Ischemic stroke is one of the leading global causes of neurological morbidity and decease. Its etiology depends on multiple events such as cardiac embolism, brain capillaries occlusion and atherosclerosis, which ultimately culminate in blood flow interruption, incurring hypoxia and nutrient deprivation. Thyroid hormones (THs) are pleiotropic modulators of several metabolic pathways, and critically influence different aspects of tissues development. The brain is a key TH target tissue and both hypo- and hyperthyroidism, during embryonic and adult life, are associated with deranged neuronal formation and cognitive functions. Accordingly, increasing pieces of evidence are drawing attention on the consistent relationship between the THs status and the acute cerebral and cardiac diseases. However, the concrete contribution of THs systemic or local alteration to the pathology outcome still needs to be fully addressed. In this review, we aim to summarize the multiple influences that THs exert on the brain and heart patho-physiology, to deepen the reasons for the harmful effects of hypo- and hyperthyroidism on these organs and to provide insights on the intricate relationship between the THs variations and the pathological alterations that take place after the ischemic injury. Stroke is one of the most frequent neurological disorders leading to chronic disability and is recognized as a major cause of morbidity and mortality worldwide. 2+ flow into the cells, thus inducing their death for excitotoxicity [Its onset depends on several risk factors including age, race, and personal habits such as cigarette smoking and alcohol consumption, socioeconomic status and comorbidities like diabetes mellitus, hyperlipidemia and hypertension ,2,3,4. Dtoxicity ,14,15. Atoxicity ,17. Thistoxicity ,19.The complex relationship between the thyroid hormones serum concentration and the insurgence, severity and recovery after stroke remains controversial because thyroid hormones exert both neurotoxic and neuroprotective effects ,21. ThyrThe deregulation of THs signaling has a strong impact during both development and adult life, representing one of the leading causes of several pathologies, such as neurological cretinism, spastic dysplasia, Alzheimer’s disease, metabolic syndromes, diabetes, obesity, skeletal muscle atrophy and multiple cancer types ,39,40,41Although, over the years, several literature data have highlighted an effect of the systemic THs alterations on the risk of stroke, neuronal damage and post-injury outcome, the results remain debatable. In the attempt to shed light on the involvement of THs in modulating the different aspects of ischemic diseases and the functional recovery of patients affected by this pathology, this narrative review aims to describe the role exerted by THs in the diverse tissues involved, with particular regard to the effects of hypo- and hyperthyroidism on stroke progression.Thyroid hormones govern neurological development and physiological brain maturation. Hence, the strict control of THs concentrations is essential in both physiological and pathological conditions, where the regulation of the deiodinases profile expression represents the principal molecular mechanism adopted by cells to orchestrate THs levels.Since the first weeks of embryonic life, a tight spatial and temporal modulation of deiodinase expression occurs, in order to limit the THs concentration in the maternal blood . In partIn the adult, the regulation of THs levels in the central nervous system (CNS) is ensured by the expression of the THs transporters on the choroid plexus of the blood–brain barrier, which exhibits a greater affinity for T4 with respect to T3 . Once crIn accordance with the key role played by the peripheral activation of T4 by the deiodinases, it has been shown that mice lacking D1 and D2 have reduced T3 levels in the brain, as well as reduced expression of several TH-target genes ,50 and aThe T3-mediated positive effects on brain development result from its dual action on astrocytes and neurons and include the stimulation of neuronal differentiation, axonal maturation, myelination and promotion of synaptic plasticity ,61. In vIf physiological THs levels are essential to guarantee proper neuronal function in the CNS, low THs levels have harmful effects and represent negative prognostic factors for stroke. Overt hypothyroidism, a clinic syndrome characterized by systemic THs deficiency, has long been associated with neuronal dysfunctions such as depression, dysphoria, attention deficit, cognitive decline, learning and psychomotor performance degeneration, as well as atherosclerosis and increased risk of cerebrovascular accidents ,72. MoreAs in the case of hypothyroidism, some epidemiological studies have shown a worsening effect of hyperthyroidism (lower levels of TSH and higher levels of free T4) on stroke prognosis and severity . ProbablAlthough both hypo- and hyperthyroidism are associated with an increased risk of stroke, the hypothyroidism is the condition that mostly affects the probability of ischemic events .Even if the condition of subclinical hypothyroidism  increases the incidence of transient ischemic attacks, recent reports suggest that it has a beneficial impact on preventing post-stroke severe outcome and reducing the mortality rate ,90. MultBesides this endogenous ischemic preconditioning, other processes may cooperate as protective mechanisms against the severe consequences of the ischemic attacks, decreasing the metabolic demand of neurons and the oxidative stress ,91,92. ATo date, clinical studies on alterations of THs levels and stroke have focused mainly on the correlation between the low T3 syndrome and a worsened clinical outcome. In acute stroke patients, the incidence of low T3 levels varied from 15.1%  to 37% . These sWhile for ischemic stroke hypothyroidism is associated with a poor clinical outcome , in caseData on T4 and stroke are conflicting. A meta-analysis by Jiang et al.  showed tData on the prevalence of low TSH (5.4–32.6%) was reported in a few studies ,106. LowTHs exert significant cardiovascular effects influencing cardiomyocytes maturation, function and metabolism, via a variety of molecular mechanisms. In the perinatal period, the systemic levels of T3 undergo a dramatic increase that elicits the Myosin Heavy Chain (MHC) switching, the hypertrophy of myofibers, the mitochondrial respiration and the induction of the calcium-ATPase pump of the sarcoplasmic reticulum (SERCA) ,111. As Multiple studies have highlighted the correlation between the circulating T3 levels and the recovery ability of the ventricular function, indicating T3 as a significant predictor of myocardial ischemia outcome ,134,135.In light of the control exerted by T3 in myocardial contractility and metabolism, and of the numerous evidence that demonstrated an important link between THs concentration and the heart stroke gravity, incidence, and recovery post-injury, THs can be considered important predictive biomarkers and major players in determining the acute coronary disease outcome.Central and local thyroid hormones dysfunctions are frequent conditions reported in case of ischemic events. THs are involved in several physiological processes and, in particular, are important players in driving the development and the maturation of multiple tissues through genomic and non-genomic actions ,115,123.Consequently, it is not surprising that disruptions in THs availability and function may result in significant variations in injury outcome. However, the impact of peripheral and systemic hypo- or hyperthyroidism can strongly differ depending on the type of pathology and, even within the same disease, distinct effects can occur in the context of specific tissues according to the function they exert. Although several studies have indicated a relevant association between THs and outcome after stroke, the potential contribution of thyroid dysregulation to the pathogenesis of cardiac and brain failure remains poorly understood.Significant knowledge gaps exist regarding the precise TH-mediated molecular and biochemical mechanisms that control brain and cardiac physiopathology and the optimal strategies for the management of thyroid dysfunction in patients with and without pre-existing cardiovascular disease. Specifically, although overt hypothyroidism and hyperthyroidism need to be treated in patients with established heart failure, it remains unclear whether subclinical thyroid dysfunction requires similar attention. The lack of epidemiological studies of cohort patients with subclinical thyroid dysfunction limits our understanding of the mechanisms by which subclinical hypothyroidism and the low T3 syndrome increase the risk of mortality in patients with established heart failure. Even in euthyroid patients, the impact of altered myocardial TH actions on heart failure remains unclear. Indeed, the specific TH-mediated myocardial effects that significantly affect the development of human heart failure are still poorly understood and their discovery may shed light on new targets for therapy with T4, T3 or thyroid hormone analogs. In the humans, the preservation of THs homeostasis controlled by the HPT system is combined with adaptive mechanisms in non-thyroidal organs that produce their own free T3 levels. Such intra-tissue TH production is also more advantageous than central regulation, because it provides peripheral organs with a certain level of regulatory autonomy to produce more or less THs to meet their different energetic, functional and metabolic needs. For this reason, the accurate quantification of TH action at tissue-level also remains a challenge. Although serum TH levels can be easily measured, there are currently no methods for their routine quantification within tissues, which may be more tightly correlated to myocardial function. Indeed, it is also not clear how TH transporters and receptors are regulated in animal models and in patients with heart failure, and there are no data to fully explain the THs effects on cardiomyocyte contractility and electrophysiology.On the basis of this multifaceted lack of awareness, areas of particular interest for further research include exploring the fundamental biology linking thyroid dysfunction to the development of cardiovascular disease and identifying novel biomarkers of THs action in the cardiac tissue.Moreover, large epidemiological studies are needed to define the subgroups of patients with thyroid dysfunction who are susceptible to cardiovascular disease and who can be treated for specific prevention. In addition, clinical trials should focus on evaluating the ability of TH or thyromimetics to improve cardiovascular performance and outcomes. Clinical and preclinical studies will clarify the role of TH in modulating endothelial function and define the cellular signaling processes by which TH regulates electric conduction, contractility and peripheral vascular function. Future research will elucidate the mechanisms underlying atrial and ventricular arrhythmias or myocyte remodeling and neuronal dysfunctions orchestrated by TH that can be exploited to improve the functional recovery in stroke patients.In conclusion, according to the available evidence and considering the vital role that THs wield in the pathophysiology of the organs affected by acute ischemia, THs regulation represents a viable therapeutic strategy for stroke management and functional recovery. Although with several discrepancies, the wide array of the mentioned studies seems to demonstrate that both conditions of hyper- and hypothyroidism, as well as the low T3 syndrome, are all associated with a poor outcome and act as adverse prognostic factors that negatively affect the regeneration process after acute stroke.However, albeit the positive effects of THs on tissue recovery are well described, still a few studies have addressed how the THs status impacts the rehabilitation of stroke patients. These studies are of crucial need and might add critical insight, considering the known positive effects exerted by THs on cardiac muscle physiology, brain regeneration and angiogenesis.


