complications and a significant reduction in terms of quality of life, it is a heavy burden not only to the patients but also for the healthcare system worldwide. Despite the development of supermicrosurgery, such as vascularized lymph node transfer (VLNT) and lymphovenous anastomosis LVA, the indications and outcomes of these complex groups of interventions remain a controversial topic in the field of reconstructive plastic surgery.This systematic review and network meta-analysis aims to assess the evidence of outcomes of LVA and VLNT in patients with lymphedema. Secondary aims of the project are to determine if for any outcomes, LVA or VLNT is superior to conservative therapy alone, and whether the available evidence favors any kind of supermicrosurgical interventions for lymphedema patients. This study will include original studies of patients with lymphedema on the extremities indexed in PubMed, EMBASE, CENTRAL, PASCAL, FRANCIS, ISTEX, LILACS, CNKI, and IndMED that reported microsurgery (supermicrosurgery) of all techniques aiming the re-functionalization of the lymphatic system. As comparators, mere observation, conservative treatment of any kind, and the other subgroups of supermicrosurgery are planned. The primary outcome of this systematic review and network meta-analysis is the difference of the limb volume, while the secondary outcomes of interest will be erysipelas rates, major and minor complications, postoperative necessity of continuous compression garments, and patient satisfaction, measured by already published and validated scores for quality of life.We will provide an overview and evidence grade analysis of the scientific literature available on the effectiveness of the subcategories of supermicrosurgical interventions for lymphedema.The online version contains supplementary material available at 10.1186/s13643-022-01885-9. Lymphedema is characterized by the development of localized swelling as a result of a compromised lymphatic system . PrimaryDespite major improvements in public health and access to healthcare, parasitic infections remain the main global cause of SL, such as filariasis or onchocerciasis . The oveThe currently accepted system of staging lymphedema is the one proposed by the International Society of Lymphology (ISL) . At stagIn early stages, compression therapy is still the workhorse approach for lymphedema. The goal of compression garments is to increase interstitial pressure and decrease capillary filtration . The litFunctional lymphedema surgery involves two sets of techniques: lymphovenous anastomosis (LVA) and vascularized lymph node transfers (VLNT) . The anaDue to the absence of strong evidence concerning the effectiveness of LVAs and VLNTs, clear guidelines concerning the use of these procedures are missing. Only two older clinical practice guidelines discussed indications for surgery in lymphedema. While the International Lymphedema Framework guideline in 2012 concluded that “peer-reviewed published literature on the surgical treatment of Lymphedema indicate that these procedures are promising for select groups of patients” , the CREThe following protocol for this systematic review and network meta-analysis adheres to the Preferred Reporting Items for Systematic review and Meta-Analysis Protocols (PRISMA-P) guidelines , in accoThis systematic review and network meta-analysis is intended to assess the evidence of outcomes of two types of supermicrosurgical interventions (LVA and VLNT) in patients with lymphedema. Secondary aims of the project are to determine if for any outcomes, LVA or VLNT is superior to conservative therapy alone and whether the available evidence favors any kind of supermicrosurgical interventions for lymphedema patients. An additional aim is to determine the areas where further evidence-based oriented reports are missing.The eligibility criteria have been established following the Population, Intervention, Comparator, Outcomes, and Study Design (PICOS) format recommended by the Cochrane Group . These aThis study will include patients with lymphedema on the extremities without exclusion of demographic factors . No specific subpopulations of secondary lymphedema will be excluded, and if possible, these subpopulations will be pooled according to the stage of the disease (following the classification of the ISL ) and/or The intervention of interest is microsurgery (supermicrosurgery) of all kinds and techniques aiming the re-functionalization of the lymphatic system. Supermicrosurgery will not be strictly defined during this network meta-analysis based on the traditional definition as procedures with anastomosis of vessels smaller than 0.8mm , but as In randomized controlled trials, inactive and active control interventions will be included. These are respectively (1) mere observation—do nothing and (2) conservative treatment of any kind . Ablative operative treatments as liposuctions or excisional-and-grafting surgeries will not be considered as an active control group during this systematic review and network meta-analysis. No exclusion based on frequencies, duration, timing of delivery, or number of interventions will be considered. A pooled comparation of two groups, LVA and VLNT, will be performed during this systematic review and network meta-analysis. Trials including only inactive or active control trials or case series, without a microsurgical intervention group for lymphedema, will be excluded. Studies including merely a supermicrosurgical intervention group, if considered as appropriate after the risk of bias assessment see the “” sectionThe primary outcome of this systematic review and network meta-analysis is the difference of the limb volume, measured by water plethysmography, circumference variation, or mathematic volumetric calculations on different scales. The limb volume difference of the limbs with lymphedema at a pre- and postoperative timepoint or the comparison of the affected and intervened limb with the contralateral extremity will be included and pooled into subgroups. Secondary outcomes of interest will be erysipelas rates, major and minor complications, postoperative necessity of continuous compression garments, and patient satisfaction, measured by already published and validated scores for quality of life Table . SubjectAny original articles, which assess the outcomes of supermicrosurgery for lymphedema, either on its own or compared to other supermicrosurgical or conservative therapies, will be included. There will be no restrictions in language, years, or geography. Our exclusion criteria include preclinic studies, case reports, consensus papers, editorials, reviews, and meta-analysis articles. Also, original publications of the same researcher’s group with overlapping time frame might be excluded if the patients are not singularized as different cohorts. In such a scenario, the authors will be contacted to confirm that the patient cohorts of the articles in question are different. If this information is not available to us, the study with the highest evidence level or the largest series, in case of equal study design, will be included. The other article, or articles, will be excluded. Since preprint servers like MedRxiv are not peer reviewed, the corresponding grade of evidence cannot be certainly determined. Therefore, preprinted reports will not be considered.A comprehensive and reproducible electronic search will be conducted by four independent reviewers in multiple electronic databases including MEDLINE via PubMed, EMBASE via Ovid, Cochrane Central Register of Controlled Trials (CENTRAL), PASCAL, FRANCIS, ISTEX, Latin American and Caribbean Health Sciences Literature (LILACS), China National Knowledge Infrastructure (CNKI), and Indian Biomedical Research Database (IndMED). An example of the search strategy and key words to be used during the systematic review and network meta-analysis is provided in Table Additionally, unpublished clinical trials will be searched by the same four reviewers independently at ClinicalTrials.gov, the International Standard Randomized Controlled Trial Number Register (ISRCTN), and the WHO International Clinical Trials Registry Platform (ICTRP). Of these trials, a back- and forward citation research will be performed by hand. Despite reviews, meta-analysis and systematic reviews will be excluded from further analysis; a manual screen of the references cited will be conducted to detect potentially overlooked articles. In order to identify all relevant studies, all the above-mentioned databases will be systematically searched from their inception with no language