Motivational and attentional processes energize action sequences to facilitate evolutionary competition and promote behavioral fitness. Decades of neuropharmacology, electrophysiology and electrochemistry research indicate that the mesocorticolimbic DA pathway modulates both motivation and attention. More recently, it was realized that mesocorticolimbic DA function is tightly regulated by the brain’s endocannabinoid system and greatly influenced by exogenous cannabinoids—which have been harnessed by humanity for medicinal, ritualistic, and recreational uses for 12,000 years. Exogenous cannabinoids, like the primary psychoactive component of cannabis, delta-9-tetrahydrocannabinol, produce their effects by acting at binding sites for naturally occurring endocannabinoids. The brain’s endocannabinoid system consists of two G-protein coupled receptors, endogenous lipid ligands for these receptor targets, and several synthetic and metabolic enzymes involved in their production and degradation. Emerging evidence indicates that the endocannabinoid 2-arachidonoylglycerol is necessary to observe concurrent increases in DA release and motivated behavior. And the historical pharmacology literature indicates a role for cannabinoid signaling in both motivational and attentional processes. While both types of behaviors have been scrutinized under manipulation by either DA or cannabinoid agents, there is considerably less insight into prospective interactions between these two important signaling systems. This review attempts to summate the relevance of cannabinoid modulation of DA release during operant tasks designed to investigate either motivational or attentional control of behavior. We first describe how cannabinoids influence DA release and goal-directed action under a variety of reinforcement contingencies. Then we consider the role that endocannabinoids might play in switching an animal’s motivation from a goal-directed action to the search for an alternative outcome, in addition to the formation of long-term habits. Finally, dissociable features of attentional behavior using both the 5-choice serial reaction time task and the attentional set-shifting task are discussed along with their distinct influences by DA and cannabinoids. We end with discussing potential targets for further research regarding DA-cannabinoid interactions within key substrates involved in motivation and attention. Because many reviews already exist that describe endocannabinoid (eCB) signaling , the ris21 terpenophenolic phytocannabinoids exist in the cannabis plant  (N-acyl-phosphatidylethanolamine (NAPE) by NAPE-specific phospholipase D (NAPE-PLD) and metabolized by fatty acid amidohydrolase (FAAH)  . It is nde; AEA) . 2AG is e (FAAH) . In this2A variant was found to be expressed in the brain and the testis, whereas mRNA for the CB2B variant was found in the spleen and immune cells . While once thought to be relegated to the immune system and spleen, recent evidence suggest that CB2 is expressed in both neurons and glial cells of the brain as a unique isoform . Specifine cells . The exaunctions . There iunctions , which hunctions . But alsunctions , who recunctions , was alsunctions . Peroxisunctions , and areunctions . In all,unctions , suggestde novo and released from post-synaptic neurons in times of sustained neuronal activity  responsible for the rapid synthesis of eCBs  that project to a variety of brain regions. Its most prominent target is the ventral portion of the striatum, or nucleus accumbens (NAc) . While ts protein subunits. D2 receptors generally exhibit high binding affinity for DA and preferential coupling to Gi or Go protein subunits  WIN using an escalating dosing regimen. To determine if this dosing regimen produced tolerance to standard cannabimimetic effects, we first tested the consequences of it using the tetrad test. As expected, we found that WIN-treated rats displayed a rightward shift in the dose-response relationship (0.002–0.8 mg/kg IV) across all behavioral/physiological measures when compared to vehicle-treated controls. We then used FSCV to investigate whether the same pharmacological history produced tolerance to the DA releasing effects of WIN and cross-tolerance to the DA releasing effects of heroin. We additionally characterized whether this dosing regimen produces cross-tolerance to the DA releasing effects of heroin because However, further studies are necessary to compare how synthetic cannabinoids, eCBs, and phytocannabinoids produce tolerance, whether each produces tolerance to the DA releasing effects of a CB1 agonist, and whether these effects vary with age, sex, or species. It is possible that a synthetic aminoalkylindole cannabinoid like WIN produce distinct effects on molecular, cellular, and/or behavioral tolerance in comparison to a phytocannabinoid like THC. Two complimentary molecular mechanism are thought to contribute to CB1 desensitization and downregulation . One invWhile many different behavioral approaches exist to study the effects of cannabinoids on behavior, this review will primarily focus on response reinforcement and operant behavior. Response reinforcement was first described by in vivo electrophysiology studies demonstrated that phasic bursts of DA neural activity respond to gambles that guide economic decision making and integrate various factors that underlie value representations to influence choice  under a variety of conditions , includie choice , 2016. We choice   schedule demonstrate that eCBs are critically involved in habit formation. As previously described, when reinforcement is delivered in fixed intervals, the animal learns to time the interval and accelerate their responding toward its culmination. By contrast, under a VI schedule, responding is reinforced after a random period of time has elapsed since the first response. Under these conditions, the cumulative response pattern is maintained at a high, constant rate—presumably because the animal is uncertain about the time of reward availability  Figure . In compCB+/– (HET) and homozygous CB+/– (HO) mice were given access to the regularly active lever in either a sugar-sated or non-sated state. As shown in ad libitum access to the sugar solution before the devaluation test resulted in both CB1 mutant groups responding less in the sated state, suggesting that habit formation is impaired in CB1 mutant mice. As illustrated in Growing evidence suggests that eCB signaling is crucial for habit formation, although the precise roles each eCB play in habit formation and whether these roles differ at distinct loci in the brain remains to be determined. The majority of aforementioned DA studies measured its release in the primary terminal field of the mesocorticolimbic pathway , the NAcTo provide cell-type and eCB specific data, ad libitum sugar, the reinforcement contingency was degraded by allowing the animal to respond as if in the VI-task but lever presses resulted in no programmed consequence. Rather, reinforcers were delivered at equal intervals, matching the total number of reinforcers earned the previous day. The FAAH inhibitor URB597 was administered to test the effects of elevated AEA levels on habitual responding; the MAGL inhibitor JZL184 was administered to test the effects of elevated 2AG levels on habitual responding. Contrary to their predictions, they found that both drugs reduced responding during the test day following contingency degradation  . FurtherThe concept of attention has long historical roots in psychology and bears several definitions. While modern terminology surrounding attention may refer to disparate concepts such as arousal, vigilance, and distractibility, it may be broadly defined as selective activation of neural representations during information processing. Through this definition, attention may be best illustrated in relation to the highly related process of working memory. Whereas attention uploads information ‘on-line’ at any discrete timepoint, working memory stores and utilizes these activated representations during recall across small spans of time . While aAlthough the neuroanatomical loci of attention are many and work as an integrated network of multiple brain regions, attentional control is largely mediated by cortical regions. Spatial and visual attentional control, for instance, have been evidenced to be strongly regulated by frontoparietal regions that filter sensory information in a top-down fashion, with injury to these areas resulting in spatial neglect despite intact bottom-up, sensory-driven networks . In termi-coupled D2 DA receptor activation on presynaptic GABA cells and a complex interplay between signaling of postsynaptic pyramidal cell DA receptors D1 (Gs-coupled), D2, and D4 (Gi-coupled)  . Pyramidcoupled) .Cortical function is also mediated by eCB signaling. In the PFC, CB1 expression has been found to be preferential to GABAergic presynaptic terminals adjacent to glutamatergic ones, both synapsing onto dendrites of mGluR5-containing pyramidal cells. This places CB1 in a position to integrate and balance excitatory and inhibitory signaling during activity-dependent eCB mobilization . This mGThe influence of cortical DA on cognition, including attentional processes, is a well-researched subject that has been intensely studied by neurobiologists and computational neuroscientists alike. Broadly, DA in the PFC facilitates integration of complex signals between sensorimotor networks by synchronizing different brain networks in response to both external signals and internal representations . This isper se, they index prioritization of attentional demand to reward-associated stimuli , attentional set-shifting and reversal learning as indices of attentional control as well as their respective deficits. Of the many factors that may influence attentional control, reversal learning – instrumental responding to swapped outcome contingencies between manipulanda – and impulsivity are both affective state-sensitive, pathology-related variables readily examined in the operant setting as adjuncts to more direct measures of attention itself . While t stimuli . Accordi stimuli . Such faThe 5-CSRTT for rodents was refurbished from a similar test of