Axenfeld-Rieger syndrome (ARS) is an autosomal dominant disorder that is primarily due to disruption of the development of neural crest cells. The onset of associated symptoms in both eyes accompanied by extraocular developmental defects is referred to as ARS. Cardiac defects associated with ARS have been reported, but the extent of the cardiac defects has yet to be defined. We report a case of a 17-year-old girl with ARS with typical facial malformations and severe mitral and tricuspid valve insufficiency. The patient was diagnosed with secondary glaucoma detected on ophthalmologic examination. Echocardiography showed severe mitral and tricuspid valve insufficiency. This case provides further evidence of the association of ARS with cardiac malformations and extends the reported range of cardiac malformations in patients with ARS. Axenfeld-Rieger anomaly (ARA) is an ocular condition characterized by extensive defects of the anterior chamber of the eye, mainly affecting the corneal structures. Anterior chamber defects include Axenfeld anomaly, characterized by prominent and anteriorly displaced Schwalbe lines and iris-corneal adhesions, and Rieger anomaly, which manifests as iris hypoplasia and pupil malformation . These oA 17-year-old girl was admitted to the hospital 1 year ago for shortness of breath after activity, dyspnea, weakness, and generalized swelling with no apparent reason. The patient then visited us for investigation of bilateral secondary glaucoma. The patient had poor vision in her left eye and could only see the optometrist's hand moving at 30 cm from her face. Ocular examination revealed visual acuity of 0.5 in the right eye, intraocular pressure of 28.2 mmHg in the right eye and 39.8 mmHg in the left eye; the left eye had 15 degrees of exotropia, a corneal diameter of 13 mm, slight edema, partial anterior adhesion of the atrial angle, iris atrophy, and localized hole formation . She hadThe patient was diagnosed with ARS and secondary glaucoma in both eyes, and anti-glaucoma surgery was recommended. Considering the patient's young age, advanced glaucoma, and the risk of non-compliance during awake surgery, anti-glaucoma surgery under general anesthesia was proposed to reduce the intraoperative stimulation of the optic nerve. Preoperative echocardiography showed a left ventricular ejection fraction of 58%, mitral valve thickening and severe mitral valve closure incompetence , total hThere are previous reports of ARS in association with congenital cardiac anomalies such as mitral, aortic, and pulmonary valve lesions, atrial septal defects, ventricular septal defects, and arterial trunk malformations –14. GripPITX2 and FOXC1, respectively  and cord defects   , 34, 35.PITX2 and FOXC1 genes.The present patient had the distinct ocular phenotype, dental anomalies, and mitral and tricuspid valve manifestations of ARS. The findings in this patient expand the range of cardiac malformations reported in ARS patients. We believe that echocardiography should be performed in patients with characteristic clinical manifestations of ARS or with alterations in the The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.JF and YW wrote the manuscript. SC collected the patient information and data. ZL, LL, and QM participated in the formulation and management of the patient's perioperative treatment plan. CZ designed and reviewed the manuscript. All authors were involved in the revision of the manuscript 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.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.


The pharmaceutical research sector has been facing the challenge of neurotherapeutics development and its inherited high-risk and high-failure-rate nature for decades. This hurdle is partly attributable to the presence of brain barriers, considered both as obstacles and opportunities for the entry of drug substances. The blood–cerebrospinal fluid (CSF) barrier (BCSFB), an under-studied brain barrier site compared to the blood–brain barrier (BBB), can be considered a potential therapeutic target to improve the delivery of CNS therapeutics and provide brain protection measures. Therefore, leveraging robust and authentic in vitro models of the BCSFB can diminish the time and effort spent on unproductive or redundant development activities by a preliminary assessment of the desired physiochemical behavior of an agent toward this barrier. To this end, the current review summarizes the efforts and progresses made to this research area with a notable focus on the attribution of these models and applied techniques to the pharmaceutical sector and the development of neuropharmacological therapeutics and diagnostics. A survey of available in vitro models, with their advantages and limitations and cell lines in hand will be provided, followed by highlighting the potential applications of such models in the (neuro)therapeutics discovery and development pipelines. The research and development platforms of therapeutics targeted to the brain or aimed to be excluded from the central nervous system (CNS) depends mainly on their accomplishment in assessing the reciprocal behavior of brain barriers and the pharmacologically active agent. This effort is made to conquer the economic and technical burden on the pharmaceutical industry attributable to high risk and poor approval rates of neurotherapeutics. During the long journey of drug development, based on the nature of the product under study and in the cases of pregnant women, neonates, and any conditions with the possibility that drugs might have short- or long-term damaging effects, substantial data are required to prove that a substance is excluded effectively from the brain. On the other hand, when the aim is efficient drug delivery to the CNS, the development of neurotherapeutics faces a number of major challenges, with difficulty in delivering them to their desired site of action in the brain being on top. The presence of highly selective barriers (the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier (BCSFB)) and the complexity of the highly regulated brain environment are among the factors responsible for the elevated attrition percentage during the development of these CNS-targeting medicines.The BBB and BCSFB are of utmost importance to pharmaceutical drug discovery, as both barriers provide obstacles to the penetration and delivery of therapeutics and diagnostics designed for the management of CNS complications and disorders. Presently, leading research has been concentrated on the brain vascular endothelium as a medicinal target to improve the delivery of brain therapeutics. However, the more poorly noticed BCSFB is also an essential gateway to the CNS, since it is broadly accepted that most substances, even macromolecules, that successfully reach the CSF, can find their way further to the brain parenchyma. Taken together with its considerable surface area, which is estimated to be ~25 to 50% the size of the inner capillary surface area of the brain , the BCSSpecies relevant in vitro model systems of the BCSFB play an essential role in enabling the understanding of this barrier’s properties and developing potential interventional techniques and therapeutics. In the pharmaceutical, pharmacological, and toxicological fields, these models harbor the potential to be applied for investigational drugs/compounds screenings, permeability and transport assays, (neuro)toxicological evaluations, and mechanistic molecular pharmacological studies and related assays at the pre-clinical level. Due to many economic concerns, ethical issues, and the necessity to comply with the three R rules  and bypassing animal testing as far as possible, there has been a great effort in past decades toward establishing robust and convenient models to study basic characteristics of the barriers and also to facilitate the research and pre-clinical investigations of neurotherapeutics in the laboratory under controlled conditions. Such models should authentically mimic the in vivo microenvironment phenotype of the BCSFB and demonstrate representative properties of functional elements of tight junctions (TJs) leading to high transepithelial electrical resistance (TEER), restricted paracellular permeability, low non-specific pinocytic activity, and the expression of receptors and transporters.With regard to the brief introduction, and despite the challenges and drawbacks inherited by these in vitro model systems, it is noticeable that the area has gained significant attention over the previous decades. These in vitro platforms offer a less resource-intensive alternative approach compared to animal models, in addition to an ease of manipulation, which gives rise to their exploitation during high-throughput end-point drug screenings for the development of drug candidates, and also for providing insights into the mechanistic principles governing CNS drug delivery. Furthermore, such models can be helpful to enlighten the principal translocation routes of small-molecule drugs and constantly increasing number of biopharmaceuticals. To this end, the present review sets out to summarize the efforts and progresses made to this research area with a notable focus on the attribution of these models and techniques to the pharmaceutical sector and the development of neuropharmacological therapeutic and diagnostic agents. It is hoped that researchers new to the field find this paper to be a straightforward guide to figure out the suitable in vitro models and optimal techniques for their particular interest.A more precise understanding of the BCSFBs physiological and anatomical attributes will pave the way toward the rational development of CNS-targeting small and large molecule therapies, aiming to treat a wide range of neurological conditions. The structure and physio-anatomical features of the BCSFB have been the subject of numerous excellent and comprehensive reviews ,5,6,7,8,The choroid plexus, as a villous connective and highly vascularized structure, is the major component of the BCSFB system that connects the two principal physiological circulatory systems, namely the peripheral blood circulation and the CSF bulk flow. The choroid plexus convolute consists of two tissue layers, an outer secretory roughly cuboidal epithelium and an inner underlying stromal core comprising an immense network of fenestrated leaky blood vessels with a rich extracellular