limitations.Two of the same review authors that performed the search will evaluate the studies, obtained and saved as abstracts in EndNote X9 , and exclude independently the publications based on the inclusion and exclusion criteria. Then, the list of the remaining studies to be included will be compared and any difference of excluded articles will be solved by consensus with a third reviewer. The same reviewers will conduct a full-text screening and exclude again unsuited articles. Once again, the result of this process will be compared, and the differences solved by mutual agreement with a third team member.A standardized data extraction spreadsheet will be developed a priori . The final studies to be included will be analyzed independently by three reviewers and the data extracted and filled into the Excel form. Inconsistencies will be resolved by unanimity after consultation with a fourth reviewing member. The following data will be extracted: year of publication, study location, type of study, cohort size, age, gender, follow-up period, risk factors for complications, supermicrosurgical intervention, control intervention, and the outcomes as outlined in the PICOS strategy   assessmeWe will summarize and describe each study narratively in tables and text by general characteristics of the study population, treatment, and design. All values that are relevant for quantitative analyses will be tabulated in an appendix. The standardized mean difference along with its 95% confidence interval (CI) will be used as an effect measure for the limb difference (the primary outcome). Depending on the scale of the secondary outcomes, we will use either the odds ratio (OR) or the standardized mean difference (SMD) along with its 95% CI, as we expect different studies to report similar outcomes using varying assessment tests and scales. We do not currently expect to identify time-to-event outcomes during the screening process.τ2 statistics as well as the I2 and the  Q-statistic. Summary of finding (SoF) tables, Forest plots , and league tables will be used to visualize estimation results. We will use P-scores to provide ranking of treatments for each outcome [A random-effects network meta-analysis will be performed for all primary and secondary outcomes if enough eligible studies are identified. A network graph will be used to visualize the connectedness for each outcome, as well as the direct and indirect comparisons. If a network meta-analysis can be performed, we will use the method based on graph theory for data synthesis  and the  outcome , which w outcome .All analyses will be performed in R  using the extensions netmeta  and metaSensitivity analyses will be performed using the design-adjusted approach for combining randomized and non-randomized evidence , as wellThe results of the systematic review and network meta-analysis will be displayed graphically and the resulting level of evidence will be determined according to the Oxford Centre for Evidence-Based Medicine . RecommeLymphedema prevalence is high in both developed and developing countries. In the former, the increasing incidence of secondary lymphedema is due to more effective oncological treatments and a higher number of patients surviving cancer. The incidence of lymphedema will vary depending on the nature of the primary tumor but is frequently associated with head and neck cancer (75%), melanoma 16%), urological (11%), and gynecological (37%) malignancies –44. Brea6%, uroloDespite conservative strategies are available and have proven to be effective , these dTo our knowledge, only Basta et al. and Carl et al. have reviewed the overall evidence of the surgical treatment of lymphedema , 49. TheThe primary aim of this systematic review and network meta-analysis is to synthesize the full body of evidence regarding the supermicrosurgical treatment alternatives to permit the development of individualized surgical strategies and guidelines for lymphedema surgery. The results of this review will quantify and statistically analyze the mean or median volume reductions of supermicrosurgically intervened limbs compared to conservative therapy, no treatment, and/or other supermicrosurgical interventions. Whenever possible, outcomes like quality of life, limb function, erysipelas rates, and postoperative complications will be pooled and analyzed. If the evidence is categoric enough, recommendations concerning supermicrosurgical interventions will be made in relation to the etiology of lymphedema and the clinical stages. On the other hand, if the results do not permit any endorsements, the data will be displayed for its interpretation and the analysis will be merely descriptive.A major strength of our planned systematic review and network meta-analysis is a robust design. Besides the systematic review of Markkula et al. , this isA possible limitation of the intended review is the inclusion of studies lacking a control group, like case series. The inclusion of non-randomized studies was decided after a pilot search in PubMed and is founded in the intention to avoid false conclusions based exclusively on the analysis of a few possible underpowered or biased RCTs. The intervened groups and their subpopulations will be pooled mathematically risking to merge heterogeneous patient populations and surgeries subtypes. To minimize the error of heterogenous pooling, the reviewing authors therefore pre-specified condition, populations, and lymphedema supermicrosurgery subgroups in advance for the data extraction and analysis.We are confident that the intended systematic review and network meta-analysis will be an exceptional contribution to the field of reconstructive plastic microsurgery. Whether the results will allow the generation of specific guidelines and recommendations for the surgical treatment of lymphedema, or identify the particular fields where more quality studies are required, this effort will be a critical contribution to evidence-based care of patients with lymphedema.Additional file 1. PRISMA-P 2015 checklist.


Tobacco use remains the leading cause of preventable disease, disability, and death in the United States, accounting for 1 in 5 deaths. Nicotine, the main psychoactive component of tobacco, is largely responsible for the addictive properties of tobacco. Although nicotine may cause feelings of euphoria or stress relief, the withdrawal state of the nicotine dependence cycle causes symptoms such as increased anxiety, irritability, stress, physical discomfort, and a profound craving for nicotine. Nicotine withdrawal symptoms often result in relapse where the person seeks and consumes nicotine even if they want to quit smoking. Identification of the neural mechanisms underlying the effects of nicotine withdrawal is an essential step in understanding tobacco use disorder and may lead to better treatments. Converging evidence suggests that manipulation of GABAergic transmission in a small midbrain region enriched in nicotinic receptors, the interpeduncular nucleus (IPN), can control the emotional, physical, and motivational aspects of nicotine withdrawal. However, a critical gap in the literature is lack of evidence that GABA transmission is dynamically regulated during nicotine withdrawal in vivo.The report by Klenowski and Zhao-Shea et al. , addressFor instance, based on the c-Fos studies, one could hypothesize that the increased IPN GABA neuron activity is due to an increase in the motivational and somatic signs of withdrawal. Indeed, the authors found that the activity of IPN GABA neurons was increased when the nicotine-dependent and withdrawn mice were exploring the open arms of an elevated plus-maze, a condition known to increase anxiety-like behavior. However, the detailed time-lock analysis by Klenowski and Zhao-Shea et al. , revealeFurther investigation on the mechanisms and neural circuits underlying these coping behaviors may provide new targets for the development of medications that mimic the coping effect of these behaviors. It will be important in the future to identify which upstream circuits control these coping behaviors during withdrawal to decrease IPN GABA neuron activity. Two candidate upstream regions are the hypothalamus and the ventral tegmental area, which have been previously identified as key brain regions for the control of grooming behavior. However, very little work has been done in nicotine-dependent laboratory animals.One limitation of the present report is that it was conducted in male mice only. The O’Dell lab has shown that female rats exhibit higher anxiety-like behavior and higher IPN GABA levels than males during nicotine withdrawaI , howeverOverall, the comprehensive study by Klenowski and Zhao-Shea et al. , provide


Biobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation—a nationwide network and registry of histo- and cytopathology in the Netherlands—was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA’s nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use. We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA’s nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (− 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA’s national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands. Biobanks play an essential role in enabling biomedical research by facilitating the collection, processing, storing, and managing of valuable human biological resources  and related clinical data  to use PALGA’s nationwide pathology network to establish an efficient, consistent, and transparent FFPE tissue sample procurement process in the Netherlands. This project was initiated by BBMRI.nl, PALGA, and the Netherlands Society for Pathology (NVVP). The governance of the DNTP is organized as follows: the PALGA foundation traditionally managed a national, nationwide pathology database. Jointly with PALGA, software tools were developed to both advance the requests for data as well as facilitate tissue sample procurement. The BBRMI.nl project installed HUB-employees, oversaw the backlog for the software, allocated the budget, and built the DNTP IT application focusing on tissue sample procurement, while jointly with PALGA a request tool for data accessibility was made. For stakeholder alignment, PALGA, the NVVP, and BBMRI.nl communicated to laboratories and science representatives. A budget of €1.2 m was allocated for software of the PALGA Portal (40%), hosting (10%), HUB-employees (10%), and program and software managers (40%). As of 2015, PALGA performed the extension, maintenance, and hosting of the tool that will be updated in 2021 in close collaboration with a BBMRI.nl sequel project. The DNTP consists of a centrally organized IT application and its complementary local HUB-mediated support for logistical processing. This paper describes the previous landscape of challenges that hindered the findability and utilization of the FFPE tissue sample collection and how we set-up efficient, consistent, and transparent exchange of FFPE tissue samples by implementation of the DNTP infrastructure, and the motifs behind it.Improve the efficiency of the FFPE material procurement and navigation by defining a central, overarching strategy.Provide practical, tailored support on the overarching strategy at a local level during the entire FFPE material procurement process.Increase the transparency of FFPE material exchange through improved monitoring (track and trace) of its procedure.Stimulate the use of FFPE tissue samples for research by making the federated database and archive accessible for all involved stakeholders across the Netherlands.Facilitate General Data Protection Regulation (GDPR) compliance through improved registration of data and material requests.Increase probability of proper return of FFPE material to the original pathology department.The DNTP was designed to stimulate the use of the countrywide, but de-centrally located FFPE material archives by improving the efficiency, consistency, and transparency of its procurement process. These aims highlight the need for a nationwide overarching infrastructure to harmonize the procurement procedures between the various local pathology departments. In addition, detailed information exchange between all involved players  can promote insight into and compliance to both ethical and legal regulations. While doing so, tailored, local support needs to be provided to optimize the procurement process for the researcher. In order to achieve these goals, specific objectives were established:The strategic decision was made to design the DNTP infrastructure as a unique combination of 1) a centrally organised IT software application for assessing, processing, harmonizing, and monitoring of FFPE sample procurement requests, complemented with (2) local, tailored support provided by, so called, HUB-employees for assistance in procurement of the requested samples while overseeing and controlling compliance  applications have been developed to address the biobanking processes, optimize workflow efficiency, and provide quality assurance . Both academic and commercial researchers can be granted access to the collections of FFPE samples. Each application is reviewed on scientific quality and compliance to privacy regulations by the review committee of the corresponding pathology department. The composition of the review committees of the providing laboratories can differ between pathology departments, including amongst other pathologists, scientists, as well as legal and ethical experts. This reviewing process takes place on a continuous basis to guarantee timely response towards the applicant. The application review process requires that the researcher specifies relevant institutional information, including the associated project leader and pathologist to allow PALGA to verify the researchers’ association with the scientific institute. More importantly, the researcher is obligated in this request proposal to specify the type and number of FFPE material needed. The filed request will then be approved by the corresponding department(s). In the case that the request proposal is assessed as insufficient, the researcher is informed and will be asked to modify their proposal to address the concerns raised. In case of a positive evaluation of the request, the researcher and the local pathology department will make further agreements for use of the material. FFPE tissue samples may be used for scientific research only. Financial expenses may be charged, varying between pathology departments, to cover the costs needed for collection, handling, and storage of the FFPE tissue samples. Procurement of the samples can then be initiated according to the terms of the agreement.Researchers can request specific FFPE tissue samples from pathology departments across the Netherlands by filing a request using the internet portal .Facilitate adherence to the legal, ethical and privacy framework due to improved documentation.Thus, the main improvements due to renewed incorporation of the centrally organised IT application software are:Although extension of the existing PALGA architecture with the internet portal helped to define a centrally organised FFPE tissue sample procurement procedure, practical support at the pathology departments was crucial and needed fostering. Therefore, a second strategic part was developed to harmonize implementation of the centrally organised procedures and to allow for practical support on a local level.The first step of FFPE sample procurement after approval through the internet portal is notification of the corresponding pathology department concerning the request. In the second part of our novel approach, dedicated employees at each pathology department of the eight university medical centres have been appointed part-time to function as so-called HUB-employees. These HUB-employees are responsible for logistical and technical support at peripheral pathology departments. Currently, eight HUB-employees are employed which provide support at their own university medical centre and, on average, an additional two to three connected non-UMC pathological centres. The HUB-employees perform a wide range of activities depending on the specific request and the pathology department involved. HUB-employees consult the requestor prior as well as during the entire request-, procurement-, and retrieval process. In addition, at the PALGA office, we have installed a so-called national HUB information point. This central PALGA-associated HUB-employee can negotiate requests in order to optimize queries. HUB-employees can take the responsibility to locate the samples in the storage of their university medical centre as well as for the oversight of the procurement in the connected institutes. Once the requested samples have been collected, quantity  will be determined for each individual sample. It is the responsibility of the HUB-employee to ensure preservation of sufficient amount of tissue to prevent compromising the ability to conduct additional clinical test for diagnosis of the patient. In order to come to a valid decision, the HUB-employee considers the lab techniques performed on the requested sample. When uncertainty exists if sufficient amount