attentional processing during discrimination of visual stimuli in humans . The parLike the 5-CSRTT, the ASST for rodents was adapted from behavioral assessments originally designed for human subjects. The most commonly cited comparison is with the Wisconsin Card Sorting Test (WCST), though a more direct comparison may be made with the Cambridge Neuropsychological Automated Testing Battery (CANTAB). Both are used to investigate ‘behavioral flexibility’ in healthy and abnormal neurological states by measuring the ability to shift attention from one reward-predictive perceptual feature to another following an unexpected switch . SpecifiThe ASST adapted for rodents has been designed as both a reward-digging task, in which exemplars comprise different combinations of digging materials and odors as dimensions , panel AIn addition to assessing attentional sets, the separate reversal learning tests allow dissociation and detection of deficits relating to this ability alone. While similar to attentional set-shifting, reversal learning is considered a less complex but important process that relies on inhibition of previously rewarding actions . BecauseWhile both mesocorticolimbic DA and eCB signaling have been shown to affect 5-CSRTT performance, their influence on sustained attention and impulsivity may be separable. DA has been shown to play a crucial role in controlling inhibitory responding, with converging data indicating elevating synaptic DA increases impulsivity by activation of both D1 and D2 receptors . In rodei-coupled muscarinic GPCRs   . Yet, muThe effects of both mesocorticolimbic DA and eCB signaling on ASST performance diverge by the task’s separate components, with DA impacting both shifting attentional set and reversal learning and cannabinergic effects restricted to reversal learning. Converging lines of evidence suggest that D1 signaling within the medial PFC in rodents and the homologous DLPFC in primates is central to attentional set formation and shifting in the ASST  while D2Insight into the cannabinergic effects on ASST, on the other hand, are far outnumbered by those of DA, though these limited studies suggest eCBs may be more important for reversal learning than shifting attentional set. Finally, several studies using rats prenatally treated with methylazoxymethanol acetate (MAM) as a developmental model of schizophrenia suggest persistent psychotomimetic effects related to mesocortical neuroadaptations may result from aberrant eCB signaling during adolescence . Both MAThese results bring into question both the locus of reversal learning and the neural mechanisms underlining its impairment by cannabinoids. Also important are considerations of dissimilarities between DA manipulations and ASST models, since like most laboratory-controlled behaviors, performance variations may be attributed to different protocols. This is underlined by cannabinoid administration having been suggested to impact visual discrimination in general, which may broadly affect performance in operant chamber-based ASST paradigms . Future To assist in clarifying DA/eCB interactions and their effects on attention, a few effective approaches may be noted. Behavioral effects of DA/eCB interactions have been demonstrated combining targeted pharmacological manipulations with measures of negative affect, showing anxiolytic effects of CB1 activation in the amygdala is D1 and D2 dependent . AdditioFinally, DA/eCB interactions may also be investigated at the level of GABAergic interneurons. Many of the previously described alterations to mesocorticolimbic functional dynamics may involve FSI that are either directly targeted by eCBs or are ultimately subject to eCB-dependent adaptations, with attentional processing being impacted by both. In the PFC, parvalbumin expressing FSIs critically gate pyramidal activity and are sensitive to DA signaling. Furthermore, levels of dendritic D2 expression in PFC parvalbumin cells have been demonstrated to be regulated by CB1 signaling  Figure . CB1-expIn this review, we describe recent scientific studies suggesting cannabinoids modulate transient DA release events in a manner that may influence motivational and attentional processes alike. While we’ve acknowledged that DA/eCB interactions still need to be better investigated across multiple overlapping neural circuits, we would like to close by further considering the intertwined relationship between DA transients, motivation and attention. And finally, we offer some speculation into the clinical implications these findings may offer the treatment of neurobehavioral symptoms in psychiatric medicine.per se—suggesting an apparent dissociation , the seeming contradiction may indicate the difficulty in separating the components of motivation and attention by performing DA manipulations in operant tasks.Transient DA release events, the neural substrates of motivation, and the neural substrates of attention interact within a tangled thicket of intertwined circuits—the nodes of which likely influence each other and can be differentially modulated by eCBs at multiple levels. First, it is important to recognize that activation of either subcortical nodes of pre-attentive visual processing  or cortical nodes of attentional visual processing  are sufficient to evoke transient DA release events in the striatum of the basal ganglia , 2018. Tociation . However 5-CSRTT . While sThe interrelated nature of motivation and attention may be an asset to research as much as a liability, and cannabinoid modulation of either may underline therapeutic targets for both constructs. In the clinical context, DA/eCB interactions may play a specialized role in ADHD patients to impact both motivation and attention. Impaired activity in both motivational and attentional networks typical in ADHD patients are stabilized by pharmacologically increasing brain DA concentration; furthermore, while cannabinoids generally negatively affect measures of impulsivity and attention, they uniquely enhance them in ADHD patients . CommonaGeneralizing this example of impulsivity to other behavioral components linked by motivation and attention, it is possible to conceive of various constructs impacted by both, and in turn, their susceptibility to eCB-modulated DA function. In this sense, interrogation of DA/eCB interactions within substrates known to modulate either motivation or attention might share explanatory potential across translatable constructs, particularly substance use disorders. Such studies may assess cannabinoid-induced changes to motivational and attentional processes through modifications of DA-mediated reward value signals, which have been shown to influence both types of measures. Yet, for clear dissociations between each measure, it will be important to characterize the separate neural representations contributing to their respective behavioral outputs. It is also important to note that while distinct measures of motivation and attention have been studied under conditions of cannabinoid exposure, there remains much to be learned about how these measures overlap within the context of DA/eCB interactions.EO and DG conceptualized this review and equally shared in the majority of the writing. LH performed a secondary writing contribution that was lesser, but significant. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Organoids are tiny, self-organized three-dimensional tissue cultures derived from stem cells that can include dozens of cell types to replicate the complexity of an organ. In contrast, MPS are highly controlled fluidic culture systems consisting of isolated cell type(s) that can be used to deconvolute mechanism and pathophysiology. Both systems, having their own unique benefits and disadvantages, have exciting applications in the field of kidney disease modeling and therapeutic discovery and toxicology. In this review, we discuss current uses of both hPSC-derived organoids and MPS as pre-clinical models for studying kidney diseases and drug induced nephrotoxicity. Examples such as the use of organoids to model autosomal dominant polycystic kidney disease, and the use of MPS to predict renal clearance and nephrotoxic concentrations of novel drugs are briefly discussed. Taken together, these novel platforms allow investigators to elaborate critical scientific questions. While much work needs to be done, utility of these 3D cell culture technologies has an optimistic outlook and the potential to accelerate drug development while reducing the use of animal testing.Kidneys are critical for the elimination of many drugs and metabolites via the urine, filtering waste and maintaining proper fluid and electrolyte balance. Emerging technologies incorporating engineered three-dimensional (3D)  In addiin vitro systems with biological relevant flow rates that recapitulate the physiology and compartmentalization in the microenvironment of organs. These microfluidic devices have been applied to therapeutic areas ranging from chronic kidney disease (CKD) to central nervous system disorders . Due to the numerous cell types present in kidneys, the various protocols that have been developed result in the production of kidney organoids with varying complexity and number of cell types present. . DiseaseOne predominant kidney condition that has been modeled and studied using organoids is autosomal dominant polycystic kidney disease (ADPKD). ADPKD is a common renal genetic condition that causes cyst formation and expansion in the kidneys and can result in kidney failure and necessitate renal replacement therapy via transplant or dialysis. The disease is characterized by mutations in the PKD1 and PKD2 genes . Kidney in vitro studies  that demonstrated active reabsorption of albumin and glucose via crosstalk between tubular epithelial and vascular endothelial homeostasis in kidney disease . Similarin vivo drug plasma or urine concentrations over time. Successful PBPK modeling requires accurate in vitro estimates of pharmacokinetic parameters for compounds of interest  modeling is a quantitative computational method that simulates the processes of absorption, distribution, metabolism, and excretion with a set of mathematical equations to predict interest . Experim in vivo . In a cotrations .In addition to the prediction of clinical dosing parameters, kidney MPS can be used to study pathways of nephrotoxicity. Recently, Imaoka et al. used a PTEC MPS to investigate the mechanism of Ochratoxin A (OTA)-induced kidney injury. In this study, PTEC MPS were perfused with clinically observed concentrations of OTA. A significant decrease in OTA-induced toxicity was observed with administration of ABT (1-aminobenzotriazole), a pan-inhibitor of P450; OTA-induced toxicity was enhanced by treatment of NBDHEX  hexanol), an inhibitor of glutathione S-transferase enzymes. Taken together, these studies confirmed the ability of the PTEC MPS system to detoxify or bioactivate OTA. In addition, VP-MPS was used to confirm that basolateral OTA uptake was mediated primarily by OAT 1/3 transporters. . Yin et in vitro–in vivo extrapolation to predict IxS renal clearance at different stages of CKD.  on OAT1 mediated active transport of the highly albumin-bound uremic solute, indoxyl sulfate (IxS). For this study, a kidney MPS was used to evaluate IxS renal uptake by OAT1 in the presence and absence of HSA or CKD-modified HSA. IxS uptake kinetic parameters, including OAT1 affinity and intrinsic renal clearance, were incorporated with  of CKD. . In a se of CKD. . Furtherin silico pharmacokinetic modeling and provide an alternative approach to guide first-in-human clinical trials.Taken together, the development of kidney MPS allows researchers to elaborate critical scientific questions and overcome the drawbacks of static 2D cell cultures and animal models. The physiologically relevant properties of MPS support further research in the contexts of drug efficacy, drug and drug interactions, and drug-induced nephrotoxicity. Potential future directions may include evaluation of the regenerative properties of the renal tubular epithelium. . Moreovein vivo proximal tubule structures. Kidney MPS are also less variable from batch-to-batch that are typically cultured with a small number of cells. This simplicity is both an advantage and disadvantage-individual enzymes or transporter proteins can be studied, but in a contrived non-biomimetic environment that does not include physiological multi-cell interactions. Like organoids, PTEC MPS are viable for more than 28 days while maintaining a functional epithelial phenotype; moreover, MPS co-cultured with podocytes and glomerular endothelial cells also show extended cell viability compared with traditional 2D cell cultures (In this short review, we have presented two models of 3D kidney cell cultures: organoids and microfluidic chip systems . Both sycultures . More wo


Congenital heart defects (CHD), the most common cause of birth defects with increasing birth prevalence, affect nearly 1% of live births worldwide. Cyanotic CHD are characterized by hypoxemia, with subsequent reduced oxygen delivery to the brain, especially critical during brain development, beginning in the fetus and continuing through the neonatal period. Therefore, neonates with CHD carry a high risk for neurological comorbidities, even more frequently when there are associated underlying genetic disorders. We review the currently available knowledge on potential prevention strategies to reduce brain damage induced by hypoxemia during fetal development and immediately after birth, and the role of erythropoietin (EPO) as a potential adjunctive treatment. Maternal hyper-oxygenation had been studied as a potential therapeutic to improve fetal oxygenation. Despite demonstrating some effectiveness, maternal hyper-oxygenation has proven to be impractical for extensive clinical application, thus prompting the investigation of specific pathways for pharmacological intervention. Among those, the role of antioxidant pathways and Hypoxia Inducible Factors (HIF) have been studied for their involvement in the protective response to hypoxic injury. One of the proteins induced by HIF, EPO, has properties of being anti-apoptotic, antioxidant, and protective for neurons, astrocytes, and oligodendrocytes. In human trials, EPO administration in neonates with hypoxic ischemic encephalopathy (HIE) significantly reduced the neurological hypoxemic damages in several reported studies. Currently, it is unknown if the mechanisms of pathophysiology of cyanotic CHD are like HIE. Neonates with cyanotic CHD are exposed to both chronic hypoxemia and episodes of acute ischemia-reperfusion injury when undergo cardiopulmonary bypass surgery requiring aortic cross-clamp and general anesthesia. Our review supports future trials to evaluate the potential efficiency of EPO in reducing the hypoxemic neurologic damages in neonates with CHD. Furthermore, it suggests the need to identify early biomarkers of hypoxia-induced neurological damage, which must be sensitive to the neuroprotective effects of EPO. Improved prenatal and neonatal detection of congenital heart defects (CHD), along with advancements in medical treatments and surgical interventions, has led to improved survival rates among children with complex CHD . PatientHIF2-α (EPO (erythropoietin), is located in chromosome seven and encodes for the protein by the same name responsible for the regulation of erythropoiesis. EPO, a glycoprotein cytokine well-known for its anti-apoptotic and antioxidant role in neuronal injury  transposition of the great arteries, 2) “functionally” univentricular hearts, and 3) hypoplastic left heart syndrome . TranspoFollowing birth, neonates with cyanotic CHD often have definitive corrective surgery later in infancy for reasons such as waiting for the infant to grow and reach a size amenable to surgery or requiring surgical repair in stages. Neonates with cyanotic CHD exhibit delayed structural brain development, appearing approximately one month less mature compared to unaffected infants . MyelinaErythropoietin (EPO) has long been used in preterm neonates to prevent and/or treat anemia of prematurity. While EPO treatment reduces the frequency and volume of red blood cell transfusions, its use is currently not recommended, pending the results of two ongoing clinical trials involving darbepoietin, a derivative of EPO . HoweverCellular adaptive response to hypoxia is mainly orchestrated by the activation of transcription factors called hypoxia inducible factors (HIFs) . HIFs arEPO gene  have some overlapping roles but also demonstrate distinct functions in different cell types . HIF-2α A growing number of pre-clinical studies in rodents suggests that the activation of HIF-1α signaling pathway prior or shortly after ischemic stroke reduces tissue damage and increases functional recovery from ischemic stroke . One exaVEGF) and EPO, and may directly protect neurons . ROS generation, mainly dependent on complexes I and III, highly relies on metabolic conditions and on the intra-mitochondrial balance between oxidative and antioxidant factors . This prIn response to excess ROS production, antioxidant tasks are pursued by enzymes as catalases, glutathione peroxidases, thioredoxins, and peroxyredoxins. These enzymes use electron donors to tackle the further formation of ROS . The celAnother important antioxidant mechanism, induced by the EPO pathway, is mediated by the enzyme superoxide dismutase (SOD), which the removal of superoxide free radicals generated from mitochondrial respiration . OverexpProlonged systemic hypoxia is known to elicit oxidative stress altering redox balance. Under hypoxia electron-transport rate in the mitochondria decreases, the binding of oxygen to the proteins/complexes changes, aerobic oxidative respiration is limited and partial reduction of oxygen leads to the formation of ROS. While the ischemic state induces anaerobic metabolism, a lower level of ATP production, failure of ion-exchange channels, cell swelling and impaired enzymatic activity in the cytoplasm, the reintroduction of oxygen during reperfusion exacerbates mitochondrial damage, electrolyte imbalance, and oxidative stress affecting the NADPH oxidase, nitric oxide synthase, and xanthine oxidase systems. ROS overproduction leads to cell death by different mechanisms, leading to progressive neurodegeneration .in utero elapses in an environment that is relatively hypoxic, however, oxygen availability to the fetus is provided by adaptive mechanisms that allow oxygen delivery to tissues for physiological growth and development. The master regulator for the cell’s adaptive responses to hypoxia are HIFs, which through VEGF and EPO activation, ensure O2 delivery to the tissue. Vascular or metabolic alterations in the mother  causes the fetus to develop under chronic hypoxia with higher risk of developing oxidative stress that may be detrimental for its fetal and postnatal development (Human life elopment .Much of what is understood regarding oxygen toxicity and ROS production comes from research in preterm neonates suffering from pulmonary diseases of prematurity (e.g. bronchopulmonary dysplasia), including injury of intestinal epithelial cells , and ophMaternal hyper-oxygenation has been studied as a potential therapeutic to improve fetal oxygenation, administered following the ultrasound diagnosis of CHD, until delivery . The relAdditionally, for the developing fetus, there are concerns regarding oxygen toxicity and radiation toxicity known to occur through a common mechanism related to increased production of ROS since the 1950s . The oxiThe formula of the oxygen delivery denotes the importance of hemoglobin concentration for optimizing oxygen delivery:Since iron is an essential component of hemoglobin, a biological response to hypoxia is to increase the demand for iron. Guidelines for iron supplementation recommend that in iron sufficient children with hematocrits below 60%, low-dose iron supplements should be given to prevent the onset of latent iron deficiency . Iron deAn ineffective hematopoietic response in newborns with CHD, or ‘‘relative anemia,’’ results in polycythemia insufficient for adaptation to hypoxia. Hematocrit increases in mildly cyanotic CHD, while hemoglobin stays within the normal range. Unlike adaptations observed in animal models of chronic hypoxia, human newborns with severe CHD appear to lack a compensatory