Contemporary biomarker collection techniques in blood and cerebrospinal fluid have to date offered only modest clinical insights into neurologic diseases such as epilepsy and glioma. Conversely, the collection of human electroencephalography (EEG) data has long been the standard of care in these patients, enabling individualized insights for therapy and revealing fundamental principles of human neurophysiology. Increasing interest exists in simultaneously measuring neurochemical biomarkers and electrophysiological data to enhance our understanding of human disease mechanisms. This review compares microdialysis, microperfusion, and implanted EEG probe architectures and performance parameters. Invasive consequences of probe implantation are also investigated along with the functional impact of biofouling. Finally, previously developed microdialysis electrodes and microperfusion electrodes are reviewed in preclinical and clinical settings. Critically, current and precedent microdialysis and microperfusion probes lack the ability to collect neurochemical data that is spatially and temporally coincident with EEG data derived from depth electrodes. This ultimately limits diagnostic and therapeutic progress in epilepsy and glioma research. However, this gap also provides a unique opportunity to create a dual-sensing technology that will provide unprecedented insights into the pathogenic mechanisms of human neurologic disease. The United States Center for Disease Control (CDC) estimated in 2015 that 1.2% of the American population has active epilepsy, including approximately 3 million adults and half-a-million children . The devParamount in the immediate surgical management of both epilepsy and glioma is optimally balancing the preservation of neurocognitive function while simultaneously achieving maximal safe resection of epileptogenic or tumor-infiltrated brain tissue. However, some gliomas and epileptic foci cannot be effectively removed without unacceptable loss of neurocognitive function. In such cases, neurosurgical avenues may still be pursued for diagnostic or therapeutic procedures, spanning biopsy, electrocorticography from subdural or penetrating electrodes (ECoG), stereotactically placed brain-penetrating electrodes (sEEG), and, less frequently, focal CNS delivery of therapies via convection-enhanced perfusion or chronic implantation of stimulation-based devices to mitigate propagation of seizures.Human neurological diseases are notoriously challenging to accurately model in vitro or in preclinical models. However, collection, identification, and quantitation of extracellular biomarkers from the diseased human brain may offer the potential to enhance our understanding of pathogenic mechanisms and thereby accelerate the development of much-needed therapies . NeuronaSince biochemistry and electrical activity are the currencies of neurophysiology, applications of purpose-built technologies could extend beyond epilepsy to include patients undergoing neurosurgical procedures for stroke, traumatic brain injury, Parkinson disease, and other neurological and neurodegenerative conditions. Since effective technologies for concurrent biochemical and electrical interaction within the CNS are a prerequisite to progress in this arena, we address the key engineering-related factors of relevance to developing optimal dual-purpose devices, first addressing microdialysis and microperfusion separately before reviewing precedent efforts to combine these with invasive EEG recording electrodes.Microdialysis and microperfusion are similar biomarker collection techniques that comprise a porous partition between the surrounding tissue microenvironment and perfusate flowing within the sampling device, allowing extracellular parenchymal compounds to diffuse down a concentration gradient across the partition  Fig. 1)1). Many A primary strength of these technologies is the ability to continuously collect perfusate using minimally invasive implantation  of probeMicrodialysis techniques progressed substantially in the 1990s and have been validated repeatedly thereafter, although with decreasing frequency in the last 10–15 years, providing foundational data and reliability as a developed science –16, 28. Other benefits of microdialysis include the relative absence of tissue damage or hydrodynamic disruption after implantation is achieved. In contrast to microperfusion, microdialysis membranes can isolate tissue structures from possibly detrimental hydrodynamic forces associated with perfusate flow and therefore can induce less overall tissue damage . FinallyConversely, microperfusion avoids the need for the dialysis membrane and maximizes biomarker collection via the use of open collection ports in the indwelling catheter. The indiscriminate pore structure of the microperfusion probe circumvents the reduced recovery rates associated with microdialysis membranes Fig.  and overAs described, both microdialysis and microperfusion have distinct advantages Table . MicrodiThe most critical design parameter in the microdialysis system is the semipermeable membrane Fig. , responsA plethora of membrane materials have been developed and successfully implemented in preclinical and clinical studies, including poly(carbonate-ether), cuprophan, and poly(acrylonitrile) . Other mApart from material, the total area where diffusion occurs as well as inlet/outlet positioning are also critical determinants of performance for both microdialysis and microperfusion. Larger diffusion areas improve recovery rate by naturally providing more surface area for concentration fluctuation, although the data is variable and dependent upon target compound . MicrofaFlow rates in each technology are commonly in the 0.5–1 µL/min range , 46 but Additionally, lower volumetric flow rates decrease the probability of tissue damage induced by impinging fluid forces as the perfusate passes the collection site . ComputaRR, and is dominated by the flow rate, The concentration of the targeted compound in the collected solution does not necessarily represent the concentration of that same compound in the tissue, because diffusion varies depending on the surrounding tissue properties and flow properties internal to the probe . Despitequations :\documenR, can be expressed as a series function:RD is the resistance of the dialysate, RM is the resistance of the membrane, and RE is the resistance of the surrounding tissue or environment [Total resistance to diffusion, ironment . In vivoironment . In vitrironment . Therefomentclasspt{minimaironment  are togeThe no-net-flux calibration method exploits repeated dialysate measurements while adding and incrementing the concentration of the targeted substance of choice into the dialysate inlet . MeasureThe reverse dialysis method, termed retrodialysis, internal standard, or delivery method, has been well validated in vivo , 27, 46.A critical point for this calibration method is that diffusion is only equivalent in both directions if the initial concentration of the analyte in the peri-probe tissue prior to calibration is zero . The preCDial is the measured concentration of the analyte, Co is the concentration of the substance around the probe, r is the mass transfer coefficient, A is the surface area of the membrane, and F is the experimentally defined flow rate [The extrapolation to zero method is executed by plotting the flow rate against the measured in vivo concentration over several different incremental flow rates . The extlow rate . Due to low rate .These calibration methods, although more popular in microdialysis, are also applicable to microperfusion, showing less than 10% variability between extrapolation to zero method and the no-net-flux method .Published literature indicates several critical parameters to reduce tissue damage during probe insertion and post-implantation that will improve strategies for successful biomarker sampling. However, despite efforts to eliminate tissue damage, the nature of probe implantation is such that damage can only be mitigated, not prevented , 25. ForFactors that skew the collection of biomarkers due to tissue damage are the size , 57, geoBiosensor failures can also stem from biocompatibility failures such as biofouling . The memNot only do the invasive consequences of probe implantation and biofouling affect the efficacy of biomarker collection, these factors may also impact underlying pathogenic mechanisms directly. Tissue sites surrounding microdialysis probes have shown elevated production of cytokines such as interleukin-1β (IL-1β) within one hour of implantation , IL6 witThere are several design and performance parameters to consider in neural depth electrode development. Depth electrodes are categorically broken into microelectrode designs capable of capturing EEG data from single neurons , 69 and Microelectrodes have benefits in precision data collection and the reduction of tissue response to implantation, just as miniature microdialysis and microperfusion probes reduce inflammatory responses that correlate with probe size . Tissue Conversely, macroelectrodes can provide similar data in terms of cellular spiking by delineating areas of cellularity , 86. MacIn terms of electrode performance, corrosion of electrodes and concomitant surface area reduction increases current densities and chemical reactivity and therefore skews data over time . RegardlModifications of the insulation surrounding the electrodes can also improve electrode functionality and biocompatibility. Specifically, lowering polyimide curing temperatures leads to more stable chronic data collection . Surfacein vivo sensing challenge of simultaneously measuring changes in biomarkers during passive electrophysiological recording or active electrical stimulation of brain tissue [in vitro or in non-human animal models, with minimal evidence obtained from clinical studies to date [Several microdialysis and microperfusion electrodes have been designed and tested to tackle the n tissue , 95–105.n tissue , 106–114n tissue –101, 103n tissue –121. The to date , 123.The default setup for concurrently measuring neurochemicals along with EEG is commercially purchased microdialysis probes located separately from implanted stimulating/recording electrodes , 