of tissue is available, the HUB-employee prioritizes preservation of the tissue and will contact the applicant. Notably, HUB-employees are in most cases either lab technicians that know the tissue limits of e.g., biopsies or have access to expertise in this field. In the majority of the requests, quantity is in accordance with the filed agreement and the requested samples will be sent to the researcher. The researcher can confirm receipt of the FFPE tissue samples by using the online IT application software. Importantly, the DNTP also allows for monitoring of timely return of the FFPE tissue samples by the researcher to the respective department.Provision of practical support service for collecting and processing FFPE samples in a hub-in-spoke approach.Increased follow-up and traceability of FFPE tissue sample requests.Relieve of the workload experienced by individual pathologists in the respective departments that helped in procurement of FFPE materials in accordance with the highest standards of stewardship, while formally not being assigned with these tasks.Active engagement in the pathology community within the Netherlands, increasing the mutual responsibility to maximize the use of pathological samples and data.Assurance of an organisation continuity in sample procurement for this biobank.The HUB-mediated local, logistical support on FFPE tissue sample procurement has several advantages:A pilot use-case was performed to assess the advantages of the DNTP during the HUB-employee-mediated tissue procurement process of PALGA’s collection . The overall aim of this project was to enrich existing Dutch cohort studies with FFPE tumor tissue samples to create tissue microarrays (TMAs) and tumor-DNA, to enable molecular pathological epidemiology and biomarker development in large cohort studies. In total, 11,706 cancer cases with tumors in one of seven different sites of interest  were selected to request archived diagnostic FFPE samples from. For each individual case, if available, 2 tumor tissue FFPE blocks and 1 healthy tissue FFPE block were collected. Because this was a nationwide project concerning 43 pathology departments throughout the Netherlands, regional phases of collection were planned and executed by the HUB-employees. The HUB-employee system allowed for an efficient collection in which each donor department was visited only once to collect all tumor tissues and cohorts combined. Tumor tissue samples were retrieved successfully for 9350 cases . This represents 79.9% of the 11,706 requested blocks. Healthy tissue blocks were retrieved for 5962 cases, which is 50.9% of the 11,706 requested blocks. TMA blocks were produced in duplicate to facilitate use of precious material by external researchers. For these cases, also tissue sections for DNA-extraction were produced. After completion of the TMA-production and DNA-section process, all collected original blocks have been returned to the respective 43 donor departments. To conclude, this use-case supports that implementation of the DNTP ensures harmonization, compliance, and quality management along the entire tissue procurement process on a nationwide scale.Following the use-case, the performance of the DNTP was evaluated over time, as indicated by the number of filed requests, the amount of provided HUB-mediated support, and the sample procurement duration time Fig. . The samThe need for large number of biological samples and its associated data is growing persistently to enable the development of precision medicines. In order to achieve this goal, researchers should have secure access to high-quality biological materials and data deriving from large patient populations. This demand can only be met when secondary or patient-derived-biobanks function on a (inter)national scale, driving biobanks to innovate their infrastructures to allow for optimal governance and increase utility of these collections.Here, we describe how we implement the DNTP to utilize PALGA’s network as an intermediate to find and access the national, federated FFPE tissue sample archive for efficient, consistent, and transparent FFPE tissue sample procurement. “The existence of PALGA as a nationwide network and registry of histo- and cytopathology in the Netherlands was of indispensable value for the design and development of the DNTP infrastructure to find and access an ever-growing collection of archived FFPE tissue samples. Importantly, access to the rich source of tissue samples would not have been possible without PALGA functioning as an intermediate between researchers and the pathology departments. Alternatively, but much more limited, one can envisage a cancer registry to start with a cancer block procurement organization if e.g., an organization like PALGA lacks in jurisdictions.” The DNTP includes a centralised online IT application as an overarching infrastructure to improve the assessing, processing, harmonization, and monitoring of the FFPE tissue specimen procurement process. Practical support while implementing this overarching strategy at the various pathology departments was achieved through appointing dedicated HUB-employees in a hub-in-spoke approach. This dual approach allows for efficient, consistent, and transparent exchange of FFPE tissue samples for healthcare decision-making as well as for research across the Netherlands. Moreover, the improved infrastructure and communication between the various pathology departments introduces a feeling of mutual responsibility over the optimal use of histopathological samples and data on a national scale.This mutual responsibility is mirrored by the European Biobanking and BioMolecular resources Research Infrastructure—European Research Infrastructure Consortium (BBMRI-ERIC) .Extension of PALGA with the DNTP has increased the utility of the federated archive and optimized FFPE tissue sample procurement procedures but necessitates continuous innovation to further improve. Future developments may include, first, nationally standardized procedures between the requestor and provider specified for each request approval procedure, defining: (A) standards regarding the type of FFPE tissue samples (tissue blocks versus—sections and its metadata) provided and the expenses charged , and (B) nationally standardized material transfer agreements (MTA). The latter can be achieved by using an MTA template that has been set up in conjunction with legal advisors of all the Dutch academic hospitals. This MTA template serves to facilitate and regulate the transfer of human materials that were previously collected by a Dutch University Hospital from patients, participants, and/or volunteers for the purpose of scientific research . This would simplify the researcher experience to use only one internet portal to find, access, and request various types of biomaterials and data deriving from a plethora of different pathologies. Furthermore, this would allow the DNTP to contribute as a FAIR-source for the continuous development of multi-disciplinary, multi-centre research to drive personalised medicine  to contribute to this common objective and to align the processes at the various pathology departments. Two roadshows have been organised in which most pathology laboratories and departments have been visited to inventory the requirements to establish the DNTP infrastructure. During these roadshows, the added benefit of HUB-mediated support—being pro bono provision of expert knowledge and competences to alleviate the pathology departments of non-core obligations—was emphasized. Pathologists in the laboratories have been pointed out to the possibility of group accounting for publications (as designed by PALGA) to give them credit for their role in maintaining access of tissues for science. Moreover, pathologists and HUB-employees are being informed and inspired on a continuous basis via various communication channels, including national pathology congresses, the NVVP magazine, and the HUB- employee annual meeting.To conclude, implementation of the DNTP infrastructure largely increases utilization of PALGA’s network to find and access the federated, national archive of FFPE tissue samples for efficient, consistent, and transparent sample procurement. The pathologists in the Netherlands aim to harmonize resource management and handling, and foster quality-assurance along the entire biobanking process. This scientific infrastructure allows the vast registry of archived diagnostic tissue and its associated data to be used as a fundamental source for precision-medicine, combining personalized medicine with population health.


