Chronic wounds remain an unresolved medical issue because of major social and therapeutic repercussions that require extensive focus. Recent related theragnostic focuses only on wound management and is not effectively promoting chronic wound healing. The rising number of patients with either under-healing or over-healing wounds highlights the ineffectiveness of current wound-healing treatments, and thus, there is an unmet need to focus on alternative treatments. To cover this gap, extracellular vesicles (EVs), for targeted delivery of therapeutics, are emerging as a potential therapy to treat both acute and persistent wounds. To address these issues, we explore the core biology of EVs, associated pharmacology, comprehension of immunogenic outcomes, and potential for long-term wound treatment with improved effectiveness and their nonacceptable side effects. Additionally, the therapeutic role of EVs in severe wound infections through biogenetic moderation, in combination with biomaterials , as well as drug carriers that can offer opportunities for the development of new treatments for this long-term condition, are also carefully elaborated, with an emphasis on biomaterial-based drug delivery systems. It is observed that exploring difficulties and potential outcomes of clinical translation of EV-based therapeutics for wound management has the potential to be adopted as a future therapy.  Mostafavi and colleagues explain that a chronic wound tends to remain an unresolved medical issue, which is due mostly to social and therapeutic repercussions. To fulfill this transition, extracellular vesicles (EVs) are recommended in targeted delivery as beneficial therapy in wounds (acute and persistent). Chronic ulcers are characterized by slow recovery of the epidermis and adjacent tissues that lasts more than three months.In the past few decades, chronic wound care has evolved into its own specialty, with doctors frequently using cutting-edge therapies such as growth factors, extracellular matrices (ECMs), and engineered skin as well as negative pressure wound therapy (NPWT). However, there is still no perfect therapy for chronic ulcers because of the expense, intensive care needs, microbial infections, and poor implementation of therapies.,,,A recent trend in wound healing field is inclined toward therapies based on extracellular vesicles (EVs). EVs are naturally produced small vesicles by cells that aid in intercellular signal transduction by carrying a variety of bioactive molecules such as lipids, metabolites, proteins, nucleic acids, and others.In the present review we explore the biogeny of EVs, their role in the regulation of immunological responses, and their contribution in various stages of wound healing. Furthermore, using biomaterials, nanomaterials, and genetically engineered EVs, we summarize existing understanding concerning EVs’ potential therapeutic application as a cell-free carrier for healing and tissue regeneration. In addition, basic research routes for maximizing the future clinical potentials of EV-based therapies are outlined.EVs are membrane-enclosed lipid double-layer assemblies with a spherical shape and a size of 40 nm to 5 μm.Apoptotic bodies are formed by cellular apoptosis and contain DNA fragments and sometimes organelles from the parent cell. The development of apoptotic bodies occurs with the contraction of the cell membrane followed by condensation of cytoplasm to reduce the size. Parallel to this, nuclear chromatin condenses and alters while the plasma membrane degrades and undergoes blebbing2+-dependent enzymes catalyze the formation of microvesicles, such as flippases in transferring lipids from external to internal leaflet and floppases in moving lipids from internal to the external leaflet.Microvesicles, or ectosomes, are the second largest type of vesicle, with a diameter of 100–1,000 nm, formed by the plasma membrane fission and outer branching.,Trauma or surgical incisions can produce acute wounds, but chronic inflammatory or diseased conditions result in recurring wounds that do not heal adequately.Hemostasis is characterized by vascular constriction and blood clotting, both of which lead to platelet aggregation and thrombus formation. Platelet-derived EVs are the most frequent in blood circulation and play a critical role during hemostasis modulation blood clotting through activation of. integrin IIb3 with a strong affinity for fibrinogen. Apart from this, these EVs also stimulate the proinflammatory p-selectin for increasing the thrombin production.The proliferative stage starts after 2–3 days of abrasion and accounts for fibroblasts migration to initiate ECM formation, collagen deposition, epithelization of keratinocyte, and ultimately wound closure  described the relationship between inflammation and burn healing. This group introduced human induced pluripotent stem cell (iPSC)-MSC-derived Exos via local injection at the burn site in rats and found that the wound healing effects depended on Exo transfer of Wnt4. Exos not only stimulated the neo-angiogenesis process, but also promoted collagen maturation required for wound healing. In addition, re-epithelialization was accelerated, and scar width was reduced.in vitro and in vivo. Exos naturally produced by adipose stem cells stimulate endothelial progenitor cell proliferation and angiogenesis in conditions of high glucose levels. This protective effect is enhanced when nuclear factor erythroid 2-related factor 2 is abundantly expressed. In vivo wound healing showed a reduction in ulcerated area, increased formation of granulation tissue, increased level of growth factor expression, and reduction in the level of inflammation upon treatment with adipose stem cells. Upon comparison with unmodified EVs, EVs isolated from UC-MSCs with miR-181c overexpression has shown a stronger suppressive effect on the Toll-like receptors 4 pathway in third-degree burn wound model.Genetic engineering is another strategy for EVs to heal chronic wounds by gene editing using genetic or biochemical procedures.,EVs extracted from various human mesenchymal stem cells may serve as therapeutic molecules for cell-free therapeutic applications.in vivo targeting by manufacturing iron oxide tagged Exos NPs and testing their therapeutic characteristics in an animal model of wound healing -BMSC-Exo promotes proliferation, migration, and emergence of new blood vessels in comparison with administration of BMSC-Exo alone. Local delivery of Mag-BMSC-Exo to the wound site increased wound closure, reduced scar size, and enhanced angiogenesis compared with BMSC-Exo delivery  improved wound closure. The experiment involved the fabrication of Exos by stimulating BMSCs with magnetic NPs and static magnetic fields to promote tissue regeneration.delivery B–3D. Intdelivery A. Overal,,,,,Because of their great biocompatibility and ability to carry and transport cargos across cells, EVs can act as vector for delivery of therapeutic drugs across cells and prevent their degradation during wound repair. At present, exogenous, and endogenous approaches are used to encapsulate drug molecules and nucleic acids into EVs.In vivo results showed that urine-derived stem cell EVs in the human acellular amniotic membrane may effectively accelerate wound healing by improving cellular senescence and decreasing senescence-associated secretory phenotype production in aged skin ulcers. Urine-derived stem cell EVs were used to grow human dermal fibroblasts in vitro to understand the process. The results proved that urine-derived stem cell EVs could regenerated senescent fibroblasts by switching the aging phenotypes of senescent human dermal fibroblasts and effectively lowering senescence-associated secretory phenotype production after triggering the NAD-dependent deacetylase sirtuin-1 pathway. As a result, urine-derived stem cell EVs are effective in improving wound treatment in elderly mice by diminishing cellular senescence.As a result, a recent study hypothesized that the human acellular amniotic membrane can serve as a vehicle for the extended release of urine-derived stem cell EVs and may enhance the wound healing of urine-derived stem cell EVs in the elderly.,in situ synthesis by adding crosslinker and polymers simultaneously.Indeed, rather than depending on EVs’ endogenous targeting to the appropriate tissues, alternative treatment approaches of therapeutic EVs is its local application. This strategy is attracting attention for tissues having external access to the environment, such as the skin. Transdermal administration of EVs has earlier been shown to promote skin healingin vitro wound-healing activity, FHE/Exo hydrogels have been found to increase the potential of human umbilical vein endothelial cells to proliferate, migrate, and form tubes. Moreover, in vivo studies revealed that FHE/Exo hydrogels dramatically improved the healing efficiency in the full-thickness diabetic model, as evidenced by improved wound closure speed, accelerated angiogenesis, re-epithelization, and increase in deposition of collagen at the wound site  hydrogels for synergistic wound-healing activity A.42 The und site B–4E. The,,in vivo study. Apart from these, in vitro analysis confirmed 100% release of EV in 24 h.Hydrogels containing EVs from various cell origins have already been used to stimulate the healing process. Hydrogels based on polysaccharides, such as chitosan, hyaluronic acid alginate, have already been given clearance by the U.S. Food and Drug Administration (FDA) for wound dressings, and have also been extensively studied for localized application of EVs.in vivo tests demonstrated that EV loading in silk/chitosan hydrogels might significantly prolong wound closure in a diabetic ulcer model  scaffold dressing, constructed out using decellularized small intestinal submucosa (SIS) combined with mesoporous bioactive glass (MBG) and Exos. In addition to enabling the sustained release of bioactive Exos, the produced SIS/MBG@Exos hydrogel 3D scaffold dressing was also observed to improve blood flow and induce angiogenesis in diabetic rats, which hastened diabetic wound healing.in situ injectable HA@MnO2/FGF-2/Exos hydrogels might speed up the healing of chronic wounds. The finding suggested that upon local injection, these hydrogels were able to create a protective barrier for covering the wounded site with subsequent rapid hemostasis and long-term antibacterial protection. These injectable hydrogels enhanced the angiogenesis and epithelialization in the wound in addition to catalyzing the conversion of excess H2O2 to O2.Delivering oxygen is another more recent technique used to heal chronic wounds. It is well established that oxygen administration can control oxidative stress and induce angiogenesis in an infection. OxOBand is an example of a wound dressing based on this method. OxOBand, an antioxidant polyurethane wound dressing containing Exos, is responsible for releasing oxygen. Upon application to a wound, this approach is used to release oxygen and adipose-derived stem cells Exos. In diabetic wounds, the dressing accelerated collagen deposition and re-epithelialization.,Likewise, proper immune responses and pH-responsive-based therapies have also been proposed as a potential and alternative way to facilitate angiogenesis and Exo-based therapy for faster wound healing.https://www.fda.gov/vaccines-blood-biologics/consumers-biologics/consumer-alert-regenerative-medicine-products-including-stem-cells-and-exosomes; reviewed on February 16, 2022). ClinicalTrials.gov  identified six separate conduct of clinical investigation trials using EVs as a treatment for chronic wounds at the time of publishing  and polymeric materials may boost the therapeutic potential of EVs. However, biocompatibility difficulties must be considered carefully while transforming EVs by genetic operations or other chemical approaches. Indeed, it has been claimed that in several diabetic model chronic wounds heal on their own, resulting in variations in results obtained from animal studies and human clinical trials. To properly examine the therapeutic benefits of EVs, standardized animal models are required for chronic nonhealing wounds.,The translation of these nanosystems into clinics poses major challenges in terms of developing a manufacturing method that provides both good quality and quantity. Researchers have worked hard to create good manufacturing practice (GMP)-grade EVs using various ways.A significant amount of research and clinical trials have been conducted over the past few decades on the detection and creation of physiological variables that influence the biological processes that occur during wound