106–114To minimize the spatial gaps between the sEEG probe and the neurochemical sampling probe, several studies have employed a commercially available microdialysis cannula epoxied to an empty guide cannula to permit simultaneous collection of small molecules in proximity to a concentric bipolar electrode in the rodent caudate and nucleus accumbens targeting GABA and other amino acids , 119–121Clinically, cerebral microdialysis has been performed with a hybrid invasive EEG electrode in neurosurgical patients with medically intractable epilepsy to delineate resectable seizure foci. In a seminal study from During and Spencer , a microA US patent from 1995  claims aA commercial version of the intra-lumen microdialysis-depth electrode system, employed by Spencer et al. , was manWhile microdialysis and microperfusion are closely related in construction, configuration, and purpose, the benefits of microperfusion generally outweigh the advantages of microdialysis. Microperfusion provides a simpler design allowing end-users to avoid limited commercial options for MWCO, thereby yielding versatility in probe geometry and perfusion port design through in-house fabrication that often requires minimal overhead equipment. Microperfusion casts a wider biomarker net and supports improved sensitivity to large compounds that have difficulty diffusing across the high-resistance dialysis membrane , but canin vivo using the no-net-flux method if time permits, or via the extrapolation to zero method, but likely not the reverse dialysis method, assuming that the compound of interest exists in the peri-probe tissue. Despite the utility of these calibration techniques under well-controlled conditions, application of these methods within the dynamic clinical setting is challenging. This is due to insufficient time to vary parameters, limited time to achieve steady-state conditions, and concerns regarding introduction of exogenous materials into the host tissue. In practice, such conditions cannot be achieved in patients and current calibration methods are inadequate to improve estimates under non-steady state conditions. Aside from the relative fluctuations in collected biomarkers, application of microdialysis and microperfusion under real-world clinical conditions – especially within the context of the CNS – will require the development of novel techniques to provide accurate estimates of absolute tissue concentrations. Both microdialysis and microperfusion provide effective ways to measure biomarker fluctuations over the course of time of epileptic events but fall short in terms of measuring absolute concentrations in the clinical setting. Furthermore, the relationship between neurochemical fluctuations and invasive electrophysiology  measurements is experimentally characterized by a lag due to the time that it takes compounds to diffuse through semipermeable brain tissue [Calibration is required and should be conducted n tissue  as well n tissue , matheman tissue  and for n tissue , 133. Adn tissue . Future Inflammatory and reactive responses to the probe remain problematic despite the reduction of biofouling in the microperfusion realm . While tIn terms of invasive electrophysiology electrodes, penetrating EEG electrodes are largely broken down into microelectrodes and macroelectrodes. Microelectrodes provide enhanced ability to monitor localized neuronal populations and single neuron activity compared to large-area macroelectrodes that sample the collective activity of large neuronal populations , 136. MaSeveral studies have been conducted to collect neurochemical biomarkers concurrently with invasive EEG data. Incorporating multiple probes, especially commercially purchased probes, can expedite experimental progress at the expense of collecting data that overlooks localized neurochemical fluctuations. Most of these published experiments have been conducted in rodents while utilizing commercially-available microdialysis probes in separate locations from the brain-penetrating electrodes , 106–114Clinically, the studies mentioned above utilized microdialysis in conjunction with invasive electrophysiology electrodes to correlate seizure activity to GABA, glutamate, and other small molecules , 123. ThDespite the plethora of previously developed and published microdialysis electrodes and microperfusion electrodes, no known microperfusion electrode system has been developed for preclinical or clinical use. Thus, there is a significant unmet need for the development of novel microperfusion electrode dual-sensing probe designs that can concurrently collect large neurochemical biomarkers such as cytokines, metabolites, cell-free DNA, extracellular vesicles, and microRNA at the precise location of brain electrophysiology measurements  monitoring. Coupled with high resolution electrophysiological monitoring spatially coincident with microperfusion ports, such a system will allow unprecedented insight into the dynamic environmental changes associated with neural function and dysfunction.in vitro settings. The lack of adequate techniques for collecting neurochemical compounds such as cytokines and other large molecules, the absence of clinical in vivo validation testing of microperfusion electrodes, and the existence of large spatial separations between electrodes and perfusate collection locations results in a significant unmet gap between the available technology and the devices needed to support novel advances in epilepsy and GBM research.Microdialysis and microperfusion are sampling techniques that may provide unique insights into neurologic disease microenvironments, especially when paired with invasive electrophysiology recordings. While both microdialysis and microperfusion have separate advantages, far more work has been done to validate preclinical and clinical use of microdialysis, leaving microperfusion as a relatively unexplored technology that may ultimately have more clinical potential and advantages. Calibration of both approaches is well documented and best conducted through the No-Net-Flux method, if conditions permit. Concerns regarding the tissue inflammatory response and biofouling may be addressed with new geometries and the development of active, rather than passive, anti-inflammatory properties built into the probe materials. Multiple previous endeavors have incorporated invasive EEG and neurochemical sampling into localized areas by fastening separate probes or designing one-off microfluidic electrodes to target dopamine and other neural biomarkers, mostly in small animal experiments and


Hydrogels are commonly used for the 3D culture of musculoskeletal cells. Sulfated hydrogels, which have seen a growing interest over the past years, provide a microenvironment that help maintain the phenotype of chondrocytes and chondrocyte-like cells and can be used for sustained delivery of growth factors and other drugs. Sulfated hydrogels are hence valuable tools to improve cartilage and intervertebral disc tissue engineering. To further advance the utilization of these hydrogels, we identify and summarize the current knowledge about different sulfated hydrogels, highlight their beneficial effects in cartilage and disc research, and review the biofabrication processes most suitable to secure best quality assurance through deposition fidelity, repeatability, and attainment of biocompatible morphologies. Hydrogels are three-dimensional (3D), chemically or physically crosslinked polymer networks with hydrophilic groups that allow for high water-absorbing capacity. Their high water content results in physicochemical characteristics that are comparable to many tissues, including the intervertebral disc (IVD) and cartilage, thus constituting promising biomaterials for tissue repair and regeneration .Furthermore, hydrogels are commonly used as drug delivery systems by providing spatial and temporal control over the release of therapeutics, predominantly via diffusion ; althougAlthough synthetic and natural hydrogels are heavily researched, natural hydrogels such as hyaluronan, heparin, and alginate are commonly favored due to their biocompatibility, biodegradability, non-immunogenicity, and overall resemblance to the extracellular matrix (ECM) of connective tissues. However, despite their similarity to the IVD and cartilage, these hydrogels lack one crucial feature that originates from the high proteoglycan content of these tissues: the presence of carboxyl and sulfonic acid groups that create the IVD/cartilage-specific negatively charged ECM microenvironment . Most reFirstly, the negative charges of sulfated hydrogels provide chemical cues to embedded cells that help induce and maintain a chondrogenic/disc-like phenotype and thus complement other signals arising from the simulated natural environment of biomimetic materials . Details throughout this article demonstrate that 3D culture models based on sulfated hydrogels are superior to non-sulfated hydrogels and outperform classical 2D culture models. Phenotypic alterations of chondrocytes and IVD cells in vitro, a process also commonly termed dedifferentiation, involve increased expression of the fibroblast marker collagen type I and a concomitant reduction in the expression of collagen type II and proteoglycans .Secondly, the negative charges of sulfated hydrogels furthermore offer the possibility to create a sustained release of positively charged therapeutic agents through electrostatic interaction ,7 and haThis review summarizes the state-of-the-art of sulfated hydrogels used in IVD and cartilage research. First, we focus on demonstrating their suitability as drug delivery systems , with su3/DMF complex.Over the past years, sulfated hydrogels have become increasingly popular in the cartilage and IVD research community due to their ability to support the chondrogenic phenotype and their potential for sustained drug delivery, as described in more detail below. However, their use either requires isolation from natural tissues or synthesis in the laboratory. While chondroitin sulfate (CS) and (to a lesser degree) heparan sulfate (HS) can be isolated from tissue, alginate, hyaluronan, and heparin are commonly used as base materials for the introduction of sulfate groups. Sulfated alginate is typically