matrix. Cellular and sub-cellular foundations of choroid plexus barrier properties are non-neuronal epithelial cells  and their adjacent TJs . From anThe choroid plexus contributes to the production and secretion of CSF  and cerebral homeostasis by regulation of the blood–CSF exchange taking advantage of numerous transport systems allocated in a polarized configuration among the apical or brush border  and the basolateral  membranes of the choroidal epithelial cells . The choAs a formidable structural and metabolic barrier, the BCSFB, similar to the BBB, prevents toxic substances in the circulation from reaching the brain. The structural elements of this barrier are dense bands of impermeable TJs near the apical (CSF-facing) surface of adjacent cells of the choroid plexus epithelium. TJs are elaborate networks of specialized membrane microdomains, which tighten adjacent epithelial cells together and consequently limit the free diffusion of hydrophilic and polar molecules. Morphologically and structurally, TJ complexes comprise transmembrane  proteins as occludin, the claudins, or junctional adhesion molecules (JAMs), and the associated cytoplasmic proteins of the membrane-associated guanylate kinase-like homologues (MAGUKs) family, termed zonula occludens proteins ZO-1, ZO-2, and ZO-3. Non-MAGUK peripheral junction proteins, such as cingulin, AF-6, symplekin, 7H6, and 4.1R have also been shown to be associated with TJs . ClaudinA large number of transport proteins and receptors are expressed in choroid plexus epithelial cells for mediating and controlling multiple functions of this tissue and acting to transduce key humoral signals between the blood and the CNS. For instance, the choroid plexus serves to transport an assortment of amino acids bidirectionally , hormones , proteins, and growth factors  and pharmacologically active agents  . The choChoroid plexus epithelial cells possess multiple structural and functional attributes of a transporting epithelium and express a broad spectrum of uni- and bi-directional transporters and ion channels, distributed in a polarized fashion at the apical and basolateral surfaces. This polarized distribution between luminal/apical and abluminal/basolateral membranes enables a heavy yet regulated and directional movement of molecules/compounds across the BCSFB. Multiple transporter proteins, classified as solute carrier (SLC) transporters, are present and functional at the epithelial cells of the BCSFB for glucose, amino acids , peptides, organic anions, monocarboxylic acids, and a myriad of solutes and ions ,30,31,32One vital physiological commitment of the choroid plexus is to furnish effectual clearance of xenobiotics and potentially deleterious metabolic end products out of the CSF back to the circulatory system, and the choroid plexus is consequently nominated as kidney of the brain . This aiThe principal choroidal drug transporters accepting a broad range of substrates belong to two superfamilies of transporters, namely ATP-binding cassette (ABC) carrier transporters and the SLC family . The supIn addition to the aforementioned efflux pumps expressed at luminal and/or basolateral membranes, diverse drug-metabolizing enzymes, responsible for inactivating endogenous and exogenous molecules, are expressed in the choroid plexus, including glutathione S-transferases (GSTs), UDP-glucuronosyltransferases (UGTs), flavin-containing monooxygenases (FMOs), epoxide hydrolases (EHs), and phase I metabolizing enzymes, namely multiple isoforms of the cytochrome P450 family ,49,50,51Given the principal role of the BCSFB and restrictions of animal and human investigations, there is a continually increasing demand for new and advanced model systems and in vitro techniques. The following sections describe and summarize the current knowledge related to in vitro models of the BCSFB and focus on their relevance to (neuro)therapeutics research and development. These in vitro model systems are arranged in a diverse range of technical, biological, and physiological complexities . It shouBasic 2D models are static systems of choroid plexus epithelial cell monocultures, predominantly based on semipermeable microporous membranes. The static culture systems are defined as those in which cultured cells are kept in the absence of physiological fluid dynamics. In this context of negligible fluid shear stress, the exchange of nutrients and waste takes place using diffusion . These iThe facile processing of filters makes this system an appropriate candidate for high-throughput screening (HTS) studies of drug permeability, binding affinity measurements, and transport kinetics. In general, this model type is well suited to explore in detail transport mechanisms  and cellular transmigration processes, and to investigate the permeability of potential drug candidates as well as the transepithelial resistance. Other applications include the analysis of the metabolism of molecules en passage and the determination of the ensuing metabolites . Based oIn spite of the advantages pertained to the ease of manipulations of static bicameral model systems, there are disadvantages mainly due to the two-dimensional nature, the absence of fluid-induced shear stress, and the lack of fundamental features, as 3D structure, cell–cell or cell–matrix interactions, comparable TEER, fluid-induced shear stress, vasculature, and the presence of other accompanying cells, necessary to establish a flawless and authentic in vitro model . The “edSpecial attention should be taken for the proper selection of the type of culture inserts for the experiments and the coating of filters prior to cells seeding.Selection of the culture insert: the choice of the cell culture insert depends on the application and desired outcomes of the study. A broad range of inserts with varying permeable membrane materials (polycarbonate (PC), polyester polyethylene terephthalate (PET), and collagen-coated polytetrafluoroethylene (PTFE)) and different porosities (pore size and pore density) are available commercially from several manufacturers. The selection of the cell culture insert is a crucial step in the design of preliminary experiments. It is recommended that the adsorption and retainment profile of investigational compounds to different types of filter materials are clarified in each experimental setting. The optical properties of filters should also be considered. Transparent membranes offer the advantage of assessing cell culture monolayers using phase-contrast microscopy. A 0.4 µm pore size is well suited to examine the transfer of solutes and a large number of proteins. Theoretically, free diffusion is not restricted in the case that the pore diameter is wider than 20-fold the effective diameter of the compound .™, and poly-D-lysine) can enhance cell attachment, growth, and spreading to form a confluent monolayer. As the most appropriate matrix component for culturing choroid plexus epithelial cells, laminin is considered the principal glycoprotein component of the basement membrane serving to expedite cell attachment, spreading, and growth -mannitol (182 Da), [14C]-sucrose (342 Da), [3H]-sucrose, [3H]-inulin (~ 5 kDa), [11C]-inulin, [125I]-albumin (67 kDa), or polyethylene glycol (~4 kDa) [Radiolabeled tracers: Small polar compounds radiolabeled with radioactive isotopes can be used, such as [(~4 kDa) . The quaBiotin tracers: Besides fluorophores and radioactive isotopes tags, biotin is another compound that can be used in conjugation with tracers to monitor the permeability of in vitro BCSFB models. Biotin-ethylenediamine (287 Da), biotin-dextran, or biotinylated-dextran amine (BDA) with various molecular sizes (3–70 kDa) are exemplary choices. Biotin-labeled exogenous tracers can be visualized both by light- and electron microscopy imaging techniques ,176,177.Analysis of the expression and localization of junctional proteins and relevant carriers/transporters and their accordance with the choroidal epithelium in vivo can reveal information regarding the quality index of the model. Depending on the individual experimental goal, the characterization of specific markers, enzymes, receptors, adhesion molecules, and specific proteins/polypeptides, can be performed. Immunostaining of cell-type-specific markers and junctional molecules leads to a qualitative confirmation of barrier integrity of an epithelial monolayer.To assess the efflux transporters’ activity, substrate accumulation assays are exploited. Various efflux inhibitors (such as cyclosporin A and MK-571) are used to perform such substrate accumulation assays, assuming that substrate-inhibitor pairs are appropriately matched for each efflux transporter. In this assay configuration, model cells are incubated with the desired substrate either with or without their respective inhibitors, and at the culmination of the experiment, normalized fluorescence of the cells is calculated. In the case of the functional expression of transporters, the uptake of substrate should be increased in the presence of a corresponding transporter inhibitor .Directional transport: in another experiment variation, transporter activities can be evaluated using directional transport assays, in which inhibitors are only added on the side of the cells grown on cell culture inserts where directional transport is being assessed. Here, again, model cells are incubated with the desired substrate either with or without their respective inhibitors, and the fluorescence signal on the opposite filter side is measured. If efflux transporters are expressed functionally at apical or basolateral surfaces, then the directional transport of substrate in the presence of the corresponding inhibitor is enhanced .The barrier-forming characteristics and functions of choroid plexus epithelial cells in vitro and their degree of differentiation are closely related to the culture conditions applied. There have been lines of evidence supporting that certain agents and conditions may induce tightening and strengthening of the barrier. The factors critical to cell type selection and culture conditions are discussed in this section.Serum withdrawal also seems to impact choroid plexus cells to reach the full barrier function. Serum deprivation can tighten the epithelial monolayer and improve the cellular polarity ,179. HowDue to the fact that barrier properties of epithelia are modulated by cAMP-dependent pathways, the