healing to improve the healing pace and quality. However, the rising number of patients suffering from persistent wounds or scarring highlights the need for more effective wound-healing treatments. The fundamental disadvantage of EV-based treatment is inadequate isolation, purification, and targeted delivery to the location because of their low stability, and lower encapsulation efficiency because of integration of parental cells. As a result, future research must focus on developing innovative delivery mechanisms with proper isolation and purification techniques for the effective administration of EVs based therapeutics.Iron oxide NPs, magnetic NPs, cationic polymeric NPs, scaffolds, hydrogels, and even inorganic materials have been found and developed as a carrier for effective delivery of EVs along with cargoes like drugs and nucleic acids. But in addition to the delivery agent, little consideration has been given to the appropriate distribution mechanism. Throughout the phases of the wound-healing process, an effective drug delivery method should discharge the therapeutic agents that match the spatial needs. Several drug delivery technologies that can manage the time-dependent drug release profile, such as passive, active, and smart systems, can be used for EV-based wound therapy. Smart materials that respond to wound biomarkers are examples of smart systems, as are integrated sensing/delivery systems.Whereas most studies have focused on the use of topical systems, in which the cargoes are delivered directly to the wound bed, there is augmented interest in the delivery of cargoes along with EVs into the deeper layers of the wound using intradermal delivery. The intradermal techniques provide for increased local availability of the EVs in the wound bed by avoiding the complete and robust exudate flow, which is rich in enzymes and can compromise the integrity of the EVs. As a result, future research will likely focus on more efficient EV delivery via intradermal devices.To conclude, the current findings are encouraging. Although the precise mechanism by which EVs induce wound healing is unknown, EVs have shown beneficial results in promoting chronic wound healing by regulating inflammatory response, enhancing neovascularization, facilitating cell growth, migration, and ECM synthesis, strengthening wound healing mechanisms, and limiting scar formation. Furthermore, EVs can be genetically manipulated, loaded with medicinal compounds, and dressed with biomaterials. Overall, the review highlights how EVs regulate different phases of wound healing and how they can be targeted using tissue engineering approaches as a summarizing remark of this paper. More study is needed to understand the precise actions of EVs in chronic wound healing and to create successful EV engineering methodologies to adapt EV-based treatments for additional therapeutic wound care in the future.


The brain is arguably the most complex part of the human body in form and function. Much remains unclear about the molecular mechanisms that regulate its normal and pathological physiology. This lack of knowledge largely stems from the inaccessible nature of the human brain, and the limitation of animal models. As a result, brain disorders are difficult to understand and even more difficult to treat. Recent advances in generating human pluripotent stem cells (hPSCs)-derived 2-dimensional (2D) and 3-dimensional (3D) neural cultures have provided an accessible system to model the human brain. Breakthroughs in gene editing technologies such as CRISPR/Cas9 further elevate the hPSCs into a genetically tractable experimental system. Powerful genetic screens, previously reserved for model organisms and transformed cell lines, can now be performed in human neural cells. Combined with the rapidly expanding single-cell genomics toolkit, these technological advances culminate to create an unprecedented opportunity to study the human brain using functional genomics. This review will summarize the current progress of applying CRISPR-based genetic screens in hPSCs-derived 2D neural cultures and 3D brain organoids. We will also evaluate the key technologies involved and discuss their related experimental considerations and future applications. Since eCRISPR knockout (CRISPR-KO) is the most common strategy used in CRISPR-based genetic screens to date. It generates efficient and precise editing of the genome using a two-component system, the Cas9 protein and a synthetic single guide RNA (sgRNA) that targets Cas9 to specific genomic loci to introduce double-stranded DNA breaks (DSBs). DNA repair machinery is activated to repair those DSBs either by homology-directed repair or non-homologous end joining. In the absence of a repair template, non-homologous end joining will result in insertions-deletions that can introduce frame-shift mutations leading to loss of function . Genome-More recently, newer iterations of CRISPR screens have emerged that utilized different forms of CRISPR-Cas9 system, which can transiently repress or activate gene expression instead of permanently editing the genome . In suchSeveral other emerging CRISPR-based genetic or epigenetic editing techniques are starting to be used for screens. CRISPR base editors, including cytosine base editors or adenosine base editors, allow precise genome editing at the nucleotide-level resolution without introducing DSBs. Collectively, they allow all four transition mutations  by using a catalytically inactive Cas9 nickase that is fused to a base-modifying enzyme . In an uA typical CRISPR-based functional genomics screen involves several key components; a tissue or a cell line, a sgRNA library to introduce the genetic perturbations, and an output assay. The Cas9 protein is either ectopically introduced with the sgRNA vector or stably expressed in the cells. The sgRNA library is usually delivered by lentiviral transduction and can be introduced in either an arrayed or a pooled format . In arraDepending on the aim of the study (positive or negative selection), the design of the screen usually varies. For example, traditional negative selection screens require a high library coverage (how many cells represent one gRNA) to detect genes that dropped out with high sensitivity, while less coverage is often needed in positive selection to determine enriched gRNAs. Recent versions of genome-wide CRISPR libraries have generated optimized sgRNA libraries with improved efficiencies and reduced off-target activities . CombineIn addition to assessing the impact of sgRNA on cell survival, cells used in a CRISPR screen can also be sorted and analyzed based on other phenotypes such as the expression profiles of cellular markers. In this type of screen, a protein of interest is either endogenously tagged with a fluorescent marker or detected with an antibody. Then, the perturbed cell populations are sorted into high- and low-expressing populations, which are later sequenced for sgRNA abundancies .in situ sequencing , Dengue (DENV), and Japanese encephalitis (JEN) can infect different brain cells including neurons, neural progenitors, and glial cells, leading to severe neurological manifestations and brain malformations in newborn children . CRISPR Genetic association studies have uncovered thousands of gene mutations linked to neurodevelopmental and neurodegenerative disorders. Understanding the molecular and cellular functions of these genes in a disease relevant context is essential to untangle the correlation between the disease genotypes and patient phenotypes. In addition, it will pave the road for developing diagnostic markers and therapeutic targets. Animal models of disease-linked mutations have shed valuable mechanistic insights, but such models may not fully translate to the human genetic landscape, and recapitulate the complex nature of the human brain. In this section, we review recent CRISPR functional screens performed in hPSC-derived 2D neural cultures and 3D brain organoids to untangle the biology of autism, microcephaly, and ALS.Xenopus, which observed an increase in the ratio of neural progenitors to differentiating neurons in the telencephalon following CRISPR knockout of 10 high-risk ASD genes (Autism spectrum disorder (ASD) represents a set of neurodevelopmental disorders that are linked to over a thousand risk genes . This coSD genes .in vivo perturb-seq study that examined the single-cell transcriptomic changes upon pooled CRISPR perturbation of 35 de novo ASD risk genes introduced in the developing mouse brain  expansion in the C9ORF72 gene represents one of the most common causes of frontotemporal dementia (FTD) and Amyotrophic lateral sclerosis (ALS) (4C2 repeat toxicity (A hexanucleotide repeat (Gis (ALS) . This exis (ALS) . Previoutoxicity . By utiltoxicity . Anothertoxicity . Anothertoxicity . These sIn 2022, Guo W. et al. conducted the first kinome-wide CRISPR screen in hPSC-derived cortical neurons to identify DPR toxicity modifiers. They found that knockouts in 113 genes, which are known to be involved in axon regeneration, dendrite development, and cytoskeletal organization, enhanced neuronal survival under DPR treatment . FurtherThere are several factors to consider when designing a CRISPR screen using hPSC-derived neural cultures .Library scale is one of the first factors to consider when designing a CRISPR screen, and it largely depends on the aim of the screen. Genome-wide screens are comprehensive because they target the majority of protein-coding genes and they can uncover novel genes involved in basic biological processes or disease development. In addition, several genome-wide CRISPR libraries are readily-available , reducinLibrary coverage reflects how often each sgRNA is represented in the targeted cellular population. Most screens utilize a library coverage between 100 and 1000 folds. Reaching this coverage for genome-wide screens usually means that hundreds of millions of cells are needed for the library transduction. Depending on the specific neural cell types, achieving this number of cells could be challenging as some hPSC-derived neural cultures may have a more limited expansion potential post-differentiation. Additionally, some neural cells such as microglia can be more difficult to transduce. To overcome these restrictions, introducing the library in hPSCs as opposed to the target cell type could be a feasible alternative as hPSCs can be readily transduced and expanded. However, one needs to be mindful that generating perturbations in the hPSC stage could potentially complicate the interpretation of data as candidate hits might arise due to defects in hPSC differentiation. The use of hPSC lines that express an inducible Cas9 gene has helped overcome such challenges by controlling Cas9 expression during or after differentiation . AdditioAnother factor to consider in the design of a CRISPR screen is the method utilized to differentiate hPSCs into neuronal or glial cells. Although some of the above-mentioned CRISPR screens utilized classic differentiation protocols based on introducing extrinsic signals , most reTo date, most CRISPR screens in hPSC-derived neural cultures utilized 2D monoculture models because they are scalable, less liable to variations, and have relatively homogeneous growth dynamics. On the other hand, hPSC-derived 2D cultures do not recapitulate the intricate organization and heterogeneity of the brain. Therefore, are not suitable for studying complex phenotypes, which include cell-cell interactions and cell migration. Such phenotypes can be examined using screens in hPSC-derived organoids because they better mimic the different aspects of early human brain development. Furthermore, organoids have been used successfully to model many disorders which include ASD, Rett syndrome, microcephaly, Alzheimer’s, and Parkinson’s, among other diseases , therebyin vivo CRISPR screens are gaining traction   . The in llenging .The choice between arrayed and pooled CRISPR-based screens in hPSC-derived cultures depends on the phenotype to be examined and the desired readout. Different readouts can be assayed in arrayed screens. In addition, minimal deconvolution is needed to link genotypes with the obtained phenotypes. However, due to challenges of analyzing readouts in arrayed formats, arrayed screens are usually limited in the number of perturbations that can be examined. Therefore, secondary CRISPR screens with arrayed formats are commonly performed in conjunction with pooled large-scale screens to validate their top hits. Pooled CRISPR screens coupled with examining sgRNA variations in surviving populations remain the gold standard to probe basic brain