synthesized through the reaction of chlorosulfonic acid in formamide, whereby the degree of sulfation can be reproducibly tuned by altering the chlorosulfonic acid concentration . SulfatiMoreover, if sodium hyaluronate is first chemically modified to include methacrylate groups prior to sulfation, free-radical initiated polymerization of the resulting sulfated hyaluronan macromer forms a crosslinked hydrogel . In a liCS is an anionic linear polysaccharide composed of sulfated disaccharide repeating units with 1–3 D-glucuronic acid and N-acetylgalactosamine linkages. It is most commonly found in connective tissue throughout the body, including the IVD and cartilage . It thusThus far, the use of CS for drug delivery has predominantly focused on nano-/microparticles (or composites thereof). Different approaches have been followed, including loading the drug into CS particles, coating particles of other origin with CS (with the drug being incorporated in the coating), and drug-loaded particles of other origin immersed in a CS hydrogel . FollowiRecent studies have demonstrated the suitability of  CS hydrogels to provide phenotypic support of chondrocytes and ultimately improve cartilaginous ECM accumulation. For instance, catechol chemistry inspired by marine mussels has been widely applied in tissue engineering due to its ability to form covalent crosslinks on the surface of any material, even wet tissue. Moreover, a catechol-functionalized CS-hydrogel was shown to create a native cartilage-like microenvironment and provide chondro-inductive signals, thus enhancing GAG deposition as well as increasing chondrogenic markers such as SOX9 and collagen type II in human adipose stem cells . SimilarCS-based hydrogels have also been investigated as a potential 3D system for IVD regeneration. For example, in a recent study conducted by Borrelli et al. (2020), an injectable biomaterial comprising functionalized CS and decellularized ECM from bovine NP tissue was developed. Interestingly, the presence of CS in this biomaterial appears to be essential to promote higher amounts of sulfated GAG deposition . In addiHS is a linear polysaccharide composed of repeating disaccharides of 1,4-linked uronic acid and glucosamine residues . HS is fDespite the fast degradation and the potential need for modifications, HS is highly sulfated and consequently has a strong binding affinity to growth factors from the FGF and TGF families , thus, mDue to their high degree of sulfation, heparin-based hydrogels have been widely studied in cartilage tissue regeneration . These sSA is created through modification of alginate, a natural polysaccharide extracted from algae that consists of linear chain structures of 1–4 linked mannuronic acid (M) and l-guluronic acid (G). Alginate has been widely used in biomedical research due to its high biocompatibility, low toxicity, low cost, ease of gelation, structural similarity to the ECM, and ability to modify and control drug release through alterations of the crosslinking process . AlginatThese changes will inherently alter the mechanical cues arising from the SA scaffold, which in turn may affect MSC differentiation . HoweverDespite these challenges, SA has been used widely for the culture of chondrocytes, with the overall goal to facilitate cartilage regeneration. Multiple papers have shown that SA generally increases cell proliferation, cell adhesion, and collagen type II expression, which creates a more hospitable physiological environment for chondrocytes. For example, bovine chondrocytes embedded in calcium-crosslinked SA strongly increased cell proliferation and RhoA activity when compared to non-modified alginate . These fSA has furthermore been extensively used for the delivery of fibroblast growth factor (FGF) but can also be applied to other drugs and growth factors. For example, chondrocytes were co-embedded with FGF in SA with different sulfation degrees  to determine FGF signaling-mediated proliferation and ECM synthesis dependent on FGF release profiles. Highly sulfated alginate (high-SA) had higher initial FGF2 encapsulation (as demonstrated by significantly increased proliferation), as well as better FGF2 retention. Notably, after two weeks, high-SA still contained about 40% of the initially loaded FGF2, while only 10–20% were maintained in the control hydrogels, resulting in significantly more collagen type II and proteoglycan deposition with high-SA .SH is based on hyaluronan, a high molecular weight GAG composed of a linear polysaccharide with disaccharide repeats of D-glucuronic acid and N-acetyl-D-glucosamine. It has been widely used for drug delivery, tissue engineering applications, and viscosupplementation due to its biocompatibility, non-immunogenic, and shock-absorption properties . HoweverDifferent types of methacrylated and, thus, photo-crosslinkable SH have been investigated for drug delivery. SH has previously been used to promote wound healing through the incorporation of Epidermal Growth Factor (EGF), whereby hyaluronan/collagen-based hydrogels supplemented with acrylated SH proved to have a prolonged EGF release and increased effectiveness on dermal fibroblasts when compared to hydrogels that did not contain hyaluronan . NotablyMSCs have also been extensively investigated for the treatment of IVD degeneration, and results by Peroglio et al. showed that their differentiation into a disc-like phenotype could be achieved in vitro and ex vivo when incorporated into a hyaluronan-based thermoreversible hydrogel (HA-pNIPAM). Notably, NP cell re-differentiation in this hydrogel was supported, as evidenced by upregulation of aggrecan and downregulation of collagen type I as well as ECM accumulation . Thus, tAside from different casting methods and bead formation, biofabrication techniques are increasingly used for sulfated hydrogels as the clinical application of implantable scaffolds requires precise sizes and shapes to match that of the defected area. 3D bioprinting, electrospinning, and electrospraying are standard biofabrication techniques  used to 3D bioprinting is a layer-by-layer material deposition technique that allows for control over scaffold pore size, geometry, and porosity. These structural characteristics are essential for promoting cell viability, proliferation, differentiation, and migration. However, bioinks play an integral role in the deposition quality that can be achieved with this technique. For this purpose, researchers are investigating CS and SA bioinks for the fabrication of 3D bioprinted cartilage scaffolds. Abbadessa et al. 3D-printed methacrylated CS scaffolds with suitable mechanical and biological properties and tunable porosity to achieve chondrogenesis . Due to −1. As compared to pure alginate, SA combined with PVA demonstrated improved electrospinnability [Electrospinning is an electrohydrodynamic biofabrication method capable of yielding polymer-based scaffolds with high surface area to volume ratios and high porosity. The high surface area to volume ratios enhance the efficiency and release of loaded drugs and increase the scaffolds’ mechanical properties . Daemi enability . Delgadonability .2+ delivery [Lastly, the electrospraying of sulfated hydrogels can be used to fabricate nano- and microparticles as drug delivery systems for positively charged therapeutics. Due to their high fluid retention and absorption capacity, hydrogels allow for a sustained and controlled release of drugs. Researchers have successfully investigated the sustained and controlled drug release of therapeutics from alginate and CS electrosprayed microbeads. Khanal et al. encapsulated magnesium sulfate in alginate through electrospraying for Mgdelivery . In addidelivery .Although sulfated hydrogels hold great promise in cartilage and IVD tissue engineering research concerning sustained drug release and phenotypic support, high costs and batch-to-batch variability associated with isolation from tissues are major limitations. Hence, extensive efforts have been made to create sulfated hydrogels in the laboratory, allowing for better control over sulfation degrees and tunability of their properties. Common limitations of sulfated hydrogels are inferior mechanical properties and fast degradation. With the ultimate goal of sulfated hydrogel research being clinical translation, future research will have to focus on circumventing these challenges. However, promising first approaches have already been developed, including the use of co-polymers or alternative crosslinking methods (including photo-crosslinking), as well as fiber- or nanotube-based reinforcement of sulfated hydrogels.Furthermore, future activities will likely target the incorporation of positively charged therapeutics other than growth factors. Slow-release systems for Etanercept, a positively charged drug used to treat rheumatoid arthritis and other inflammatory diseases, could be investigated in the future. Other positively charged agents including Hydrocortisone (anti-inflammatory drug) and Lidocaine (anesthetic) have already been tested using different types of hydrogels and could next be embedded in sulfated hydrogels. Lidocaine, for example, is occasionally used for intra-articular injections but holds a high risk of toxicity that could be limited through controlled release by using sulfated hydrogels as a carrier material.Lastly, upon development of improved sulfated hydrogels, their use as bioinks will require the identification of 3D bioprinting process parameters that help replicate the morphology of the envisioned tissues. As detailed before, electrospraying and electrospinning can be used to generate stand-alone structures. However, additional consideration should be made to incorporating electrospinning/-spraying in the process of 3D bioprinting to ultimately fabricate hybrid tissue engineering constructs for cartilage and IVD regeneration. Such constructs may hold promise to better address the mechanical and morphological characteristics of native cartilage and the IVD.In summary, sulfated hydrogels are promising biomaterials that may help overcome some of the current limitations in cartilage and IVD tissue engineering, repair, and regeneration, especially if sophisticated  biofabrication methods are employed.