presence of membrane-permeable cAMP analogs such as 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) or the adenylate cyclase activator forskolin can have an augmenting effect on TEER values of choroid plexus epithelial cell layers ,180,181.Corticosteroids  application to in vitro models may increase the barrier tightness of the epithelial and endothelial cells monolayer. This phenomenon is proposed to be a consequence of the regulation of the expression and distribution of tight junction proteins upon corticosteroids treatment . DexametSeveral lines of evidence propose that unique transport and barrier attributes of the choroid plexus, in conjunction with the blood–brain barrier, are among the decisive factors establishing cerebral bioavailability of therapeutics and diagnostics. A large number of innovative and highly specific active compounds and investigational pharmaceutical preparations are continuously invented/developed/manufactured during the research and development cycles by the pharmaceutical industry, yet the necessity to discover a practical strategy for the effective delivery of them to the CNS target site remains to be addressed properly. Leveraging a robust and authentic in vitro model of the BCSFB can diminish the time and effort spent on unproductive or redundant development activities by a preliminary assessment of desired physiochemical behavior of an agent toward this barrier. In addition, these model systems can prove helpful in elucidating the nature of permeability and transport mechanisms, with a resolution at the cellular and molecular level, as a focal step during the long journey of neurotherapeutics research and development.Any rational drug discovery project dealing with candidates targeted to the brain or requiring exclusion from the CNS to prevent possible side effects should contemplate permeability measurements and prediction studies. Hence, profiling brain/CSF permeability of investigational novel molecular species at preliminary stages of the drug development track is game-changing. To this end, models can predict the CNS permeability of novel or known compounds in relation to both the route and rate.−1 or cm min−1 unit, can be calculated according to the following equation:Most in vitro BCSFB models used for permeability studies/screenings and understanding the pharmacokinetics of CNS active compounds are variants of 2D static bicameral inserts, employed to measure compound movement across the cell monolayer cultured on the porous membrane support. When using these models, a possible disturbance of the mass balance of the initial amount of drug added to the donor chamber with the amount recovered on the opposite reservoir would provide rough evidence of either cellular accumulation or intracellular metabolism of the compound (unspecific adsorption to filter membrane or well plastic should be ruled out). Measuring the concentration of the test compound, using an appropriate method , as a function of time in one of the compartments and knowing the initial concentration on the opposing compartment yields to the clarification of active transport rate and direction. Subsequently, the permeability coefficient substance added to the basal (mimicking blood side) channel toward the luminal (presenting CSF side) channel, the permeability coefficient The permeability coefficient of a cell layer-free device VolumAn advantage, in the case of measuring the permeability using microfluidic devices compared to static bicameral cell culture filter inserts, is that the drug or substance can be supplied to the basal channel at a constant flow rate and the transported amount can be sampled from the apical/luminal channel with a constant flow rate. Consequently, the assumption that the concentration differences beyond the filter membranes remain constant throughout the experimental read-out is met, albeit in the static bicameral devices this difference decreases over time .The permeation rates of various drugs  ,188,189,Thanks to their physicochemical nature, hydrophobic/lipophilic substances of a low molecular weight are capable of free diffusion across cell membranes. In contrast, as mentioned in previous sections, the unrestricted diffusion of hydrophilic chemical species is substantially hindered due to the effective closure and sealing of the paracellular shunt by TJs. Accordingly, access to the CSF is granted exclusively to those compounds that are transported actively by the corresponding transport systems in the plasma membrane of choroid plexus epithelial cells. Theoretically, therapeutic- and diagnostic agents that are effectively and successfully transported by the choroid plexus and remain to some extent unaffected by the metabolizing enzymes and efflux transporters are rapidly distributed throughout the CNS using the bulk flow of CSF. This is due to the fact that at the brain ventricles, the extracellular/interstitial fluid (ISF) and the CSF are separated from each other by the non-barrier/non-restrictive permeable layer of ependymal cells, leading to a direct continuity of these two components and the subsequent free exchange of substances within the extracellular space of CNS .Models expressing the specific transporters found in choroid plexus epithelium could be harnessed to establish whether the permeation of a compound of interest is impacted by a specific carrier system  and to provide comprehensive knowledge on the physiology and modulation of such transporters. From another point of view, several neurological disorders are assumed to be associated with the dysfunction of transporters in the brain, and the detailed region-specific knowledge of transporters and their interaction with investigational therapeutics can prove helpful. In the case that transporters govern the permeability or uptake of a test compound, Michaelis–Menten kinetics can be obtained using nonlinear regression analysis of the concentration dependence influx. In vitro models have proven advantageous and constructive in studies of thyroxine , leptin Therapeutic and diagnostic agents can pass the BCSFB via the transcellular route by employing one of the possible pathways, which are passive diffusion, facilitated diffusion, or vesicular transfer or transcytosis mechanisms. Generally, cellular uptake mechanisms based on endocytosis/transcytosis are the preferred cell entry route for many compounds. The endocytosis process can be categorized into two broad divisions of phagocytosis and pinocytosis. While the former is restricted to specialized cell types, the latter occurs in all cell types and can be subdivided into macropinocytosis, clathrin-dependent endocytosis (CDE), and clathrin-independent endocytosis (CIE) . ContrarDesigning novel CNS therapeutics and pharmacologically active candidates that exploit the intrinsic transporting capacity of choroid plexus epithelial cells will prove helpful toward the generation of CNS drug delivery approaches through the BCSFB. Nevertheless, the choroid plexus has received little attention as a potential gateway for drug delivery to the brain, and there remain debates about its eligibility for this aim . An evalTargeting transcytosed receptors (receptor-mediated transcytosis (RMT)): From a pharmacological viewpoint, although most efforts have been concentrated on the BBB, yet the epithelial barrier of the choroid plexus is of considerable importance as a potential target for drug delivery to the CNS, and the subsequent maintenance of effective drug concentrations needed to treat conditions and disorders. Hitherto, transcytosis-mediated transport by interacting with hormone/protein receptors expressed on choroid plexus epithelial cells seems to be the most favorable mechanism exploited for this purpose. The conjugation of BCSFB-penetrating antibodies/peptides with drugs or the engineering of bispecific antibodies, harboring both a therapeutic arm and a BCSFB-transcytosing arm, are among the most promising attempts toward neurotherapeutics-developing pipelines. Other alternatives are vectors based on the native ligand or a fragment of the ligand of the transcytosed receptor.CNS shuttling of therapeutic and diagnostic macromolecules can be achieved through RMT, an active transport mechanism involving transcytosed receptors, including transferrin receptor (TfR), insulin receptor (IR), insulin-like growth factor receptors (IGF1R and IGF2R), folate receptor FRα, and receptors responsible for lipoprotein transport, namely the low-density lipoprotein receptors (LDLRs) ,205,206.Contrary to solute carrier-mediated transcytosis, targeting an RMT pathway circumvents the size constraints of therapeutic cargos, since it employs a vesicle-based transport rather than a stereoselective carrier. This Trojan horse approach, although being currently studied by many research groups, holds a few technical drawbacks, which need to be addressed. The saturation of transferrin receptors with endogenous transferrin from the circulation requires the transferrin receptor-targeted compound to compete with the natural ligand . As wellThere are reports of enhanced CNS penetration of antineoplastic drugs (methotrexate), peptides , and tracer protein native horseradish peroxidase using anti-transferrin receptor antibody OX26, the mouse monoclonal antibody against the TfR ,211. TheSolute Carrier-Mediated Transcytosis (CMT): Harnessing in vitro BCSFB models, mechanisms pertaining to solute CMT exploited as a drug delivery approach to the CNS, using various solute carrier transporters (SLC) present on apical and/or basolateral membranes of choroid plexus epithelial cells, can be substantiated. In this context, the SLC proteins experience a conformational alteration from an outward to inward-facing orientation and consequently translocate their substrates across the cellular barrier. Generally, small di- and tripeptides can be transferred using this mechanism through the SLC15A family members ,215. AltNano-therapeutics: Nanotherapeutics represent a promising area of CNS drug delivery approaches and have revolutionized the concept of drug delivery across brain barriers with some successful exemplars currently in the clinic ,217,218.In principle, all the BCSFB in vitro model systems described so far have the potential to be explored to shed light on the mechanisms of nanotherapeutics interactions with the BCSFB, identify the impact of biological variables on