Quercus ilex may be considered the queen tree of the Mediterranean Basin, dominating coastal forest areas up to 2000 m above sea level at some sites. However, an increase in holm oak decline has been observed in the last decade. In this review, we analysed the current literature to answer the following questions: what are the traits that allow holm oak to thrive in the Mediterranean environment, and what are the main factors that are currently weakening this species? In this framework, we attempt to answer these questions by proposing a triangle as a graphical summary. The first vertex focuses on the main morpho-anatomical, biochemical and physiological traits that allow holm oak to dominate Mediterranean forests. The other two vertices consider abiotic and biotic stressors that are closely related to holm oak decline. Here, we discuss the current evidence of holm oak responses to abiotic and biotic stresses and propose a possible solution to its decline through adequate forest management choices, thus allowing the species to maintain its ecological domain. Quercus ilex (holm oak) is one of the most widespread arboreal sclerophylls in Mediterranean forests. Despite its dominance in Mediterranean forests, many dieback episodes have been reported for this species in southern Europe. Holm oak dieback episodes are generally associated with extreme events such as wildfires, heat waves and droughts; however, we hypothesized that an increase in winter drought spells might have contributed significantly to the decline of this species. Among the biotic factors threatening holm oak, root rot induced by Phytophthora cinnamomi can directly result in tree mortality. Mitigation practices that control holm oak decline include adaptive silviculture, integrated pest management, chemical control and management of understory vegetation, grazers and browsers. Furthermore, the use of holm oak genotypes tolerant to drought stress and P. cinnamomi provides a valuable opportunity to restore declining holm oak forests. Climate change refers to variations in the mean values and properties of the climate that persist over an extended period, typically decades or longer . ExtremeThe Mediterranean Basin is characterized by high climatic variability and includes the highest number of Mediterranean-type ecosystems. The peculiarity of Mediterranean climate is the seasonality of temperature and rainfall that generates cold and wet winters, opposed to warm and dry summers (Quercus ilex L. (holm oak) may be considered the queen of the Mediterranean Basin and is one of the most widespread arboreal sclerophylls in Mediterranean forests  Shoot death and leaf detachment.(ii) Production of epicormic shoots.(iii) Fine root loss.(iv) Decreased growth and increased mortality .Forest dieback has been attributed to increased temperatures, reduced soil moisture and increased vapour pressure deficit  and biotic stresses , that despite their sensitivity to water loss, possess a great chance of establishing thanks to their large size, large mass and extremely rich metabolome profile . Terpenes are also important during the recovery from drought, as reported by Another class of secondary metabolites produced by holm oak leaves are terpenes, among which monoterpenes are the most abundant . Indeed,Concerning wood traits, holm oak may adopt xylem anatomical adjustments in response to dry conditions to avoid drought-induced hydraulics disfunctioning (Pinus spp.) as well as isohydric (e.g. when compared to other co-occurring angiosperms such as Phillyrea latifolia L.)  . Holm oaP. cinnamomi, intensified occurrence of extreme climatic events (such as heat waves and droughts), and reductions in precipitation associated with climate change are likely to undermine the holm oak’s domain. Notably, recent assessments in Italy, Portugal, and Spain have elevated the holm oak’s status to threatened, with its conditions deemed unfavourable or inadequate (U1) in accordance with the Habitats Directive—Article 17 (https://www.eionet.europa.eu/article17/habitat/summary/?period=5&group=Forests&subject=9340&region=/). Consequently, given the observed instances of holm oak dieback, the species has been classified as moderately tolerant to mild drought , increasing the risk of embolism formation due to freeze-thaw events and drought  on the same site or even on the same tree can result in a more rapid decline of holm oak forests   and attack of secondary insects (e.g. Lymantria dispar) (Scolytus spp. (Curculionidae), Xylotrechus spp. (Cerambycidae) or Agrilus spp. (Buprestidae)) whose attacks on already weakened plants are usually difficult to prevent  or fosetyl-aluminium  that reduce disease by implementing the host plant’s natural defence mechanisms to arrest pathogen development , despite the difficulties of clonal propagation of oaks (P. cinnamomi-affected areas using tolerant holm oak plant material has greater applicability, as previous greenhouse and field experiments have highlighted that P. cinnamomi tolerance can vary according to plant provenance and plant constitutive defences (P. cinnamomi infection on Q. ilex progeny provide opportunities for the long-term natural recovery of holm oaks.The use of defences . FurtherP. cinnamomi-tolerant genotypes, should be considered when selecting seed-bearing plants for the conservation of holm oak forests in agro-silvo-pastoral settings (Furthermore, proteomic approaches have addressed various aspects of holm oak resistance to both biotic and abiotic stresses. Therefore, the inclusion of drought-tolerant provenances, in addition to Despite the morpho-anatomical, biochemical, and physiological traits that allow the holm oak to dominate the Mediterranean basin, many dieback episodes have been reported for this species in southern Europe. Extreme events such as wildfires, heat waves and droughts, pose a serious threat to holm oak domain and have been reported to cause holm oak dieback through both carbon starvation and massive xylem hydraulic dysfunction. However, the progressive loss of resilience revealed for holm oak might have to deal with the timing of drought events. Because holm oak is adapted to summer heat and drought stress, we hypothesized that an increase in winter drought spells might have contributed significantly to the loss of resilience of this species. In this context, despite the high number of studies dealing with drought and heat stress in holm oak, there are a limited number of studies on the impact of winter drought on its physiology, which deserves further investigation.P. cinnamomi can directly result in tree mortality when the infection is sufficiently high. Furthermore, the possibility of multiple Phytophthora species living on the same site or plant, difficulties in the detection of early stages of the disease, and the increase in the spread of Phytophthora species expected in the next decades, make Phytophthora management very difficult. However, in the case of non-lethal infections, P. cinnamomi can make holm oak even more vulnerable to drought-induced mortality or pave the way for other opportunistic pathogens and attacks by secondary insects involved in the decline phenomenon.Among the biotic factors threatening holm oak, root rot induced by P. cinnamomi provides a valuable opportunity to restore declining holm oak forests. Finally, accurate management of understory vegetation, grazers and browsers would improve natural oak regeneration thus improving holm oak forests and agro-silvo-pastoral forest conservation.Mitigation practices that control holm oak decline include adaptive silviculture, integrated pest management and chemical control. Furthermore, the use of holm oak genotypes tolerant to drought stress and


Heart transplantation, the gold standard treatment for end-stage heart failure, is limited by heart graft shortage, justifying expansion of the donor pool. Currently, static cold storage (SCS) of hearts from donations after brainstem death remains the standard practice, but it is usually limited to 240 min. Prolonged cold ischemia and ischemia-reperfusion injury (IRI) have been recognized as major causes of post-transplant graft failure. Continuous ex situ perfusion is a new approach for donor organ management to expand the donor pool and/or increase the utilization rate. Continuous ex situ machine perfusion (MP) can satisfy the metabolic needs of the myocardium, minimizing irreversible ischemic cell damage and cell death. Several hypothermic or normothermic MP methods have been developed and studied, particularly in the preclinical setting, but whether MP is superior to SCS remains controversial. Other approaches seem to be interesting for extending the pool of heart graft donors, such as blocking the paths of apoptosis and necrosis, extracellular vesicle therapy, or donor heart-specific gene therapy. In this systematic review, we summarize the mechanisms involved in IRI during heart transplantation and existing targeting therapies. We also critically evaluate all available data on continuous ex situ perfusion devices for adult donor hearts, highlighting its therapeutic potential and current limitations and shortcomings. Heart transplantation (HT) serves as the gold standard therapy for advanced heart failure . The gloConsequently, grafts sourced from extended criteria donors are now under consideration. These donors are typically over the age of 55 years, possess mild left ventricular hypertrophy, exhibit non-obstructive coronary artery disease, are recipients of high doses of vasopressors/inotropes, or show indications of left ventricular dysfunction due to brain death interaction . In addiThe evolving scenario of marginal graft acquisition and the growing complexity of recipients have extended the duration of cold ischemia and subsequent ischemia-reperfusion injury (IRI), both of which contribute to a heightened risk of primary graft failure (PGF) , 3. PGF Current standard practice involves static cold storage (SCS) for hearts from donors following brain death. This method combines cardioplegia and hypothermia, which substantially reduces the energy needs of the donor heart. Nevertheless, an ischemic time (between aortic clamping in the donor until aortic declamping in the recipient) exceeding 240 min is linked to an elevated risk of PGF (OR 3.01) , 16.Allograft injury can manifest as transient myocardial stunning, which lasts for 12–24 h post-HT , or as dCurrently, the standard approach involves utilizing SCS for hearts obtained following brainstem death (BSD). However, this method is linked to significant occurrences of IRI , 18. RecThe cellular and molecular mechanisms underlying IRI are intricate and diverse. Among these, impaired mitochondrial function and the depletion of energy metabolites are particularly important for the heart given its close interdependence with energy metabolism. A synthesis of the principal mechanisms and some of their resultant effects is presented in Brain death has a significant impact on heart graft function and transplantation. It precipitates an intense systemic inflammatory response often referred to as the “cytokine storm”. This inflammatory response is characterized by an excessive activation of the immune system, leading to the release of inflammatory mediators  into the blood.Myocardial damage resulting from this cytokine storm can compromise the heart's ability to function properly after transplantation. This can manifest as reduced ventricular function, decreased contractility, and an increased susceptibility to IRI during transplantation.Additionally, the period of brain death may be associated with hemodynamic and metabolic disturbances in the donor, which can also impact cardiac function.Ischemia is traditionally defined as a mismatch between the supply of oxygen and energy substrates, and the cellular requirements for proper function and survival. This disparity can lead to various forms of cellular damage. In the context of heart transplantation, the ischemia phase is initiated after the donor's aortic clamp, when the heart is no longer perfused.