Moreover, we describe interlinkages between these pathophysiological mechanisms as well as “vicious cycles” involving cytokines and inflammation that may contribute to explain the chronic nature of these illnesses. This paper summarizes and expands on our previous publications about the relevance of findings from critical illness for ME/CFS. New knowledge on diagnostics, prognostics and treatment strategies could be gained through active collaboration between critical illness and ME/CFS researchers, which could lead to improved outcomes for both conditions.We propose an initial explanation for how myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) could originate and perpetuate by drawing on findings from critical illness research. Specifically, we combine emerging findings regarding (a) hypoperfusion and endotheliopathy, and (b) intestinal injury in these illnesses with our previously published hypothesis about the role of (c) pituitary suppression, and (d) low thyroid hormone  Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating illness that affects millions of people worldwide  , 2. Impaacute phase of critical illness—in the first hours or days following severe trauma or infection; and the chronic or prolonged phase—in the case of patients who survive the acute phase but for unknown reasons do not start recovering and continue to require intensive care  for as of yet unexplained reasons  hypoperfusion and endotheliopathy, and (b) intestinal injury in these illnesses with our previously published hypothesis about the role of (c) pituitary suppression, and (d) low thyroid hormone r ME/CFS –20 and br ME/CFS . The genIn the following sections we describe four central pathophysiological mechanisms in critical illness, including their relationship to inflammation. We also provide initial arguments for suggesting that similar mechanisms may underlie ME/CFS. Readers are referred to our prior publications for additional details about these mechanisms in critical illness (including heat stroke) and possible lessons for understanding ME/CFS –20.It has long been suggested that inadequate oxygen circulation is central to critical illness . SpecifiEndothelial dysfunction appears to occur in parallel with circulation disturbances during critical illness. Probable drivers of distortions in the structure and function of endothelial lining  are cytokines , inflammWe propose that similar alterations of the vascular system in response to a physical, infectious and / or emotional stressor  may also contribute to explain the emergence of ME/CFS. This is consistent with recent hypotheses describing vasoconstriction in muscle and brain as a principal element of ME/CFS –46, and Critical illness researchers have found profound intestinal alterations within hours following a physiological insult: a dramatic shift in the composition and virulence of intestinal microbes –59, an eCritically, this intestinal injury may lead to bacterial translocation from the gut into circulation  and/or the formation of toxic gut-derived lymph , 60. ThiWe propose that the sequence during critical illness—from splanchnic hypoperfusion to hypoxia, redox imbalance, altered gut microbiome, intestinal injury, gut-related endotoxemia, pro-inflammatory cytokines and systemic inflammatory—may also contribute to explain the emergence of ME/CFS following a physiological insult. Our proposal is in alignment with others' findings that intestinal injury and resulting inflammation are central to ME/CFS –81 and cprolonged critical illness the pituitary's pulsatile secretion of tropic hormones (unexpectedly) remains suppressed.Almost immediately after a physiological insult, endocrine axes experience profound alterations considered a vital response to severe stress or injury to allow for a shift in energy and resources to essential organs and repair –97. Wherprolonged critical illness continues to be debated. Inflammatory pathways likely play a role irrespective of the nature of the original injury or infection. For example, cytokines increase the abundance and affinity of glucocorticoid receptors (GR) at the level of the hypothalamus / pituitary, thereby enhancing the negative feedback loop of the hypothalamic-pituitary-adrenal (HPA) axis, and consequently suppressing pituitary release of adrenocorticotropic hormone (ACTH) (active thyroid hormone (T3), thereby enhancing the hypothalamic-pituitary-thyroid (HPT) axis' negative feedback loop and consequently suppressing pituitary secretion of thyroid stimulating hormone (TSH) irrespective of circulating thyroid hormone concentrations  , 98. Simtrations –101. Cyttrations , 103 conecretion .pulsatile pituitary secretions has important implications for the autonomic nervous system, metabolism, and the immune system. Without sufficient pulsatile stimulation by ACTH, adrenal glands begin to atrophy (pulsatile pituitary production of growth hormone (GH) leads to an imbalance between catabolic and anabolic hormones, resulting in loss of muscle and bone mass, muscle weakness, and changes in glucose and fat metabolism  relative to the inactivated thyroid hormone (rT3)  —consider tissues , 170.acute phase of critical illness, it is increasingly seen as maladaptive and hampering the recovery of patients in the case of prolonged critical illness  create the condition necessary for persistent inflammation which depresses the activity of thyroid hormones during critical illness  could contribute to explain the persistence of chronic ICU and ME/CFS.There are several pathways linking the activity of the pituitary with that of thyroid hormones. Firstly, GH secreted by the pituitary co-regulates the activity of the deiodinase enzyme (D3) responsible for the conversion of thyroid hormones into inactive forms  , 201. Retrations , 198. In illness –152. In ecretion , 203. Infunction in endotheliopathy in ME/CFS, including as it relates to the new finding that plasma from ME/CFS patients inhibits eNOS and NO production in endothelial cells  activate the endothelial nitric oxide synthase (eNOS) responsible for nitric oxide (NO) production , which ial cells . Relatedal cells , 210; thal cells .function and inflammation appear to collectively affect mitochondrial activity in critical illness   , 211–213protein) . Some haprotein) , 213. Thprotein) –216. Lowprotein) .function—may provide therapeutic avenues for ME/CFS  is acknowledged for support.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.


Recent review articles provided an extensive collection of studies covering many aspects of format thought disorders (FTD) among their epidemiology and phenomenology, their neurobiological underpinnings, genetics as well as their transdiagnostic prevalence. However, less attention has been paid to the association of FTD with neurocognitive and functioning deficits in the early stages of evolving psychosis. Therefore, this systematic review aims to investigate the state of the art regarding the association between FTD, neurocognition and functioning in the early stages of evolving psychotic disorders in adolescents and young adults, by following the PRISMA flowchart. A total of 106 studies were screened. We included 8 studies due to their reports of associations between FTD measures and functioning outcomes measured with different scales and 7 studies due to their reports of associations between FTD measures and neurocognition. In summary, the main findings of the included studies for functioning outcomes showed that FTD severity predicted poor social functioning, unemployment, relapses, re-hospitalisations, whereas the main findings of the included studies for neurocognition showed correlations between attentional deficits, executive functions and FTD, and highlighted the predictive potential of executive dysfunctions for sustained FTD. Further studies in upcoming years taking advantage of the acceleration in computational psychiatry would allow researchers to re-investigate the clinical importance of FTD and their role in the transition from at-risk to full-blown psychosis conditions. Employing automated computer-assisted diagnostic tools in the early stages of psychosis might open new avenues to develop targeted neuropsychotherapeutics specific to FTD.The online version contains supplementary material available at 10.1007/s00406-021-01295-3. HoweverRecent review articles provided an extensive collection of studies covering many aspects of FTD among their epidemiology and phenomenology, their neurobiological underpinnings, genetics, their neurological correlates, as well as their transdiagnostic prevalence. , 11, 12 Therefore, we conducted a systematic review regarding the state of the art of the association between FTD, functioning outcomes and neurocognitive impairments in the early and prodromal stages of psychotic disorders. We summarized the main findings of the included studies providing evidence related to the early diagnostic and prognostic potential of FTD in association with functioning and neurocognition. Furthermore, we discussed possible reasons for the paucity of studies investigating the clinical relevance of FTD in early psychosis and presented new perspectives for potential future investigations on FTD with the help of modern computational analytical techniques that might serve as computer-assisted early diagnostic tools. We concluded with an outlook for future research targeting the potential preventive and predictive role of FTD in psychosis trajectories.We conducted two separate systematic literature searches; (i) association between FTD and functioning outcomes and (ii) association between FTD and neurocognition, both in the early and prodromal stages of psychosis, by following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  statemenFor the literature search regarding the association between FTD and functioning outcomes, we used the following search terms combination:  AND (child OR adolescent OR young adult) AND (psychosis OR “early-onset schizophrenia” OR “first episode psychosis” OR “recent onset” OR “high risk” OR prodrom) AND  AND (child OR adolescent OR young adult) AND (psychosis OR “early onset schizophrenia” OR “first episode psychosis” OR “recent onset” OR “high risk” OR prodrom) AND (neurocognition OR neurocognitive or cognition or cognitive or neuropsycho OR memory OR executi OR process).Articles had to meet the following inclusion criteria: (1) reported statistically significant associations between FTD and either functioning outcomes, or neurocognitive measures; (2) did include high-risk groups  such as Based on these criteria, as a first step, two authors have screened the titles and abstracts separately in three different databases , and the results of included and excluded articles were discussed among co-authors. Based on the abstracts screening, records for which an inclusion or exclusion decision could not be taken were listed. Thus, in a second round, the same two authors have screened the full texts of these articles to check their eligibility for inclusion. Records for which both authors agreed on their inclusion were then proved for their eligibility again through further full-text investigation.The procedure used for the selection of studies for each literature search is reported in Figs. . [We screened 106 studies in total and included 8 of them. These studies were included due to the presence of significant associations between FTD in adolescents or young adults and their reports, and functioning outcomes measured with different scales. Overall, the included studied showed that higher severity of FTD predicted poor social functioning, unemployment, relapses, re-hospitalisations. Three studies, namely Harrow. , Marengo. , reporte. [. [Kotov et al.  intervie. [.  