development and the mechanisms underlying neuropathology. As we discussed, several reports have successfully used this technology in hPSC-derived neurons, glial cells and even organoids and assembloids to identify regulators of neuronal cell fate specifications, cell survival, and disease phenotypes associated with ASD and microcephaly. Previous functional genomics screens in immortalized human cell lines provided valuable mechanistic insights into the modulators of different pathologies associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease . Future Utilizing pooled screens in 3D brain organoids with readouts that depend on sgRNA enrichment/depletion has been challenging, probably owing to the large library coverage needed to overcome noise associated with the heterogenous growth dynamics in 3D cultures. Luckily, recent CRISPR screens in brain, colon, and intestinal organoids have helped overcome these challenges by employing clonal tracing and single organoid sequencing technologies . The devß-amyloid, and a-synuclein among others (The introduction of CRISPR screens with single-cell transcriptomic readouts have unleashed unprecedented research avenues that can utilize 2D and 3D hPSC-dervied neural cultures. By integrating findings from genome-wide association studies, such screens can provide valuable insights into the complex biology of polygenic neuropsychiatric disorders, which is difficult to discern with single-mutant studies. For example, It is estimated that over a hundred loci and more than a thousand risk genes are linked to schizophrenia . By emplg others .in vitro will continue to drive significant progress in our understanding of basic brain biology and the genetic underpinnings of neurological disorders. In the future, the findings obtained from CRISPR screens in hPSC-derived cultures are anticipated to extend beyond basic science and will be utilized in translational research to target genetic modifiers of disease-linked proteins or reactive cell states, which can improve treatment outcomes for various neurological diseases.In conclusion, the combination of CRISPR-based screens and hPSC-derived neural systems has revolutionized our knowledge of the complex interactions between genetics, development, and disease. It is expected that the persistent advancements in CRISPR technologies, high-content readouts, and methods to model human brain development


Global surgery broadly refers to a rapidly expanding multidisciplinary field concerned with providing better and equitable surgical care across international health systems. Global surgery initiatives primarily focus on capacity building, advocacy, education, research, and policy development in low- and middle-income countries (LMICs). The inadequate surgical, anesthetic, and obstetric care currently contributes to 18 million preventable deaths each year. Hence, there is a growing interest in the rapid growth of artificial intelligence (AI) that provides a distinctive opportunity to enhance surgical services in LMICs. AI modalities have been used for personalizing surgical education, automating administrative tasks, and developing realistic and cost-effective simulation-training programs with provisions for people with special needs. Furthermore, AI may assist with providing insights for governance, infrastructure development, and monitoring/predicting stock take or logistics failure that can help in strengthening global surgery pillars. Numerous AI-assisted telemedicine-based platforms have allowed healthcare professionals to virtually assist in complex surgeries that may help to improve surgical accessibility across LMICs. Challenges in implementing AI technology include the misrepresentation of minority populations in the datasets leading to discriminatory bias. Human hesitancy, employment uncertainty, automation bias, and role of confounding factors need to be further studied for equitable utilization of AI. With a focused and evidence-based approach, AI could help several LMICs overcome bureaucratic inefficiency and develop more efficient surgical systems. The exponential growth of computing power in the 21st century has led to the application of artificial intelligence (AI) in the field of healthcare. AI is used as an umbrella term for the scientific discipline that focuses on the wide range of algorithmic programs and models to perform numerous complex tasks constituting the application of mathematics, statistics, and computer sciences to mimic the simulation of intelligent behavior .Global surgery broadly refers to a rapidly expanding multidisciplinary field concerned with providing better and equitable surgical care across international health systems. Global surgery initiatives primarily focus on capacity building, advocacy, education, research, and policy development in low- and middle-income countries (LMICs) -6. In 20Thereafter, The Lancet and the Financial Times launched a collaborative commission focused on the confluence of digital health, AI, and universal health coverage in October 2019 . The rapIt is also imperative to acknowledge the disparities of global health coverage particularly in the provision of surgical procedures between developed regions and LMICs. A recently published study found that there was significant gender inequity among global surgery researchers with limited involvement from low- and middle-income countries . In the Types of AI and its applicationsThere is a wide spectrum of AI technologies that are being used in healthcare services with broad predominant techniques being machine learning (ML), artificial neural networks (ANNs), deep learning (DL), computer vision (CV), natural language processing (NLP), reinforcement learning (RL), fuzzy logics, robotics, and cybernetics. Through the use of algorithms and pattern recognition, ML can analyze real-world data and estimate an outcome ,14. The ANN is an advanced subset of ML . SimilarMultiple layers of interconnected neurons have led to the development of DL . This suThe applications of these subsets of AI have further led to the development of advanced technologies. Intraoperative video can be analyzed with CV which can help in the reliable identification of surgical phases (steps) and tools across a range of operations. CV can even detect operative phases with similar accuracy as surgeons in some circumstances . The useReinforcement learning has the potential to assist in surgical decision-making by recommending actions at predefined intervals which mimics a human trial-and-error learning process to calculate optimum recommendation policies . Fuzzy lPillars of global surgery and AI: opportunitiesEducation and TrainingThe shortage of doctors in LMICs has been a long-standing challenge for several key reasons such as poor working conditions, brain drain, insufficient training opportunities, and uneven distribution of resources. For instance, India, the world's most populous nation, faces a nearly 80% shortage of surgeons, gynecologists, and other specialist doctors in rural areas . With suFurthermore, AI is a useful aid for people with special needs and helps them train accordingly taking into account their disabilities, therefore, providing individualized learning . AI alsoThe usage of simulation training allows for cost-effective surgical training and a potential avenue for training non-surgical healthcare workers to provide basic surgical care. In a comparative study done in low- and high-resource educational centers, simulation was shown to successfully enhance surgical training in low-resource settings . AI-assiGovernance and Infrastructure DevelopmentOverall, surgical interventions are among the most cost-effective procedures in global health. For example, the treatment of congenital surgical conditions costs approximately 12 to 59 United States Dollars (USD) per disability-adjusted life years averted (DALY), in comparison to HIV prevention efforts, which cost between 103 and 302 USD per DALY averted -38.Within each National Surgical, Obstetric, and Anesthesia Plan (NSOAP) domain , data drAI systems that analyze the data transparently may be used in better-allocating system resources and streamlining supply chains . Poor urResearch and Quality ImprovementResearch using AI-augmented technology is becoming increasingly popular in a multitude of different industries including the surgical field. To the researcher, AI assistance will reduce the amount of time spent on a project by automating redundant tasks in a cost-efficient manner, completing tasks more rapidly, reducing errors, and discovering trends and meaning in data more rapidly. Recent advancements include AI-enabled multispectral wound imaging devices that can provide real-time pathogen detection. Numerous projects especially in high-income countries (HICs) have studied AI models in various specialties to improve clinical efficiency and quality improvement with mutual benefits to doctors and patients ,42. The With a shortage of doctors and inequality between rural and urban health, such AI tools provide already overburdened healthcare systems a unique opportunity to improvise and use these advancements to their benefit in LMIC . AccessiLogistics and Surgical Service DeliverySystems requiring the importation of components, complex supply chains, or expertise that is not locally supported face significant barriers in providing surgical services. AI has previously been shown to streamline the process of medical equipment delivery and has been a game-changer for developing countries, Zambia and Tanzania, to close the immunization gap . SimilarSimilarly, in the case of local unavailability of superspecialists, AI-assisted tools including telemedicine and augmented reality can be used to interact with a healthcare professional virtually and use their assistance. In Peru and Vietnam, for example, the Proximie app was used to connect local surgical teams with academic centers in the United States and the UK . With thChallenges aheadRepresentation of Minorities and EthicsAs machines are “trained” by the number of inputs, minority populations will be at inherent risk of underrepresentation as there will be fewer datasets compared to the majority of the population. This may lead to discriminatory bias due to models aimed at representing the majority and not necessarily the minority subgroups . Also, tFinancing, Human Hesitancy, and Employment UncertaintyRising automation comes at a time when economic inequality is widening, increasing worries of mass technological unemployment and renewing calls for policymakers to address the impact of technological transition. Despite conflicting opinions, there is a possibility that AI may obviate human employment, which naturally causes considerable reluctance to embrace the implementation of AI at the outset even among the medical fraternity who feel unaware and unprepared to incorporate AI in their daily practice -52. In pRegulation and Oversight of High-Quality DataFor machine learning models directed at patients, special regulations and framework is needed as a patient may not be able to verify or interpret the results . AutomatConfounders, Manipulation of Findings, and the Black-Box ProblemAdversarial attacks may also lead to the manipulation of findings. Machine learning models may exploit unknown confounders to achieve the best prediction which may lead to erroneous results later . The advIn some machine learning models, the emphasis is put to correlate input and output data without a focus on causality. This leads to an inherent flaw that correlation does not imply causation. For these models, it can only be used as an approximation and not as a trusted answer . In addiArtificial intelligence in surgical care systems is expected to undergo substantial growth post the COVID-19 pandemic. The global healthcare infrastructure has observed that to develop and maintain a sustainable healthcare setup, the utilization of computational technologies such as artificial intelligence becomes crucial. In addition, several research centers and governments have actively participated in the building of robust AI technologies which are assisting healthcare professionals to work efficiently even under a shortage of resources. These factors will eventually drive surgical education and training growth. AI is expanding its footprint in clinical systems ranging from databases to intraoperative video analysis. The unique nature of surgical practice leaves doctors and healthcare stakeholders well-positioned to help usher in the next phase of AI, one focused on generating evidence-based, real-time clinical decision support designed to optimize patient care and workflow.