increase in hematocrit and hemoglobin, suggestive of an anemic-like state . On the The number of transfusions received during the neonatal intensive care unit stay  also correlates with increased white matter injury observed by MRI. A reasonable “double hit” hypothesis resulting in white matter injury involves the hypoxia of low hemoglobin leading to the transfusion and the inflammation associated with transfusion .If the formula of the oxygen delivery is considered, in the presence of neonatal hypoxemia, means to improve the oxygen delivery to the brain, and subsequently the neurological conditions include 1) increase of the cardiac output, 2) increase of the oxygen saturation. Increase in cardiac output can be achieved either with interventional catheter procedures, such as the balloon atrial septostomy (= Rashkind procedure), or with surgery, aimed at improving the intra-cardiac mixing and the effective pulmonary blood flow. Additionally, there are medications utilized to increase cardiac output and optimization of respiratory support . In ordeHIF2-α stabilization and hence EPO production. Both EPO and its receptor, primarily secreted by kidney and, in the fetal period, by the liver  in neuroblastoma cell line. This mechanism seems to be one of the main causes for the neurotrophic, neuroprotective, antioxidant, and antiapoptotic effects  might be effective against hypoxic, ischemic, and traumatic brain injury, as well as chronic and progressive degenerative diseases . An incrammation . The neueriments . The efferiments . While sTo cross the blood-brain barrier, rhEPO must be administered in high doses of 2000–5000 IU/kg of body weight . The neeEPO has been proposed as a potential therapeutic option in CHD in previous reviews , even ifEPO administration to improve oxygen delivery and treat anemia in infants have been proposed by several studies, sometimes together with iron administration . A very in vivo animal studies in adult rats , as adjunctive treatment to hypothermia therapy, and in preterm infants who do not qualify for hypothermia therapy . While Eult rats .in vitro and in vivo studies suggest the neuroprotective effects of both hypoxia-induced and administrated EPO in infants. The studies published so far suggest the need for further clinical investigations to evaluate the potential efficiency of EPO administration in reducing the hypoxemic neurologic damages in neonates with CHD.Neurological damages in children with CHD are associated with elevated mortality and morbidity, seriously affecting the patients, their families, the care providers, and society at large. In this review available knowledge on potential prevention and reduction of the severity of neurological damages due to hypoxemia have been analyzed, in particular the potential role of EPO administration. Several


Characterizing perceived physical fatigability enables researchers to quantify an individual’s susceptibility to experiencing fatigue in the context of a standardized physical task. This approach eliminates self-pacing, and is a less-biased, more sensitive means to measure the degree to which fatigue may limit activity. Our previous work with two validated measures of perceived fatigability, the Pittsburgh Fatigability Scale (PFS) and Borg Rating of Perceived Exertion (RPE) at the end of a standardized 5-minute treadmill walk, are prognostic indicators of phenotypic aging. This symposium will present new directions related to greater fatigability as a marker of biological aging, organ system health and functioning, as well as brain pathology and structure. Specifically, Mr. Katz will explore the relationship between leukocyte telomere length, a marker of biological aging, with PFS fatigability in participants from the Long Life Family Study. The other four papers use data from the Baltimore Longitudinal Study of Aging (BLSA) and RPE fatigability (RPE). Drs. Simonsick and Karikkineth investigate fatigability as an early marker of aging and disease related impacts on key organ systems, specifically diminished renal function as reflected in estimated Glomerular Filtration Rate and cardiovascular health evaluated as vascular stiffness. Ms. Liu and Dr. Schrack will share whether there are associations of perceived fatigability with brain health, specifically Alzheimer’s disease-related pathology (PiB) and changes in brain structure. Lastly, our Discussant, Dr. Eldadah, will critically review the presentations in the context of new directions in fatigability research.


Stem cell-laden three-dimensional (3D) bioprinted cardiac patches offer an alternative and promising therapeutic and regenerative approach for ischemic cardiomyopathy by reversing scar formation and promoting myocardial regeneration. Numerous studies have reported using either multipotent or pluripotent stem cells or their combination for 3D bioprinting of a cardiac patch with the sole aim of restoring cardiac function by faithfully rejuvenating the cardiomyocytes and associated vasculatures that are lost to myocardial infarction. While many studies have demonstrated success in mimicking cardiomyocytes' behavior, improving cardiac function and providing new hope for regenerating heart post-myocardial infarction, some others have reported contradicting data in apparent ways. Nonetheless, all investigators in the field are speed racing toward determining a potential strategy to effectively treat losses due to myocardial infarction. This review discusses various types of candidate stem cells that possess cardiac regenerative potential, elucidating their applications and limitations. We also brief the challenges of and an update on the implementation of the state-of-the-art 3D bioprinting approach to fabricate cardiac patches and highlight different strategies to implement vascularization and augment cardiac functional properties with respect to electrophysiological similarities to native tissue. I.Cardiovascular diseases (CVDs), which are well recognized as a non-communicable killer disease, account for 32% of the global deaths and comprise a broad terminology, assigned for a set of pathologies that affect the heart.Existing pharmacological approaches with drugs  are often considered as the first treatment options offered to the patients.in vivo or exogenously provide new cardiomyocytes to replace the necrotic tissue.An ideal approach to regenerate a damaged or diseased myocardium is to either promote the proliferation of resident cardiomyocytes et al. demonstrated functional recovery of the infarcted myocardium in a porcine MI model using clinical-sized large cardiac tissue sheets. The sheets consisted of various human-induced pluripotent stem cells (iPSCs)-derived cardiac cells, including cardiomyocytes, vascular endothelial cells (ECs), and vascular mural cells.Bioengineering approaches have been developed as alternative treatment strategies to increase the efficacy of transplanted cells by enhancing their retention or engraftment during the early integration phase.This review discusses the types of candidate stem cells with cardiac regenerative potential  and elucidates their applications and limitations. Furthermore, we provide challenges and an update on the implementation of the state-of-the-art 3D bioprinting approach to fabricate the cardiac patch based on the use of either types of stem cells or a combination of cells. In addition, we highlight the different approaches to implement vascularization and augment cardiac functional properties with respect to electrophysiological similarities to the native tissue, which can further aid in myocardial repair.II.in situ post-transplantation.Interest in stem cell-mediated cardiac repair has significantly increased with accumulating evidence from preclinical and clinical studies. Stem cells possess properties such as plastic adherence, self-renewal, immunomodulation, and multilineage transdifferentiation.A.Multipotent adult stem cells can be generated from different sources . Because adult stem cells possess cardiogenic differentiation capability, the focus is now shifting toward the development of stem cell sources for repairing or regenerating the damaged myocardial tissues.in vivo upon transplantation to the myocardium in both non-injury and MI models. When characterized with immunohistochemistry, differentiated MSCs show specificity for both cardiac and ECs and gap junction proteins.in vivo studies have reported improvements in cardiac function.Mesenchymal stem or stromal cells (MSCs) are characterized by their (1) self-renewal ability, (2) low immunogenicity, (3) multipotency to transdifferentiate, (4) plastic adherence characteristics, (5) homing ability to tissue injured areas, and (6) participation in vasculogenesis and myogenesis.et al. transplanted autologous BM-MNC in patients with coronary artery disease or LV dysfunction and found no differences in any of the outcomes, including cell therapy and clinical improvement between placebo and BM-MNCs.et al. demonstrated improved therapeutic benefits of primary skeletal myoblasts in a rat MI model by co-culturing the myoblasts with MSCs; the addition of MSCs to skeletal myoblasts' cell sheets led to enhanced angiogenesis due to increased secretion of a hepatocyte growth factor (HGF) and a vascular endothelial growth factor and consequently augmented cardiac functional recovery in the MI model, thereby suggesting it as a strategy for clinical applications.Another cell source selected for cardiac regeneration is BM-derived mononuclear cells (BM-MNCs), because these cells are widely available and also feasible to isolate from patients through BM aspiration.in vitro, and secrete cytokines and growth factors that stimulate endogenous stem cells and regeneration mechanisms through a strong paracrine signaling.in vitro.et al. successfully isolated c-kit+ CSCs from a rat heart and showed their multipotency in vitro and the ability to regenerate cardiomyocytes and blood vessels following MI.6 c-kit+ CSCs were infused into swine hearts via the intracoronary route.Similar to MSCs, resident CSCs are multipotent and self-renewing, differentiate Although the risks in terms of immunogenicity and possible arrhythmic occurrence related to adult stem