assessed. [.  and spee. [. [. [Roche et al.  evaluate. [.  tested t. [. [.  examined. [. [.  and lingWe screened 106 studies in total and included 7 of them. These studies were included due to the existence of significant associations between FTD in adolescents or young adults, and neurocognition. The study from Minor et al.  was incl. [Minor et al.  explored.  and spee. [Nuechterlein et al.  investig. [. [Xu et al.  reported.  subdivid. [.  evaluate. [. [Caplan et al.  examined. [.  assessed. [Ilonen et al.  explored. investigated in a few studies the course of psychosis after the first episode with a focus on FTD and formalized the following questions already in 1986 [Our literature survey showed that there was an increased interest for FTD in the early stages of psychosis from the late 70 s to late 80 s. Harrow et al in 1986 : “Is tho in 1986 , 11, 12  in 1986 , 51 Furt in 1986 , and neg in 1986  and over in 1986 .. [. [. [. [. [. [Moreover, given the significant associations between FTD, neurocognition and functioning impairments here reported, and given that these impairments very often precede the onset of full-blown psychosis and persist after the acute phase is resolved , 25, we .  reported. [.  found th. [. [.  found th[. [. [.  found th[. [. [.  found th[. [. [.  found a [. [. [.  showed t. [. [. [Consistently with findings from our review, disorganization seems to be the most notable FTD symptom predicting transition to psychosis in high-risk groups. The POC was another FTD symptom that commonly associated with social and role functioning as well as with transition to psychosis. Not only these findings are consistent with the associations between FTD, functioning and neurocognition we have discussed, but they also further speak in favor of the clinical relevance of FTD in the pathophysiology of psychosis and encourage future research to longitudinally investigate their role in the clinical trajectories of individuals at-risk for psychosis. A potential way to methodologically accomplish this future research direction could be represented by further efforts in designing computational methods that might help clinicians in evaluating risk categories of patients at their first presentation also through the presence of FTD, especially in the disorganization domain. For example, some machine learning and computational pattern recognition studies provided promising results in automated computational speech or text analyses to improve the clinical utility and objective quantifiability of FTD. The earlier effort has appeared already in 2009 in the literature: Strous et al.  showed t. [.  reported. [.  Furtherm. [. [.  showed t. [. [. .Nevertheless, overall, these finding and those from our systematic review would support the relevance of FTD for several psychosis-associated outcomes, from neurocognition, to functioning, to transition to psychosis, and that applying cutting-edge methodological techniques to further FTD investigations may be a promising way to deliver clinicians with time-efficient and objective clinical tools supporting diagnostic and prognostic procedures. Many more future studies are warranted.This review has some limitation. Mainly, studies have employed different assessment strategies to evaluate FTD, neurocognition and functioning. As a matter of fact, this high variability did not allow us to conduct any direct comparative analysis. Therefore, the results in this review can be only partially interpreted. We also acknowledge that our results are limited only to the published studies and that there was no study excluded due to non-significant associations. Therefore, a possible bias through studies showing non-significant associations between FTD, neurocognition and functioning could not be excluded.The reviewed studies showed that FTD severity is significantly associated with poor social functioning, unemployment, relapses of psychosis and re-hospitalisations, as well as transition to psychosis in high-risk groups. The results also showed significant associations between FTD and attention performance, executive functions, and verbal abilities. Machine learning algorithms show good potential for understanding the prognostic value of FTD in the risk trajectories of psychosis and encourage the development of computer-assisted early diagnostic tools targeting FTD, especially disorganization. Further studies taking advantage of the acceleration in computational psychiatry using methods such as unsupervised, supervised machine learning algorithms, deep learning techniques, natural language processing, sound, and rhythm analyses in records of patients’ clinical evaluations, would hopefully allow researchers to develop novel clinical markers for FTD. Such automated and time friendly computer-assisted diagnostic tools could give researchers and clinicians the chance to re-investigate the clinical importance of FTD starting from high risk and early stage of psychosis, and to fill the gap in the literature that might open new avenues to develop targeted neuropsychotherapeutics specific to FTD.Supplementary file1 (DOCX 16 KB)Below is the link to the electronic supplementary material.


This review summarizes current applications of advanced diffusion magnetic resonance imaging (MRI) throughout the glaucomatous visual system, focusing on the eye, optic nerve, optic tract, subcortical visual brain nuclei, optic radiations, and visual cortex. Glaucoma continues to be the leading cause of irreversible blindness worldwide and often remains undetected until later disease stages. The development of non-invasive methods for early detection of visual pathway integrity could pave the way for timely intervention and targeted treatment strategies. Principles of diffusion have been integrated with MRI protocols to produce a diffusion-weighted imaging modality for studying changes to tissue microstructures by quantifying the movement of water molecules in vivo. The development and applications of diffusion MRI in ophthalmology have allowed a better understanding of neural pathway changes in glaucoma. The feasibility of translating diffusion MRI techniques to assess both humans and experimental animal models of glaucoma and other optic neuropathies or neurodegenerative diseases is discussed. Recent research focuses on overcoming limitations in imaging quality, acquisition times, and biological interpretation suggest that diffusion MRI can provide an important tool for the non-invasive evaluation of glaucomatous changes in the visual system.Glaucoma is a group of ophthalmologic conditions characterized by progressive retinal ganglion cell death, optic nerve degeneration, and irreversible vision loss. While intraocular pressure is the only clinically modifiable risk factor, glaucoma may continue to progress at controlled intraocular pressure, indicating other major factors in contributing to the disease mechanisms. Recent studies demonstrated the feasibility of advanced diffusion magnetic resonance imaging (dMRI) in visualizing the microstructural integrity of the visual system, opening new possibilities for non-invasive characterization of glaucomatous brain changes for guiding earlier and targeted intervention besides intraocular pressure lowering. In this review, we discuss dMRI methods currently used in visual system investigations, focusing on the eye, optic nerve, optic tract, subcortical visual brain nuclei, optic radiations, and visual cortex. We evaluate how conventional diffusion tensor imaging, higher-order diffusion kurtosis imaging, and other extended dMRI techniques can assess the neuronal and glial integrity of the visual system in both humans and experimental animal models of glaucoma, among other optic neuropathies or neurodegenerative diseases. We also compare the pros and cons of these methods against other imaging modalities. A growing body of dMRI research indicates that this modality holds promise in characterizing early glaucomatous changes in the visual system, determining the disease severity, and identifying potential neurotherapeutic targets, offering more options to slow glaucoma progression and to reduce the prevalence of this world’s leading cause of irreversible but preventable blindness. Glaucoma is one of the leading causes of irreversible blindness worldwide, with current estimates indicating 1 in 25 people aged 40–80 years old live with glaucoma . GlaucomOptic nerve damage and resulting vision loss are, as of now, irreversible and the probable multiple etiologies make glaucoma difficult to treat. The effects of glaucoma-related damage are further exacerbated by age-related nerve fiber loss, estimated at 4000–5426 nerve fibers per year in the non-diseased state . TreatmeNon-invasive medical imaging offers the potential to resolve some gaps in our understanding of glaucomatous damage in the visual system. The microscopic nature of optic nerve damage presents a challenge, mainly with most conventional medical imaging modalities having resolutions on the order of millimeters while optic nerve axons average about a micrometer in diameter ,22. SignResearchers and ophthalmologists are turning to more advanced MRI techniques to image beyond the anatomical structure of the optic nerve and explore biomarkers of early glaucomatous states, potentially before the onset of detectable clinical changes . MRI is MRI is often preferred over other imaging modalities for brain assessments due to no-depth limitation, non-ionizing radiation, and the availability of various image scanning protocols that can result in a range of resolutions and specificity. For instance, functional MRI and structural MRI are effective in deciphering abnormalities in the interactions between the eye and brain’s visual system in disease states ,39,40. IDespite prominent investigations of macro structures in the central nervous system of glaucoma patients, challenges remain when imaging structures