Adverse childhood experiences (ACEs) can have harmful, long-term health effects. Although primary care providers (PCPs) could help mitigate these effects, no studies have reviewed the impacts of ACE training, screening, and response in primary care.This systematic review searched four electronic databases  for peer-reviewed articles on ACE training, screening, and/or response in primary care published between Jan 1, 1998, and May 31, 2023. Searches were limited to primary research articles in the primary care setting that reported provider-related outcomes  and/or patient-related outcomes . Summary data were extracted from published reports.Of 6532 records, 58 met inclusion criteria. Fifty-two reported provider-related outcomes; 21 reported patient-related outcomes. 50 included pediatric populations, 12 included adults. A majority discussed screening interventions (n = 40). Equal numbers (n = 25) discussed training and clinical response interventions. Strength of evidence (SOE) was generally low, especially for adult studies. This was due to reliance on observational evidence, small samples, and self-report measures for heterogeneous outcomes. Exceptions with moderate SOE included the effect of training interventions on provider confidence/self-efficacy and the effect of screening interventions on screening uptake and patient satisfaction.Primary care represents a potentially strategic setting for addressing ACEs, but evidence on patient- and provider-related outcomes remains scarce.California Department of Health Care Services and the Office of the California Surgeon General.The  In a preliminary search of PubMed, Web of Science database, APA PsycInfo, and CINAHL, we assessed existing evidence on training, screening, and response to addressing adverse childhood experiences (ACEs) in primary care between January 1998 and May 2023, not restricted to the English language. Our search terms included primary care setting terminology  separated by OR statements, conjoined using AND statements with ACE-related terminology  also separated by OR statements. We identified a number of studies on scale-up of screening for ACEs in primary care settings, as well as epidemiologic studies on ACE prevalence and demographic differences in these settings. A 2022 systematic review on patient-to-provider communication of childhood adversity in primary care identified five studies from 2011 through 2021 and concluded that effective communication is essential for addressing ACE exposure; however, it did not link implementation of ACE screening in primary care with patient or provider outcomes.This study uses a comprehensive search strategy to synthesize evidence on the effects of ACE training, screening, and clinical response in primary care—from the vantage point of provider- and patient-related outcomes. We found 58 studies that met inclusion criteria, collectively providing information of the state of the science on addressing ACEs in primary care settings. We found that the current strength of evidence to support interventions is low or very low with respect to most outcomes, including enhanced provider knowledge, changes in clinical behavior, improved patient-provider rapport, and altered health outcomes. Evidence was strongest (moderate) for intervention effects on provider confidence when discussing composite ACEs and patient acceptability and satisfaction with care. We did not find any evidence of iatrogenic effects.This systematic review demonstrates that there is a dearth of evidence linking screening for ACEs and clinical response interventions in primary care settings with respect to patient and provider outcomes. We found few studies outside high-income countries, despite the vast majority of children and adolescents living in low- and middle-income countries. Furthermore, only 12 studies evaluated ACE-related assessments of adults, compared to 50 studies that included pediatric populations. Given the rapid and widespread scale-up of ACE screening and response in primary care settings, this study provides impetus for more rigorous evaluations. Although the limited evidence to-date suggests addressing ACEs in primary care may yield benefits, more rigorous investigations would help clinicians discern which types of interventions are most effective in addressing ACEs outside specialty care settings.,Adverse childhood experiences (ACEs) represent a constellation of potentially traumatic life stressors that occur during childhood and adolescence.,,A growing literature has delineated the potential utility of screening for and providing services that countervail the effects of varying ACE exposures. For example, a recent review classified seven types of interventions shown to have varying levels of benefits for those with a history of ACEs.Primary care settings are meant to be routine entry points into the health system, where primary care providers (PCPs) can gradually cultivate rapport and trust with their patients.32,,,,ACE screening and clinical response in primary care could also come with challenges. For example, PCPs have reported insufficient time to incorporate new screenings and interventions.To our knowledge, no studies have systematically reviewed the effects of implementing ACE training, screening, and clinical response in primary care settings for a composite of ACE exposures. In this study, we present findings from a systematic review of the peer-reviewed literature on this topic, in which studies identified forms of adversity as ACEs. Primary outcomes of interest were provider knowledge, confidence, screening behavior, and clinical care, and patient satisfaction, engagement in referrals, and health outcomes.1.,Pertain to one or more of the categories of ACEs described by the landmark CDC-Kaiser Permanente study2.Focus on ACE training, screening, and/or response with respect to composite ACE measurement—using a tool such as the CDC-Kaiser Permanente Questionnaire—rather than measurement of a single ACE exposure such as child sexual abuse or intimate partner violence.3.Involve implementation of ACE training, screening and/or response in a primary care setting, defined in terms of one of the following clinical areas: pediatrics, family medicine, internal medicine, obstetrics and gynecology, or geriatrics.4.Focus on PCPs or staff typically present in primary care settings, rather than specialty providers.5.Analyze the effects of implementation on one or more pre-specified provider and/or patient outcomes (see Data Abstraction and Synthesis).6.Constitute empirical research that underwent peer review. We excluded commentaries, viewpoints, guidelines, conference abstracts and proceedings, and reviews.7.Be written in or translated into English.We searched the literature from January 1, 1998 through May 31, 2023 and reported results following PRISMA guidance.For a study to be included, it needed to contain one or more search terms within two domains: first, an indication the article pertained primary care and/or primary care providers; second, an indication the study focused provider and/or patient outcomes related to ACEs or toxic stressors that could arise during childhood or adolescence. These criteria were applied to four databases: PubMed, Web of Science, APA PsycInfo, and CINAHL. The complete search strategy can be found in Each title and abstract were independently screened by two members using DistillerSR,Each article was assigned a primary and secondary abstracter: the primary abstracter was responsible for reviewing the article and abstracting data into a standardized form. The secondary abstracter reviewed accuracy and quality. We abstracted the following study characteristics: title, authors, publication year, geographic setting, study design, study period, study population(s), sample size(s), and provider-related outcomes and patient-related outcomes. We also clarified whether the study focused on pediatric or adult populations. Given that a large majority targeted pediatric populations, study findings are focused on children and adolescents unless explicitly stated otherwise in the Results or Discussion.For the purposes of this review, assessments of ACEs included those focused on measurement of multiple ACEs, as captured by composite screening and assessment tools. We limited studies to those assessing composite ACE exposures because we were targeting articles that conscribed to the ACEs conceptual framework—i.e., that recognized that varying forms of childhood adversity converge on a common set of physiologic stress-response systems, and that this in turn may lead to negative health outcomes.To be coded as an ACE training intervention, a study needed to describe implementation and measure outcomes associated with training PCPs and staff on the epidemiology of multiple ACEs, identification of multiple ACEs in clinical settings, and/or appropriate responses once ACEs were identified. To be coded as an ACE screening intervention, a study needed to describe implementation and measure outcomes associated with the introduction, scale-up, or strengthening of composite ACE screening in primary care settings. To be coded as an ACE response intervention, a study needed to describe implementation and measure outcomes associated with behaviors in response to identified ACEs in primary care. Studies could be coded to one, two, or all three intervention categories.For provider-related outcomes, we indexed provider knowledge, confidence and self-efficacy, screening behavior, and clinical response—inclusive of changes in clinical care, reporting, and referral practices. For patient-related outcomes, we quantified acceptability and satisfaction, engagement with referrals, and health outcomes. To the extent possible, we reported measures indicating the magnitude of effects, strength of associations, and statistical significance.We rated strength of evidence (SOE) according to GRADE criteria. Two team members independently assigned scores each article, based four domains: risk of bias, inconsistency, indirectness, and imprecision. Observational studies received a starting score of “low”, while randomized studies received a starting score of “high”. Studies could be upgraded or downgraded based on individual assessments within each domain. In the event two team members arrived at different scores, each evaluation was shown to all team members for collective deliberation. All team members collectively deliberated the weight of evidence across studies for each outcome of interest. SOE was assigned an ordinal value: very low (+), low (++), medium (+++), or high (++++).The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. RKM had access to all data and was responsible for the decision to submit for publication.The literature search generated 6532 articles, of which 1577 were duplicates, yielding 4955 unique studies see . Of thesAmong included studies, 41 used samples from the United States, 3 from the Netherlands, two from the United Kingdom, and one each from Australia, Canada, Denmark, Japan, Kenya, and Saudi Arabia. Twelve studies did not list location. Studies contained an average of 73 providers and 1781 patients and represented a broad age range and both sexes. Seven studies were randomized controlled trials (RCTs) and two were quasi-experimental. Of the remaining, 20 used mixed methods, 13 used a prospective cohort design, 11 were cross-sectional, and seven were qualitative.,,,,,Ten of 13 studies in this category examined provider knowledge