In this review, we will discuss zebrafish as a model for studying mechanisms of human fetal alcohol spectrum disorders (FASDs). We will overview the studies on FASDs so far and will discuss with specific focus on the mechanisms by which alcohol alters cell migration during the early embryogenesis including blastula, gastrula, and organogenesis stages which later cause morphological defects in the brain and other tissues. FASDs are caused by an elevated alcohol level in the pregnant mother’s body. The symptoms of FASDs include microcephaly, holoprosencephaly, craniofacial abnormalities, and cardiac defects with birth defect in severe cases, and in milder cases, the symptoms lead to developmental and learning disabilities. The transparent zebrafish embryo offers an ideal model system to investigate the genetic, cellular, and organismal responses to alcohol. In the zebrafish, the effects of alcohol were observed in many places during the embryo development from the stem cell gene expression at the blastula/gastrula stage, gastrulation cell movement, morphogenesis of the central nervous system, and neuronal development. The data revealed that ethanol suppresses convergence, extension, and epiboly cell movement at the gastrula stage and cause the failure of normal neural plate formation. Subsequently, other cell movements including neurulation, eye field morphogenesis, and neural crest migration are also suppressed, leading to the malformation of the brain and spinal cord, including microcephaly, cyclopia, spinal bifida, and craniofacial abnormalities. The testing cell migration in zebrafish would provide convenient biomarkers for the toxicity of alcohol and other related chemicals, and investigate the molecular link between the target signaling pathways, following brain development. Ethyl alcohol (ethanol) is the most widely used and consumed drug in humans’ daily lives. Alcohol addiction became common in 1700s when the increased distillation of potent alcohol made vast quantities readily accessible to the masses . During Alcohol poses a broad range of irreversible side effects on the human fetus. The maternal consumption of alcohol is considered to have a highly teratogenic effect on the fetus. No studies have reported a safe time period and quantity for alcohol intake during pregnancy. Its consumption has been widely reported to be teratogenic in all three trimesters of pregnancy. The higher consumption of alcohol during the first trimester is associated with brain and facial anomalies, whereas alcohol consumption during the second trimester can cause sponataneous abortions, and alcohol consumption during the third trimester is linked with reduced weight, brain volume, and height. Therefore, it could be concluded that the fetal exposure to alcohol at any point of pregnancy can cause irreversible damage and potentially lead to fetal alcohol spectrum disorders (FASD) and their related neurobehavioral deficiencies including the developmental and learning disorders . Women oAs an alternative model, zebrafish have also been used to study. Embryo development in normal and disease conditions due to their high fertility, size, and external development of the transparent embryo, which can be visualized easily. These features of zebrafish can be crucial for defining key times of ethanol exposure and allowing easy analyses of tissue morphogenesis, cell movement, and gene expression . Recent The zebrafish brain development initiates from the process of neural induction during the gastrula stage of the embryo. At this stage, two neural inducers represented by chordin and fibroblast growth factor (FGF) emanate from the dorsal organizer and marginal mesoderm, inducing the fate of the central nervous system (CNS) in the dorsal- and ventral-vegetal ectoderm, respectively . At the When the gastrulation is complete, the newly formed neural plate is transformed to the neural tube by the process of neurulation, folding, and intercalation neural cell movement  Figure . At the It has been shown in zebrafish that ethanol alters the gene expression and cell movement during the blastula stage and gastrula stage . Early echordin, wnt11, and eve1, and delayed the migration of gsc-positive prechordal plate cells. The result indicates that the influence of ethanol on the gastrula cell movement is not restricted to one tissue but rather broadly affects the multiple cell fates and movement including the neural ectoderm and underlying mesoderm.Blader and Strahle reported that ethanol exposure at the gastrula stage suppressed the migration of the chordal mesoderm, prechordal plate, causing failure of eye field separation and the cyclopia . Zhang eEthanol also reduces the cell adhesion activity and cell directional movements during gastrulation. Cell movement at the gastrula stage is regulated by cell adhesion molecules such as Protocadherin 18a (pcdh18a)  and E-caAs discussed above, The ethanol exposure during zebrafish embryogenesis can also lead to morphological malformations. The reduction of eye diameter, body length, and pericardial edema has been reported in zebrafish embryos after the ethanol exposure , resultiBesides brain malformation, developmental abnormalities are also observed in the craniofacial structures in the neurula stage of the zebrafish embryos due to the abnormal development of the neural crest . The neuEmbryos of the transgenic zebrafish strain, Tg(HuC:KAEDE), express fluorescent protein in a range of neurons in the central and peripheral nervous systems. A decreased number of KAEDE-positive neurons in the spinal cord were found when embryos were exposed to 1% ethanol . Isl1 [TMultiple factors contribute toward birth defects, but FASD caused by utero ethanol exposure has been thought to be the most common reason for abnormalities . A betteThe zebrafish provides an ideal model for studying the effects of alcohol as many phenotypes show similarities to human FASDs, including microcephaly, holoprosencephaly, spinal bifida, cyclopia, neural crest defects, craniofacial defects, and cardiac defects. Unlike human and mammalian models, it is possible to trace the development of living embryos being exposed to the controlled concentration of ethanol. It is also possible to screen drugs and mutations that can enhance/suppress the symptom of FASDs using high throughput large-scale screening. On the other hand, potential disadvantages may be that size and structure of the brain are much smaller and simpler than those of mammalian models; therefore, detailed symptom of FASDs may show some differences . As zebrafish develop externally without a gestation period, it is not possible to investigate the influence of the transfer of alcohol from the mother to fetus. It might also be conceivable that genetic mechanisms for metabolizing alcohol may have some variation between species, considering that even within the same mouse species, different strains show different sensitivity and symptoms to alcohol .FASD is a frequent birth defect syndrome that is related to alcohol consumption by pregnant mothers, affecting embryos and leading to birth defects. Research on zebrafish facilitates the investigation of ethanol-related defects during embryogenesis. It can be concluded that ethanol produces multifactorial defects during blastula, gastrula, and organogenesis stages, which are the key stages of embryogenesis. There are many target pathways of ethanol during the development including the stem cell gene expression, cytoskeletal regulation, and gastrulation cell movement, neurulation, eye morphogenesis, neural crest migration, and neurogenesis. With its sensitivity and simple morphology, it is possible to design high throughput testing and screening methods for assessing the dose-dependent effects of ethanol and other toxicants on brain development and associated human birth defects. It would also be important to investigate long-term effects of moderate dosage using zebrafish embryos to detect weak but significant effects to the development, growth, and behavior in the later life stages.