on the order of microns, such as the optic nerve partly due to the trade-off between spatial and temporal resolution in MRI. Susceptibility to image artifacts is also increased by natural eye movements as well as partial volume effects from the surrounding cerebrospinal fluid and neighboring structures . Rather The combination of glaucoma disease prevalence and global burden, its degenerative nature, and the challenges of detecting changes in optic nerve integrity and beyond triggered research into dMRI applications within the visual pathway of both human patients and glaucoma animal models. This review is centered on dMRI analysis of abnormalities of the eye and the visual pathway in glaucoma. Additionally, novel research methods employing dMRI variants for improving specificity for glaucoma interpretation are discussed. We expect that this review can better inform biologists, radiologists, and clinicians alike of the prospectives of dMRI for non-invasive and longitudinal understanding of glaucomatous neurodegeneration and neurotherapeutic options in both basic research and clinical translations.This review documented dMRI applications, as well as associated methods applicable to the study of glaucoma from journal articles and conference proceedings. Sources were found using the PubMed database. Search results for journal articles that described glaucoma applications were generated by searching “glaucoma” coupled with the following search terms: “MRI”, “Structural MRI”, “Diffusion Tensor Imaging”, “Diffusion Kurtosis Imaging”, “Diffusion Weighted Imaging”, “diffusion MRI”, “white matter tract integrity”, and “tractography”. Data were excluded if research was not quantitative and did not provide a breakdown of cohort ages and MRI field strength. Data were also excluded if the focus of the research was not on either the optic nerve, optic tract, eye, subcortical visual brain nuclei, lateral geniculate nucleus, optic radiation, or the visual cortex. Journal articles that described methods potentially translatable to glaucoma were included if the same search query showed results for study on optic neuropathies other than glaucoma or on white matter neuropathies outside the visual system on a limited basis with respect to the values and relevance to prospective research designs in glaucoma study.Conventional anatomical MRI can be useful for the detection of atrophy in the glaucomatous visual pathway, such as the progressive reduction in the human optic nerve and optic chiasm volumes toward advanced glaucoma stages ,51 Figu, and theWhile elevated intraocular pressure could result in mechanical damage to the optic nerve, mechanical damage could also occur from other phenomena. Demer et al. used structural MRI to image the optic nerve in the presence of pulling, tractional forces . QuantifThe macroscopic changes detectable by structural MRI are often observed near advanced stages of glaucoma when vision loss is already underway and neural damage is evident. Conversely, diffusion weighting can assist in detecting microstructural changes as these methods appear to be sensitive to inflammation , demyelib-values. Apparent diffusion maps, made from normalized diffusion values, are generated by acquiring data from multiple b-values during MRI scanning to quantify the diffusion weighting along one diffusion direction. The apparent diffusion coefficient (ADC) denoted D in mm2/s is calculated with the Stejskal–Tanner equation. The ADC simplifies the average trace  of the diffusion tensors at every voxel. The tensors are obtained by estimating from the ADC taken in different directions [S), the b-value, and the ADC is summarized by Equation (1), where 0S is the baseline signal intensity without diffusion weighting, i.e., at b = 0 mm2/s (b0) [Diffusion-weighted images are acquired at varying diffusion gradient strengths and timing factors that are termed diffusion weighting factors or rections . The rel2/s (b0) :S = S0·e0 images in differentiating loss of integrity in a mouse model of retinal degeneration (rd1) versus wild-type mice [Within the eyeball, DWI was used to observe age-dependent changes in mean ADC in the vitreous of healthy human subjects from toddlers to over 71 years old . The resype mice . Glaucomype mice , apart fIn the brain’s visual system, with the assumption of homogeneity in every diffusion direction, DWI on its own is more commonly applied in grey matter analysis when average diffusion values per voxel are sufficient since the grey matter is relatively isotropic . White miλ) of the diffusion tensor can be used to describe the magnitude of the diffusion and level of directionality, whereas the eigenvectors can be used to describe the orientation. Ultimately, the DTI model can derive rotationally invariant biophysical parametric maps of fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD). FA represents the variance of the eigenvalues and is a normalized value that ranges from 0 to 1, where 0 indicates isotropy [Diffusion tensor imaging (DTI) is a technique that uses diffusion-weighted scans acquired in different directions to provide microstructural information based on measures of anisotropy and restricted water diffusion within physiological structures ,69. The isotropy . AD descisotropy ,70. Tablrd1 mouse model of retinal degeneration, which was subsequently confirmed via histology, indicative of photoreceptor cell damage . The three main DKI parametric outputs include mean kurtosis (MK), radial kurtosis (RK), and axial kurtosis (AK), which are kurtosis analogs inversely proportional to the tensor parameters in DTI [Another way to conceptualize the differences between DTI and DKI models is to consider that DTI assumes homogenous water diffusion, whereas, in true biological systems, diffusion is highly heterogeneous in varying directions. DKI measures the deviation from the Gaussian distribution akin to homogenous diffusion, and this calculation can be observed in Equation (6), where is value . For theis value . Higher is value . By conts in DTI .ln(S(b)b-values for image acquisition over conventional DTI, kurtosis parameters offer higher specificity and may potentially serve as more reliable biomarkers for characterizing early glaucomatous damage in both white matter and gray matter [With an increased number of gradient directions and higher y matter . DKI meay matter  and lowey matter . Xu et ay matter  and decry matter  reportedy matter . Their ry matter . In normy matter . The lowBoth DTI and DKI are based on diffusion value transformations and offer little information regarding the (patho-)physiological processes in different structural compartments . A biophEASα) [n becomes (7) [Measures obtained using the WMTI model include tensors for the intra-axonal space (IAS) and extra-axonal space (EAS), the axonal water fraction (AWF), and the tortuosity of the extra-axonal space (αEAS) . AWF is αEAS) . In ordeomes (7) :(S(b))/, and EASD can be estimated by (9). WMTI follows the DTI model using DKI parameters, resulting in the eigenvalues of the matrices that represent the tensors corresponding to estimated physiological measurements [Here, urements .DIAS = EASD, λEAS1,, corresponds to the axial EAS diffusivity. The average of eigenvalues λEAS2, and λEAS3, corresponds to the radial EAS diffusivity. The ratio between the axial EAS diffusivity and the radial EAS diffusivity corresponds to the tortuosity, denoted EASα. EASα can also be thought of as the ratio between the path or curve length and the total displacement. The trace of the IASD tensor, the sum of all eigenvalues, corresponds to the intra-axonal diffusivity. At each voxel, the AWF, f, can be fundamentally estimated by (10), where Kmax is the maximal kurtosis calculated from the diffusion in every direction [The primary eigenvalue of irection .fKmax =WMTI was coupled with DKI and DTI measurements for investigating the effects of aging , traumatdMRI can be used to create reconstructions of fiber tracts along the visual pathway. Tractography methods for visualizing nerve fiber tracts and architecture using dMRI data allow for a non-invasive means for tracking orientation and directionality of white matter across the central nervous system in 3D space and may provide greater accuracy in the region-of-interest analysis . Tract rYou et al. suggested that probabilistic dMRI tractography could be ideal for modeling trans-synaptic neurodegeneration along the visual pathway . Using tRecent dMRI of experimental animal models of glaucoma may represent certain aspects of glaucomatous optic neuropathy. However, to date, there is no single animal model that can fully represent primary open-angle glaucoma, normal-tension glaucoma, or other glaucoma types. Thus, the forward and reverse translations between dMRI of humans and experimental animal models of glaucoma should be interpreted with caution. Future studies may take the recent consensus recommendation for experimental glaucoma models into consideration when devising appropriate research questions relevant to glaucoma . On the The main overarching pitfall of dMRI results is the over-extraction of information for biophysical and pathophysiological interpretations. The dMRI methods described whether mathematical or compartmental systems all represent a simplification of a stochastic biological system, which could lead to overfitting of results . Though Robust post-processing imaging protocols such as optimized motion correction and registration methods are necessary to facilitate image analysis of the visual system , especiaq-space imaging (QSI), also known as diffusion spectrum imaging (DSI), shows promise in resolving intravoxel fiber crossing that DTI cannot at the expense of larger field gradients and time-intensive sampling requirements [Another limitation of dMRI is the inability of DTI to resolve fiber crossings, which could result in the emergence of inaccuracy in visual pathway tractography and false detection of FA changes in the optic chiasm . The proirements . QSI meairements .Due to its lengthy acquisition time, QSI is predominantly used in small organisms and non-biological applications. Novel QSI, namely q-ball imaging, resolves some of the sampling burden of QSI through the calculation of an orientation distribution function (ODF) rather than a single diffusion tensor from high-angular resolution diffusion imaging (HARDI) . High-deb-values, for example, may result in varying DTI parametric measures in the same brains [It is challenging to compare dMRI results between studies due to the reproducibility from numerous confounding conditions in patients with glaucomatous abnormalities. In addition, currently, no standard DTI protocol exists, evident by the various acquisition parameters across studies. Different e brains . dMRI cae brains .There is growing evidence to