cell transplantation are usually rare and safety and feasibility have been proven in various preclinical and clinical studies, the cardiac output or function is rather inferior.B.in vivo and continue to maintain the regional and global contractile function after MI. Nevertheless, further studies are warranted for understanding mechanistic insights, and tests should be conducted for analyzing the occurrence of arrhythmic events in species with slower heart rates. Similarly, human ESC-CMs demonstrate the capability to couple electromechanically with recipient's cells, which enables synchronous contraction between the transplanted cardiomyocytes and the host tissue.in vitro testing platform for cardiomyocytes.Pluripotent stem cells  are characterized by their unlimited self-renewal (stemness) and multilineage differentiation abilities and are among the most attractive cell sources for cardiac research.in situ.In order to circumvent the ethical concerns and legal debates surrounding the use of ESCs, induced pluripotent stem cells (iPSCs) have been developed by cellular programming through retroviral transduction of a defined combination of transcription factors.in vitro and in vivo -modified alginatein vivo engraftment over time. Similarly, Wang et al. demonstrated 3D bioprinting of cardiomyocytes loaded in a fibrin-based bioink into cardiac tissues that showed contractility with cellular organization and uniformity.et al. It is a porcine heart-derived dECM-based hydrogel that has appropriate rheology for delivery. In their study, VentriGel was applied in both early and late post-myocardial patients, from 60 days to 3 years post-MI, having LVD by transendocardial delivery, and it caused revascularization. There were no discontinued patients, side effects related to mapping and injection, or significant ventricular arrhythmias. This clinical research showed the safety and feasibility of VentriGel by percutaneous coronary intervention in the patients.et al. who demonstrated the concept and success of 3D bioprinting using specific tissue-derived dECM-based hydrogels, efficiently utilizing its thermoresponsive phenomenon to bioprint cell-laden tissue analogs with engineered porosity.et al. reported an easy, versatile, and biocompatible two-step process to improve its printability.In vivo implantation in rat hearts demonstrated good retention of the cardiac patch with evidence of vascularization over 14 days. Hence, the potential therapeutic application of these patches has been suggested for pediatric patients with right ventricular heart failure or as an allogeneic reparative patch for adult cardiac dysfunction.3D bioprinting enables the possibility to biofabricate clinically applicable, viable, and organized cardiac tissue analogs in vivo, the fabricated cardiac patch needs further contemplations in terms of cellular maturation, oxygen and nutrient diffusion avoiding creation of hypoxic condition, integration with host tissues, to name a few.et al. demonstrated a concomitant method to examine the synergistic effects of two types of stem cells to promote cardiac repair.in vivo; collectively rejuvenating the injured myocardium and vessels post-MI .in vivo priming can be an efficient approach to maximize the therapeutic potential of BM-MSCs and enhance cardiac repair of infarcted hearts.Considering the efficacy and avoiding the potential undesired effects  post-MI . Howeverin vivo implantation of a bioprinted pre-vascularized cardiac patch to the rat MI model demonstrated cell infiltration into the infarcted area and evidenced the formation of neomuscle and capillary, in addition to an enhanced cardiac function [in vivo analysis showed vascularization and successful integration of the 3D bioprinted cardiac patch into the native rat myocardium. This experimental study is a good example to show that along with the 3D bioprinting technique, co-culture of different cell types may aid in improving cell maturation, neovessel formation, and cell viability. However, there are certain limitations associated with the fabricated cardiac patch, in terms of its short-term culture, concerns related to vascularization, slow conduction velocity (due to immatured hiPSC-CMs), and inferior mechanical properties that could be attributed to the fabrication of a single layer of spheroid cells. Maiullari et al. reported multicellular 3D bioprinting of the cardiac patch comprising of human umbilical vein ECs (HUVECs) along with hiPSC-CMs.in vivo grafting [et al. contributed substantially to the field by reporting the fabrication of a patient-specific thick and functional perfusable vascularized cardiac patch, eliminating the need for immunosuppression treatment.in vitro tissue-engineered cardiac patch with improved electrical conduction and generating electrophysiological properties similar to those from a native heart.in vitro. In native, cardiomyocytes are uniquely organized to perform synchronous and spontaneous beatings and contain mitochondria, densely packed sarcomeres, sarcoplasmic or endoplasmic reticulum, and transverse tubules.et al. produced endothelialized myocardium tissue based on 3D bioprinting. A hydrogel lattices scaffold with microfibers was fabricated using a low viscosity bioink developed by mixing alginate and GelMA. HUVECs are encapsulated in this bioink and printed directly. The cells move to the edge of the microfiber to form a layer of confluent endothelium. The migration and layering of HUVECs within the hydrogel lattices microfiber scaffold were made possible by the low viscosity composite bioink and facilitated by intrinsic polarization tendency and nutrient gradients. Thereafter, hiPSC-derived cardiomyocytes were cultured on a 3D anisotropic endothelial microfiber scaffold to create an aligned myocardium tissue capable of spontaneous and synchronous contraction.et al. developed a nanocomposite bioink, gold nanorods incorporated gelatin methacryloly (GelMA), for 3D bioprinting of functional cardiac tissue. In general, conventional bioinks are composed of polymeric biomaterials, which have low conductivity and interfere with an efficient electrical coupling between adjacent cardiac cells. To solve this problem, the gold nanorods in the nanocomposite bioinks connect the electrical resistive pore walls of the polymer, improve intercellular bonding, and promote the synchronized contraction of the 3D bioprinting structure to enhance electrical propagation and functionality in cardiac tissue.in vitro may not conform to the developmental paradigm in vivo. As differentiation progresses, the response of hiPSC-CMs to the induced physical stimuli declines; therefore, the electromechanical stimulation should commence during the period of cell plasticity.et al., in which the early stage hiPSC-CMs that were exposed to myocardium-like physical conditioning , with gradually increasing intensity (from 2 to 6 Hz in 4 weeks), promoted cells to attain the unprecedented maturation level.With respect to printing, a convincing example can be found in a study that demonstrated the multicellular and multilayered 3D spatial patterning of a pre-vascularized cardiac patch by using a porcine hdECM loaded with hCPCs and MSCs and growth factors.function . Anothergrafting . This stAltogether, these studies indicate that stem cell-laden 3D bioprinted cardiac patches have the potential to be used as a model platform to gain insights into human cardiac functions with respect to their force, pacing activity, contractile properties, and electrophysiological parameters. In addition, with more in-depth analysis and research on its applicability for further preclinical studies and clinical trials, a 3D bioprinted cardiac patch strongly represents a promising modality or resource for the upcoming future cardiac regenerative therapies.IV.in vivo. Significant uncertainties remain, and improvements in terms of (1) rapid vascularization, (2) functional and mechanical properties of the engineered cardiac patch, (3) perfusion by host's coronary circulation, and (4) enhanced integration with host tissue are mandatory before a bioprinted cardiac patch can be considered for routine use in relevant clinical settings for treating patients with myocardial diseases. Another challenging aspect is identifying factors that drive cardiomyocytes' synchronous electrical activity, keeping in pace with that of the host tissue as it is highly desirable in order to further ameliorate the bioprinted cardiac patch. However, as the enthusiasm for cardiac regeneration charges and science continues to advance, the 3D bioprinted cardiac patch will soon become an increasingly feasible, viable, and functional option, unblocking the barriers to achieve cardiomyocytes properties. This will open new avenues for cardiac research, paving the way toward clinical translation for treating patients with CVDs.Stem cell-laden bioprinted cardiac patches are a promising treatment modality for cardiac repair and regeneration for patients with ischemic cardiomyopathy. Multiple types of stem cells are suggested as candidates for cardiomyocytes regeneration. Nevertheless, there is no gold standard on the specific cell type to be used, and the search is ongoing for cardiac repair. Given the complexity of the myocardial tissue that accommodates diverse cell types, its regeneration process demands multiple coordination from varied cell types with synergistic effects, along with an established ECM microenvironment. Globally, researchers have made substantial progress in developing the hiPSC-CMs for better understanding the mechanistic aspects of these cells. In addition, advances in bioengineering approaches (3D bioprinting), along with the potentially powerful autologous cell source (hiPSC-CMs), offer a new realistic option with the capacity to resuscitate injured cardiomyocytes and vasculatures and may provide new insights into the pathogenesis of CVDs. Despite the promising preclinical results, the approach is still at its infancy for subsequent clinical translation, demanding further in-depth analyses to develop as an effective and efficient strategy to achieve cardiac regeneration The authors have no conflicts to disclose.S.D. and H.N. contributed equally to this work.