before versus after ACE training.,45,49,,,,,Eight of 15 studies in this category examined changes in confidence and/or self-efficacy from before to after training.,,,,,,,,,,,,All nine studies in this category—three of which were RCTs,,,,Five studies discussed changes in clinical care following training interventions.Four studies examined reporting and documentation practices, following training efforts. Three focused on child protective services (CPS). The first observed an increase in providers’ understanding of their reporting responsibilities following training,,,,Lastly, four studies inspected changes in referral patterns after ACE training.,,,,,,,Five studies found that screening pilots led to improvements in knowledge on effective ACE screening.,,,,,,,,,,,Of five studies that conducted pilot programs,,,,,,,,,,59,,,,,,,,,,59,,,,Of 24 articles measuring screening behavior, 20 evaluated interventions,,,,,,Of 12 studies that examined post-screening clinical care, which ranged from providing resources to motivational interviewing to treatment planning, six studies found either high rates of clinical responses or significant improvements after an intervention,,,,,,,,,,,,77Of 14 studies identifying referrals as a clinical response outcome, seven concluded implementation of screenings was associated with increased referrals.One study examined mandated reporting.,,,,,Five studies examined provider rapport with patients.,,,,One of seven articles,,,,,Four of five studiesOf five studies that examined onsite clinical responses to ACEs, two evaluated Practicing Safety, finding that provider participation was associated with a higher likelihood of offering evidence-based guidance,,,70,,,We identified eight studies that examined external referrals as a clinical response.,,,Four of seven clinical response studies,,,Three studies examined provider-patient rapport.SOE assessments of individual studies can be found in ,,Three of five studies in this category were RCTs. One found that patients served by providers randomized to training reported significantly greater satisfaction with their visit.Of two studies in this category, one RCT found caregivers served by providers assigned to training were 17-times more likely to contact community resources.,Two RCTs found that caregivers who were served by providers assigned to the training group reported fewer instances of psychological aggression or physical violence, compared to those served by providers in the control group.,,,,,,,,,,,Eleven of 14 studies in this category comprised surveys (n = 10) or interviews (n = 1) of caregivers or patients after screening.,,,,Among five studies examining referral acceptance,,Of three studies, two RCTs focused on reductions in household violence. Each found that physical violence or psychological aggression was reduced among parents served by intervention providers.Five observational studies examined patient satisfaction. One reported high levels of satisfaction (>90%) with an intervention involving motivational interviewingTwo observational studies examined acceptance of referrals. One found 39% of patients accepted behavioral health referrals one-month post-intervention.,,,Three studies reported patient health outcomes.SOE assessments of individual studies can be found in This systematic review indicates that evidence on the benefits of expanding ACE training, screening, and response in primary care remains nascent. Where available, studies have typically indicated positive associations. Yet, for the most part, we failed to observe consistent linkages between specific interventions and outcomes. Additionally, studies were predominately observational and subject to a variety of limitations.,With respect to ACE training interventions, a preponderance of studies focused on self-reported changes in provider knowledge and confidence. In most instances, studies recorded positive, short-term gains. Whether gains were maintained, and the extent to which they altered clinical practice, were unclear. Thirteen studies—including two RCTs—also reported large increases in ACE screening rates following training. The magnitude of these gains was comparable to documented rates on the scale-up of behavioral health screenings more generally.A common pattern here and elsewhere pertained to lack of insight on referral practices. In a recent analysis, Hartveit and colleagues (2017) articulate several quality indicators for assessing referral processes, including whether the referral is appropriate and results in timely access to care.,,,Only nine studies examined the association between ACE training interventions and patient-related outcomes, primarily patient satisfaction with care and acceptability of ACE screening. Three RCTs found patients in intervention arms reported higher levels of comfort,With respect to the 29 screening interventions we reviewed, 25 quantified subsequent changes in providers’ screening behavior. Collectively, these studies provided medium SOE, similar to the quality of evidence and risk of bias documented in previous systematic reviews on uptake of screening for cancers and mental health conditions.,,,,,Twenty-one studies examined how ACE screening interventions influenced patient-related outcomes. Fifteen quantified patient or caregiver acceptability and/or satisfaction, reporting high levels in each category—ranging from 72%,,,With respect to ACE response interventions, we only identified seven that addressed provider-related outcomes. Three outcomes—provider knowledge, confidence, and rapport with patients—possessed very low SOE: multiple studies found little or no relationship with improved knowledge,A larger group of studies (n = 14) with moderate SOE found ACE response interventions led to meaningful changes in clinical care and increased referrals. For example, Gerlach and colleagues (2021) found that—following the ACE intervention—over three-quarters of providers reached out to on-site clinical social workers or therapists to engage with patients after positive screening.,,,,,,,It was uncommon for studies to document ways in which altered provider behavior affected patient outcomes: we identified only seven studies, all focused on patient satisfaction or acceptability. In most instances, feedback was positive.,,,,,,Only 12 studies specifically focused on ACE screening and response among adults, compared to 50 focused on pediatric services see . Adult-fOverall, our analysis indicates a critical gap in knowledge regarding the association between ACE training, screening, and response in primary care and provider- and patient-related outcomes. Studies were inconsistent in terms of the ACEs they investigated, interventions they employed, and outcomes they measured. Studies were also inconsistent in their own terminology, and we excluded studies that did not include ACE-specific language. Future studies that aim to be more comprehensive—by including studies that do not self-ascribe as focused on ACEs—would benefit from collating an inventory of clinical screeners that assess ACEs. This could then be used as the basis for future assessments of the ACEs literature.Of the 58 studies we identified, only seven were RCTs. Among the observational studies, roughly half employed survey instruments that involved self-reporting, often with small sample sizes and low response rates. A strategic next step would be to generate expert consensus on which interventions in primary care may be the most likely to mitigate the sequelae of specific adversities, and then conduct experimental investigations. This includes investigations that employ implementation science frameworks to map relationships between processes and outcomes in the context of clinical practice. It also includes investigations in resource-limited settings where adversities—particularly economic and food insecurity—may be widely prevalent but are significantly understudied.We note several study limitations. First, although our search strategy assessed over 6000 articles, it is possible we failed to capture relevant studies. We reviewed studies that self-identified as focused on training, screening, and response to multiple ACEs, rather than the broader literature on toxic stress physiology and pediatric clinical screeners, or the narrower literature on individual exposures such as child sexual abuse. Second, heterogeneity of sampling frames and study designs limited our ability to draw broad inferences. Likewise, interventions may have entailed components that were not explicitly described in the text and we were therefore unable to reflect particular nuances. Lastly, to the extent publication bias influenced this investigation, it would imply that strength of evidence may be weaker than that conveyed by our assessment.Notwithstanding these limitations, this systematic review is the first we know of to show that high-quality information on the effects of ACE-related interventions in primary care settings is limited, and that the field is currently relying primarily on observational studies that have small sample sizes. At the same time, the extant literature indicates high rates of patient acceptability, improvements in provider knowledge and referrals, and reductions in household violence following specific interventions. We did not find any evidence indicating that ACE screening and response led to adverse outcomes, apart from possible tensions in caregiver-provider rapport when discussing ACEs—particularly in the context of suspected child abuse. Moving forward, researchers should endeavor to conduct rigorous studies to evaluate best practices for addressing the health consequences of ACE exposure.RKM, NKE, JSL, and GMS conceived and designed the study. SM and NQ led data extraction, with FKM and JSL verifying extracted studies. Differences in opinion on study inclusion were resolved by discussion among RKM, NKR, JSL, SM, and NQ. RKM, SM, NQ, and JSL accessed and verified the underlying data. All authors  contributed to interpretation of the results and manuscript writing, and they approved the final manuscript for submission.rmcbain@rand.org).All extracted data that correspond to the findings of this systematic review will be made available upon request after approval of a proposal from the corresponding author, beginning the publication date of this article  over the prior 36 months, including since the initial planning of work, as well as consulting fees from UCAAN. RG also declares consulting fees from the Center for Youth Welnness and payment for expert testimony from Eglet Adams and Nevada State ACEs and Toxic Stress Abatement Plan. RG also declares participation in three data safety monitoring/advisee boards: for Pathways to Resilience Expert Advisory Committee–Aurrera Health, Expert Review Panel, Safe Spaces: Foundations of Trauma-Informed Practice for Educational and Care Settings–Office of the California Surgeon General, and National Early Relational Health Network. RG declares roles as the co-founder of the National Committee on Asthma and Toxic Stress and board membership of the Board Member, California American Professional Society on the Abuse of Children, as well as stock options in Stronger Brains Inc. KK declares support from California Department of Health Care Services and UCAAN since the initial planning of the work, as well as grants/contracts and consulting fees through Aurrera Health Group, Hillcrest Advisory, and the Population Health Innovation Lab in the prior 36 months.