suggest that glaucoma involves disruption to brain networks beyond the visual system. Visuomotor and postural coordination were shown to be altered in the glaucomatous state, resulting in falls and injuries beyond healthy aging . A DTI sThough not necessarily a neurodegenerative disease, there is dMRI evidence to suggest astronauts experience changes to white matter microstructure in zero-gravity environments, resulting in impaired balance post-space flight . Gait anAnother potential comorbidity that warrants further investigation is the incidence of sleep disorders in glaucoma patients. Vision loss can disrupt inputs to the suprachiasmatic nucleus, thereby impacting circadian rhythms . The unddMRI was proposed as a complimentary assessment when monitoring changes associated with traumatic brain injury such as emotion, cognition, and motor control, with DTI being sensitive to the acute phase of mild traumatic brain injury . A mouseThe transition from dMRI preclinical investigations to clinical applications depends on optimized imaging sequences that are within a clinically practical acquisition time. Moreover, though single-shot echo planar imaging is a common technique for diffusion dMRI data acquisition, it is susceptible to magnetic field distortions, and innovation is required in this regard. Paul et al. improved the anatomical accuracy of imaging the eye through a multi-shot diffusion-weighted Rapid Acquisition with Relaxation Enhancement (RARE) echo planar hybrid pulse sequence at 3 Tesla and 7 Tesla . Their eWith recent advancements such as multiband imaging, current DKI and WMTI sequences may allow whole-brain acquisitions of 2–3 mm spatial resolution within 10 min with good image signal-to-noise ratios in clinical MRI scanners . Other nMRI allows multimodal assessments of the structural, metabolic, and functional aspects of the visual system in health and disease . In ordedMRI may offer the capability for the non-invasive evaluation of neuroprotective drug interventions targeting retinal ganglion cells and their axons. Based on histological data, brimonidine, an alpha-2 agonist with intraocular pressure-lowering properties, was shown to reduce the rate of retinal ganglion cell loss in rat models of optic nerve injury . A recenAnother example of longitudinal MRI evaluation of glaucoma neurotherapeutics involves oral citicoline treatment in experimental rodent models . ThroughDiffusion MRI of the visual pathway, from the eye to the visual cortex and beyond, may contribute to key evidence characterizing glaucoma as a trans-synaptic disease involving both the brain and the eye, and may inform the neurodegenerative mechanisms that implicate the disorder. Due to the level of complexity of the biological system as well as the glaucoma pathogenesis, further basic and translational investigations are needed to improve this imaging modality and the analysis models, and to relate imaging results to the underlying biological processes better. Despite existing technical and clinical challenges, the inherent flexibility of MRI sequences supports the development of novel dMRI methods for improving the specificity of tissue microstructure characterization for a more comprehensive description of glaucomatous changes in humans and experimental animal models. DTI offers a key and sensitive tool for detecting and investigating white matter changes in the glaucomatous brain in vivo, while the higher-order dMRI models may lead to more robust gray matter characterization and better interpretation of the biological significance of the DTI findings in the glaucoma literature. The increasing availability of dMRI data may also help develop better pathological profiles and to determine the similarities and differences for diseases affecting the eye and visual pathways. Overall, advanced dMRI of the glaucomatous visual system may play an important role in identifying novel biomarkers and neurotherapeutic targets, as well as in promoting early detection, timely intervention, and outcome monitoring so as to reduce the prevalence of this irreversible but preventable disease.


Numerous human chronic pathological conditions depend on epigenetic modifications induced by environmental triggers throughout sensitive stages early in development. Developmental malnutrition is regarded as one of the most important risk factors in these processes. We present an overview of studies that the initiation and progression of many diseases are largely dependent on persisting epigenetic dysregulation caused by environmental insults early in life. For particular disorders, candidate genes were identified that underlie these associations. The current study assessed the most convincing evidence for the epigenetic link between developmental malnutrition and adult-life disease in the human population. These findings were obtained from quasi-experimental studies , i.e. naturally occurring environmental conditions in which certain subsets of the population have differing levels of exposure to a supposed causal factor. Most of this evidence was derived on the DNA methylation level. We discussed DNA methylation as a key player in epigenetic modifications that can be inherited through multiple cell divisions. In this Perspective article, an overview of the quasi-experimental epidemiological evidence for the role of epigenetic mechanisms in the developmental programming by early-life undernutrition is provided. Compelling evidence indicates that developmental exposure to adverse environmental conditions can programme the risk for chronic disorders in adult life. According to the Developmental Origins of Health and Disease (DOHaD) concept that was proposed to explain this phenomenon, such exposure may reprogramme an individual for immediate adaptation to potentially stressful postnatal experiences but affect the risk for adult-life pathological conditions such as cardiometabolic and neurodegenerative disorders and cancer, as well as longevity , 2. DeveIn human populations, most convincing evidence for the epigenetic link between developmental malnutrition and adult-life disease was obtained from quasi-experimental studies , i.e. naturally occurring environmental conditions in which certain subsets of the population have differing levels of exposure to a supposed causal factor . Most ofIn this opinion article, an overview of the epidemiological evidence obtained from quasi-experimental studies for the developmental programming by early-life malnutrition and for the role of epigenetic mechanisms in these processes is provided.in utero etiologic determinants, and present-day epidemiologic studies tend to directly examine the effects of certain intrauterine factors on postnatal health outcomes, irrespective of birth weight [Multiple findings from animal models indicate that inadequate nutrition during critical developmental periods can cause permanent structural and/or functional impairments of various tissues and organs and lead to long-term adverse health consequences . These fh weight .Presently, quasi-experimental  design is generally regarded as the most reliable approach for determining cause–effect relationships between early-life exposures and adult-life health outcomes. Famine  has apparent eligible features in order to be applied as a natural experiment. The studies we review have all been observational. Moreover, it should be taken into account that the effects of social, economic and family conditions on adult health outcomes contribute to the same extent as nutrition itself. Currently, long-term health outcomes of prenatal exposure to the Dutch famine, which affected the western Netherlands during the German occupation from November 1944 to May 1945, are studied most thoroughly in quasi-experimental setups. Among these adverse outcomes, there are obesity, T2D, and also multiple other cardiometabolic and mental disorders , 23. FurIGF2) gene playing a fundamental role in growth and development in exposed subjects aged ∼sixty years compared with their non-exposed siblings [IL10, INSIGF, LEP, MEG3, ABCA1 and GNASAS [PIM3, a gene involved in energy metabolism, mediated about 13% of the association between prenatal famine exposure and BMI. DNA methylation at six cytosine-phosphate-guanine dinucleotides (CpGs), including TXNIP affecting β-cell function and ABCG1 influencing lipid metabolism, together mediated about 80% of the association between prenatal famine exposure and serum TG level. Analyses restricted to those persons who were exposed to famine during early gestation identified additional CpGs mediating the relationship with TG near PFKFB3 (glycolysis) and METTL8 (adipogenesis). Moreover, in a genome-wide analysis of DNA methylation, differentially methylated regions were found to extend along pathways related to growth and metabolism in subjects periconceptionally exposed to the Dutch famine [IGF2/H19 region were shown to be dependent on single-nucleotide polymorphisms in the exposed population [in utero and unexposed controls [Convincing evidence indicates that epigenetic mechanisms such as DNA methylation can mediate the association between prenatal famine exposure and adult-life health outcomes. Most of this evidence was obtained from the Dutch famine birth cohort, although no significant association between prenatal exposure to the Dutch famine and global DNA methylation in adulthood was reported . Howeversiblings  , and pathways implicated in neurogenesis and development of nervous system were identified as the most significantly affected by developmental nutritional deprivation [INSR gene involved in growth and metabolism has been found in late adulthood [INSR gene and larger adulthood waist circumference [DUSP22, was observed in the Chinese famine-exposed schizophrenia patients [Recently, evidence for the possibility of undernutrition-mediated epigenetic programming was also obtained in several Asian populations. For example, early-life exposure to the Chinese famine of 1959–61 was found to be related to elevated levels of methylation of ult life . In a gerivation . In peopdulthood . More remference . From thmference , 41. Regmference . Howevermference . Howeverpatients . There ipatients . The datInterestingly, differential adult-life epigenetic signatures, in particular, differential methylation in gene pathways involved in cell cycle, development and apoptosis, were shown to be associated with birth seasonality . It seemOn the basis of information provided in the previous section, it can be concluded that epigenetic mechanisms may substantially contribute to long-term outcomes of developmental famine exposure. This seems all the more likely given that early gestation, a period when epigenome is most labile and susceptible to environmental stimuli , was fouData might be available under request to corresponding author.