Pilot/feasibility studies play an important role in the development and refinement of behavioral interventions by providing information about feasibility, acceptability, and potential efficacy. Despite their importance and wide-spread use, the approaches taken by behavioral scientists to scale-up early-stage studies to larger-scale trials has received little attention. The aim of our study was to understand the role that pilot studies play in the development and execution of larger-scale trials.We conducted interviews with childhood obesity researchers who had published pilot behavioral interventions and larger-scale trials of the same or similar interventions. Questions were asked about the role of pilot studies in developing larger-scale trials and the challenges encountered when scaling-up an intervention based upon pilot findings. Data were coded and analyzed using an inductive analytic approach to identify themes.n = 15). When asked to define a high-quality pilot study, interventionists described studies that allowed them to evaluate two independent factors: components of their intervention  and study parameters . Interventionists expressed that more process implementation measures, different study designs, and additional iterations could improve decisions to scale-up. Most agreed that pilot studies were likely to produce inflated estimates of potential efficacy though only nine interventionists provided potential solutions for decreasing inflated measures of efficacy. Suggested major causes of inflated effects included high levels of oversight in pilot studies , reliance on subjective measures, and utilizing convenience or highly motivated samples. Potential solutions included designing pilots for real-world implementation, only conducting randomized controlled pilot studies, and pre-registering pilot studies.Twenty-four interventionists  completed a total of 148 pilot studies across their careers , of which 59% were scaled-up. Scaling was described as resource intensive and pilot work was considered essential to successfully competing for funding by 63% of the sample (Pilot studies purposes are multifaceted and deemed essential to obtaining funding for larger-scale trials. Clarifying the form and function of preliminary, early-stage research may enhance the productive utilization of early-stage studies and reduced drops in efficacy when transitioning to larger scale studies.The online version contains supplementary material available at 10.1186/s40814-022-00991-8. To achieve widespread improvements in non-communicable diseases rates, interventions shown to be efficacious in smaller-scale studies need to be effective when “scaled-up” for widespread impact –5. SucceThough the terminology and purpose of preliminary early-stage studies, commonly referred to as pilot or feasibility studies is debated –17, theyHow behavioral scientists actually approach intervention development from early-stage studies to larger-scale trials remains unexplored in the literature. Understanding the experiences of senior researchers who conduct pilot studies and subsequent larger trials could help elucidate the purpose, motivations, and difficulties of piloting behavioral interventions and add depth and contextual significance to our understanding of the scaling processes. A clear understanding of the purpose and function of pilot studies can help the field adopt and integrate improved practices in pilot study execution . The purWe developed a semi-structured interview guide using a collaborative process. Questions were aimed at eliciting participants’ personal experiences in the design and scaling of behavioral interventions and were based on the research team’s experiences as interventionists as well as theories of program implementation . QuestioA preliminary interview was conducted to ascertain the length, pace, and the suitability of question sequence for the interviews. For the preliminary interview, one individual was selected from the research team who had also provided input in question development (DL). Based on feedback from the preliminary interview, questions were amended for additional clarity and a semi-structured format was chosen to facilitate a conversational interviewing style, allowing the interviewer to follow-up with additional questions as needed. The finalized formal interview guide consisted of 10 predetermined open-ended questions.Thirty-eight previously identified authors of published pilot/feasibility studies and subsequent larger-scale trials of behavioral interventions targeting childhood obesity were eligible for participation. Procedures used to identify all possible qualifying pilot studies and their subsequent well powered trials have been described elsewhere .All 38 authors were invited to participate in the study by email in November of 2019. The initial recruitment email introduced the study and the study’s purpose. Each email provided potential participants with citations for the specific pilot/feasibility and larger-scale studies that had led them to be included in the study. Emails included definitions for “pilot/feasibility study” and “behavioral intervention” to help orient participants. Participants were encouraged to respond to the email and select one of three listed time/dates for an interview lasting approximately 30 to 60 min in length. After the initial email, non-responding participants were contacted up to three more times to participate. After no response from the third contact, no more attempts to recruit participants were made.All procedures were approved by the first author’s institutional review board (registration number Pro00086876) prior to enrollment of the first participant and were consistent with the ethical standards outlined by the Helsinki Declaration of 1975, as revised in 2000 .Interviews took place through video conference software  for participants living outside of the USA and/or via phone for U.S. residents. All interviews were conducted by one of the authors (MB). During the interview, the interviewer guided the conversation through the predetermined questions attempting to not make any leading statements. Questions were followed up with probing questions for clarification and to explore new ideas as necessary. All interviews were audio recorded and notes were taken throughout the interview to capture initial thoughts and observations. Participants were read an IRB-approved script which informed them of the interview’s purpose and the use of the data. Participants provided verbal consent to have their interview recorded and used for the study.All transcription was done verbatim using Otter.ai  and verified by trained research assistants. Transcriptions were uploaded into NVivo 12 Plus  for coding and synthesis.Two trained research assistants  coded, analyzed, and synthesized the data using analytic induction , 27. BotModified analytic induction was appropriate for this study, given that we expected interventionists who are interviewed to explain successful/unsuccessful translation of pilot to large-scale intervention results by citing factors that can be traced to both well-documented mediating variables in implementation science in addition to novel, less documented experiences expressed by participants.This process was comprised of four key steps: data immersion, creating codes, arranging themes, and formulating and refine the explanatory hypothesis. The first step was immersion which allowed for a detailed examination of the data during which emergent themes began to surface. Once the data had been examined, the two independent coders read the transcripts in batches of 2–3 transcripts and compared coding line by line, discussing and resolving disagreements. Themes were identified a priori, and emergent themes identified during data emersion were incorporated. Themes were identified in the literature as prevalent features of scaled-up interventions . Coders Guided by Shenton’s provisions to ensure credibility, transferability, dependability, and confirmability in qualitative research , intentiOf the 38 individuals eligible to participate, a total of 24 interviews were completed (62%). The remaining eligible participants did not respond to the email invitations. Interviews varied in length ranging from 32 to 68 min (mean 47.45). The interviews were conducted between November 2019 and March 2020. Participants predominantly self-identified as White (92%), were employed by universities (92%) and currently or formally held tenured positions (87%) with an average of 20 years of research experience (range 11–35 years). The average age of participants was 50.5 years (SD 8.8) and 46% were male. Most participants resided in Australia (35%), the USA (38%), and the UK (19%).Through modified analytic induction 20 themes were generated containing 111 subthemes which were organized into four broad categories, or stages: (1) conceptualization of an intervention, (2) interpretation of results, (3) scaling, and (4) reflection , while simultaneously providing evidence that the intervention has potential impact on either primary or secondary  outcomes, referred to as a preliminary signal. The following elements were identified by interviewees as metrics that would provide valuable information for later interpretation when designing a pilot study, prior to scaling-up an intervention.In the initial design of the interventions, researchers sought multiple sources of information to inform intervention components. These included reviews of the literature and input from key stakeholders to inform specific intervention content (quote 1a). Emphasis was placed on generating new or novel ideas for testing, the integration of existing evidence-based practices, and targeting the mechanisms of behavior change (quote 1b).Pilot studies were described as an opportunity to determine whether study measures would result in sufficient amounts of quality data. For instance, investigators described wanting to gauge whether participants would tolerate particular measures, such as dual X-ray absorptiometry, return rates of measurement tools , complete self-report questionnaires, determine if measures were age appropriate, and/or refine the content/focus of certain measures . Study specific contexts drove this component of the pilot study, such as delivering mHealth interventions where measures would be completed without direct contact with research staff (quote 2b).A key indicator for moving forward with the scale-up of an intervention was positive results on trial-related parameters. Trial-related parameters were described as target population recruitment and whether they could be recruited in sufficient numbers, retention of participants over the duration of the intervention  and participant engagement in the intervention-related activities  to receive an adequate dose for an effect. Receptivity of being randomized to conditions was also considered (quote 3a). Other trial-related parameters involved process implementation measures such feasibility and fidelity that were embedded within pilot studies, along with measures of acceptability and satisfaction (quote 3b) from both participants, and where appropriate, delivery agents.n = 3; quote 4a) while others placed emphasis on aligning conceptual components between the pilot and the larger-scale trial. This included hypothesized mechanisms of behavior change  or intermediary behaviors related to outcomes of interest   because those components were within the control of the investigator (quote 4b).Making an intentional effort to design pilot studies a priori as if they were smaller versions of the anticipated larger, future trial was consistently mentioned. Several investigators were adamant that trial and intervention protocols be as close as possible to the future larger-scale, more well-powered trial  and participant engagement  were also considered crucial sources of information for decision making from pilot studies (quote 5b). Investigators also sought evidence of favorable participant reception including qualitative feedback (quote 5a).n = 5) though specific benchmarks were not provided. Several interviewees commented that interpreting preliminary statistical tests were prone to biased interpretation (quote 6d).Opinions varied on the importance and appropriate use of hypothesis testing and statistical significance when interpreting evidence of efficacy in pilot studies. When expressing their personal views, interviewees tended to view statistical testing as either inappropriate, or not a primary concern, relying instead on intuition in conjunction with the direction and magnitude of the effects along with process measures to make decisions about judging the value