The miniaturization, stimulated tissue mechanical forces, and microenvironment of OoCs offer unique properties for biomedical applications. However, the large amount of data generated by the high parallelization of OoC systems has grown far beyond the scope of manual analysis by researchers with biomedical backgrounds. Deep learning, an emerging area of research in the field of machine learning, can automatically mine the inherent characteristics and laws of “big data” and has achieved remarkable applications in computer vision, speech recognition, and natural language processing. The integration of deep learning in OoCs is an emerging field that holds enormous potential for drug development, disease modeling, and personalized medicine. This review briefly describes the basic concepts and mechanisms of microfluidics and deep learning and summarizes their successful integration. We then analyze the combination of OoCs and deep learning for image digitization, data analysis, and automation. Finally, the problems faced in current applications are discussed, and future perspectives and suggestions are provided to further strengthen this integration.Microfluidic-based organs-on-chips (OoCs) are a rapidly developing technology in biomedical and chemical research and have emerged as one of the most advanced and promising  The most widely used experimental models in biological research are cell-based and animal models; however, both these models have many limitations. Traditional cell-based models lack essential features, such as complex multiple cultures, physiological microenvironments, and tissue mechanical forces . Animal in vivo target organs. These novel in vitro biological models can replicate the local characteristics of a disease and control the environmental parameters of cell survival, making them a cost-efficient and high-throughput platform for biological research. Polydimethylsiloxane (PDMS) and poly(methyl methacrylate) (PMMA) are the most commonly used materials for the fabrication of OoC devices. Owing to the transparent nature of these materials and its high compatibility with fluorescence microscopy, OoC applications usually generate a large number of images, leading to a large amount of image-based data. This has traditionally been accumulated and processed by manual methods, which are typically inefficient . Th. Th92]. In this section, we discuss various applications of deep learning in OoCs. By discussing examples of each type of application in detail, the power and versatility of the integration of deep learning with OoCs were illustrated.Dendritic cells play a critical role in the recognition of tumor cells by absorbing tumor antigens and presenting them to T cells. The effectiveness of immune therapy therefore relies heavily on the interaction between the tumor and dendritic cells in the tumor microenvironment to induce an effective antitumor response . Parlato+ tumor cells  approach tumor cells. To monitor the interaction between PBMCs and tumor cells, Biselli et al. utilized OoC technology and reconstructed a tumor-on-a-chip that cocultured the PBMCs with HER2or cells  96]. Th. Th+ tumor cells  97]. Us. Us+ tum+ breast tumor microenvironment in the tumor-on-a-chip , which can accept input images of arbitrary size and perform pixel-by-pixel classification of images . The U-NFinally, the real-time visualization of cell morphology and trajectory is crucial for medical research. Zhao et al. combined a classic U-Net architecture with glass–air Anderson localizing optical fibers (GALOFs) . This imIn recent years, deep learning has completely changed many traditional industrial systems. Moreover, researchers in different fields are trying to integrate deep learning technology into their respective fields. In particular, many research advances have shown the promise of the integration of OoC technology and deep learning. The main reasons for this are as follows.First, the open-source projects implementing deep learning have gradually improved, including the open-source code based on TensorFlow, Pytorch, and Keras frameworks. These allow researchers to quickly reproduce open-source engineering code in different fields and apply it to their own specific tasks. Second, novel and better deep learning models with excellent performance are constantly emerging. These will ultimately be applied to the rapid development of industries that are extensively integrated with deep learning. Lastly, the multitype data generated by OoC experiments are not only complex but also large in quantity. Deep learning technology can be introduced to simplify the labor-intensive data analysis and feature extraction steps, alleviate the huge challenges brought by massive biomedical big data, and solve tasks that were previously considered infeasible.Current applications of deep learning in cell biology mainly focus on the morphological changes of whole cells; however, the analysis of subcellular structures or organelles can provide additional and important information , 111. VeTissue mechanical force control is one of the most important features of OoCs. For example, in the case of a lung-on-a-chip, human airway epithelial cells were cultured in a computer-controlled two-phase microfluidic system. The system can simulate the propagation and rupture of fluid embolism that occurs during airway injury in obstructive lung disease . As the in vitro human-related disease models, which can be an excellent platform for the study of pharmacokinetics, drug toxicity, and pharmacology. For example, Boos et al. tested embryoid bodies, influenced by human liver metabolites, in an organoid system [in vitro tests, thus increasing the predictive power of adverse drug effects. Thus, OoCs can be used to investigate embryotoxicity. Several examples [OoCs have been widely explored as d system . The plaexamples , 117 havin vitro disease models [in vivo or clinical samples. These data can be further analyzed by deep learning in real time, thus enabling the analysis of changes in the development of such diseases at the molecular level and ultimately obtaining the specific mechanisms of disease occurrence.OoC technology has been widely utilized to build various e models . Howevere models , 119. OoHuman-on-chips, a further development of OoCs, consist of interconnected compartments. Each compartment  contains specific cell types that represent different organs. All compartments are connected by a microfluidic circulation system , which iAlthough deep learning technology has excellent performance in feature representation and data mining, its internal mechanism and calculation strategy still need to be optimized for specific applications. Therefore, the development of a highly automated OoC system to provide a convenient, reliable, and integrated intelligent platform for researchers is the main development direction and challenge in the future.Data Augmentation. The most advanced methods such as GAN and unsupervised data augmentation  can be introduced to generate simulation data with real data characteristics, expand the capacity of high-value data samples, and reduce the cost of data acquisition.Automatic Data Annotation. The development of automatic data annotation algorithms and tools enables the automatic labeling of massive unlabeled data, reducing the huge cost of manual labeling, and improving the efficiency of labeling and development.Semisupervised Learning. Semisupervised learning [learning  can be ilearning , 126, a learning .Transfer Learning. Transfer learning can also be used as an excellent technical means to reduce dependence on a large volume of labeled data. The network is pretrained using the labeled data from other domains. Then, the fine-tuning of the network can be completed with a small amount of labeled data in a specific domain.At present, the ability to acquire experimental data on OoCs has been greatly improved, and massive amounts of big data have been accumulated. However, reducing the huge cost of manual labeling and automatically mining and refining the inherent characteristics of massive data are key challenges that urgently need to be overcome. The following aspects of this can be explored. Customized Design of Deep Learning Models. Facing the diverse application requirements in the field of OoCs, the deep learning models that are used in the field of natural optical images cannot be directly applied. Therefore, customized designs are required, and improvements must be made in terms of the model architecture and deep network layout.Automatic Network Design. For specific application scenarios of OoCs, neural architecture search technology can be used to realize the automatic construction and the optimal intelligent design of deep models, which avoids the complexity and limitations of traditional deep models design based on expert experience.Iterative Upgrade of Deep Models. Facing the increasingly updated OoC data, deep learning models usually have the problem of “catastrophic forgetting.” Therefore, enabling the deep learning model to handle new data and maintain robust performance requires further exploration of the iterative upgrade technology of deep learning models.Interpretability. Deep learning models usually have the problem of the “black box effect”; that is, the internal mechanism is not clear. This limits the understanding of the underlying mechanism and interaction principle of the specific OoC, which then lacks interpretability and reliability. Therefore, there is an urgent need to develop interpretable deep learning technology to transform the “black box” of deep learning into a “white box” and enable meaningful physical explanations from a biological point of view.Model Compression and Acceleration. For online scene applications, the accuracy and inference speed of the model need to be balanced. Therefore, it is necessary to thoroughly study the compression and acceleration technology of deep learning models, and under the premise of ensuring accuracy, compress the model volume to the extent possible to improve the inference speed.At present, the application of deep learning models is mainly based on computing hardware including GPU, CPU, FPGA, and other devices. The GPU and CPU are used for offline training of deep learning models. In particular, owing to the efficient computing power of the GPU, it has become the main hardware used for deep learning model training, including many products made by NVIDIA. FPGAs are mainly used in online applications and are edge devices for real-world applications.With the development of distributed technology, current devices such as GPUs and FPGAs have been able to meet the application of deep learning models. In the future, it will be necessary to combine hardware resources to carry out research to improve the inference efficiency of deep learning models and enhance the reliable transfer and rapid deployment of models to meet additional application scenarios.