+/K+-ATPase pump. Consequently, cytoplasmic sodium (Na+) levels increase, with a corresponding reduction in potassium (K+). This heightened Na+ concentration triggers cellular swelling, loss of structural integrity, and the activation of Na+/Ca2+ channels, causing an elevation in cytoplasmic calcium (Ca2+) levels . Consequently, anaerobic glycolysis becomes the primary means of generating ATP, but the levels are inadequate for fulfilling the cellular energy demands. Prolonged hypoxia results in an ATP deficit that hampers the activity of essential transporters, such as the Na) levels . The excOxidative stress is characterized by an excessive production of reactive oxygen species (ROS), which is often observed during re-establishment of the oxygen supply. The generation of ROS triggers substantial cellular damage, primarily through processes such as lipid peroxidation, which in turn leads to sterile inflammation, causing vascular permeability. Furthermore, ROS production contributes to impairment of mitochondrial function. The modifications in mitochondrial structure, coupled with calcium accumulation and the generation of free radicals, initiate apoptosis via mechanisms involving increased membrane permeability, resulting in opening of the mitochondrial transition pore; the release of cytochrome C; and the subsequent activation of caspase 9, followed by other apoptotic proteases. These processes are modulated by the Bcl-2 protein family situated within the mitochondrial membrane. Nevertheless, when ischemic conditions prevail, adaptive responses to hypoxia manifest . An enzy2+) levels, or the composition of graft preservation solutions . Under normal physiological conditions, the endothelium facilitates the release of molecules that regulate the homeostasis of the vascular wall. These molecules include nitric oxide (NO), prostaglandin (PGI2), and endothelium-derived hyperpolarizing factor. However, ischemia disrupts the endothelium's ability to regulate these functions, particularly by altering the expression and activity of nitric oxide synthases (NOSs), leading to a reduction in NO production , leukocyte infiltration (neutrophils and macrophages), myocardial edema (impairing cellular metabolism and ion channel function), allograft rejection (inflammation and immune response leading to tissue damage), fibrosis , cytokine storm , microvascular dysfunction (compromising oxygen delivery to cardiac cells), impaired electrical conduction (leading to arrhythmias and conduction disorders), and impaired metabolic balance.Overall, the impact of inflammation on heart graft function is a complex interplay of immune responses, cytokine signaling, oxidative stress, and cellular interactions. Strategies aimed at mitigating inflammation, such as immunosuppressive therapies and anti-inflammatory agents, may be crucial for maintaining graft function and promoting successful HT outcomes.In the context of HT, the reperfusion phase initiates upon aortic declamping in the recipient, at which point the heart is perfused with warm blood, enabling it to restore contractility.Reperfusion involves the reinstatement of oxygenated blood supply to the tissue. During this phase, two mechanisms contribute to reperfusion injuries. Firstly, the no-reflow phenomenon arises due to inadequate microvascular reperfusion, often linked to hemostasis disorders marked by increased platelet and complement activation, as well as leukocyte aggregation. Microvascular dysfunction emerges from an imbalance between vasodilator and vasoconstrictor agents. Secondly, the sudden surge in oxygen levels gives rise to an overwhelming production of oxidative stress, surpassing the capacity of cellular antioxidant systems that were already compromised by the preceding ischemic phase. Though oxidative stress is accentuated during reperfusion, its initiation occurs during brain death and is exacerbated by ischemia. The concurrent generation of ROS and NO yields peroxynitrite ions (ONOO-), which are recognized for their cytotoxic effects, as they curtail the availability of NO, a potent vasodilator. The detrimental consequences of reintroduced oxygen also involve the opening of the mitochondrial permeability transition pore, leading to mitochondrial edema, complete inhibition of mitochondrial functions, including ATP production, and the release of cytochrome C. This sequence of events activates the proapoptotic pathway . ConsequThough ischemic damage predominantly results in necrosis, reperfusion injury can also trigger apoptosis , 30. In The reperfusion phase is also marked by the initiation of an innate immune response within the initial days post-transplantation, followed by an adaptive immune response that plays a role in the development of a chronic immune reaction , 34. ThuThese intricate processes intersect with the recruitment of T cells and the ensuing adaptive immune response, which occurs a few days following reperfusion and intensifies in response to the degree of graft allogenicity . This adClinical presentations of IRI encompass arrhythmias, microvascular dysfunction, and myocardial stunning. Reperfusion-induced arrhythmias may be influenced by mitochondrial dysfunction. After prolonged ischemia, the mitochondria may struggle to restore or maintain their inner membrane potential, leading to destabilized action potentials and heightened vulnerability to arrhythmias .Myocardial stunning denotes a temporary myocardial dysfunction that arises post-reperfusion. It is thought to stem from a combination of factors, such as oxidative stress, myocardial edema, and persistent calcium overload, even after reperfusion . This coInitially, the inflicted damage is reversible and, if blood flow is reinstated during this period, the structures and functions can recuperate to their normal state. However, if ischemia persists for an extended duration, the damage becomes irreparable, culminating in cell death. Before cell death is a vulnerable window during which ischemic myocytes are viable but prone to further injury upon blood flow restoration . The restoration of blood flow triggers three events that can inflict additional harm on myocytes: microvascular obstruction, deposition of red blood cells (hemorrhage), or localized inflammation that could exacerbate microcirculation damage. Myocardial edema, both intracellular and interstitial, exhibits a bimodal pattern following reperfusion. The initial surge occurs shortly after reperfusion and diminishes within a few days, whereas the second wave emerges days later and coincides with the healing process .Over the past few decades, numerous strategies have been proposed to mitigate IRI, capitalizing on a deeper understanding of its mechanisms and its repercussions . These sSCS is the most widely employed technique for heart preservation. This method involves the prompt removal of blood from the organ, meticulous cleansing of the vascular system with a cold preservation solution and maintaining the heart in a hypothermic state of rest until it is ready for transplantation. Typically, the heart is placed within a sterile bag filled with the preservation solution, then nestled in a container equipped with ice for transportation. These measures are implemented to induce diastolic arrest in the heart, thereby curtailing its metabolic demands and mitigating the adverse impacts of ischemia during transport.+/K+ ATPase activity, resulting in cellular swelling. Under this condition, sodium is unable to be effectively removed and, instead, passively enters the cell. This creates a hyperosmolar intracellular environment, causing water to flow into the cell, resulting in cell edema. To counteract this swelling, colloids are introduced into preservation solutions.Although hypothermia does not bring cellular metabolism to a complete halt, it does decelerate the degradation of vital compounds essential for cellular viability . FurtherCold storage within the temperature range of 0–4°C triggers rapid intracellular ATP depletion. Within 4 h, nearly 95% of ATP is hydrolyzed, leading to adenosine monophosphate becoming the predominant nucleotide. This metabolic shift results in acidosis, with the production of two lactic acid molecules . The impSCS has been associated with the promotion of ROS production, likely due to mitochondrial impairment. Free or chelated iron catalyzes the formation of ROS and directly contributes to hypothermia-induced damage by causing mitochondrial dysfunction and initiating apoptosis. ROS rapidly react with other molecules, causing extensive damage to lipids, nucleic acids, and proteins . The ensThe process of rewarming can also induce harmful effects. Systemic collapse and cellular lesions may occur due to the elevation in temperature and oxygen pressure. Upon restoration of blood flow, the oxidation of hypoxanthine and xanthine produces ROS. These free radicals lead to lipid peroxidation, increased membrane permeability, oxidation of membrane proteins, and DNA damage, culminating in enzyme dysfunction. In addition, the accumulated hypoxanthine diffuses out of cells, impeding the cell's capacity to replenish its energy reserves , 49. TheParagonix Technologies  has developed the single-use, disposable Paragonix SherpaPak® Cardiac Transport System for non-perfusing storage. Its purpose is to sustain donor heart temperatures between 4°C and 8°C for extended durations. The system involves suspending the donor heart in a preservation solution, which is de-aired to allow complete submersion of the heart to facilitate even cooling. Subsequently, the inner cannister is inserted into the outer cannister and surrounded by disposable cooling packs. These cooling packs, in contrast to regular ice that undergoes a phase change at 0°C, undergo a phase change at 5°C, thereby maintaining the desired preservation temperatures. In preclinical and clinical studies, various preservation solutions, such as Celsior, University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK), have been employed with this system –52. PrelStrategies centered around ischemic conditioning encompass both preconditioning and postconditioning. The mechanisms underlying ischemic conditioning remain somewhat elusive due to their multifaceted nature. Ischemic preconditioning (IPC) serves to prevent the uncoupling of NOS and the subsequent generation of reactive oxygen and nitrogen species. In addition, it boosts signaling through proteins, such as protein kinase G, reperfusion injury rescue kinase, and survivor activation factor, in reperfused cardiomyocytes . IPC alsin vivo, where the viral vector is directly administered to the recipient after transplantation. This approach allows for targeted gene delivery to the transplanted heart. The transferred genes induce cellular responses that mimic the effects of IPC: reducing oxidative stress, improving cellular energy metabolism, promoting anti-inflammatory processes, and inhibiting apoptotic pathways. By inducing these protective pathways, the transplanted heart becomes more resistant to the detrimental effects of ischemia and reperfusion, leading to improved graft survival, reduced ischemic injury, and enhanced overall function.Preconditioning's effects can be triggered by pharmacological agents that act on identified targets and have demonstrated protective effects in animal models. Among agents that specifically open mitochondrial K-ATP channels, diazoxide has demonstrated promising outcomes, though it can induce hypotension due to its broad effects. Another potential avenue involves inducing these mechanisms through gene transfer. Gene transfer can be used to induce protective pathways akin to IPC in the context of HT. Certain genes are associated with protective mechanisms that mimic IPC. For example, genes encoding heat shock proteins (HSPs), adenosine receptors, and protein kinase C (PKC) isoforms have been identified as key players in mediating the protective effects of IPC. These genes can be transferred to the graft to induce similar protective pathways. In some cases, gene transfer can also be performed However, the practical applicability of preconditioning in clinical settings remains uncertain. Effective administration of the required substances would necessitate their use prior to donor death, which is currently prohibited by ethical and legal considerations. Furthermore, the impact of such treatment would be systemic and could affect all donor organs, potentially compromising the retrieval of specific organs due to their toxicity. A possible alternative is the in-situ administration of pharmacological agents via the preservation solution to replicate the effects of preconditioning.To mitigate the adverse effects of IRI, a range of heart preservation solutions have been developed, each containing varying concentrations of cellular nutrients, metabolites, electrolytes, and antioxidants. The first solution, Euro Collins, was formulated in 1960, followed by the introduction of the UW solution in 1988 . Subsequ•Nutrient and electrolyte provision: Preservation solutions must contain essential nutrients, such as carbohydrates, amino acids, and electrolytes to sustain fundamental metabolic processes and maintain electrolyte balance in cardiac cells during the storage period.