of an intervention and whether it should be scaled-up . When commenting on perceived external expectations from grant or manuscript reviewers about inferential statistics in interpreting pilot study outcomes, authors felt expectations were confusing or impractical (quotes 6c). Setting a priori thresholds for effect sizes was mentioned by several participants . Only nine interventionists provided potential solutions. Solutions to address inflated effects focused on designing pilot studies to more closely resemble the conditions under which the larger-scale trial would be conducted. For example, if the classroom teachers will be the delivery agents in the larger-scale trial, then the pilot should also have the classroom teachers deliver it .Once pilot testing was complete, interventions were often implemented on a larger scale. While some elements of the pilot interventions and trial parameters were maintained in the next phase of research, many were adapted. It was not uncommon for investigators to report changing the duration, typically from a shorter pilot  to longer timeframe in the larger-scale trial  or reducing the intensity of the intervention in the larger-scale trial from what was provided in the pilot. Notably, some investigators indicated using pilot studies to evaluate the logistics of deploying and collecting measures, though a challenge repeatedly mentioned when discussing larger-scale trials was the ability to collect measures with a substantially larger number of participants.n = 3), while others (n = 5) mentioned re-piloting interventions in hopes of achieving stronger effects on their specified outcomes (quote 8a). Complete lack of engagement, inability to recruit participants, and substantial changes to intervention content were noted as signs that re-piloting was not worthwhile .Investigators were asked to describe, either from their own experience or hypothetically, circumstances where re-piloting an intervention would be necessary based upon the results of a pilot study. Some investigators expressed rarely or never re-piloting studies , allowing for more flexibility during intervention implementation (quote 9b) as well as adapting the dissemination model for an increased number of participants or delivery agents (quote 9c).n = 18). Funding was described as necessary for conducting to a pilot study, with outcomes from pilot studies deemed critical to securing additional funds to conduct a larger-scale trial of the same or similar intervention (quote 10b). Thus, pilot studies were consistently viewed as a necessary prerequisite to obtaining funding for larger-scale trials. Some interviewees believed that publishing pilot studies increased their odds of successfully obtaining large-scale funding (n = 9). Conversely, an equal number believed that publishing pilot studies was a difficult and disincentivized process, because pilot studies were unlikely to produce significant effects or were considered lower quality studies . Other strategies for successfully obtaining funding included conducting only pilots perceived to have a greater likelihood of success at obtaining larger-scale funding (10c) and choosing primary outcomes in pilot studies likely to indicate positive change (change quote 10a).Funding played an essential role in the execution of both pilot studies and subsequent larger-scale studies (n = 17). Pilot study outcomes were mentioned as useful in the identification of areas for improvement when taking the intervention to scale. Notably, investigators mentioned changing intervention intensity, duration, and study design . These changes were driven by participant feedback in the pilot studies, funding priorities, methodological requirements of larger-scale trials, or the desire to increase efficacy of the intervention (quotes 11a–11c). Insufficient recruitment, retention, or participant satisfaction  were indicative of intervention adaptations, in addition to addressing practical limitations of space , personnel , intervention protocol, and material  when delivering the intervention to a larger audience.Intervention components and study designs often differed between the pilot and larger-scale trials (n = 18). Common challenges encountered included decreased stakeholder engagement, changes in delivery personnel, and logistical difficulties collecting outcome measures (quotes 12a–12c) in a substantially larger sample of participants. According to investigators, this resulted in less meaningful results, logistical difficulties that impeded data collection, less fidelity in intervention delivery due to a change in intervention delivery agents, ultimately rendering the impact of the scaled intervention smaller than originally anticipated.Most investigators encountered challenges when scaling-up their pilot studies  investigators highlighted the role of collaborating with colleagues in developing interventions, though they differed on the perceived benefits. Some investigators found additional contributions detracted from intervention’s primary purpose (quote 14f) while others felt they had capitalized on collective brainstorming to improve the interventions approach (quote 14a).The experience of scaling pilot interventions provided investigators lessons to improve future interventions (quote 13a). Several investigators mentioned including more process implementation measures in subsequent studies (quote 13b) while others prefer more pre-development or iterative pilot work (quote 13c). Designing interventions to be simple, with fewer moving parts and complexities, were mentioned as key takeaways . Several .To better understand the process of scaling-up early-stage trials, we conducted interviews with 24 investigators who had a published pilot study and subsequent trial on topics related to childhood obesity. Investigators reported that pilot studies were used to preemptively address challenges such as engaging key stakeholders, establishing trial parameters and measurement logistics, nonetheless, these same elements were identified as reasons scaled-up trials were less successful. These findings suggest that, while pilot studies may result in favorable trial-related parameters and evidence of preliminary efficacy, this evidence may not accurately reflect the conditions encountered during the implementation and evaluation of the subsequent scaled-up intervention.The current study builds on previous findings indicating some components of early-stage studies have a high potential to produce exaggerated or inflated effects which diminish during subsequent, scaled-up iterations , 14. ComFindings from this study suggest that potential conflict may be present between the need to obtain funding and the need to assess key uncertainties when piloting interventions. Investigators expressed that obtaining funding for a larger intervention was a key definition of success. To attain funding, investigators may introduce artifacts within their pilot studies that lead to inflated effects and it is these inflated effects that may be viewed more favorably from a funding perspective. Minimizing potentially inflated effects obtained from pilot studies was not mentioned as a key consideration when executing early-stage work by the investigators even though they recognized inflated effects often occur. Early-stage studies that minimize artifacts associated with inflated effects, such as ensuring the sample in the pilot study matches the sample in the larger-scale trial, have a smaller reduction in their effectiveness when scaled and a greater likelihood of demonstrating statistically significant effects in the larger-scale trial , 14. WitThe conflict between piloting for key uncertainties and the need to obtain funding may also be exasperated by the common two-step process for intervention development—initial piloting with small sample sizes followed by a larger-scale trial with a substantially larger number of participants. This may inadvertently force investigators to choose between fostering evidence of effects over addressing key uncertainties related to feasibility and acceptability. In other words, investigators may only have a single opportunity to establish the “value” of the intervention being piloted. Funding structures may add to this conflict by offering few mechanisms that support a multi-phased developmental approach for early-stage studies which offer the opportunity to pilot and refine an intervention in progressively larger sample sizes, as opposed to conducting a single small study and using this as the only basis for which to judge the readiness of an intervention to be tested on a much larger-scale , 30. ThuExisting models for scaling behavioral research describe an iterative sequencing of formative work which may help address this concern, though they vary in the number and purpose of each specified step or phase of the process . SimilarInvestigators who perceived the results of their pilot studies to be lacking, often re-piloted or abandoned the next stage of their intervention development. However, those who felt their pilot results were promising often adapted elements of their interventions in their larger trial, sometimes involving a major reworking of the intervention. These reworkings included changing the intervention protocol and/or utilizing different locations, settings, or delivery agents. Investigators often perceived the process of interpreting pilot-produced evidence as nebulous, involving both quantitative markers of preliminary efficacy and process measures such as acceptability collected via mixed-methodology. This perspective of interpreting feasibility markers alongside preliminary signals of efficacy is reflective of changes in the field of behavioral research where investigators suggest testing for statistical effects in pilot studies is no longer recommended , 32.There a several imitations in the current study. First, we purposively sampled only individuals who had conducted and published pilot research of behavioral interventions and subsequent scaled-up trials of the same or similar intervention. While early-stage research is widely used to develop behavioral interventions, there are a number of larger-scale behavioral trials that do not explicitly reference early-stage research that informed the intervention. Additionally, there are a large number of published pilots that do not inform later scaled-up trials. The viewpoints of authors who did not explicitly cite pilot trials and authors who have not scaled-up pilot trials are therefore not represented. Second, response bias may be present among the interviewees. Investigators who felt more comfortable discussing their early-stage work, those who had more positive experiences executing pilot studies, or who spent more time engaging in reflection on preliminary work, may have been more likely to participate. Though the 24 participants interviewed represent 63% of the entire eligible population of PIs, the potentially unique perspectives of non-respondent investigators are not represented. Additionally, it is possible that the inclusion of authors as participants may have skewed findings . However, it should be noted that the conceptual/theoretical approach employed for this study allowed for the inclusion of participant-authors  and intentional, pre-planned methodological steps were employed to ensure the trustworthiness of the data. Lastly, it was not practical to conduct interviews face-to-face as the participants were located across four continents. Thus, contextual information like body language were not collected.In conclusion, our study suggests that careful planning during the early-stage testing of an intervention can lead to pilot studies that provide important information about whether an intervention should be scaled-up. This could be achieved by supporting behavioral interventionists in designing pilot studies that closely mimic the condition encountered at scale. Additionally, guided interpretation of pilot-produced information could assist interventionists in making prudent, informed decisions regarding the necessity and appropriateness of re-piloting their interventions prior to scaling such as transparency in the rationale and a priori progression criteria in pilot studies. High-quality pilot studies have the potential to inform effective, impactful interventions to address important public health problems. However, the design, execution and interpretation of high-quality pilot studies should not be assumed to be self-evident or intuitive but rather the result of careful planning, execution, and thoughtful interpretation.Additional file 1. Semi-Structured Interview Guide.Additional file 2. A Priori Themes and Emergent Themes.