A laparoscopic approach is suggested with the highest grade of recommendation for acute cholecystitis, perforated gastroduodenal ulcers, acute appendicitis, gynaecological disorders, and non-specific abdominal pain (NSAP). To date, the main qualities of laparoscopy for these acute surgical scenarios are clearly stated: quicker surgery, faster recovery and shorter hospital stay. For the remaining surgical emergencies, as well as for abdominal trauma, the role of laparoscopy is still a matter of debate. Patients might benefit from a laparoscopic approach only if performed by experienced teams and surgeons which guarantee a high standard of care. More precisely, laparoscopy can limit damage to the tissue and could be effective for the reduction of the overall amount of cell debris, which is a result of the intensity with which the immune system reacts to the injury and the following symptomatology. In fact, these fragments act as damage-associated molecular patterns (DAMPs). DAMPs, as well as pathogen associated molecular patterns (PAMPs), are recognised by both surface and intracellular receptors of the immune cells and activate the cascade which, in critically ill surgical patients, is responsible for a deranged response. This may result in the development of progressive and multiple organ dysfunctions, manifesting with acute respiratory distress syndrome (ARDS), coagulopathy, liver dysfunction and renal failure. In conclusion, none of the emergency surgical scenarios preclude laparoscopy, provided that the surgical tactic could ensure sufficient cleaning of the abdomen in addition to resolving the initial tissue damage caused by the “trauma”. Why should laparoscopy be preferred in emergency?The history of laparoscopy in emergency and urgent surgical scenarios, both traumatic and pathological, began immediately after the implementation of the technique itself.Less than 20 years after the first experiment of laparoscopy on living dogs by Kelling in 1901 . In 1924In the 1940s, laparoscopy was considered to be contraindicated for acute abdominal illnesses and stab wounds, gunshot wounds, and acute perforations of viscera, for fear of spreading infection .The modern concept of diagnostic laparoscopy for trauma began in the 1960s, when Heselson proclaimed its safety, efficacy and economic benefits, and demonstrated decreased hospitalisation, avoiding unnecessary laparotomies ,8,9.The advantages of laparoscopy in several emergency situations are clearly stated, undoubtedly whenever the surgical procedure is standardised. On the other hand, the benefits are lesser or unclear when only a basic plan of the procedure is definable and the surgical technique strictly depends on the intra-abdominal findings. Our aim is to review all of the evidence concerning the choice of laparoscopy in emergency settings and to clarify the pathomechanism underlying the choice.The available evidence clearly demonstrates the superiority of a laparoscopic approach in many emergency situations, with the grade of recommendation outlined in In these scenarios, laparoscopy guarantees the main advantage of having a shorter hospital stay that is associated with a quicker surgery, which is that patients experience a faster and uneventful recovery.Earlier enteral feeding is related to such a fast recovery. Adequate nutritional support contributes to maintaining the homeostasis and, consequently, optimises the immune response, minimising the risk of post-surgical infection .In terms of contraindication, these do not differ from those clearly defined for elective surgery in the case of an abdominal emergency. Laparoscopy should cautiously be considered whenever there is suspected difficulty in accessing the abdomen, such as in cases of organomegaly, adherence syndrome, or bowel distension.However, the additional systemic and local effects of laparoscopy must be mentioned because these may represent limitations in some emergency scenarios.In fact, the creation of the pneumoperitoneum is a key part of the procedure. The main consequences are due to increased intra-abdominal pressure, which causes respiratory, cardiovascular, and neurological alterations. Cardiovascular effects depend on intra-abdominal pressure and carbon dioxide uptake into the systemic circulation. At intra-abdominal pressures greater than 15 mmHg, the inferior vena cava is compressed, causing a reduction in venous return and resulting in a decrease in cardiac output and blood pressure. Increased intra-abdominal pressure elevates the diaphragm, and diminishing lung volume may cause basal atelectasis. The effects of the pneumoperitoneum and patient positioning may also raise intracranial pressure, reducing cerebral perfusion ,39. ThusOn the other hand, the pneumoperitoneum could precipitate pre-existing abnormalities in cardiac output or gas exchange, as well as intracranial pressure, liver dysfunction or coagulopathy.Acute cholecystitis is an acute inflammatory disease of the gallbladder, most commonly due to gallstones. Cholecystectomy is the accepted treatment for acute calculous cholecystitis and is considered to be the standard of care for gallstone disease for the majority of patients . The lapGastroduodenal perforation is an acute abdominal disease most commonly caused by peptic ulcers with an incidence of 2–10% . The treAcute appendicitis is the leading cause of emergency surgery. In developed countries, occurs in 5.7–50 patients per 100,000 inhabitants per year, especially at the ages of 10–30 years ,23. The In cases requiring emergency surgery the laparoscopic approach for gynaecologic conditions has the advantage over laparotomy of shorter hospitalisation and a faster recovery. In addition, it permits a complete exploration of the abdominal cavity, allowing the treatment of different diseases through using the same access, reducing the risk of post-surgical adhesions and surgical site infections. The main gynaecologic conditions that may present to the general surgeon in an emergency scenario are pelvic inflammatory disease (PID), ovarian diseases, ectopic pregnancy and endometriosis. Different treatments are available for PID, although surgery is required especially if there is no improvement in clinical conditions or in the presence of a pelvic abscess. The laparoscopic approach is indicated to perform the lavage and drainage of the infected collections, adhesiolysis and debridement of necrotic tissue . In the Nonspecific abdominal pain is not a defined disease; it refers to an abdominal or pelvic pain of a duration of less than one week for which the diagnosis remains uncertain even after clinical examination and baseline investigations ,34. NSAPWhen clinical examination and radiological findings can only suspect the disease responsible for the acute abdomen, laparoscopy allows for wide exploration of the abdomen to ascertain the diagnosis. In the international literature, accuracy is reported with a rate of 89–100% ,68,69,70In such situations and whenever, despite a clear intra-abdominal picture, the surgical management cannot be standardised, but the tactic must be adjusted to the intraoperative findings, the therapeutic role of laparoscopy is a matter of debate .A laparoscopic approach might guarantee significant advantages only if performed by experienced teams and surgeons, guaranteeing a high standard of care.The laparoscopic approach can also be advantageous in abdominal trauma in hemodynamically stable patients. In fact, it could allow for a quicker diagnosis and could also limit blood loss and reduce the risk of contamination. In recent years, indications for laparoscopy in abdominal trauma have widely increased, but the procedure is still reserved for hemodynamically stable patients. The laparoscopic approach is useful in treating bleeding from minor injuries of the liver or the spleen ,117, mesThe laparoscopic approach has gained importance in treating paediatric patients, not only in terms of elective procedures, but also in emergencies scenarios. With an experienced surgical team, a minimally invasive approach could be used safely in children, with low rates of morbidity and mortality . LaparosTo understand the reasons that guide the surgical tactic, pro or contra laparoscopy, it is fundamental to know the pathological