Reproductive function is critical for species survival; however, it is energetically costly and physically demanding. Reproductive suppression is therefore a physiologically appropriate adaptation to certain ecological, environmental, and/or temporal conditions. This ‘allostatic’ suppression of fertility enables individuals to accommodate unfavorable reproductive circumstances and safeguard survival. The mechanisms underpinning this reproductive suppression are complex, yet culminate with the reduced secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, which in turn suppresses gonadotropin release from the pituitary, thereby impairing gonadal function. The focus of this review will be on the role of RFamide-related peptide (RFRP) neurons in different examples of allostatic reproductive suppression. RFRP neurons release the RFRP-3 peptide, which negatively regulates GnRH neurons and thus appears to act as a ‘brake’ on the neuroendocrine reproductive axis. In a multitude of predictable  and unpredictable  situations in which GnRH secretion is suppressed, the RFRP neurons have been suggested to act as modulators. This review examines evidence for and against these roles. Reproductive function is fundamental for species survival. However, it is physiologically costly—especially in mammals—due to the high energetic demands of pregnancy, lactation and rearing of offspring. When environmental or physiological conditions are not conducive for reproduction, neuroendocrine pathways that can exert allostatic control over reproductive function cause acute reproductive suppression. This ‘allostatic’ reproductive suppression is adaptive, as it enables individuals to accommodate unfavorable changes in their environment and/or in their physiology in order to safeguard survival. According to McEwen and Wingfield , allostaThere are many examples of allostatic reproductive suppression. For example, in seasonal breeders, reproductive suppression is predictable, such that fertility is coordinated to ensure offspring are born when environmental conditions are most favorable for survival. During different life cycle stages, such as pre-puberty and post-menopause, reproductive function is also predictably suppressed. However, unpredictable events can also cause reproductive suppression, such as certain environmental and/or physiological states. During times of severe metabolic stress, for example due to famine, energy demand exceeds energy supply, which will initiate an allostatic response that directs an individual away from energetically costly breeding and into survival mode. During times of psychosocial trauma or severe illness, which are also unfavorable times to reproduce, sufficient energy may be present, but the activation of the stress systems can induce a facultative physiological and/or behavioral response that ultimately defers reproduction ,2. AllosTo understand the mechanisms underpinning allostatic reproductive suppression, it is necessary to first understand how the reproductive axis—the hypothalamo-pituitary-gonadal (HPG) axis—is regulated. The hypothalamic gonadotropin-releasing hormone (GnRH) neurons governing the HPG axis serve as the final integrators of a multitude of neuroendocrine and hormonal inputs . GnRH redecrease in RFRP neuronal activity is the preovulatory GnRH/LH surge [Rfrp gene expression, RFRP cell numbers, and RFRP + cFos coexpression  all decline on the afternoon of the surge in naturally cycling or surge-induced hamsters and mice [RFamides form a class of neuropeptides characterized by an arginine–phenylalanine–amide motif at their C terminus. They were first identified in 1977 in molluscan cerebral ganglia, but almost another decade passed before they were purified and chemically characterized in any mammalian species. There are now five RFamide families known to exist in the mammalian central nervous system, where their widespread fiber distribution implies varied roles including regulation of energy balance, behavior, and reproduction . The RFRLH surge . Hypothaand mice ,26. Thisand mice ,4.As mentioned previously, RFRP neurons appear to be involved in both the temporal regulation of reproductive function, which causes predictable cycles of reproductive activation and inactivation, as well as in stress-related reproductive regulation, which allows for facultative reproductive suppression. RFRP neurons are thus emerging as important integrators of environmental and physiological cues within the neuroendocrine reproductive axis, such that they release RFRP-3 peptide to act as a ‘gate’ or ‘brake’ on reproductive function when the afferent inputs they receive suggest conditions are not optimal see . HoweverMany studies have explored the impact(s) of exogenous central RFRP-3 administration on reproductive function. In contrast to the variable results obtained from peripheral administration, these studies mostly support the concept that RFRP-3 acts as a brake on GnRH-induced LH release and reproductive drive. For example, intracerebroventicular (ICV) injection of RFRP-3 to male rats significantly suppressed all facets of male sexual behavior and also reduced plasma levels of LH . ICV RFRRfrp gene silencing using targeted shRNA alleviated various stress-induced infertility measures in female rats [Rfrp, thereby inhibiting its effect, show elevated gonadotropin levels [To further investigate the in vivo role of RFRP-3 on the HPG axis, our lab generated a novel RFRP-Cre mouse line, which we then crossed with Cre-dependent designer receptor (DREADD)- or diphtheria toxin receptor-expressing mice to investigate the effects of endogenous RFRP-3 excitation and inhibition, as well as RFRP neuronal ablation . Chronicale rats , suggestale rats  . WhiSeasonal breeding is crucial to the survival and reproductive success of the vast majority of vertebrate species inhabiting temperate regions. Seasonal changes in environmental factors, such as photoperiod, lead to alternating periods of photosensitivity and photorefractoriness in small birds and short-lived mammals to bring about alternating periods of reproductive activity and quiescence, the latter being maintained by an increased potency of negative feedback regulation. In larger long-lived animals, the environmental cues interact with the underlying endogenous rhythm, producing the same reproductive outcome. The timing of reproductive activity, which precedes the birth period by the duration of gestation, ensures that the young are born during a season in which the environmental conditions most favor neonatal survival. Photoperiodic cues can act directly on photoreceptors on the GnRH neuronal network in birds, whereas in mammals they are encoded and communicated to reproductive (and other) brain control centers in the form of elevated secretion of the pineal hormone melatonin during the hours of darkness, which are extended in duration during winter and shorter in summer in temperate regions ,50.It would be reasonable to hypothesize that RFRP neuronal activity would be higher during reproductive quiescence than during the breeding season. Consistent with this hypothesis, more RFRP-3 immunoreactive cell bodies were observed during the non-breeding season  than the breeding season in male macaques  . SimilarRfrp mRNA and immunoreactive RFRP neurons compared to virgin diestrus controls, and this effect was attributable to the presence of elevated circulating prolactin concentrations in the lactating animals . In another study that employed a different experimental design (lactating rats with pups removed were used as controls), the suckling stimulus exerted the opposite effect: it increased Rfrp mRNA levels, and the authors suggested this might be a cause of lactational infertility [Rfrp expression.There are currently very few studies in which the role of RFRP-3 in lactational infertility has been tested. Conceptually, the output of RFRP neurons might increase to cause lactational amenorrhea since they can inhibit reproductive function. Alternatively, it might decrease since lactation is associated with stress hyporesponsiveness  and RFRPertility . ClearlyStress-related reproductive dysfunction is perhaps the most notable form of allostatic reproductive suppression. The reproductive impairments can be subtle, such as reduced hormone secretion, or severe, such as the complete suppression of puberty onset and/or fertility . While mRfrp gene expression in vitro [Rfrp gene expression in male and female rats [Rfrp gene silencing using a targeted shRNA prevented stress-induced infertility in female rats [Importantly, approximately half of RFRP neurons express GR , and corin vitro . Physiolale rats ,67, but ale rats . Data frale rats . Similarale rats . A singlale rats , indicatRfrp mRNA levels [Metabolic challenges, such as starvation, inhibit fertility and sexual behaviors. Many of the neurons that provide important input to the reproductive axis are also involved in control of metabolic functions, and this allows the availability of metabolic fuels to be coordinated with fertility . For exaA levels  or no deA levels  relativeA levels . TogetheIn Syrian hamsters, the percent of RFRP cells co-expressing c-Fos  was significantly increased at 8 and 12 days after food restriction . The effRfrp and Npffr1 mRNA levels, and these were negatively correlated with Gnrh mRNA and serum LH levels [Rfrp mRNA despite causing a reduction in serum LH levels [Immune stress, such as infection and inflammation, can also have a negative impact on fertility ,78,79. EH levels . In anotH levels . HoweverH levels , suggestH levels ,67,69, bRfrp mRNA, which correlates with a downregulation of serum LH in male rats [Rfrp mRNA, since it was prevented by adrenalectomy [Rfrp expression associated with suppressed sexual maturation in non-dominant naked mole rats, RFRP-3 administration was sufficient to sustain their suppressed sexual maturation, even when the individuals were removed from the dominance cues [As with metabolic and immune stress, which are physiological stressors, psychosocial stress  can likewise cause allostatic reproductive suppression. However, unlike metabolic and immune stress, which directly threaten an individual’s survival, psychosocial stress is purely a perceived threat. In humans, depression and other mood disorders are examples of psychosocial stress associated with reproductive dysfunction . One comale rats . GC relealectomy . We havealectomy . Furtheralectomy ,31,83,84alectomy  and othealectomy . Not onlnce cues . CollectThe mechanisms underpinning adaptive reproductive suppression are complex and involve a multitude of factors. The RFRP-3 neuropeptide system appears to play a modulatory role in integrating many of these factors and acting as a ‘brake’ on reproductive drive when conditions are unfavorable. RFRP neurons appear to contribute to stress-induced and (in some species) seasonal reproductive suppression, while their role in mediating changes in reproductive competency during different life cycle stages  is less clear and would benefit from functional data from manipulating RFRP neuronal activity see . With th