•Oxygenation and antioxidants: Preservation solutions may incorporate antioxidant agents to shield cardiac cells from damage caused by free radicals produced during ischemia-reperfusion. Adequate oxygenation in the solution can also support aerobic metabolism of cells.•Acid-base balance and pH maintenance: Preservation solutions need to uphold acid-base equilibrium and appropriate pH levels to prevent metabolic acidosis that can occur during ischemia.•Cellular edema reduction: Some solutions contain osmotic agents to minimize water accumulation in cardiac cells, reducing the risk of cellular edema and structural damage.•Inhibition of inflammatory responses and apoptosis: Certain solutions may include anti-inflammatory agents or substances that inhibit apoptotic pathways to mitigate cellular damage induced by ischemia-reperfusion.•Support of mitochondrial function: Mitochondria are critical for ATP production and cell survival. Some solutions may contain energy substrates and cofactors to support mitochondrial function.•Prevention of endothelial injury: Preservation solutions aim to preserve vascular endothelial integrity to promote adequate blood flow and minimize the risks of thrombosis or endothelial dysfunction.Ultimately, the compositions of heart graft preservation solutions aim to optimize cellular viability and cardiac function during the storage period to mitigate ischemia-reperfusion-related damage and maximize the success of HT. Each solution may offer specific advantages based on its composition, route of administration, and capacity to protect the heart against various consequences of ischemia and reperfusion.The different solutions for heart graft preservation are designed with specific compositions to optimize the viability and function of the heart during the storage period prior to transplantation. Each solution aims to maintain cellular integrity, prevent IRI, and support the necessary metabolic processes for effective recovery post-transplantation. The main reasons underlying the distinct compositions of heart graft preservation solutions include:n = 3,107) demonstrated a significantly higher 1-year survival rate than Celsior solution , though this difference may not have clinical relevance   . ComparaRegardless of the preservation solution used, optimal results with SCS are achieved when the ischemia time remains under 4 h. Each additional hour of ischemia beyond 4 h amplifies the risk of graft failure by 43% and increases the 1-year post-transplant mortality risk by 25% .Targeting cellular signaling pathways that contribute to IRI outcomes is promising as a therapeutic approach. Numerous potential therapies are being explored. Antioxidants and NO transport agents are of particular interest due to the pivotal role of oxidative stress and endothelial damage , 49. In in vitro  or ex situ normothermic perfusion (ESNP), have demonstrated successful preservation of animal and/or human hearts . NotablyThese perfusion devices are designed to be portable and enable an extended and secure preservation window, facilitating the use of hearts from donors located at a distance. Currently, a single commercially available perfusion system is available for clinical applications, known as the Organ Care System®  (ESNP). In Europe, a multicenter randomized clinical trial is currently underway to explore the safety and potential advantages of the XVivo® technology for heart preservation  (HMP) (NCT03991923) , 76. TheFollowing the retrieval of the heart graft previously preserved using a hypothermic solution, the graft is prepared and positioned on a dedicated module designed for isolated perfusion see . The OCSThe anterograde coronary perfusion is regulated by maintaining a minimum pressure of 40 mmHg to ensure sufficient coronary flow and avoid inadequate perfusion at lower pressures –77. PseuAfter the arterial shunt is activated, the heart is gently massaged during the rewarming process. The heart may regain its spontaneous rhythm or fibrillate, necessitating an external defibrillator shock. When electrical activity resumes, the inferior vena cava is tied off. Gradually, the assisted flow is increased to reach a target range of 650–850 ml/min measured at the return to the pulmonary artery, indicating the coronary flow. This is achieved to maintain a desired mean arterial pressure of 70–85 mmHg. Adjustments are made to the mean aortic pressure and coronary flow to ensure the extraction of myocardial lactate, which is evident by a positive arterio-venous difference.Several prospective non-randomized trials comparing SCS with the use of the OCS® demonstrated similar survival rates at 30 days, 1 year, and 2 years , 78, 79.Initial research efforts in the field of machine heart perfusion concentrated on hypothermic oxygenated perfusion methods . NumerouThe HMP procedure involves the use of a serial roller pump, an oxygenator, a leukocyte filter, and a heater/cooler component. To sustain the perfusion circuit, 2.5 L of perfusion solution and 500 ml of compatible irradiated and leuko-reduced donor/recipient blood are required, with a target hematocrit of 18%. The heart is connected to the HMP system using an aortic cannula. Once the heart is in position, the ex-situ circulation process is initiated. Oxygenated blood is introduced into the aortic root, aiming for a perfusion pressure of 20 mmHg and a coronary flow rate of 150–250 milliliters per minute. The heater/cooler mechanism maintains the temperature at 8°C and the pH at 7.4.HMP studies in the context of HT are limited, but they have shown promising results, particularly in the field of lung preservation and evaluation of expanded criteria lung transplant donors. Early clinical applications of the HMP system have also yielded positive outcomes for donor heart preservation. In a nonrandomized open-label phase 2 trial , 6-monthOverall, though HMP studies in HT are relatively limited, the outcomes observed thus far are promising and suggest that HMP could play a crucial role in improving donor heart preservation and transplantation outcomes.However advantageous these techniques may be, they are not devoid of substantial limitations. In addition to their high cost and the need for specialized transportation, these methods also demand intricate technical expertise for graft retrieval, device implementation, and oversight during transit as outlined in The current assessment of graft performance during ESNP is confined to observing lactate kinetics and clearance within a perfused, beating heart devoid of afterload conditions. This evaluation approach restricts the comprehensive appraisal of heart function, limiting it to a Langendorf perfusion model, which falls short in terms of sensitivity and specificity. Critical aspects, such as systolic and diastolic functions, cannot be accurately gauged in the absence of preload and afterload considerations, constraining the scope of graft evaluation.Preclinical studies are imperative to comprehensively understanding the intricate dynamics of IRI across different levels, ranging from the tissue to the microcirculation and endothelium, as well as cellular calcium homeostasis. For example, a porcine model demonstrated a correlation between lactate kinetics and the success or failure of normothermic ex situ preservation, as determined by left ventricular contractility measurements . In anotGiven these complexities, there is a pressing need for additional comparative data to elucidate the inflammatory and immunohistochemical phenomena between ex vivo normothermic perfusion (ESNP) and the conventional cold static preservation approach. Such data could provide a more comprehensive understanding of the underlying processes and aid in refining these preservation techniques.Ex situ perfusion systems present a significant opportunity to serve as a prominent conduit for the administration of therapeutic interventions to donor hearts without causing adverse effects on other donor or recipient organs. The central objective of these interventions is to mitigate the impact of IRI, with a particular focus on the potential to rejuvenate marginal organs for viable transplantation. Although these therapeutic applications are still in the preclinical phase and have not yet undergone clinical testing, their preliminary outcomes are promising. The prospective integration of ex situ therapies has the potential to not only enhance graft viability, but also contribute to expanding the existing donor organ pool, a prospect that has significant potential for the field of transplantation.-Pharmacological agents: Various pharmacological agents, such as antioxidants, anti-inflammatory drugs, and vasodilators, can be administered to the donor heart during ex situ perfusion. These agents aim to counteract oxidative stress, reduce inflammation, and improve vascular function, ultimately protecting the heart from IRI-related damage.-Gene therapy: Gene transfer techniques can be utilized to introduce specific genes into the donor heart during ex situ perfusion. These genes can encode for protective factors or enzymes that counteract IRI processes. For example, introducing genes that promote antioxidant production or inhibit apoptosis could enhance the heart’s resistance to IRI.-Cell-based therapies: Ex situ perfusion systems can facilitate the delivery of therapeutic cells, such as stem cells or genetically modified cells, directly to the donor heart. These cells have the potential to promote tissue repair, regeneration, and immunomodulation, further enhancing the heart’s viability.-Metabolic modulation: Manipulating the metabolic pathways within the donor heart during ex situ perfusion could promote energy production and reduce the negative consequences of metabolic disruption during IRI.