mechanisms underlying the outcome of the patient. We support the idea of minimising post-surgical infection, not only with the use of antibiotic prophylaxis, but also through the optimisation of the immune response by maintaining the homeostasis with nutritional support  and reducing surgical trauma (as is done with laparoscopy). These measures consequently reduce the stress response and immune suppression .To this concern, ten years ago the European Association for Endoscopic Surgery (EAES) stated, as subsequently reported:“Changes in systemic inflammatory and anti-inflammatory parameters (mainly cytokines) as well as in stress response parameters are less pronounced after laparoscopic surgery than conventional surgery (grade A).”However, the EAES notes that it remains to be proven if this leads to clinically relevant effects.In fact, the extremely broad range of symptomatology developed after trauma or any significant severe acute disease, seems to be correlated with the intensity through which the immune system reacts, this is also related with the severity of the initial tissue damage caused by the “trauma”.In other words, any abdominal urgency, as well as traumatic injury events, almost instantaneously (or at least extremely quickly) causes a high level of cell death, with the consequential release of cell debris fragments. These events deploy the so-called damage-associated molecular patterns (DAMPs) derived from cellular necrosis, and the pathophysiological consequences follow the mechanistic insight depicted in the so-called “danger model”.The “danger model”, described by Matzinger in 2002, is based on the idea that the immune system does not distinguish between self and non-self, but rather between events that cause damage and events that will not ,136,137.This model represents the last evolution of the scientific concept regarding the activation of the immune system, which takes its origin from the traditional “non-self model”, developed in 1959, and passes through the “infectious non-self model” after the rediscovered concept of co-stimulation by Jenkins and Schwartz in 1986 . For both the “infectious non-self model” and the “danger model”, antigen-presenting cells (APCs) play an important role in the activation of both the innate immune system and the acquired immune response.As proposed by Janeway in 1989 , APCs arConcurrently both PAMPs and DAMPs bind to the nucleotide-binding oligomerisation domain (NOD), which together with leucine-rich repeat (LRR) and pyrin domains containing protein 3 (NLRP3) constitute the subunit of the inflammasome. The activation of the NOD domain causes the oligomerisations of NLRPs that is the first step towards the activation of the inflammasome. Once activated the NLRP3 inflammasome leads to caspase 1-dependent cleavage of pro IL-1β and pro IL-18 into their active forms, as well as to gasdermin D-mediated pyroptotic cell death ,143.The mediator of lung inflammation, fever and fibrosis is IL-1β. DAMPs spreading after pyroptosis amplifies the cascade and potentiates the inflammation.Mt-DNA is one of the most investigated and best described DAMPs , being bThe activation of APCs up-regulates costimulatory signals, processes the foreign antigens, and presents them to passing T cells. In addition, cytokines or pathogen-associated molecular patterns (PAMPs) leads to the transcriptional upregulation of the canonical and non-canonical inflammasome components .Between the causes of triggering the inflammatory response and beside the danger molecules, the formation of neutrophil-associated extracellular traps (NETs) seems to play an emerging role.Neutrophil function and NETs are critical components involved in human immune defence. This was first described by Brinkmann et al. as a cell death pathway, which is distinct from apoptosis and necrosis ,149, wheNETosis leads to the dissolution of the nuclear envelope and cytoplasmic granules, leading chromatin to mix with granular antimicrobial proteins via the NADPH oxidase and RAf- MEK-ERK pathway . NETs consist of neutrophil DNA, granular proteins, and several cytoplasmic proteins with antimicrobial activity, including histones, neutrophil elastase, MPO, pentraxin (PTX), lactoferrin, cathepsin G, and bactericidal permeability increasing protein ,149,154.Although NETs play important roles in host defence by trapping pathogens, extensive formation of NETs with increased amounts of extracellular DNA may contribute to the perpetuation of inflammation and tissue damage ,160,161.Several studies have shown that for severe trauma or burn injury the expression of the leukocyte transcriptome will be altered during the first 28 days after injury. This “genomic storm” involves genes implied in the systemic inflammation, in the innate immunity response and the simultaneous suppression of those involving adaptive immunity, especially in terms of T cell function and antigen presentation. Much later, alteration of gene pathways occurs, which implicates B cell proliferation and immunoglobulin synthesis. Only at a later time will the pathway responsible for compensatory anti-inflammatory response, which is involved in suppression of adaptive immunity, be redefined. Interestingly, according to the severity of the injury, the magnitude and the duration of this physiological derangement differs and will become responsible for the development of multiple organ dysfunction syndrome (MODS), as the result of excessive pro-inflammatory response (systemic inflammatory response syndrome [SIRS]) followed temporally by compensatory anti-inflammatory response syndrome (CARS) and the suppression of adaptive immunity ,165,166.The patho-mechanisms are consistent with a genomic storm that is neither chaotic nor erratic, but rather highly coordinated and reproducible. This storm likely represents a common transcriptional response to severe stress in humans regardless of its origin, with far more similarities than differences .In summary, in critically ill surgical patients a deranged response to the addicted infectious insult and to the first amount of PAMPs and DAMPs is a trigger for subsequent activation of downstream pathways. The results are the development of a progressive and multiple organ dysfunctions manifesting with ARDS, coagulopathy, liver dysfunction and renal failure. The predominant presentation depends on the underlying prevalent pathomechanism: thrombotic microangiopathies and coagulopathies and/or complement pathways’ hyper activation that may lead to disseminated microvascular thrombosis; pathologic immune activation and following hyper inflammation; immune paralysis with associated secondary bacterial or fungal infections and an inability to combat inflammation due to CD4+, CD8+ and dendritic cells deficiency. In the light of this overview and following the basic principles of any efficacious treatment after an abdominal acute emergency, there is not only the ability to resolve the injury itself, but also the capability to clear the damaged tissues which limits the release of DAMPs.In addition, a longer surgical procedure might be justified to obtain this goal, enabling a faster recovery.Taking into account all of these considerations, none of the emergency surgical scenarios preclude laparoscopy. In fact, the main advantage is its capability of limiting tissue damage, which, if associated with adequate cleaning of the abdomen, could ensure its superiority to an open approach ,169,170.Laparoscopy should not be recommended only when unable to control the whole abdominal cavity due to both the extent of the disease and technical limitations due to the surgical instruments or to the experience of the team.In conclusion, laparoscopy must be considered a safe, extremely versatile and prompt surgical approach, through which we could achieve a quicker diagnosis, avoiding large preoperative studies, minimising morbidity and requiring shorter hospital stay.To this end, laparoscopic training in elective scenarios should be recommended, not only for the surgeon but for the entire team, including scrub nurses and the other members of the staff in the operating theatre must become confident.Routine practice, combined with adequate equipment, could guarantee a high standard of care for laparoscopy in almost all emergency scenarios.