Correlative studies should leverage clinical trial frameworks to conduct biospecimen analyses that provide insight into the bioactivity of the intervention and facilitate iteration toward future trials that further improve patient outcomes. In pediatric cellular immunotherapy trials, correlative studies enable deeper understanding of T cell mobilization, durability of immune activation, patterns of toxicity, and early detection of treatment response. Here, we review the correlative science in adoptive cell therapy (ACT) for childhood central nervous system (CNS) tumors, with a focus on existing chimeric antigen receptor (CAR) and T cell receptor (TCR)-expressing T cell therapies.We highlight long-standing and more recently understood challenges for effective alignment of correlative data and offer practical considerations for current and future approaches to multi-omic analysis of serial tumor, serum, and cerebrospinal fluid (CSF) biospecimens. We highlight the preliminary success in collecting serial cytokine and proteomics from patients with CNS tumors on ACT clinical trials. In the USA, there are currently more than 30,000 children living with central nervous system (CNS) tumors, making it the most prevalent solid tumor and leading cause of cancer-related death in children , 2. PatiThe challenges and perceived opportunities for ACT against CNS tumors have been well documented –5. HematDue to the variety of antigens targeted, along with differences in eligibility criteria in pediatric cellular therapy trials, identifying common features in immune responses across trials likely requires tight alignment of correlative data analyses. Correlative studies can be broadly defined as investigations that utilize serially collected biospecimens  or blood) or clinical data  to uncover an underlying bioactivity and effectiveness of the trial intervention. Challenges in collecting shareable data have inspired the creation of cooperative working groups to develop guidelines for upstream sample operations and to determine the best metrics to measure tumor evolution, on-target and off-target toxicity, and disease response. The Consortium for Pediatric Cellular Immunotherapy (CPCI) is a network of collaborating institutions delivering cutting-edge cellular and gene therapies for children through multicenter clinical trials. In addition to coordinating clinical practice, the consortium fosters a real-time review of the current state of the literature and lessons learned from ongoing trials to better inform the next generation of ACT approaches ••. Here,All pediatric CNS ACT trials incorporate standard neuroimaging correlates; however, there is an acknowledged challenge in interpreting these studies in the setting of acute immunotherapeutic delivery. This is compounded in cross-trial comparisons due to the inherent variability in timing, radiographic sequences, and neuroaxis coverage. Traditionally, radiographic response criteria for CNS tumors have been based on the historical experience evaluating cytotoxic therapies, leading to unreliable response data for immunotherapeutic approaches , 25. SpeThe response assessment in neuro-oncology (RANO) criteria were initially developed to better evaluate imaging response assessment in glioma considering possible radiation- and temozolomide-induced pseudoprogression as well as pseudoresponse following anti-angiogenic therapies such as bevacizumab , 27. A lNovel imaging technology, using both functional and molecular imaging techniques, may provide clarity in therapeutic response assessment for patients treated with cellular therapies , 31, 32.As current neuroimaging is insufficient to assess disease response, and as serial tumor biopsies are frequently ethically and practically challenging, alternative biomarker-based strategies may provide greater insight into therapeutic endpoints and allow real-time clinical decision-making. In particular, correlative assessments of cytokine levels and circulating cells in the CSF and peripheral blood are beginning to reveal key insights into CAR bioactivity and anti-tumor immune responses ••, 21••.The route of CAR T cell administration may be associated with unique cytokine and toxicity profiles. Intravenous (IV) CAR T cell administration for CNS tumors is generally associated with higher proinflammatory cytokine levels in the blood, particularly IL-6, than is observed with intracerebroventricular (ICV) delivery ••. In coBeyond circulating chemokines/cytokines, paired peripheral blood and CSF sampling also enables an informative evaluation of immune cell populations during therapy. The routine use of flow cytometry permits robust multiparameter characterization of cells recovered from these compartments. Importantly, flow cytometry also allows identification of CAR T cells in these samples, although with less sensitivity than targeted PCR . MultiplFlow cytometry with single-cell technologies enables highly multiplexed characterization of cell state and identity, including at the level of TCR sequence, transcriptome, and epigenome. Pseudotime and time course analyses permit tracking of clonal evolution and cell fate in the context of the CNS tumor environment, providing deep insight into the anti-tumor immune response . These assays also can provide quantitative analysis of variable immune populations that may be driving CAR T cell efficacy or antagonism. Through multiplexed analyses that are consistent among trials, there is the potential to unlock variability in immune populations based on tumor biology, tumor location, and ACT characteristics, which would otherwise be undetectable in an underpowered single-institution study.It is important to note that variations in sample collection, handling, flow cytometry panel design, and single-cell techniques can make cross-study comparisons difficult. Standardizing collection, processing, data warehousing, and analysis pipelines will be important for ensuring that future trials are as informative as possible. Technical publications on processing and data alignment, such as the recent report from the CPCI outlining practices in flow cytometry and cytokine/chemokine analysis, are essential in this endeavor ••. FigurMonitoring of the immune state will be critically important for improving ACT. This is especially true in CNS tumors, where the Munro-Kellie doctrine imposes a narrow therapeutic window for immune-mediated treatments. Increasingly, it is becoming clear that this monitoring must be both broad and deep if we are to extract meaningful information from these first trials.Current standard practice typically incorporates multiplex cytokine analysis, which permits the measurement of scores of cytokines (often up to 50). However, there are many biologically active small polypeptides in blood and CSF, and most of them bear important functional post-translational modifications that cannot be detected by conventional analysis. Another developing platform for this purpose is targeted proteomics, which takes advantage of the exquisite sensitivity of liquid chromatography/mass spectrometry (LC–MS/MS) , 43. TheThis approach was piloted in two children with DIPG who received a combined 28 doses of intracerebroventricularly delivered B7-H3 CAR T cells and through analysis of multiple timepoints was able to capture modulation of 50 CSF and 59 serum proteins ••. CorreThere have been significant advances in the use of cell-free tumor DNA (cfDNA) in CSF as a “liquid biopsy” technique to detect CNS tumors –51. By iPediatric CNS tumors have been stratified into molecular subtypes through transcriptomics and methylomics –57. WhilImmune cell characterization is feasible through single-cell sequencing techniques but with the caveat that single-cell RNA-sequencing is less feasible due to its requirement for viably disaggregated tumors. Further, immune cell single-cell RNA sequencing requires further validation to determine immune cell function within the tumor/TME. Similar to flow cytometric studies, single-cell RNA-sequencing protocols are not standardized across institutions which can limit the comparison of data between studies conducted in multiple institution.Additional limitations with single-cell techniques are the loss of spatial orientation that occurs through the dissociation of tissue, as well as inadequate amounts of material to process. When surgical material is available, spatial proteomics can readily be performed on formalin-fixed, paraffin-embedded (FFPE) tissue. Multiplexed immunofluorescent staining, of FFPE samples, using up to 9 antibodies can detect cells within the TME, and subsequent analytics can be applied to the images to (1) measure distances between cell types, (2) quantify the cell subtype, and (3) identify architectural features within the TME and the cell subtypes within the features. In addition, recent work has shown how orchestrating the spatially resolved transcriptomics, and the data from immunohistochemistry, H&E staining, and RNA transcriptomics can be aligned to delineate the cellular subtype, the heterogeneity with the tumor, and the tumor microenvironment with the spatial orientation intact . UtilizaHowever, unlike CSF and blood collection, where it is feasible to perform serially throughout a trial, serial CNS tissue specimens may not be practical, and there are ethical questions to be asked in serial tumor collections that require neurosurgery and most likely will not change the treatment outcome for that particular child. For studies enrolling patients with recurrent tumors where surgical resection is often performed, it may be possible to give ACT treatments prior to surgery and use historical data as a comparison to changes within the TME. Ultimately, real-time evaluations via this method are impossible with current technologies, and so while direct tissue evaluation is important, timepoints will inherently be limited. Regardless, protocols for tissue collection could still be standardized even in the setting of autopsy, as many patients generously donate tissue at the time of death due to a selfless appreciation for the critical nature of these studies and the potential benefit to future children. Considering the trust that they bravely place into these early phase, and sometimes first-in-human, clinical trials, we have a mandate to maximize our learning from every biospecimen available, especially donations at the end of life.The growth of early-phase ACT clinical trials in pediatric neurooncology holds great promise for the future and reflects tremendous recent preclinical advances. Many of these trials are small and at single institutions, demanding standardized collaboration across clinical efforts as the most efficient way to improve cancer care for children; this was the motivating principle behind the CPCI. Foremost, we strongly advocate for continued cross-institutional alliances that prioritize scientific discovery and excellence in clinical care while defying barriers in the sharing of biospecimens, data, and advances.Correlative assessments will be exponentially more valuable if they are collected, processed, and analyzed in a fashion that allows harmonization Fig. . While sFor radiographic endpoints, pediatric ACT clinical trials currently may be best served to incorporate iRANO criteria with a goal of centralized review within immunotherapy consortiums. Early adoption of machine learning and advanced imaging techniques may be critical, enabling the obsolescence of imaging sequences that are less helpful for these patients. Ultimately, considering the lack of validation of iRANO for children and the inherent complexity of determinant progression-free survival due to pseudoprogressive symptoms, we advocate that overall survival be the central response and survival outcome for pediatric CNS ACT trials. Hopefully a future state will allow incorporation of other prospectively validating imaging and correlative modalities based on the frameworks we and others aim to provide. This near future of even more sophisticated pediatric CNS ACT trials with broadened correlative science objectives will demand data warehousing and infrastructure to become even more vital. Initiatives like the Children’s Brain Tumor Network (CBTN), a partnership among international researchers to collect the rich supply of molecular, imaging, and clinical data available through consortium-led trials, will allow the ACT field to better share and interpret collective data.The field of ACT is advancing rapidly, particularly for children with CNS tumors. As initial pioneering trials reach the first clinical crossroads, we should embrace the opportunity to collaborate rather than compete. Current early phase trials offer hope and inherently strive for therapeutic success, but these studies also importantly serve as the seed for enhanced future studies. To this aim, a critical step in the maturation of our field will be developing standards for the systematic collection, analysis, storage, and understanding of serialized correlative assessments of, in particular, CSF biomarkers. With parallel progress surely coming in genomics and neuroimaging, we have the ability to collect patient biospecimens now to maximize the learning from this first generation of pediatric CNS ACT clinical trials. Through technological progress, and most importantly broad cooperation, there are innumerable opportunities to optimize future trials and ultimately reach the most significant goal: curing the currently incurable tumors of childhood.