-Targeted drug delivery: Nanoparticles and other drug delivery systems can be designed to target specific cellular pathways and deliver therapeutic compounds directly to the heart tissue, minimizing systemic effects and maximizing efficacy.Though these strategies have shown great promise in preclinical studies, it is important to note that translating them into clinical practice requires rigorous testing and validation. Clinical trials are needed to assess the safety, efficacy, and long-term outcomes of these interventions in human donor hearts. If successful, ex situ perfusion-based interventions could revolutionize the field of organ transplantation by expanding the pool of viable donor organs, reducing the incidence of IRI-related complications, and improving overall graft quality.There are several potential therapeutic strategies that can be explored within the ex-situ perfusion context:Interception of the apoptosis pathway in porcine hearts using hypothermic perfusion solutions infused with small interfering RNA molecules targeting key apoptotic and inflammatory enzymes has had notable outcomes in diminishing cellular apoptosis and mitigating myocyte injury. Furthermore, this approach has resulted in enhancement of donor myocardial function –95. In aYet another preclinical investigation unveiled the efficacy of a therapeutic regimen that couples two inhibitors of the necrosis pathway, resulting in a discernible reduction in IRI among rat cardiomyocytes , 96. Thein vitro and in vivo studies, including immunomodulation, antioxidant activity, anti-inflammatory effects, and tissue repair capabilities. These EVs possess the ability to enter target cells and modulate signaling pathways that contribute to tissue healing. EVs have also demonstrated the capacity to hinder left ventricular dilatation and enhance cardiac contractility. At the cellular level, EVs have been effective in mitigating energy depletion, reducing oxidative stress, and curtailing the infiltration of neutrophils and macrophages by activating the protein kinase B and glycogen synthase kinase 3 (AKT/GSK3) pathways. Various animal models have revealed that the administration of EVs prior to, during, or after IRI can counteract inflammatory responses, diminish infarct size, and promote reparative processes (Extracellular vesicles (EVs) derived from various cell lines have exhibited a range of beneficial properties in both rocesses  while oprocesses .In the context of myocardial IRI in murine models, EVs derived from cardiac progenitor cells (CPCs) have demonstrated significant cardioprotective effects . When ad13 total viral particles of an adenoviral firefly luciferase vector harboring a cytomegalovirus promoter into porcine donor hearts before heterotopic implantation. The comprehensive assessment encompassed levels of expression, protein activity, and dispersion of the firefly luciferase protein, and was conducted over a series of three heart transplants with a post-transplant endpoint of 5 days. Notably, though the perfusion solution and ex situ circuitry had no influence on viral vector transduction, the serum or plasma components of the donor blood significantly hindered the transduction process  during normothermic ex situ perfusion to facilitate transgene delivery to porcine cardiac allografts. A myocardially enhanced variant known as SASTG was utilized to assess the transduction efficiency in OCS perfusate. Employing normothermic ex situ perfusion, the introduction of SASTGs containing the firefly luciferase transgene into porcine donor hearts was executed across four heterotopic transplantation procedures. This approach yielded sustained, dose-dependent transgene expression in allografts within 30 days, showing no indications of off-target transgene expression. This investigation underscores the feasibility and efficacy of using AAV vectors during ex situ perfusion to achieve gene delivery to a large animal allograft .Subsequent experiments pertaining to gene delivery to the explanted pig heart involved a method of blood washing prior to the secondary infusion of blood into the perfusate, thereby removing undesired plasma or serum components from the donor blood before its incorporation into the circuit. The enzymatic assessment of luciferase activity within tissues  obtained on days 5  and 30  post-traTaken together, these two investigations collectively establish the feasibility of employing two distinct types of viral vectors to achieve potent and comprehensive transgene expression through administration utilizing the OCS®. This innovative approach to viral vector delivery introduces the prospect of biological modification of the allograft prior to implantation, which could potentially enhance post-transplant outcomes.As the demand for donor hearts continues to rise, transplant centers are increasingly considering the utilization of expanded-criteria donors. This resurgence in interest also extends to the population of donation after circulatory death (DCD), which has the potential to substantially augment the pool of available transplants. Furthermore, DCD can offer grafts from donors who typically possess lower comorbidity levels and are often younger . NonetheEncouraging outcomes are also being observed with direct harvesting of DCD hearts combined with ex situ reperfusion.A recent preclinical study  utilizinp = 1.00] and 1 year  were comparable between the two groups. The US Food and Drug Administration granted approval for application of the OCS® in heart preservation after DCD donation in April 2022, and the results of a dedicated clinical trial (NCT03831048) are anticipated.Messer and colleagues shared their 5-year experience involving DCD donation, encompassing 79 transplants derived from DCD donors (22 with NRP and 57 with direct harvesting using OCS®). This approach notably amplified the transplant activity of the center by an impressive 48% . Survival rates at 30 days [DCD: 97% vs. brainstem death (BSD): 99%; p < 0.001 for noninferiority)  .Early outcomes stemming from DCD donation have been promising and present a potential avenue for tapping into an underutilized donor pool. Current projections suggest that widespread adoption of DCD donation could elevate the annual count of adult heart transplants in the US alone by 300–600 . Given tTransplantation stands as the most efficacious approach to addressing end-stage heart disease, yet the insatiable demand for donor organs is anticipated to persistently outpace the available supply. The success of HT hinges upon the preservation of grafts against the detrimental effects of IRI. Capitalizing on the ongoing advancements in techniques and technologies, transplant centers are actively seeking to enlarge the donor pool by pushing the boundaries of established practices and constraints on ischemia time. In this pursuit, the evolution of ex situ perfusion machines has emerged as a pivotal development. These systems offer a dual advantage of enhanced preservation and more precise assessment of organs procured from expanded criteria and donation after circulatory death. Furthermore, the versatility of these ex-situ perfusion platforms extends to their potential as conduits for targeted therapies directed at donor organs prior to transplantation. As the landscape of HT continues to evolve, these innovations promise to redefine the limits of graft viability and optimize outcomes for both recipients and donors.


The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), published in 2013, includes an alternative model of personality disorders (AMPD) focusing on a maladaptive trait model utilized to diagnose several personality disorders. Borderline personality disorder (BPD) and antisocial personality disorder (ASPD) are two conditions categorized by AMPD that exhibit high rates of violence and aggression. Several of the traits outlined in the AMPD, including hostility, impulsivity, risk-taking, and callousness, have been previously linked to aggression in BPD and ASPD. However, to the best of our knowledge, there has never been a synthesis of neuroimaging studies that have investigated links between these traits and aggression in BPD and ASPD. To overcome this gap, we conducted a systematic review under the PRISMA framework to locate neuroimaging articles published since the release of AMPD linking trait anger/hostility, impulsivity, risk-taking, and callousness to aggression in BPD and ASPD. Key findings included the following: i) anger/hostility, associated with alterations in the interplay between prefrontal and subcortical regions , may be a common factor explaining aggressive reactions to response to interpersonal threat or provocation; ii) alterations of fronto-temporal-limbic regions and serotonergic and endocannabinoid signaling systems may link impulsivity to aggression in BPD and ASPD; iii) weaker cortico-striatal connectivity could relate to greater risk taking and greater proclivity for violence. Insufficient evidence from neuroimaging articles was discerned to describe a relationship between callousness and aggression. Overall, results of this review reveal a relative paucity of neuroimaging studies examining AMPD traits relevant to aggression in BPD and ASPD. In addition to encouraging further investigation of neuroimaging markers of AMPD traits linked to aggression, we recommend multi-methodological designs, including the incorporation of other biomarkers, such as hormones and indices of physiological arousal, to fully expand our understanding of aggression in BPD and ASPD. Aggression is a common clinical feature of antisocial personality disorder  and borMost of the initial neuroimaging research in BPD and ASPD failed to consider the potential link between specific personality disorder traits and neural markers of aggression. However, early in the last decade, a shift towards a transdiagnostic, trait-based approach was reflected by the inclusion of an alternative model of personality disorders (AMPD) in Section III of the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). This demonstrated that while the field was not yet prepared to abandon categorical diagnoses, a framework for researching the trait-based nature of these disorders had emerged see Box . To advaWe, therefore, sought to systematically review the literature since 2013, using a trait-based approach. As previous evidence suggests that anger/hostility –26, impuBox 1 AMPD personality disorderAs noted above, DSM-5 contains an alternative model of personality disorders (AMPD) in Section III that was introduced as a candidate to replace categorical classification of personality disorders (PDs) . The dimThe AMPD was originally designed to mitigate shortcomings of the current diagnostic approach to PDs, for example, poor reliability, heterogeneous clinical presentations, and high rates of comorbidity . A substIn deciding on which traits to focus on in relation to aggression, we were guided by a recent publication that sought to compare PID-5 associations with self-report and collateral reports of aggressive behavior, one of the few studies to examine PID-5 associations in relation to aggression . This stBox 2 Intermittent explosive disorder and overlap with BPD and ASPDIntermittent explosive disorder (IED) captures individuals with recurrent, problematic, and impulsive aggression  and is hA systematic literature search was undertaken according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guide. The search was restricted to papers published between 2013 and 2022 and the following databases were searched in October 2022: MEDLINE® ALL, Embase (via Ovid), APA PsycInfo®, EBSCO CINAHL, and APA PsycArticles® (via ProQuest). We searched for papers from 2013 onwards, as this was the date when DSM-5 was published and the AMPD was first introduced.The search was conducted by an information specialist , using a combination of free text terms (searching the title and abstract) and relevant controlled vocabulary headings customized for each database, as well as advanced search syntax , to ensure all relevant studies were identified. The search terms included the following themes, with synonyms to describe each: borderline personality disorder or emotionally unstable personality disorder or antisocial personality disorder or psychopathy, traits , aggression, and neuroimaging.or included incarcerated offenders with a Psychopathy Checklist- Revised (PCL-R) score for psychopathy; (3) included a quantifiable, standardized metric of at least one of the following: risk-taking, impulsivity, hostility, or callousness/lack of empathy; and (4) investigated the link between at least one of these traits and a metric of aggression using a neuroimaging technique. Exclusion criteria were the following: 1) review articles; 2) dissertations; 3) letters to the editor; 4) opinion articles; 5) editorials; and 6) case reports or case series.Studies were initially included if they were (1) published as a peer-reviewed article with original data in adult samples using structural MRI (sMRI), functional MRI , positron emission tomography (PET), magnetic resonance spectroscopy (MRS), and diffusion tensor imaging (DTI); (2) included individuals with BPD and/or ASPD (with or without psychopathy), defined using standardized classification tools (DSM or ICD criteria for BPD and ASPD) Our PRISMA diagram see Fig.  illustraSeveral studies have examined the relationship between neural correlates of anger/hostility and aggression. We identified five studies including individuals with BPD and four studies with violent offenders with at least the majority having a personality disorder diagnosis – mostly ASPD.In one study , 48 femaAnother study  measuredA further study  used a sUsing fMRI, another group compared a sample of 25 healthy controls with 33 aggressive BPD patients (20 receiving specific anti-aggression group psychotherapy and 13 patients taking part