NPs transport across the BCSFB, evaluate the biocompatibility of nanomaterials, help design nanomaterials interacting selectively with the epithelium of the choroid plexus, assess the safety and efficiency of pre-clinical formulations, model the effects of social/stress/immune/environmental factors on NPs transport across BCSFB, and meet the ongoing need of research in neurotherapeutics’ research and development ,226.The fate of drugs and xenobiotics metabolites, affiliated to several chemical classes, generated at choroidal epithelial cells and the relative contribution of various efflux pumps present at this barrier can also be investigated using these in vitro models. In addition, the potential effect of multiple metabolites, xenobiotics, and compounds on both influx into and efflux out of the CSF can be studied. Referring to static bicameral device models, efflux transport assays can be performed, and the efflux ratio is estimated by dividing the permeability value in the apical to basolateral (A−B) direction by the permeability value in the basolateral to apical (B−A) direction. With respect to efflux transport assays, the transport of the compounds under study is assessed in the presence of inhibitors of relevant transporter proteins and compared to the non-inhibited conditions. Namely, verapamil, GF120918, PSC833, and N-desmethyl-loperamide as inhibitors of ABCB1/P-gp, Ko143, and fumitremorgin C as an inhibitor of ABCG2/BCRPs, tariquidar and elacridar as inhibitors of both P-gp and BCRP, or MK571 as an inhibitor of various MRPs are examples that can be applied for such experiments . As an aOne of the main thrusts behind the development of in vitro BCSFB models has been to shed light on the pharmacological molecular mechanisms of compounds toward this interface. Using cell-based BCSFB models, in vitro activity, potency, and mechanisms of actions of investigational CNS therapeutics and compounds under study can be demonstrated in preclinical drug evaluations. Excerpts of case studies from distinct pharmacological categories describe investigations of the anti-inflammatory activity of synthetic matrix metalloproteinase (MMP) inhibitors , and eve1c (5HT1c) -1--2-aminopropane as an agonist) [Cell cultures of the rat choroid plexus have been used to elucidate the regulation mechanisms and phenomena of various receptors, including 5-hydroxytryptamineagonist) .Investigational anti-viral compounds can be tested in vitro in the BCSFB model platforms. In these experiments, the choroid plexus cells lines could be used as hosts and cellular substrates for viruses to perform a wide range of assays, including the cytopathic effects inhibition assay, the virus titer reduction assay, cytotoxicity assays, for the determination of the effective concentration of compounds under study, and even molecular mechanistic assays. For instance, the activity of nucleoside cytidine analogs as possible anti-HIV compounds was investigated in sheep choroid plexus cells as the cellular substrate for the Maedi-MVV virus . In anotHarnessing appropriate in vitro BCSFB models, the ever-increasing concern regarding hazardous compounds, such as environmental toxins, metals, pesticides, solvents, as well as the broad field of study of neurotoxicants, can be the target of both exposure (amounts permeated) and impact (effects exerted) investigations. The models have proven valuable in evaluating the influx/efflux of (neuro)toxins, understanding the associated neuroprotective mechanisms, elucidating the alteration of the BCSFB function by toxic compounds, and the involvement of the BCSFB in neuroinflammation .Choroid plexus cells and models have been used to study underlying mechanisms of metal-induced toxicity and the transport of neurotoxicants metals including, but not limited to, mercury (Hg), as presumably the most studied neurotoxic metal, lead (Pb) ,234,235,Transport data acquired through pharmacological interventive measures and modulation by hormones and drugs contribute to understanding the BCSFB homeostatic phenomena and barrier/transporting functions. In addition, establishing approaches for restoring aberrant choroid plexus and CSF dynamics during disease has been of great interest to researchers in pharmacological, neurological, and neurosurgical fields. In this sub-section, major pharmacological mediations used to study this barrier are reviewed.2 to produce carbonic acid (H2CO3), which spontaneously dissociates into HCO3ˉ and H+ [Carbonic anhydrase inhibitors : Pharmacological agents of different classes altering CSF dynamics are exploited to study CSF production and secretion. Prototype pharmacological agents to reduce the CSF production rate in the range of 50–100% are inhibitors of carbonic anhydrases, the enzymes responsible for the hydration of COˉ and H+ ,245,246.ˉ and H+ .+-K+-ATPase pump and consequently ion and fluid transport. This cardiac glycoside reduces the CSF production rate to a half basal level when placed on the luminal side of the choroid plexus epithelial cells. This aspect along with its neurotoxic side effects hinders its clinical application [Ouabain is a potent inhibitor of the Nalication ,22,248.+-H+ exchanger) inhibitor, blocks Na+ distribution into the choroid plexus from the blood-facing side [Amiloride, the NHE  likewise attenuate Nasporters ,251,252,− transport into the CSF. DIDS is a stilbene-type inhibitor of anion exchangers, which can be used as a pharmacological tool to evaluate the activity of these ion exchangers at the choroid plexus. However, as a disadvantage, its effects are not specific, and a wide range of other transporters can be affected [Disulfonic stilbene (DIDS) is an anion transporter blocker, inhibiting Claffected ,254,255.Ouabain, acetazolamide, furosemide, bumetanide, and DIDS inhibit CSF secretion from the luminal membrane of choroid plexus. Acetazolamide, DIDS, and amiloride are inhibitors acting on the basolateral/blood-facing membrane ,256. In The regulation of apical ion channels of choroid plexus by various substances, including serotonin, mianserin, and mesulergine, has been the topic of research ,257,258.In this subheading, the role of choroid plexus dysfunction in various diseases and in conditions where itself is a target, for instance, choroid plexus carcinomas, is discussed. Choroid plexus breakdown has been proposed and hypothesized in a wide range of neurological conditions, including aging, Alzheimer’s disease, Parkinson’s disease, epilepsy, stroke, neoplasms, perhaps psychiatric disorders, intracranial hypertension, and certainly varying types of hydrocephalus. Therefore, targeting this tissue may offer opportunities to translate neurotherapeutics from the lab to the clinic, and provide insights toward rational drug therapy to restore barrier function ,263,264.Strategies to diminish choroidal fluid turnover or to develop efficacious agents, which can manage altered CSF dynamics in disorders, including hydrocephalus, ventriculomegaly, cerebral edema, and intracranial hypertension, can be designed based on the obtained transport data at choroid plexus. In spite of a large body of knowledge of choroid plexus transporters and ion channels that mediate CSF secretion, effective pharmacological agents are sparse, and a gap regarding pharmacological substances capable of interfering still remains.Supportive evidence proposes the involvement of the choroid plexus in neuroinflammation and neurodegeneration ,266,267.This review set out with the aim of rendering an overview of the up-to-date status of in vitro BCSFB models currently exploited, with a special emphasis on their applicability for research and development of CNS-affecting therapeutics and drug delivery to the brain. Taken together, a suitable model should exhibit the features of the restricted paracellular shunt, the expression of TJ proteins and choroidal markers, and the functional and polarized expression of BCSFB-specific transport mechanisms. In addition, the model should meet the features of availability, convenience, predictability, reproducibility, and extrapolation to human and in vivo settings.Although a large body of knowledge on transporters, receptors, and drugs pharmaco-kinetics and -dynamics are derived from in vivo animal models and invasive techniques, the fragility of choroid plexus tissue, its small mass, and hardly accessible location along with ethical constraints leads to technical challenges during isolation and further experimental interventions. On the other side, a challenge to reproduce the complexity of the in vivo environment with in vitro models as much as possible still exists. It is generally recognized that the reproducibility of cell-based models can be imperfect, with day-to-day and lab-to-lab inconsistencies being observed. In the case of static monoculture/coculture bicameral devices models, technical challenges may prevail, e.g., the cells can have an incomplete surface coverage or even overgrow and subsequently produce multiple layers instead of monolayers.Nevertheless, despite all the mentioned shortcomings, in vitro models remain desirable workhorses for routine applications in industrial and academic pharmaceutical studies. Still, it should be emphasized that since no single model can imitate the absolute in vivo physiological environments, for each experimental condition based on the desired outcomes, the question under investigation and the throughput level, a suitable configuration of model system-cell type-validation criteria should be chosen. In other words, by weighing the pros and cons of various platforms, a translationally relevant model of the choroid plexus appropriate for the desired research needs should be selected.Despite the fact that in vivo, ex vivo, and in situ models  may be considered as gold standards and ultimate test systems for exploring drugs transport and pharmaceutically related events, integrated and physiologically related in vitro models are more promising to eliminate the effect of species-specific differences. Furthermore, these in vitro models can be captivating when the aim is to survey transport and other pharmacological mechanisms solely at the choroid plexus in the absence of interfering biochemical effects and traces of the BBB and brain parenchyma. In these model systems, secondary effects originating from drug pharmacokinetics, distribution, and tissue blood flow are eliminated exceedingly.