At the outbreak of the COVID-19 global crisis, diverse scientific groups suggested that this unprecedented emergency could act as a ‘blessing in disguise’. In the aftermath of the pandemic, cancer immunotherapy is unexpectedly ranked among the particularly benefited fields. Lessons learned from the real-life safety and efficacy profiles of the vaccines developed against SARS-CoV-2 are currently being applied in the field of cancer. At the same time, the introduction of relevant vaccine technology in clinical cancer trials has pinpointed a number of novel challenges, including the selection of appropriate targets, the prediction of responses, and the improvement of tolerance and specificity. In this Editorial, we draw attention to important recent articles that describe these tectonic changes in cancer immunotherapeutics and indicate the perspectives for human endogenous retroviruses (HERV) regarding further improving personalized patient management.Immunotherapy has induced a rapid paradigm shift in clinical oncology. A key reason for the success of immunological drugs is that, instead of directly attacking tumors as cytotoxic drugs do, they ally with a person’s own immune system and train it to recognize and destroy cancer cells, thus establishing durable responses. A well-known blockbuster, linked with the Nobel Prize in Medicine in 2018, is the FDA-approved immune checkpoint inhibitors (ICIs), which release the brakes that prevent T lymphocytes from killing cancer cells. Intriguingly, the pandemic propelled mRNA vaccines, a distinct class of immunotherapeutics, into the limelight as a cost-effective means to harness our immune systems against this global viral threat ,4, as weNevertheless, responses to immunotherapeutics vary widely, since some patients achieve superior outcomes, while others do not really benefit. Long-term updates show a sustained clinical benefit for melanoma patients treated with ICIs such as nivolumab or pembrolizumab or a combination of nivolumab and ipilimumab. At the polar opposite, about 40–50% of patients acquire resistance within five years from the onset of ICI therapy . On a siCancers in July 2023 comprehensively analyzed the expression of several classes of HERVs in hepatocellular carcinomas (HCC). By applying RNA sequencing on 254 HCC tissues and their 34 matched normal tissue controls, the authors described 3357 locus-specific activations of HERVs, 180 of which were correlated with poor patient survival [HERVs constitute 8% of the human genome and have attracted increasing attention due to their association with cancer development and immunotherapy. They are remnants of ancient retroviruses, which entered the germline by infecting our distant ancestors. In the largest in silico survey on full-length HERVs across 49 primates, Li Y et al. traced their origin back to nonhuman primates and reported that some HERVs originated before the speciation of Hominidae, Hylobates, and Old-World monkeys . Over tisurvival . Similarsurvival .Strikingly, HERVs have developed a multifaceted crosstalk with the immune system of their human hosts ,17. DuriSeveral groups have, independently, underlined that tumor-specific reactivation of HERVs generates signatures that predict responses to immunotherapy. Clinical studies have associated the expression of HERVs and response to ICIs not only in NSCLC patients , who exhIt is noteworthy that HERVs’ re-activation not only predicts but also increases responsiveness to ICI therapy. For example, a vaccine encoding for a region of the envelope (Env) protein of HERV  has been developed and tested in the preclinical setting. Mutated ISD enhanced T-cell immunogenicity in both prime and prime-boost vaccinations and, when combined with ICI therapy, it eradicated colorectal tumors in mice . AdditioIn conclusion, HERV products could be invaluable as predictors of ICI response and/or targets for breakthrough immunotherapeutic formulations. Intriguingly, therapeutic responses increase when ICIs are combined with mRNA vaccines. In KEYNOTE-942 NCT03897881), an ongoing open-label phase IIb trial on completely resected stage III/IV melanoma, patients who received a combination of a mRNA vaccine encoding 20 different mutated neoantigens (mRNA-4157/V940) plus ICI (pembrolizumab) showed 44% lower risk of disease relapse or death versus those who received pembrolizumab monotherapy 897881, a, the mRN


Neuronal signalling is a key element in neuronal communication and is essential for the proper functioning of the CNS. Astrocytes, the most prominent glia in the brain play a key role in modulating neuronal signalling at the molecular, synaptic, cellular, and network levels. Over the past few decades, our knowledge about astrocytes and their functioning has evolved from considering them as merely a brain glue that provides structural support to neurons, to key communication elements. Astrocytes can regulate the activity of neurons by controlling the concentrations of ions and neurotransmitters in the extracellular milieu, as well as releasing chemicals and gliotransmitters that modulate neuronal activity. The aim of this review is to summarise the main processes through which astrocytes are modulating brain function. We will systematically distinguish between direct and indirect pathways in which astrocytes affect neuronal signalling at all levels. Lastly, we will summarize pathological conditions that arise once these signalling pathways are impaired focusing on neurodegeneration. Astrocytes are increasingly accepted as “Master-regulators” of brain function, mainly due to their ability to modulate various neuronal processes via direct and indirect pathways at the molecular, synaptic, cellular, and network levels . Mountin2+ ions are one of the most important secondary messenger molecules in the brain. They play a key role in various signalling cascades and processes that account for both normal physiological functioning (2+ concentration (termed Ca2+ oscillations or waves). Indeed these Ca2+ signals are referred to as the main readouts of astrocytic function  in neurons can be suppressed by dopamine-induced Ca2+ signals in astrocytes and could be modified by stimulation of astrocytic Ca2+ rise leading to the release of GABA or adrenaline    and is fceptors) .2+ oscillations in mediating synaptic plasticity including LTP in the hippocampus.  inhibition of pyramidal cells through the release of ATP , exchangers and cotransporters  which are essential for developing ionic gradients required for Na+ signalling on the one hand, and maintain Na+ homeostasis on the other, reviewed by  for the transportation of neurotransmitters, ions, amino acids, and molecules across the membrane through the “lactate shuttle”, which is essential to form long-term memory   . Astrocylutamine .+ currents to Ca2+ signals in the astrocytic soma, perisynaptic cradle and thin astrocytic processes and thus, can potentially regulate the excitatory and inhibitory activity of neurons as discussed above  channels (+/K+-ATPase (NKA) and Na+/K+/2Cl- (NKCC) cotransporters . . .Taken toexcitotoxicity and neuronal death  and their release is driven by astrocytic intracellular Ca2+ concentration. This conversion is vital to avert glutamate spill over events and unnecessary peri/extrasynaptic NMDAR-excitatory postsynaptic currents in pyramidal cells  that is usually accompanied during LTP  and firing of nearby pyramidal neurons . Moreovering LTP .B receptor and GATs activity to induce synaptic depression in pyramidal cells  can trigger astrocytic GABAal cells . It is ial cells , monoamial cells , and diaal cells . The dirte gyrus . Neverthte gyrus . Additiote gyrus . Moreovete gyrus . Astrocyte gyrus .vice versa through diverse mechanisms. For instance, Heja and colleagues described one of the mechanisms wherein, upon the intense excitatory activity of the neurons, astrocytes uptake glutamate which leads to the synthesis of GABA from polyamine putrescine and releases GABA via reverse operation of GAT-1/3 that result in neuronal tonic inhibition  specifically in astrocytes, Perea and colleagues reported that Ca2+ events initiated by astrocytic GABAB receptors contribute to interneuron induced synaptic potentiation via activation of mGluRs , a region responsible for visceral-pain-cognitive interactions is due to the astrocytic release of L-lactate, resulting in improved decision-making . In addiet al showed that astrocytes can potentially modulate the synchronous and asynchronous states of neurons by adjusting the threshold value of transition  are associated with learning, memory and cognitive functions  and astrour test . Furtherour test .+o which is closely regulated by astrocytes, indicating its indirect control over neuronal oscillations  and GqGPCR (sleep duration) elucidating the contribution of network cortical astrocytes in modulating the sleep length and quality , Parkinson’s Disease (PD), Huntington’s disease (HD), and Amyotrophic lateral sclerosis (ALS), all of which are associated with the loss of a specific type of neurons confined to a particular region . In thisß-Amyloid (Aβ) plaques, intracellular neurofibrillary tangles consisting of hyperphosphorylated tau, neuronal loss, and high levels of reactive astrocytes  that have lost their ability to secrete synaptogenic factors and thus synapse elimination in AD and aged brain, which might be implicated in disease pathology  -a protein essential for vesicle exocytosis, accompanied by reactive astrocytic morphology and increased expression of IL-6  led to elevated persistent sodium inward currents, increased intracellular Ca2+ transients, and hyperexcitability of MNs that ultimately result in their death (+]o and glutamate concentrations make the MNs vulnerable to excitotoxicity and worsen the disease progression (ALS is a type of motor neuron disease (MNs) that is characterized by the gradual loss of upper and lower motor neurons which are responsible for muscle movement, speech, and breathing . Astrocyir death . In addigression .G93A mice , viral vector delivery systems, optogenetic and chemogenetic tools (in situ to target specific astrocytic channels and proteins in specific brain areas, as recently reviewed by (+ indicators by Dong group (Traditionally, most research about neural signaling took a neuro-centric approach, focusing on neuronal dysfunction, connectivity, and morphology. Our understanding of the role astrocytes play in the modulation of neuronal signalling has come a long way in the past two decades, mainly due to the development of new techniques, including two-photon laser scanning microscopy, electron microscopy reconstruction, genetically encoded Caic tools . Neuronaiewed by . Indeed,iewed by  and the ng group  have greng group .Neuronal astrocytic interactions are highly dynamic and span a wide spectrum ranging from molecular to network levels. While some of the impacts astrocytes convey on neuronal signalling are carried via direct pathways, including downregulation of synaptic receptors and LTP, other astrocytic processes, such as heterosynaptic depression and regulation of hyper-synchronised activity of neurons are carried indirectly. Elucidating the involvement of astrocytic dysfunction during ageing and neurodegeneration has great potential to develop future CNS-related therapeutic targets. Indeed, as astrocytes are dividing cells and more adaptable than neurons, therapies aimed at astrocytic dysfunction rather than at neurons may prove superior in treating CNS disorders.