in a non-specific control psychotherapy) . FindingTwo studies, using an overlapping sample of violent male offenders, provided further insight into the link between neural correlates of anger/hostility and aggression in ASPD. In one , a correTogether, these studies suggest altered processing of anger-inducing situations in aggressive individuals. This seems to involve both more automatic, limbic reactions as well as prefrontal processes of cognitive control. However, the interplay of these regions, dynamic patterns, and the precise situational triggers remain unclear.Several studies of BPD and ASPD have examined the neural correlates of impulsivity and the relationship between impulsivity and aggression using imaging techniques. In general, neuroimaging investigations in BPD have reported structural, metabolic, and functional alterations of fronto-limbic networks that provides a neural basis for emotional dysregulation and impulsive and aggressive behavior –45. One A different analysis of the same sample of 51 mixed sex BPD participants compared impulsivity (BIS-11), aggression (LHA), and brain structure using sMRI in high and low lethality suicide attempters . No sign18F]altanserin PET to quantify whether sex had a significant effect on the associations between 5-HT2A binding; personality traits, such as impulsivity and aggression; and suicidal behavior in BPD. Thirty-three BPD patients (mixed sex) and 27 healthy controls (mixed sex), all unmedicated, were examined. The group had previously found effects of sex among healthy volunteers on the association between 5-HT2A binding and aggression [18F]altanserin binding potential (BPND) in medial frontal cortex, while aggression was negatively related to BPND in medial OFC. There were no significant relationships between these traits and BPND in male BPD subjects. Additionally, among BPD subjects, aggression, cluster B comorbidity, ASPD, and childhood abuse each related to altanserin binding. Therefore, region-specific differences in 5-HT2A binding related to diagnosis, sex, and history of childhood abuse may relate to the clinical expression of aggression and impulsivity in BPD.The same group  used CURB PET study [An [ET study  that invFinally, one study demonstrated that in 27 violent offenders, gray matter volume in multiple prefrontal regions including superior frontal gyrus and superior orbital gyrus was negatively associated with PCL-R Factor 2 traits . HoweverTaken together, these studies suggest that abnormalities of fronto-temporal-limbic regions are implicated in the impulsivity of BPD and ASPD and may predispose to aggressive behaviors. Neurochemically, alterations in serotonergic and endocannabinoid system signaling pathways may also give rise to impulsivity and aggression in these populations. It should be noted that most of the reviewed studies of impulsivity and aggression in BPD and ASPD used questionnaire-based measures of impulsivity, as opposed to neuropsychological paradigms, to assay these constructs.We found two studies linking neural correlates of risk-taking to metrics of aggression, both in incarcerated male offenders. In one of study , 49 aduln = 11) and hyper-reactive offenders . Thirteen male healthy controls without a criminal or psychiatric history also participated. Results revealed that hypo-reactive offenders differed from healthy controls by exhibiting decreased neural activation in rostral ACC in response to uncertainty and decreased activity in the prefrontal cortex when consistently choosing safe alternatives. There was a positive correlation in hypo-reactive offenders between right inferior frontal gyrus activity preceding a “stock” choice and subscale scores on a questionnaire measuring aggression as well as the number of risk-seeking mistakes, which was interpreted as a measure of behavioral dyscontrol.Another study of violent criminal offenders explored emotion-related mechanisms leading to risky decisions using an fMRI paradigm, where respondents were required to choose between low-risk bonds and high-risk alternatives, such as stocks . While bOne study in 27 violent offenders and 27 healthy controls  linked sTo explore how trait-based approaches to neuroimaging research in BPD and ASPD have progressed since publication of DSM-5, we conducted a systematic review of neuroimaging studies investigating key traits linked to aggression across these disorders. While a lack of methodological consistency in the field remains and limits the scope of our findings, our study identified some important considerations for future work.First, evidence from studies identified in our review suggests that anger/hostility associated with alterations in the interplay between prefrontal  and subcortical regions  could be a common factor explaining aggressive reactions in response to perceived interpersonal threat or provocation. Interestingly, findings indicate that a proneness to act out aggressively may be linked to a reduced differentiation  between threatening and non-threatening interpersonal cues, in line with the hypothesis of a hostile filter that biases the perception of the entire social environment, thus increasing the likelihood for aggressive encounters. This is also in line with the findings of an earlier PET study, which revealed stronger amygdala responses to high as well as low provocation in individuals with comorbid BPD and IED . HoweverSecond, there remains an overall lack of clarity about the respective links between neural correlates of impulsivity and aggression. This is surprising, given prior evidence suggesting that impulsive behavior and reactive aggression may share common neural underpinnings . One potAnother potential explanation for this lack of clarity is the conceptual overlap between impulsivity and risk taking. This may explain in part why our search yielded so few studies specifically examining links between risk-taking and aggression. Whereas impulsivity may reflect acting on the spur of the moment in response to immediate stimuli, acting on a momentary basis without consideration of outcomes, or difficulty establishing and following plans, risk-taking may involve poor representation of the degree of risk, especially in situations where the degree of risk gradually increases combined with the magnitude of a potential favorable outcome when the precise odds of negative outcomes are unknown or not explicit. However, some imaging reports in healthy populations have argued for a clear dissociation between high-risk behavior tendency as a construct distinct from that of impulsivity . We propThird, we found a notable lack of studies that investigated potential neuroimaging correlates for both callousness/lack of empathy and aggression. This was also surprising, considering considerable previous evidence suggesting a neurobiological component to callousness –69 and tOur review identified further gaps in the existing literature. First, the included studies used a variety of different measures for aggression, ranging from different self-report trait questionnaires, interviews assessing aggressive behavior within the past weeks, to aggressive responses in experiments. Although acceptable reliability has been shown for most of these measures, their ecological validity remains questionable. There are several reasons for this: i) the correlations between different measures of aggression are often small to moderate; ii) most of these measures are subject to social desirability effects; iii) measuring aggression in a highly standardized yet ecologically valid experiment is a particular challenge in a neuroimaging setting; and iv) most of these measures do not provide information about which particular situation a particular person acts out aggressively in real life. Hence, future studies combining neuroimaging methods with ecological momentary assessment are needed.Second, there was a marked lack of specificity about forms of aggression in most studies in our review. This is especially important as previous evidence suggests that reactive aggression is more likely be associated with anger and impulsivity  and proaThird, none of the studies in our review investigated more than one of the personality traits potentially related to aggression within a single sample. This limits inferences on the specificity of findings from individual studies. It also precludes investigation of the interplay between these specific traits in a causative model of aggression. Future studies will benefit from a principled approach to exploring links between the relative contributions of individual traits (selected based on prior work demonstrating links to aggression) - and their neural underpinnings - to aggression. Analytical models that can explore whether particular traits, or combinations of traits, mediate the correlation between neural signatures of aggression, or vice versa, would be particularly beneficial. Such studies will likely require more specific measures of aggression, as well as large and heterogeneous samples of aggressive individuals with personality disorders .Fourth, with few exceptions, the majority of neuroimaging studies of aggression in BPD samples involve women only, while neuroimaging studies of aggression in ASPD and psychopathy focus on men. To some extent, these patterns reflect the prevalence of each condition by sex under certain scenarios. For example, females with BPD are over-represented in clinical settings , while APrevious reviews of clinical studies using AMPD have suggested that this model demonstrates acceptable interrater reliability, largely consistent latent structures, substantial convergence with relevant external measures, evidence for incremental validity when controlling for categorical PD diagnoses , and cliFurthermore, some consideration should be given to the potentially loaded and negatively-valenced descriptors of certain personality traits. Consistent terminology about antisocial symptom domains remains important and has utility in inter-professional communication . HoweverMethodological issues also warrant further consideration. The lack of a consistent approach to inclusion criteria and stratification of groups of antisocial and violent offenders in studies could be addressed by consensus approach to defining both categorical measurements  and symptom domains . Second, since aggression is a complex phenomenon, future studies will need to include multi-methodological designs: hormones and physiological measures indicating the level of peripheral arousal should accompany neuroimaging and self-reports. Several studies have suggested that arousal, for example, resting state heart rate  shows a This systematic review examined trait-based approaches to aggression in neuroimaging research in BPD and ASPD published since the introduction of AMPD. While there were relatively few neuroimaging studies examining AMPD traits relevant to aggression in BPD and ASPD, several key themes emerged. First, a variety of different measures for aggression exist, but studies combining neuroimaging methods with ecological momentary assessment are needed to better understand under what situations particular individuals act out aggressively in real life. Second, very few of the studies differentiate between proactive and reactive forms of aggression, which has relevance for understanding how subtypes of aggression relate to AMPD traits. Third, the existing neuroimaging studies are limited to the study of only one particular trait in relation to aggression, when in reality most individuals will likely endorse multiple AMPD traits. Fourth, most studies of BPD focus on females, while those of ASPD sample males. This lack of heterogeneity makes it difficult to parse the neuroimaging markers of aggression in male BPD patients and female ASPD subjects. We have also highlighted methodological inconsistencies across the existing literature and emphasized the importance of a consistent approach to categorical and trait specification. We conclude that multi-methodological designs incorporating a range of biomarkers hold the most promise for understanding how a relatively new maladaptive trait model of personality disorders can better inform on the biological underpinnings of aggression in BPD and ASPD.