Moreover, the weight of AN-induced plasticity against other mechanisms of brain plasticity is not known, adding to the controversy. In this review, we would like to show that recent technical advances in brain MR imaging methods combined with improved models can resolve the debate.Adult neurogenesis (AN) can be defined as the birth and development of new neurons in adulthood. Until the 1990s, AN was deemed not to happen after birth. Gradually, several groups demonstrated that specific zones of the brain of various species had a neurogenic potential. AN could be the key to treating a large range of neurodegenerative, neuropsychiatric, and metabolic diseases, with a better understanding of the mechanisms allowing for regeneration of new neurons. Despite this promising prospect, the existence of AN has not been validated  During the twentieth century, the prevalent dogma was that there was no generation of new neurons after birth. Then, gradually, different studies showed that adult neurogenesis (AN) induced plasticity in some specific regions of the brain of various species including humans. Changes occurred following learning, exercise, environmental enrichment, or stress. If validated, AN could have enormous implications for human health Gage, .in-vivo functional and metabolic roles of AN have not been established prompting to the non-existence of AN in the brain of adult humans , the subventricular zone (SVZ) of the hippocampus, and the olfactory bulb  was extensively used in the late 1990s to demonstrate the presence of AN in post-mortem SVZ brain samples of patients with cancer  is, by far, one of the most translatable imaging techniques currently available for estimation of non-invasive biomarkers of development and progression of diseases and for investigation of responses to treatment in various species. MRI offers a large panel of qualitative, semi-quantitative, and quantitative parameters for visualization and understanding of both healthy and pathological features.To establish the existence of AN For the past 20 years, crucial technological developments of MRI machines have permitted important advances of structural, functional, and metabolic imaging methods that are now widely used in both experimental and clinical settings. As a matter of fact, a wide range of identical imaging sequences  exists for small animals and for humans, while more and more animal-dedicated scanners propose similar hardware and software functionalities. Moreover, subsequent image analysis of animal or human scans often uses identical platforms, although programs need to be adapted.in vivo necessitates acquisition of images at cellular resolution. With conventional magnetic resonance imaging (MRI) techniques, the in vivo in plane spatial resolution is rarely below 100 μm2. Direct detection of AN is therefore difficult with MR techniques, and many studies mention possible effects of AN without demonstrating its presence. In in vivo MR studies on AN.Of course, direct detection of AN in vivo. Among them, vascular markers such as cerebral blood flow (CBF) and cerebral blood volume (CBV) have been used as potential surrogate markers of AN both in the rodent and human hippocampi after prolonged exercise  make them irrelevant for pharmaco-therapeutic challenges. In terms of MRI, the differences in spatial and temporal resolutions can be difficult to transpose to the humans in the clinical setting despite the use of higher magnetic field strengths for research only purposes. Finally, physiological and behavioral responses to anesthesia in rodents may differ substantially from the humans, which nevertheless are rarely anesthetized for routine MRI scanning.On the other hand, non-human primates (NHPs) are considered the most relevant animal model for translational purposes, being the closest animal models to humans regarding their genetic background, physiology, and behavior. Most often, they are considered as the last criterion for establishing the efficacy of a drug before transition to humans. However, recent studies have also provided evidence that NHP models have also provided disappointing contributions to medical advancements. Moreover, ethical issues such as suffering and deaths of NHPs in biomedical research are frequently raised. The validity of some studies regarding cognition and behavior assessments of captive NHPs has also been questioned.Despite the ethical issues mentioned previously, establishment of relevant NHP models of various diseases in combination with non-invasive MRI techniques is a growing area of research holding great promises for development and assessment of various therapeutic protocols. Moreover, MRI investigation of NHPs can be useful to validate the characterization of deeper brain structures and specific nuclei  and describes neuronal activity in an indirect manner by neurovascular coupling. The underpinnings of BOLD responses remain difficult to apprehend with conventional methods. Therefore dissociating the BOLD signals due to newborn neurons from already existing neurons requires cutting-edge techniques involving the use of ultra-high magnetic fields as well as the combination of MR techniques with genetic tools  to access the single cell function , dynamic susceptibility contrast MRI, myelin MRI, and 1H-MRS to hypothalamic AN. All these MR techniques are widely used in the clinical setting. These examples were extracted from data, which are still under analysis but appear to show some consistent response among all the different MR modalities used with structural, functional, and hemodynamic changes occurring during the period of AN.Therefore, s 1H-MRS  of the hs 1H-MRS . We deveex vivo investigations with direct possible applications to study the human brain. Finally, diffusion-weighted MR spectroscopy (DWS) uses the diffusion of endogenous metabolites to probe the microstructural features of the intracellular and extracellular spaces. DWS approaches have been shown to be very effective to probe the intra-neural space as well as astrocytic morphology with N-acetyl-aspartate (NAA), glutamate (Glu), and inositol (Ins) both in animal models and human subjects  has become an inevitable neuroimaging technique, which has proven to be uniquely sensitive to the presence of various cell types  and their density, orientation, and permeability. dMRI provides unprecedented microstructural information on a micrometer scale enabling the investigation of underlying subtle microstructural changes in various disorders and physiological mechanisms. It represents therefore a technique of interest to investigate brain neuroplasticity both in animal models and in the clinical setting Gatto, . As for How brain metabolism influences AN is a question that had gained more and more insights during the past decade. In particular, neuroenergetic mechanisms controlling neural stem cells and progenitor cells and their differentiation, proliferation, and maturation require vast amounts of energy involving various steps and various metabolic mechanisms such as lipogenesis and mitochondrial activity . The methodology proposed by Manganas et al.  and 31P-MRS could be of interest for investigating AN. For example, Koush et al.  in the human brain [despite remaining controversies .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.


Here, we review the current knowledge of common and divergent mechanisms shaping the forebrain in vertebrates, and causing cortical malformations in humans. We next address the utility, benefits and limitations of whole-brain/organism-based fish models or neuronal ensembles in vitro for translational research to unravel key genes and pathological mechanisms involved in neurodevelopmental diseases.The variety in the display of animals’ cognition, emotions, and behaviors, typical of humans, has its roots within the anterior-most part of the brain: the forebrain, giving rise to the neocortex in mammals. Our understanding of cellular and molecular events instructing the development of this domain and its multiple adaptations within the vertebrate lineage has progressed in the last decade. Expanding and detailing the available knowledge on regionalization, progenitors’ behavior and functional sophistication of the forebrain derivatives is also key to generating informative models to improve our characterization of heterogeneous and mechanistically unexplored cortical malformations. Classical and emerging mammalian models are irreplaceable to accurately elucidate mechanisms of stem cells expansion and impairments of cortex development. Nevertheless, alternative systems, allowing a considerable reduction of the burden associated with animal experimentation, are gaining popularity to dissect basic strategies of neural stem cells biology and morphogenesis in health and disease and to speed up preclinical drug testing. Teleost vertebrates such as zebrafish, showing conserved core programs of forebrain development, together with patients-derived  Since the early comparative studies in animal models , developmental biologists have shed light on the precise choreography of genetically controlled events that shape the vertebrate brain in different domains along the AP (antero-posterior) and D-V  axes. These events produce the conserved organization of the forebrain anteriorly, morphologically and functionally distinguished in different domains . The palWithin the developing pallium, a precise balance and timing of signaling drive neural fate commitment and patterning, as well as the rate of progenitor pools’ expansion (level 1), migratory behavior and differentiation (level 2), and the establishment of connectivity patterns (level 3). Fine changes in local patterning schemes and precursor cells’ behavior across evolution underlie the array of diversity in vertebrate cognitive capabilities and the formation and expansion of the multilayered cytoarchitecture of the mammalian cortex, responsible for high-order functions typical of humans . From a The continuous identification of new genetic lesions affecting these different levels of neurogenesis and resulting in heterogeneous neurodevelopmental disorders with malformation of cortical development (MCD) highlights the importance of these processes during cortex formation. Improving our knowledge of the specific biology and genetics of pallium patterning, regionalization, precursor behavior and cell type specification and its variation on the main vertebrate theme can potentially impact our ability to model, understand and manage MCD. Indeed, these heterogeneous conditions can manifest with various degrees of microcephaly, lissencephaly and cortical dysplasia, originating from impaired proliferation, migration and/or white matter and circuits establishment. They are often associated with cognitive impairments and with at least 40% of non-treatable epileptic conditions . The cliNevertheless, given the current acceleration in the identification of candidate genetic lesions in previously undiagnosed MCD conditions, scientists are exploring alternative systems. These should (i) be more readily implementable than mammalian species in healthcare-oriented institutes, (ii) offer possibilities for fast candidate gene variants validation serving differential diagnosis, while (iii) parallelly allow the search of basic patho-mechanisms, and (iv) via methodologies which reduce the overall burden related to animals experimentation. To this aim, teleost fish can be advantageous . They rein vitro alternatives in conjunction to the in vivo models to recapitulate species (i.e. human)-specific traits of early neurogenesis, differentiation, connectivity and even cytoarchitecture at small scale  or from the minimal tissue complexity and lack of whole-organismal context (organoids).A different and complementary approach, quickly gaining popularity and reducing further the burden on animal models, is the establishment of induced pluripotent stem cells (iPSCs) directly from patients’ bioptic material and iPSCs-derived cerebral organoids (COs). The latter are becoming attractive ll scale . Direct ll scale . This rein vivo have been improved and the breakthrough of organoids – including COs- is revolutionizing the study of the healthy and diseased mammalian brain.Since the experiments of Spemann and Mangold in the early 20th century, developmental biologists used animal models to demonstrate that brain development is achieved by tightly controlled gene expression schemes that generate specific, recognizable local fingerprints. These events depend on the ability of cells in the early embryological fields to respond to varying doses of inducing and inhibiting molecules (morphogens) secreted by neighboring cells, which sequentially restrict the fate of each previously homogeneous domain, leading to patterning, regionalization, and cell type specification. These events step up the global brain architecture and its functional sub-specialization in different domains, and are globally conserved in evolution . UnderstBf-1  territories, while a fine-tuning of Bmp, Shh, and Wnt signals from axial mesodermal and non-neural ectodermal cells contribute to define instead the D-V  embryo axis . In thisBf-1 , Emx, anBf-1 . The Emxretectum .The development and plasticity of the neocortex, originating from the dorsal