Brain organoids are proving to be physiologically relevant models for studying human brain development in terms of temporal transcriptional signature recapitulation, dynamic cytoarchitectural development, and functional electrophysiological maturation. Several studies have employed brain organoid technologies to elucidate human-specific processes of brain development, gene expression, and cellular maturation by comparing human-derived brain organoids to those of non-human primates (NHPs). Brain organoids have been established from a variety of NHP pluripotent stem cell (PSC) lines and many protocols are now available for generating brain organoids capable of reproducibly representing specific brain region identities. Innumerous combinations of brain region specific organoids derived from different human and NHP PSCs, with CRISPR-Cas9 gene editing techniques and strategies to promote advanced stages of maturation, will successfully establish complex brain model systems for the accurate representation and elucidation of human brain development. Identified human-specific processes of brain development are likely vulnerable to dysregulation and could result in the identification of therapeutic targets or disease prevention strategies. Here, we discuss the potential of brain organoids to successfully model human-specific processes of brain development and explore current strategies for pinpointing these differences. Homo sapiens)-specific molecular and cellular processes of brain development that are not shared even with our closest Hominidae relatives like chimpanzees (Pan troglodytes). While mammalian neurodevelopment mutually exhibits apicobasal organization of radial glia cells (RGs), timed cellular specification, and maturation of heterogeneous cell populations resulting in functional neural circuits  (Macaca)  . Neotenymulatta) . Pre- anmulatta) . The timmulatta) . Synaptomulatta) . Studiesmulatta) . Humans mulatta) . Additio(Macaca) . Altoget(Macaca) ; Table 1Uniquely human features of neocortical development and maturation are not only intriguing for their implications in human-specific cognitive abilities, but they are also vulnerable to dysregulation which could cause or contribute to distinctly human brain disorder pathophysiology. The human cerebral cortex is essential for both cognition and emotional processing and dysregulation of these processes of the cortex are associated with a wide range of brain disorders including schizophrenia (SZ), autism spectrum disorder (ASD), Parkinson’s disease (PD), and Alzheimer’s disease (AD) . Much rePan paniscus) show large sets of differentially expressed genes between human and NHP iPSCs. Perhaps the most compelling differentially expressed genes are those related to increased long interspersed element-1 (LINE-1) mobility in chimpanzees and bonobos, which could have implications on the rates of genetic divergence among species, and alternative mechanisms of pluripotency maintenance in chimpanzees , PSC-derived structures that display complex radial organization of expanding neuroepithelium following embedding in an extracellular matrix like Matrigel and can recapitulate some subsequent processes of neurodevelopment including neurogenesis, gliogenesis, synaptogenesis, heterogenous cytoarchitecture, cell and axon migration, myelination of axons, and spontaneously-active neuronal networks . It is lIn this review we discuss the application of human and NHP-derived brain organoids, and the implementation of genome editing technologies in these model systems, to investigate human brain evolution and pinpoint human-specific processes of neurodevelopment and maturation vulnerable to dysregulation . We furtin vitro access to elaborate, physiological relevant model systems of human-specific neurodevelopmental. Brain organoids have the advantage of retaining the species-specific genetic background of the PSCs from which they are derived while supporting the orchestration of dynamic processes of neurodevelopment as transcriptomic changes occur in a 3D space. PSCs have been established from a variety of NHP species including chimpanzee, bonobo, gorilla (Gorilla gorilla), orangutan (Pongo abelii), and rhesus monkey  and 84.6 billion non-neuronal (NeuN-negative) cells . Althougin vivo brain development; however, although some cell types were reproducible generated, others appeared inconsistently which was partially attributed to bioreactor-based batch effects  to analyze human, chimpanzee, and macaque cerebral organoids at several timepoints from the PSC stage to 4 months of organoid development, researchers found that human neuronal development occurs at a protracted rate compared to chimpanzees and macaques and differentially expressed genes related to proliferation, neuronal migration, and neurite formation are enriched in human radial glial cells (RGs), intermediate progenitors (IPs), and neurons. Furthermore, accessible chromatin profiling demonstrated that differential chromatin accessibility corresponded to human-specific gene expression. Four of the 23 differentially expressed genes were specific to G2M phase progenitors of the telencephalon and included ARHGAP11B, FAM72B, FAM72C, and FAM72D . An addiscRNA-seq is powerful in characterizing known and unknown cell types, comparing tissue compositions, and understanding evolutionary differences at the cellular level; nevertheless, it requires the loss of spatial information which is necessary for elucidating the effects of transcriptomic differences on function and complex, physical cellular interactions as cells must be dissociated from their location in tissues. Through scRNA-sequencing analysis of four different lobes of the human neocortex, researchers found asynchronous cell development and a priority for regional maturation in the different areas emphasizing the importance of spatial resolution in conjugation with transcriptomic information . Furthercis and trans acting regulation on gene expression. Human and chimpanzee iPSCs were fused so that the tetraploid genomic information contributed by each species shared the same nuclear space. Neural organoids were then established from the hybrid iPSCs and allele specific expression in hybrid organoids was compared to chimpanzee and human organoids to reveal cis regulatory divergences. Human somatostatin receptor 2 gene (SSTR2), responsible for neuronal calcium signaling regulation and associated with neuropsychiatric disorders, was found to be upregulated in these studies  and early Homo sapiens brains found that Neanderthals, when averaged, had smaller cerebellar hemispheres than Homo sapiens which could have influences on cognition and social functions -like structures; however, cortical organoid protocols have further demonstrated more distinct oSVZ-like regions and upper and deep cortical layers containing neurons and astrocytes reminiscent of third trimester human neocortical development  populations when compared to primary neocortex tissue and the continued loss of GPCs as organoids develop . Preservin vivo . Observiin vivo .via the expression of the mature oligodendrocyte marker, myelin basic protein (MBP), and the pinpointing of axons that are insulated by 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) positive oligodendrocyte myelin sheets  and from unedited isogenic control hiPSCs (NOVA1Hu/Hu). NOVA1Ar/Ar organoids were smaller in diameter than NOVA1Hu/Hu organoids during the proliferation and maturation stages and demonstrated increased surface rugosity. Additionally, NOVA1Ar/Ar organoids had less VZ- and SVZ-like areas, increased apoptotic cells, and slower proliferation compared to NOVA1Hu/Hu organoids. The researchers then performed RNA sequencing and found 277 genes differentially expressed between NOVA1Hu/Hu organoids and NOVA1Hu/Hu organoids with the top three differentially expressed genes being FEZF1, PAX6, and LHX5. Single-nucleus RNA-seq then revealed differences in cell-type proportions at one and 2 months of development and variations in gene splicing. Finally, NOVA1Ar/Ar organoids had lower levels of pre- and postsynaptic proteins with less colocalized synaptic puncta likely resulting in reduced synchrony and increased variability based on observed firing rate and coefficient variation when evaluated by MEA  as a gene with differences between human and archaic hominin genomes that could play a role in human-specific neurodevelopment. Using CRISPR-Cas9, they replaced the human allele of NOVA1 in hiPSCs with an ancestral allele associated with Neanderthals and Denisovans. They then generated cortical organoids from edited hiPSCs with the archaic variant (NOVA1d by MEA .Denisova hominins) were thought to be our closest evolutionary relatives. The discovery of Homo longi, suspected to be an even closer extinct relative of humans than Neanderthals, could open the possibility for a new understanding of divergence in human brain evolution  levels have been observed in patients with schizophrenia (SZ) . Additiovia pruning , and gorillas, demonstrate diffuse amyloid plaques, neuritic amyloid plaques, and vascular amyloid, in addition to hyperphosphorylated tau expression in neurons, they most often do not present with tau positive neurofibrillary tangles (NFTs) or neuropil threads and very few NHPs have been reported with both amyloid beta pathology in combination with NFTs  and found a reduction in both TBR1+ and CTIP2+ cortical neurons, and a subsequent reduction in the size of the CP-like structure of human organoids following infection, but they did not observe these effects in chimpanzee organoids after ZIKVBR infection suggesting human-specific neurotropism adaptations of ZIKVBR (In addition to the genetic and molecular basis of brain disease, the influence of external factors on brain development might also be compared in human- and NHP-derived brain organoids. For example, researchers infected human and chimpanzee cerebral organoids with the Brazilian Zika virus (ZIKVf ZIKVBR . These sf ZIKVBR . Finallyf ZIKVBR . Human aSF wrote the manuscript. DK designed and generated the figures. MM conceived and supervised the work. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.