This retrospective case-control study aimed to investigate associations between disease severity of Alzheimer’s dementia (AD) and macular thickness. Data of patients with AD who were under medication (n = 192) between 2013 and 2020, as well as an age- and sex-matched control group (n = 200) with normal cognitive function, were included. AD patients were divided into subgroups according to scores of the Mini-Mental State Examination (MMSE) and Clinical Dementia Rating (CDR). Macular thickness was analyzed via the Early Treatment Diabetic Retinopathy Study (ETDRS) grid map. AD patients had significant reductions in full macula layers, including inner circle, outer inferior area, and outer nasal area of the macula. Similar retinal thinning was noted in ganglion cells and inner plexiform layers. Advanced AD patients (MMSE score < 18 or CDR ≥ 1) showed more advanced reduction of macular thickness than the AD group (CDR = 0.5 or MMSE ≥ 18), indicating that severe cognitive impairment was associated with thinner macular thickness. Advanced AD is associated with significant macula thinning in full retina and inner plexiform layers, especially at the inner circle of the macula. Macular thickness may be a useful biomarker of AD disease severity. Retinal imaging may be a non-invasive, low-cost surrogate for AD. Alzheimer’s dementia (AD) is a progressive neurodegenerative disorder that is the most common cause of dementia . CurrentBrain imaging, including magnetic resonance imaging (MRI) and positron emission tomography (PET), cerebrospinal fluid (CSF) biomarkers such as amyloid beta (Aβ) and tau, and serologic testing, including genetic markers, are reported to have diagnostic value . HoweverThe retina is an integral part of the central nervous system, and the concept of the retina as a surrogate of the brain has received increasing interest in recent years . RetinalThe primary aim of the present study was to analyze the associations between the disease severity of Alzheimer’s dementia and macular thickness through retinal OCT images. We aimed to identify the decisive retinal layer and sectors of the retina for detection of AD, and we also correlated the results with clinical cognitive exams, including the MMSE and CDR scores. The study also demonstrates the possibility that macular OCT may address several clinically pertinent challenges in dealing with AD, including disease screening and the evaluation of AD disease severity.This retrospective case-control study extracted data from the hospital electronic medical records of Kaohsiung Medical University Hospital Database (KMUHRD), which includes various patient medical records from one medical center, two regional hospitals, and one local hospital in southern Taiwan. The KMUHRD data used in this study included patient data of outpatient visits, inpatient hospitalization, retinal spectral-domain OCT imaging and reports, and patient neuropsychological examination reports.The Institutional Review Board (IRB) and Ethics Committee of Kaohsiung Medical University Hospital reviewed and approved the study protocol, which also adheres to the Declaration of Helsinki. Since this study was retrospective in design and used existing patient information that was deidentified, the IRB also waived patients’ informed consent.Patients diagnosed with AD based on ICD-9-CM and ICD-10 diagnosis codes and who received prescribed medication by neurologists between 2013 and 2020 in one medical center and one regional hospital of the KMUHRD database were included. The cognitive examination was obtained from the department of Neurology. Patients with diabetic macular edema (DME), proliferative diabetic retinopathy (PDR), glaucoma, optic neuropathy, and ocular disease that may lead to significant changes in retinal thickness were excluded.The included patients had records of OCT exams at ophthalmic visits, as well as OCT images taken close to the date of AD diagnosis within the time interval of 1–2 years. OCT images were generated by the Spectralis OCT . Initial quality control was performed by experienced ophthalmologists and technicians during the clinical OCT examination, to filter out images with low resolution or improper format as well as eyes with age-related macular disease (AMD), macular holes (MH), VMT (vitreomacular traction), and epiretinal membrane (ERM), even chorioretinal atrophy or prominent posterior staphyloma. Precise matching of patients’ age (by birth year) and sex was done to generate the comparison group (control group) of non-AD patients. Patients receiving OCT exams between 2013 and 2020, filtered by the exclusion criteria cited above, were included. Of these patients, under initial matching with sex and age and exclusion of outliers, 175 patients had AD under medication and 188 patients had no cognitive impairment. The data augmentation procedure was performed by exporting the macular thickness map into different retinal layers, including full layers of macula, nerve fiber layer, ganglion cell layer, and inner plexiform layer, in the total four categories of thickness map. The entire thickness map was exported into the Early Treatment Diabetic Retinopathy Study (ETDRS) grid map, which was divided into nine sectors , as previously described . Figure Patients with AD were then divided into subgroups according to MMSE scores and CDR scores . MMSE ist-tests or ANOVA tests to compare continuous variables between groups. Multivariable linear regression models were used to compare the differences in ETDRS grid macular thickness between groups  while controlling for age and sex. A two-tailed p < 0.05 was considered significant in all statistical tests. We further used the Bonferroni approach to calculate adjusted p-values among multiple site comparisons to handle multiple testing concerns [Frequencies were used to present the proportions of demographic characteristics. Chi-square tests were used to compare differences between categorical variables. We first did the hypothesis testing for normality for the measures of ETDRS grid macular thickness using the Anderson-Darling test , which iconcerns . All stap-value < 0.001), inner temporal area , inner inferior area , inner nasal area , outer inferior area , and outer nasal area . The results were similar in the inner plexiform layer (IPL) and ganglion cell layer (GCL), which showed significantly decreased macular thickness in AD, especially in the inner circle part and outer inferior area in the ETDRS grid. However, when the data were defined solely on the NFL layer, no significant differences were noted in macular thickness between the AD group and the control group. Thus, when we converted the statistical results into plots of the ETDRS grid map, the area marked with a star represented a significant reduction of macular thickness in different retinal layers in AD , Bonferroni p-value = 0.006), inner temporal region , Bonferroni p-value 0.015), and inner inferior region , Bonferroni p-value = 0.006)  , and theThe present study showed that retinal thickness was thinner in patients with AD in comparison to that of people without AD. Significant differences were found in the thickness of full macular layers and IPL thickness, but not in NFL thickness. In addition, major differences were found at the inner sectors of the macula in the ETDRS grid map.The retina shares similarities with the brain, and the concept of the retina as an exceptionally convenient window by which to assess the neuronal and vascular changes in the central nervous system (CNS) started decades ago. Various retinal changes in AD have been investigated in the last three decades. In 1986, Hinton et al. discovered in postmortem histological research that AD patients possessed prevalent axonal degeneration, decline in a variety of retinal ganglion cells (RCGs), and decline in the thickness of the retinal nerve fiber layer (RNFL) compared to those of age-matched controls . This fiResults of the present study were compatible with previous reports demonstrating thinning of the retina, although in variable sectors ,25,26,27It is highly expected that changes in retinal thickness of AD patients correlates with the severity of AD. Currently, the relationship between macular thickness and the severity of cognitive function impairment is still not well established. A systematic review supported the presence of RNFL thinning and mild cognitive impairment (MCI), particularly amnestic MCI, though less remarkable than the associations with AD . The preThis study has several limitations. First, it was a retrospective study, and OCT images taken around the time of AD diagnosis were retrieved, suggesting there may be a gap between the diagnosis or disease severity and the images taken. Some patients might not visit neurology department and ophthalmology department around the same time, and this was the possible reason leading to the gap in the retrospective study. Retrospective design also limits generalization of results to other populations and does not allow the ruling out of selection bias. Second, nearly 40% of cognitive examinations were missing from patients’ records at the time of switching from paper to electronic medical records or were simply not done. We are planning to conduct a further prospective longitudinal study where the patients may receive OCT imaging in a timelier manner during the initial diagnosis of AD, which will allow associations between the trend of macular thickness and the change of MMSE or CDR scores within several years to be detected prospectively. However, it should be noted that the present study included large case numbers, and the other systemic or ocular diseases were excluded or matched before analysis.In conclusion, significant retinal thinning was noted in the retinas of AD patients, especially in full retinal layers and inner plexiform layers at the inner circle of the macula. Results of this study support that macular thickness may be a useful biomarker of the disease severity of AD. Retinal images may be non-invasive, low-cost surrogates for AD.