pallium in mammals as a late evolutionary innovation  depends Importantly, the developing cortex of lissencephalic models, such as mice, have little bRG cells which divide both symmetrically and asymmetrically  and are Next, the basal projections from progenitor cells allow newborn neurons and glia produced by asymmetric division to migrate radially to the destination in the cortical plate (CP) by somal translocation or active locomotion . The firThe high complexity and specialization of the molecular and cellular events governing neocortex development and function in space and time make them particularly vulnerable to genetic alterations. A large group of malformations of cortical development (MCD) has been described which are characterized by various defects at the level of brain size (decreased or increased), final position of neurons, and formation of cortical layers and foldings . DiffereARF3, encoding a small GTPase involved in post-Golgi trafficking , or postnatally (secondary), as an isolated condition or in combination with other clinical features, i.e., developmental delay, epilepsy, and CNS malformations , as wellfficking . More reocephaly .Impaired migration of newborn neurons from the ventricle to the CP along the RG scaffold causes different forms of neuronal migration disorders (NMDs). This is a group of highly heterogeneous conditions, which can show mixed features, including microcephaly or other cerebral malformations ; Table 2LIS1, DCX, KIF2A, TUB1A,  on one side and the consensus on minimizing the animal distress associated with the invasive nature of experimentation in these large animals, quest for possible alternative models. These might be used for fast and large-scale functional validation of variants of unknown impact. Ideally, valid model systems successfully complementing the established and emerging mammalian ones should: (i) be rapidly implementable and manageable in medical research institutes, (ii) bring to a fast validation of the candidate genetic variants, (iii) while reducing the reliance on invasive animal procedures and still (iv) allowing scientific and translational gains in terms of mechanistic understanding of the disease. In this sense, despite not harboring the multi-layered cortex and given the conservation of fundamental mechanisms, the arsenal of genetic, imaging, and behavioral tools, teleost fish  are actiin vitro approach is also gaining increasing popularity, which is the use of iPSCs and COs derived from patients’ fibroblasts  affecting cortex formation in humans have already contributed to our understanding of pathogenic variants and mechanisms involved in the disease .Numerous are the features and benefits that favor the use of zebrafish in neurodevelopmental biology and disease modeling  establish fast quantitative platforms to screen and investigate the increasing number of candidate pathogenic variants, (2) perform an initial investigation of the underlying mechanisms of disease, and (3) examine potential therapeutic targets. Indeed, from a translational point of view, zebrafish models are useful to validate previously undiscovered rare neurodevelopmental diseases within integrated functional genomics pipelines. Here, different pathogenic variants are first identified via NGS screening in patients, their differential impact on cellular and molecular events is often assessed in in vitro and in vivo zebrafish modeling was carried out in a single study to assess the pathogenicity of newly discovered mutations in the aminoacyl-tRNA synthetase-encoding VAR2 gene. Mutations in this gene lead to microcephaly, intellectual disability, and epilepsy   , and indin vivo, rapid path toward the off-label employment of lorcaserin. This FDA-approved compound antagonizing serotonin signaling pathway was transferred from preclinical discovery to potential clinical treatments of Dravet syndrome. From here, the compassionate use of commercial Belviq® is now approved for a small population of children affected by the syndrome, who successfully exhibit a reduction of generalized tonic-clonic events.Generally, examples exist also of direct use of zebrafish embryo models of rare (neuro)developmental diseases in the search of possible candidate drugs to counteract impaired signaling cascades [such as Ras/MAPK ] or amelin vivo zebrafish models should be complementary to other feasible alternatives closer to large mammals and humans , as well as iPSCs-derived COs for better recapitulating the structural and functional complexity of the human brain  for functional validation and classification of rapidly emerging heterogeneous MCD in humans, experimental designs, questions, and data interpretation in these models should be carefully confined by the limitations discussed. In translational workflows dedicated to solving the underlying biology of MCD d humans . Of partan brain . These aan brain . iPSCs can brain  as 3D-sean brain .iPSCs can be an informative and renewable source of cells to model human and mammalian neurogenesis. Their use already helps reduce the number of animals and the possible distress caused by studying cortical development and function in mammals. Furthermore, a precious source of information derives from iPSCs directly obtained from residual bioptic material of rare patients. In addition, among the main advantages of using iPSCs to obtain simplified models of neural networks , one shoNevertheless, these cells are in a 2D environment that hardly recapitulates the elaborated, multi-layered architecture of the human brain. They might show a low programming efficiency and can be very expensive to obtain. 3D systems, such as embryoid bodies (EB) first and cerebral organoid models (COs) later, are obtained via different protocols  and theyComparative iPSCs and COs preparations are also useful to understand the ontogeny of species-specific similarities and differences in proliferative potential, cortex size and complexity, underlying the variability in cognitive specialization across taxa . For insin vitro also holds promises to study drug delivery into the human brain  mammals just began. From a technical point of view, compared to classical and innovative mammalian models, in both the context of brain EvoDevo and disease, teleosts offer a distinctive readiness for generating affordable models with a broad and fast interrogation capacity of nano-scale dynamics within precursor cells and up to neuronal circuits logics of entire brain territories at the whole-organism scale. Furthermore, quantifiable behavioral readouts can be performed simultaneously, creating an ideal setting for scalable preclinical drug testing. Specifically, for disease modeling, limitations exist, however, when researching biological processes which could be altered in MCD using zebrafish and other teleosts, given the differences discussed so far and the clear lack of a patients . It is wisorders  and to cisorders . A robusAL conceived the manuscript and generated the illustration. AL, GF, and CC wrote the manuscript. MT and BD revised the final manuscript. GF and AL conceived and generated the tables. All authors contributed to the article and approved the submitted version.


Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airflow limitation, and is associated with abnormal inflammatory responses in the lungs to cigarette smoke and toxic and harmful gases. Due to the existence of common risk factors, COPD is prone to multiple complications, among which cardiovascular disease (CVD) is the most common. It is currently established that cardiovascular comorbidities increase the risk of exacerbations and mortality from COPD. COPD is also an independent risk factor for CVD, and its specific mechanism is still unclear, which may be related to chronic systemic inflammation, oxidative stress, and vascular dysfunction. There is evidence that chronic inflammation of the airways can lead to destruction of the lung parenchyma and decreased lung function. Inflammatory cells in the airways also generate reactive oxygen species in the lungs, and reactive oxygen species further promote lung inflammation through signal transduction and other pathways. Inflammatory mediators circulate from the lungs to the whole body, causing intravascular dysfunction, promoting the formation and rupture of atherosclerotic plaques, and ultimately leading to the occurrence and development of CVD. This article reviews the pathophysiological mechanisms of COPD complicated by CVD and the effects of common cardiovascular drugs on COPD. Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease with persistent airflow limitation. The airflow limitation is usually gradual and not fully reversible. It is mainly related to smoking and inhalation of other toxic and harmful gases . COPD isCardiovascular disease is one of the most common comorbidities of COPD , which iChronic systemic inflammation plays an important role in the pathogenesis of CVD , 16 and In addition to inflammation, oxidative stress is another feature of COPD that plays an important role in the development of the disease and is also associated with CVD . OxidatiCardiovascular events are one of the leading causes of hospitalization in patients with COPD and contribute significantly to the cost burden of the disease. One study found that among smokers with mild to moderate COPD, CVD accounted for 50% of hospitalizations . InflammVascular endothelium is widely distributed in the body and can maintain the tension of blood vessels and the structure of blood vessels by mediating vasodilation, contraction, production inhibition and production promotion, as well as the balance of anti-inflammatory and pro-inflammatory, and can synthesize and release vasoactive substances., to regulate vascular tone, thereby regulating platelet function, inflammatory response, and vascular smooth muscle cell growth and migration . VasculaVascular endothelial injury in COPD patients mediates the occurrence of CVD, which is the result of enhanced oxidative stress and inflammatory response, thereby significantly increasing the risk of vascular diseases such as atherosclerosis, myocardial infarction, and stroke . In the in vivo, all promote pulmonary arterial remodeling, vessel narrowing, and subsequent increased pulmonary arterial pressure. Later, it can lead to right heart enlargement, followed by pulmonary heart disease and ventricular failure  reductase inhibitors, potently lower not only total cholesterol and LDL but also triglycerides to a certain extent, can also increase high-density lipoprotein, so statins are more comprehensive lipid-lowering drugs . The mecin vivo . A laterin vivo . The resin vivo . Howeverin vivo . The resin vivo . Of courin vivo . Severalin vivo –101. Thein vivo . Statinsin vivo .The renin-angiotensin-aldosterone system (RAAS) is a blood pressure regulating system produced by the kidneys in the body, causing vascular smooth muscle contraction and water and sodium retention, resulting in a blood pressure boosting effect. Renin-angiotensin-aldosterone system inhibitors, including angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARBs), are currently widely used in the prevention and treatment of cardiovascular diseases, while RAAS may promote the progression of COPD and pulmonary fibrosis by inducing the production and release of inflammatory factors and reactive oxygen species . There aThrombosis is one of the main causes of cardiovascular diseases. The adhesion, release and aggregation of platelets in blood vessels are the main causes of thrombosis. Therefore, antiplatelet therapy is widely used in some vascular diseases. Platelet activation is increased in COPD patients , it may Diuretics, mainly divided into loop diuretics, thiazide diuretics, and potassium-sparing diuretics, are important components of the treatment strategy for patients with heart failure. The use of thiazide diuretics in COPD patients with hypertension has not been found to affect respiratory function and does not increase the risk of acute exacerbations in COPD patients. Thiazide diuretics are not contraindicated in COPD patients . There aBy reviewing the current literature, in general, most CVD drugs are relatively safe in COPD patients, and active drug intervention is encouraged for COPD patients with CVD.The comorbidities of COPD are very common and may even be fatal, and such patients should be actively intervened and managed. CVD is a common complication of COPD, among which atrial fibrillation, heart failure and ischemic cardiomyopathy are the most common, and many factors such as inflammation, oxidative stress, hypoxia, and vascular endothelial dysfunction are involved in this process. Due to overlapping symptoms and signs such as dyspnea and fatigue, clinical treatment is often insufficient, and acute respiratory symptoms are often caused by confounding factors in the lungs and heart, and most cardiovascular drugs do not increase the risk of COPD exacerbation, or even can improve prognosis. Therefore, comprehensive management and individualized treatment should be done for COPD patients with CVD.X-FL and C-QW designed the study. X-FL searched the literature and wrote the manuscript. Y-MM revised the manuscript. All authors contributed to the article and approved the submitted version.