As the prevalence of allergy and autoimmune disease in industrialized societies continues to rise, improving our understanding of the mechanistic roles behind microbiota-immune homeostasis has become critical for informing therapeutic interventions in cases of dysbiosis. Of particular importance, are alterations to intestinal microbiota occurring within the critical neonatal window, during which the immune system is highly vulnerable to environmental exposures. This review will highlight recent literature concerning mechanisms of early-life microbiota-immune homeostasis as well as discuss the potential for therapeutics in restoring dysbiosis in early life. The neonatal intestinal microbiome, composed of bacteria, fungi, viruses, protozoa and archaea, is in constant flux from initial microbial seeding to approximately three years of age, when the microbiota stabilizes and more closely resembles adult-type microbial composition , 2. DuriThe timeline of initial microbial seeding is a matter of debate. The sterile womb theory, adopted over a century ago, suggests that seeding does not occur until birth . In receBifidobacterium infantis  and retinoic acid exposure in combination with bacterial antigen presentation by MHC class II (MHCII) on dendritic cells  cells, innate B-1 cells, dendritic cells, innate lymphoid cells (ILCs), γδ T-cells, and mucosal-associated invariant T (MAIT) cells . Many of+ populations suggesting that the time frame for microbial influence is specific to early life  via polysaccharide A (PSA)  is a pleiotropic cytokine which regulates IgA production and promotes the suppressor functions of both pTregs and tTregs , 61. TGFpression . Recent pression . Another A (PSA) .While this section primarily focuses on Tregs during development, other cell types such as myeloid-derived suppressor cells (MDSCs) and B regulatory cells can also contribute to immune tolerance in the neonatal period and future studies may demonstrate how their development is impacted by early-life microbiota.Given that this phase of development is centered around tolerance, vaccines administered at birth generally have reduced efficacy. Additionally, immunization at birth with diphtheria, pertussis and tetanus (DTaP) was associated with a significant reduction in antibody response to subsequent boosters compared with a routine immunization schedule suggesting that early neonatal vaccination might impair the response later in life . This beBoth protective and tolerogenic immune responses are important for healthy neonatal development but their imbalance may increase infection susceptibility or increase propensity to develop allergy and autoimmune disease. Alterations to microbiota exposure or composition in the neonatal period can have long-lasting consequences that impact immunity in adult life. Importantly, these disruptions cannot always be resolved once the critical window of opportunity closes. Multiple factors may result in deviation from the carefully constructed developmental pathway, so what options are there to restore early-life microbiota in the face of dysbiosis?This section will discuss attempts to restore normal microbiota in common scenarios which may alter microbial composition during early life such as antibiotic treatment, preterm birth, cesarean section delivery (CSD) and formula-feeding.th century China where patients with food poisoning or severe diarrhea were given a “yellow soup” composed of human feces. Today, FMT has been approved by the FDA for use in scenarios of recurrent Clostridioides difficile infection and ongoing research may highlight additional clinical applications.Fecal microbiota transplantation (FMT) is a procedure during which fecal matter is transferred from a healthy donor to a recipient in order to restore microbial homeostasis in the gut. The technique’s origins can be traced back to 4Staphylococcus as well as increases in Clostridia spp  is a gastrointestinal disease characterized by ischemic necrosis of the intestinal mucosa and inflammation which can lead to bowel perforation and sepsis in severe cases. NEC is associated with increase abundance of pathobionts within the phylum Proteobacteria (family Enterobacteriaceae) . Probiotic administration in neonates is most popular in the context of preterm birth with the most common bacterial species reintroduced being riaceae) . Studiesriaceae) , 78. Desriaceae) .Ongoing clinical studies hope to shed more light on the efficacy of these microbial restoration strategies to help inform best clinical practice when faced with early-life dysbiosis –89.While many studies focus on reversing the progression of allergy and autoimmune disease later in life, prevention and early correction of dysbiosis during the neonatal window is important for minimizing the public health burden of disease. Despite major advances in this field of research, many outstanding questions remain. As the utilization of neonatal microbial restoration interventions becomes more popular, future studies should seek to better understand their impact on the developing immune system and subsequent potential to reduce the risk of developing dysbiosis-related disease. Finally, although the timeline for the window of opportunity in mice is more or less defined, how that translates to the specific timeline of human neonatal development remains unclear given that the human neonate has a more mature immune landscape at birth compared with neonatal mice . DefininHR: Conceptualization, Funding acquisition, Writing – original draft, Writing – review & editing. MB: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.


Attention deficit Hyperactivity Disorder (ADHD) is the commonest childhood neurodevelopmental disorder, affecting 3 to 9% by school age, and often persists into adulthood. ADHD in children and young people (CYP) has wide ranging multi-modal impacts on the affected CYP, their carers and the society. Co-morbidity with other neurodevelopmental, behavioural and emotional disorders is the rule rather than exception. Pharmacological treatment is not recommended as the sole therapeutic intervention, and several other non-pharmacological interventions have been advocated within a framework of Multi-modal strategy as the norm, to address both the core symptoms as well as the behavioural and other related difficulties. All paediatric professionals need to be familiar with the principles of different modalities of non-pharmacological or behavioural interventions for managing ADHD in CYP. Most published up-to-date evidence for behavioural interventions both for the core ADHD symptoms and other outcome measures are summarized in this article, including the peculiar problems related to their research. The most effective evidence-based strategies for controlling ADHD core symptoms are combination of stimulant medications with Behavioural therapy (BT) or Cognitive behaviour therapy (CBT), as well as group-based parental Psychoeducation. Standalone BT, CBT, Mindfulness, Neurocognitive training and Neurofeedback cannot currently be recommended for controlling core symptoms due to limited evidence. Other Behavioural interventions could lead to improvements in ADHD-related outcomes, including parenting skills, CYP's social skills, academic performance and disruptive behaviours. School-based non-pharmacological interventions have been shown to reduce disruptive behaviours. Executive skills are also significantly improved with use of computer-based Neurocognitive training and regular physical Cardio exercises. It is disappointing that combinations of different types of psychosocial interventions have low efficacy on both the core ADHD symptoms and other related outcomes. The readers are welcome to test their knowledge and learning efficacy through an accompanying quiz. Its core symptoms are persistent and pervasive inattention and/or overactivity/impulsiveness impairing functioning in multiple settings. It is now classified under the umbrella of “Neurodevelopmental disorders” within the DSM-5 and ICD-11 standards. Furthermore, 65–80% of patients with ADHD have conduct problems and other comorbidities, in addition to low academic achievement and poor social and organizational skills. Parents of children and young people (CYP) with ADHD often experience poor self-concept and emotional problems Pharmacological treatment is not recommended as the sole therapeutic intervention, and several other non-pharmacological interventions have been advocated within a framework of Multi-modal strategy as the norm. In the UK, the National Institute for Clinical Excellence - NICE guidance recommends only psychosocial treatment for ADHD CYP with mild or moderate levels of symptoms and impairment, escalating to additional pharmacological treatment for those with severe symptoms and impairment, for those refusing non-pharmacological interventions or if the response is poor We have assessed and summarise here the non-pharmacological interventions for ADHD which could be used as first line treatment for patients with ADHD. The implementation of non-pharmacological strategies has become particularly crucial during the COVID-19 pandemic crisis and thereafter, with restricted contact with healthcare professionals The readers are welcome to test their knowledge and learning efficacy through the accompanying quiz.Clinical Scenario: A 14 year old boy was recently diagnosed with ADHD. He is reluctant to use stimulant medications because he and his parents have reservations about their side-effects. He has come to discuss with the Clinician about the effectiveness of non-pharmacological options for managing his symptoms.2.We conducted a systematic search of the following electronic databases: Pubmed, PMC, CINAHL, Embase, PsycINFO, Ovid, Database of Abstracts and Review and the Cochrane Database of Systematic reviews, with no limitations in terms of language and date of publication from inception to Dec 2021. Search terms included a combination of the following: “non-pharmacolog?”, “childhood”, “psychoeducation”, “behavior?”, “parent?”, “ADHD”, “impairment” or “psycholog?”. The relevant publications were summarised, and the emerging themes are presented. Over 5000 abstracts and titles were retrieved from various databases searched, out of which relevant full text articles were independently identified by each author and included in the review by agreed consensus.3.3.1.Multimodal treatment approaches are recommended by several national and international clinical guidelines Problems of public scepticism/cynicism about ADHD managementPublic opinions have become divided, and several controversies have emerged around the wide variations in medical and non-orthodox practices that have been promoted and implemented in the management of ADHD. Some people have gone as far as querying the existence of ADHD as a bona fide medical diagnosis ADHD medications do not result in universal effective responses.It has been reported that 10–30% of CYP would not respond to ADHD stimulant medications Efficacy of stimulant medications is lower in younger children and is associated with a higher burden of side effects Parental and some Clinicians' perceptionsSome parents and clinicians are reluctant to recommend ADHD medication use due to concerns about undesirable long-term side-effects and express preference for non-medication options Problematic low therapeutic compliance during adolescence.Pharmacotherapy is less effective for the management of commonest co-morbidities of oppositional and conduct problems or challenging behaviour.Pharmacologic treatment is less effective among preschool children Adverse effects of pharmacological treatmentsMild to moderate adverse effects are common with all pharmacological agents used in the treatment of ADHD, including disturbances of sleep, growth, appetite, blood pressure and heart rate. More severe side effects such as psychotic symptoms are also