This literature review evaluates the mechanisms and efficacy of different types of antimicrobial photodynamic therapy (aPDT) for treating peri-implantitis by reviewing existing experimental studies to provide guidance for the clinical application of antibacterial photodynamic therapy (aPDT) in oral implants.  From February 2001 to February 2021, we have collected 152 randomized controlled trials of aPDT for peri-implantitis by searching the experimental studies and clinical trials published in PubMed, Embase, Web of Science, and Google Scholar databases via online search. After screening the retrieved literature, we finally selected 10 statistically significant literature for evaluation and review. P < 0.05). Furthermore, the combination of the aPDT and other treatments shows the synergistic antibacterial effect, signifying better clinical effect in many aspects (P < 0.05). In these 10 papers, by comparing the probe depth (PD), bleeding on probing (BOP), synosteosis, and periodontal pathogenic bacteria detection, etc., obtained after treating peri-implantitis by application of the antimicrobial photodynamic therapy, and using the SPSS data analysis software for statistical data processing, we found that the antimicrobial photodynamic therapy combined with other periodontal treatments has a more prominent postoperative effect. Meanwhile, the antibacterial photodynamic therapy with targeted action of photosensitizer has strong specificity to some bacteria, while the synthetic photosensitize for antibacterial photodynamic therapy can show good inactivation effect on broad-spectrum periodontal anaerobes without side effect.  Compared with the traditional nonsurgical treatment of peri-implantitis, the aPDT was superior to the traditional mechanical irrigation treatment group in terms of periodontal indexes PD, BOP, PLI, and postoperative effect, and the difference was statistically significant ( The experimental studies and clinical data of antibacterial photodynamic therapy for treating peri-implantitis show a good postoperative treatment effect. In addition, it did not develop resistance due to the use of antibiotic drugs. Owing to multiple advantages from combining antibacterial photodynamic therapy and other treatments, it is applicable for clinical treatment. Peri-implantitis is an inflammatory pathological state of the organism that occurs in the hard and soft tissues around the oral implants, whose clinical manifestations mainly involve the peripheral gingival soft tissues and alveolar bone, which may cause bleeding in the gingival soft tissues, broken and absorbed alveolar bone, and loosening implants and cause other risks. It is one of the main factors to cause the failure of implant operation .According to current studies, some believes that it is the bacterial plaque that is the root cause of the peri-implantitis, while other studies have shown that patients with periodontal disease can also show the same pathogen groups around their own implants as those in periodontal disease, such as Staphylococcus aureus, Streptococcus, and subgingival with actinomycetes . TherefoBased on findings in recent years, antibacterial photodynamic therapy (aPDT) effectively treats a local microbial infection. For the pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, Porphyromonas gingivalis, and multidrug-resistant bacteria , RajendrAs a result, we searched the published experimental studies and clinical trials on PubMed, Embase, Web of Science, and Google databases and collected 152 randomized controlled trials of antibacterial photodynamic therapy (aPDT) for treating peri-implantitis. After screening, we selected 10 papers of statistical significance to go through the test for significance.Given the lack of clarity related to the effectiveness of nonsurgical treatments, this study is made to conduct a systematic review of controlled and randomized clinical trials related to the efficacy of peri-implantitis and its different adjuvant therapies.2.We searched the literatures published on PubMed, Embase, Web of Science, and Google Scholar databases between 2001 and February 2021 and included the retrieved clinical studies on antimicrobial photodynamic therapy for the treatment of peri-implantitis. There are no obvious conflicts between the retrieved articles.Study on the clinical treatment of oral peri-implantitisClinical treatment and application of antibacterial photodynamic therapyClinical studies that have passed ethical review and have reliable dataExperimental studies on peri-implantitis in animalsExistence of significant flaws and errors in the design of the implants used in the studyClinical study on peri-implantitis leading to implant lossThe search strategy applied was as follows: (((Periimplantitis OR periimplantitis OR peri-implantitis) OR ) AND (bone-to-implant contact)).Animal studies were excluded and language limits (English) were imposed. The obtained results were combined with manual searches of the bibliographies of all full-text articles and related reviews selected from the electronic search.Among the 152 relevant articles, 10 articles passed the data analysis of the consistency test (Kappa = 1) after excluding the duplicates and articles failing to meet the inclusion conditions. We found that there was no conflict relationship among these 10 articles. The antibacterial photodynamic therapy group had different results from those obtained by the mechanical therapy and normal saline irrigation group in all clinical trials. A total of 481 patients and 663 implants were included .The reason why we excluded other literature is that only part of the antimicrobial photodynamic therapy for peri-implantitis is based on animal experiments, lacking clinical data support. In addition, in some literatures, peri-implantitis may be caused by improper design of implant surgery.P < 0.05), which showed statistical significant.In the analysis of antimicrobial photodynamic therapy and mechanical irrigation debridement for peri-implantitis , generalAccording to the studies by Rakašević et al., the clinical adhesion level (CAL) of the PDT group recovered more quickly after 3 months of treatment, showing a significant advantage over the 0.1% chlorhexidine gel group; the bleeding was significantly improved after treatment . In paraThe photodynamic therapy for the treatment of peri-implantitis also has obvious advantages on periodontal pathogenic bacteria , 21. AccMeanwhile, in the study of Romeo et al., the recovery effect of the aPDT group was better than that of the traditional mechanical defibrillation group in terms of the periodontal probing depth (PD) of the implant after the treatment. The aPDT group at the end of treatment was statistically significant .We can compare a large number of literature studies in terms of the effects of antibacterial photodynamic therapy, traditional mechanical therapy, and drug therapy. Through a systematic review of the literature, we have collected reliability analysis of the efficacy of antimicrobial photodynamic therapy to treat peri-implantitis.Abduljabbar compared it with traditional mechanical therapy; the photodynamic therapy was proven to have a more effective effect on the regulation of periodontal microflora, which also significantly improved the recovery of peri-implantitis soft and hard tissue .For the application of the photodynamic therapy for peri-implantitis, the effect of treatment recovery also depends on the selected photosensitizer. In most cases, we use toluidine blue as a photosensitizer for antibacterial photodynamic therapy. Alqahtani et al. have achieved an ideal therapeutic effect using the toluidine blue photosensitizer in photodynamic therapy . In addiSince antimicrobial photodynamic therapy has been widely used for that treatment of peri-implantitis, a group of literature data showed that the antibacterial photodynamic therapy combined with traditional mechanical therapy for peri-implantitis is superior to a single treatment scheme . The expAfter the statistical analysis of retrieved literatures, the combined application of antibacterial photodynamic therapy and traditional mechanical treatment for peri-implantitis can achieve an obvious therapeutic effect . Besides