rarely reported. A few observational studies have also reported increased risks of seizures, tics, depression and suicidal attempts associated with pharmacologic treatment of ADHD Higher level of acceptabilitySome studies have confirmed high levels of acceptability Behaviour therapy alone when compared to pharmacology treatment, and there are higher risks of patients discontinuing ADHD medication use compared to placebo 3.2.Four types of Behavioural or Psychosocial interventions are available . Each moBox 1.Psychosocial interventions: Body-focused interventions: Cognitive/neuro-Cognitive Therapy (CT): CT/computer attention training/working-memory training/attention training/Neurofeedback (NF) training/Electromyographic (EMG) biofeedback interventionsCognitive-behavioural training: Box 2.Psychoeducation is defined as an intervention with systematic, structured and didactic knowledge transfer for an illness and its treatment, integrating emotional and motivational aspects to enable patients to cope with the illness and to improve its treatment adherence and efficacy. Psychoeducation empowers patients and families to better understand and cope with the illness more successfully and to commit to more long-term involvement.Neurocognitive training involves repeated practice of ADHD-specific deficient neuropsychological processes such as working memory or attention. Neurocognitive training typically involves digital, automated training exercises for strengthening any deficient neurocognitive function.Neurofeedback enhance beta (15–18 Hz) and inhibit theta (4–7 Hz) brain activity.Neurofeedback uses video visualization or sound representations of brain activity via EEG recording to teach children to increase attention and impulse control. The most frequently used frequencies in Mindfulness-Based Interventions enhance self-regulation and capacity to pay attention to experiences in the present moment through three processes: enhanced attention control, improved emotional regulation and altered self-awareness (meditation).Parent training encourages behaviour control strategies designed to enhance desirable and discourage inappropriate child behaviours, improve positive and constructive adult–child interactions, enhance Parenting self-concept and increase parenting confidence.Behavioural therapy includes interventions aimed at changing child's behaviours (enhancing appropriate behaviours and decreasing undesired behaviours), based on social learning and other cognitive principles.Cognitive Behaviour Therapy (CBT) integrates a combination of both cognitive and behavioural learning principles to teach coping skills and encourage desirable behaviour, emotions and thought patterns.3.3.Several problems have been identified as barriers to efforts in carrying out high quality research into the effectiveness of non-pharmacological and psychological treatment modalities, which are not encountered in research with pharmacological agents. These problems include difficulties with running truly blinded trials, high costs, a dearth of specialist therapists and homogeneity of ADHD phenotypes Box 3.It is difficult to ensure the principles of gold-standard randomized trials with blinded outcome assessment , with the consequent bias risk of inflated efficacy estimates.Behavioural interventions produce outcomes that are difficult to maintain over long durations, and research projects are generally more expensive to run than pharmacological studies The outcomes of behavioural interventions only affect specific targeted functions, do not appear to be generalised to other behaviours and may not be applicable in other settings There is a general shortage of trained community-based behavioural specialists to match the number of CYP with ADHD.Significant improvement in some key areas of desirable functioning such as academic achievement, social skills, family relationships and executive abilities may not be achievable thorough non-pharmacological therapies There are limited studies to confirm the sustenance of immediate treatment outcomes for many behavioural therapies  ADHD is an heterogeneous condition with impaired functioning in several neurocognitive domains, which are difficult to measure accurately.3.4.I. Treatment of ADHD core symptomsThe research evidence for non-pharmacological / psychological treatment for ADHD core symptoms is summarised in Box 4.Psychoeducation in group of parents improved ADHD symptoms and cognitive levels significantly Behavioural therapy combined with stimulants seems superior to psychostimulants or non-stimulants alone.Stimulants alone seem superior to behavioural therapy, cognitive training and non-stimulants.Positive effects of Behavioural therapy are more likely to be reported for assessments made by unblinded parent ratings.Effectiveness of Cognitive behaviour therapy (CBT) for treating ADHD symptoms and functional impairment is mixed in several studies.Cognitive Behaviour Therapy (CBT) combined with stimulants improves core ADHD symptoms The evidence for the effect of working memory training programmes in producing a significant decrease in ADHD symptomatology is mixed.Effectiveness of Neurocognitive training to improve ADHD symptoms and related functional impairments has been mixed in several studies.Cognitive training had significant effects on total ADHD and inattentive symptoms for reports by raters most proximal to the treatment setting , while the effect size of cognitive training decreased to near 0 and became non-significant for blinded measurements made by observers.Meta-analytic evidence on the efficacy of Neurofeedback for ADHD core symptoms is currently mixed.The reported effectiveness of standalone non-pharmacological or psychological modalities of BT, CBT, working memory training, Mindfulness (meditation), NGT and NF for controlling ADHD core symptoms is mixed, and they currently cannot be recommended as stand-alone therapies.II. Effects on other ADHD-related behavioural problems.The research evidence for non-pharmacological /psychological treatment in controlling other ADHD-related behavioural problems is summarised in Box 5.Cognitive Behaviour Therapy (CBT) improves problematic and antisocial behaviours Combination of stimulants with Computer-based attention training game systems significantly improve attention scores, and evidence of F-MRI captured Functionality of both the salience/ventral attention network (SVN) and dorsal attention network (DAN).Cognitive training improves Executive functions (EF), matching with the healthy control children Behavioural therapy leads to significant improvements in 3 areas of parenting quality: positive parenting, decreased negative parenting and increased parenting self-concept.Parent training and other Behavioural therapies have shown positive results for parental knowledge and children's emotional, social and academic functioning - mostly from unblinded studies.Parent training improves effective parenting skills, reduces oppositional and noncompliant behaviours and may improve other functioning areas.Parent training has no significant treatment effects on self-rated parental mental health .Behavioural interventions are effective with low effect sizes on Child Psychopathology and functionality including conduct problems, social skills and academic performance Meta-analysis of 100 studies showed that classroom interventions reduce off-task and disruptive classroom behaviour in children with symptoms of ADHD, with largest effects for consequence-based and self-regulation interventions Cardio exercise improves executive functions based on event-related brain potentials; attention and Academic performance Parent-led and other adult/child-led Psychosocial interventions are associated with relatively low effect sizes, but they significantly improve Parenting skills, including positive parenting self-concept and improved parental knowledge Box 6.Classroom-based behavioural and teacher-based psychoeducation interventions are reported to reduce disruptive classroom behaviour in CYP with ADHD.Executive functioning in CYP with ADHD including attention and Academic performance are significantly improved with use of digital attention training game systems and other NCT interventions and active involvement in regular Cardio exercises III. Study of combined non-pharmacological modalities.Research studies on combined modalities, e.g., Computer-based Cognitive Training (CT) and parent training and combined CT, physical exercises and classroom behaviour management strategies, have yielded disappointingly no significant improvements on either the ADHD core symptoms or other related outcomes 4.There is an accumulated body of published evidence to prove the efficacy of some behavioural / psychosocial strategies for managing CYP with ADHD, with measurable benefits on several domains of daily patient and parent functioning. The only non-pharmacological intervention that has consistently shown significant benefits in several studies is parent- or teacher-child dyad Behavioural therapy Several ADHD-related outcomes, including parenting skills, child's social skills, academic performances and challenging behaviours, have been shown to respond significantly to non-pharmacological, psychological and Behavioural treatment strategies. School-based non-pharmacological interventions have been shown to reduce disruptive behaviours. Executive skills are also significantly improved with use of computer-based Neurocognitive training and regular physical Cardio exercises It is important to acknowledge that most clinical studies of ADHD in CYP are based on a predominantly male population, which has been partly attributed to gender stereotyping and symptomatology differences 5.Standard procedures for evaluating quality of the published evidence were not applied in this brief paper because it was not designed to be a systematic review. This is partly due to the dearth of high-quality research studies available for many non-pharmacological therapies and interventions. This article is mainly addressed to busy clinicians making daily judgment about appropriate psychosocial interventions for their young ADHD patients and their carers.6.With new advancements in computer science and wider implementation of Artificial Intelligence and Machine learning in many sectors of human endeavours, we could anticipate that more sophisticated neurocognitive enhancement programmes will be based on new advances in ADHD Neurobiology and consequently lead to better targeted improvements in both the core symptoms and other behavioural effects of ADHD We need to explore further ways to individualise optimal treatment for CYP with such a heterogenous condition as ADHD and in the future tackle potential public scepticism and cynicism regarding its management 7.Combining behavioural therapy with stimulants has shown significant effectiveness in enhancing attentional processes, reduces impulsiveness and may potentially lead to reduced dosing and duration of medication treatment and also help to reduce the risk of side effects. It also improves problematic and antisocial behaviours Standalone BT, CBT, Mindfulness, Neurocognitive training and Neurofeedback cannot currently be recommended  for contParenting CYP with ADHD can be very stressful, and targeting parental mental health problems with appropriate additional service provisions may enhance the effects of behavioural interventions The limitation of this paper should be noted because it was not designed as a systematic review, with no strict evaluation of the quality of the published evidence. The recommendations of this article are designed as a quick reference for busy clinicians for making quick day-to-day decisions for managing CYP with ADHD and supporting their carers. The recommendations will continue to evolve in the future as more robust research evidence becomes available.Click here for additional data file.