In order to prevent new pathogen outbreaks and avoid possible new global health threats, it is important to study the mechanisms of microbial pathogenesis, screen new antiviral agents and test new vaccines using the best methods. In the last decade, organoids have provided a groundbreaking opportunity for modeling pathogen infections in human brains, including Zika virus (ZIKV) infection. ZIKV is a member of the Flavivirus genus, and it is recognized as an emerging infectious agent and a serious threat to global health. Organoids are 3D complex cellular models that offer an in-scale organ that is physiologically alike to the original one, useful for exploring the mechanisms behind pathogens infection; additionally, organoids integrate data generated in vitro with traditional tools and often support those obtained in vivo with animal model. In this mini-review the value of organoids for ZIKV research is examined and sustained by the most recent literature. Within a 3D viewpoint, tissue engineered models are proposed as future biological systems to help in deciphering pathogenic processes and evaluate preventive and therapeutic strategies against ZIKV. The next steps in this field constitute a challenge that may protect people and future generations from severe brain defects. The zika virus (ZIKV) is a vector borne-flavivirus, with a single serotype, but two differentiated lineages: the East/West African and the Asian genotypes .Aedes is capable of carrying and transferring ZIKV to humans, with Aedes aegypti and Aedes albopictus being the main vectors as they feed almost exclusively on humans. Although most ZIKV infections are transmitted by infected mosquitoes, ZIKV transmission can occur through sexual intercourse contact, blood transfusion, laboratory exposure, and both intrauterine and intrapartum transmission. The viral transmission via breastfeeding has not been reported.ZIKV is closely related to several other pathogens that cause disease globally, including Dengue (DENV), yellow fever (YFV), West Nile (WNV), Japanese encephalitis (JEV), and tick-borne encephalitis (TBEV) viruses . Among mZIKV was first isolated in sentinel rhesus macaque monkeys in 1947 in Uganda and later in humans in 1952 . For a lIn general, the most relevant feature of ZIKV with respect to other of Flaviviridae family relies on its ability to target the nervous system and induce the onset of neurological symptoms in fetuses and infants.Infections during pregnancy are critical for fetuses, especially when intrauterine transmission occurs during the first trimester and the immature neurons are susceptible to ZIKV . In addiDifferent in vitro and in vivo models confirmed that the neural progenitor’s infection in the mammalian brain constitutes the pathogenetic key leading to the hypomorphic effect, but more complex experimental models are needed to address subcellular pathogenesis events, allowing the trials of new therapeutic interventions.Up to date, a total of 86 countries and territories have reported evidence of mosquito-transmitted Zika infection and the current epidemic is caused by ZIKV from an Asian lineage  and therIt is well-known that Zika spreads similar to other arboviruses  and, as Zika virion has a diameter of 50 nm with a 11 kb positive-stranded RNA molecule that encodes three structural and seven non-structural proteins. The structural proteins: capsid (C), membrane (prM/M) and envelope (E) aim at assembling the viral particle, whereas the other seven are non-structural proteins  are used by the virus to orchestrate viral replication, avoid host defenses and exploit the host replication machinery. Furthermore, while the sequences of ZIKV NS3 and NS5 proteins are highly similar to other flaviviruses, the sequences of prM, E and NS3 are significantly different . For thiThe ZIKV life cycle depends on the cellular secretory pathway for complete virion formation, maturation and release. After the mosquito bite, ZIKV infects nearby skin cells, and then it is delivered to the draining lymph nodes after being picked up by skin-resident dendritic cells. In the lymph nodes, ZIKV infects monocytes and macrophages and then disseminates to other body sites, including placenta and testes.Translation occurs within the ER membranes. The nascent virions accumulate within the ER lumen and are delivered to the next secretory compartment, the Golgi complex. Immature virions accumulate in the ER-Golgi hybrid compartment where the viral surface glycoproteins undergo final glycosylation and maturation. Virions can be secreted by a secretory autophagy-related pathway as membrane-wrapped clusters of virions (pathway 1), alternatively virion clusters could be released via direct fusion of the membranous compartments with the plasma membrane (pathway 2). However, the major mechanism of virion egress is via a secretory granule-like mechanism as individual virions (pathway 3) .Extracellular vesicles (EVs) represent an important mode of intercellular communication between cells by delivering cytosolic proteins, lipids, and RNA. In the same extent of hepatitis C virus (HCV), it has been recently reported that Zika virus also hijacks EVs pathways to package viral components that are infectious and potentially less immunogenic than mature virions. As EVs have been shown to cross blood–brain and placental barriers, it is possible that Zika virus could gain access to the brain or developing fetus via this mechanism. In particular, Zika virus-infected cells secrete distinct EV subpopulations with specific viral protein profiles and infectious genomes with respect to those released from non-infected cells .It is known that most pantropic viruses employ similar dissemination strategies. At the inoculation site virions replicate in tissue’s macrophages and dendritic cells, which traffic the virus to the draining lymph nodes and other lymphoid tissues. In these latter, large numbers of macrophages are recruited and amplify the virus spreads to monocytes, macrophages or dendritic cells in multiple tissues reaching the remaining tissues. In fact, in rhesus macaques, in situ hybridizations have demonstrated that neurons, macrophages, neutrophils, and dendritic cells are permissive ,22,23. NIndeed, ZIKV RNAs can be found in many biological fluids, so it can be diagnosed by detecting its RNA in blood, urine, and other body fluids by reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay. However, the length of time that it remains detectable in such fluids is not clear. ZIKV can also be diagnosed by assessing the presence of anti–ZIKV IgM antibodies in the blood. .Toxoplasma gondii, other, rubella virus, cytomegalovirus, herpes simplex virus, parvovirus B19) [The placenta is the main organ appointed to filter toxic molecules, allowing the exchange of nutrients and solutes and acting as a specialized interface between the mother and the developing fetus , as wellrus B19) , an acrorus B19) . The plarus B19) .The sequelae of infections in pregnancy usually include teratogenic effects, that cause congenital anomalies which the most common are ventriculomegaly, lissencephaly, hydranencephaly, microcephaly, developmental delays  and oculSimilar to ZIKV, neonatal herpes-simplex-virus and congenital-cytomegalovirus infections are associated with neurological deficits such as microcephaly . In factThe knowledge of viral entry receptors and cellular tropism is fundamental for understanding the mechanisms of viral immunity and pathogenesis.While the E protein has been appointed as the viral receptor for the binding and fusion, several are the candidate molecules as entry receptors, which many are shared with dengue virus (DENV).In clinical samples putative target receptors have been identified in glial cells, amniotic epithelial cells, fetal mesenchymal cells, neural progenitors’ cells, glial cells, and the macrophages of the placenta (Hofbauer cells) ,35,41,42Flavivirus infections implicate a role of the innate immune system in controlling their infections. Usually, the type I interferons  system is triggered within hours of viral infection upon recognition of the virus by pattern recognition receptors (PRRs) such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and toll-like receptors (TLRs). Their engagement results in the activation of multiple transcription factors, including the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and interferon regulatory factors (IRFs), that enhance interferons and various other inflammatory cytokines and chemokines orchestrating the innate and adaptive immune response. RNA viruses that replicates in the cytosol are recognized by RLRs RIG-I and melanoma-associated differentiation antigen 5 (MDA5), activating downstream IRF3 and NFKB to induce type I interferon production [A short course of illness and self-limiting febrile symptoms in most cases of oduction .For ZIKV the infectious capacity seems to be primarily dependent by prM, a structural protein known as pre-membrane protein in ZIKV, and secondly by the ability to evade type I IFN responses, achieving long-term infection. It seems that the protein NS acts in modulating IFN signaling in favor of the viral immune escape. In summary, NS1 stabilizes caspase-1, NS2A down-regulates the promoter activity of IFN-β, NS3 protein blocks RIG-I and MDA5, while NS4A at the mitochondrial level and NS5 guarantees methyltransferase and RNA-dependent RNA polymerase activities .Viral entry may occur also via the process of ADE of infection, where the quantity and the avidity of the antibodies produced, at the second viral encounter, are extremely important for tilting the table from protection to disease. In fact, antibodies generated form the primary infection that are sub-neutralizing in quality (low avidity) or quantity (low concentration) can bind a cross-reactive virion and, as immune complex, gains access to permissive cell bearing immunoglobulin Fc receptors (FcRs) such as FcRγ. This event can lead to increased viremia and viral burden in other tissues, occurring to increased disease severity for the host. On the contrary, when high avidity and concentration antibodies are produced the neutralizing response determines protection. Recently, a study showed ADE during ZIKV infection when a murine model lacking STAT2 passively received DENV-immune human sera . FurtherUltimately, highly cross-reactive antibodies that target the fusion loop in E-DII were not efficiently neutralizing in vitro and showed no protective activity in vivo ,57,60. IAlthough these have been studied for DENV, it does not take that far to assume that those mechanisms could be used by ZIKV. Systemic studies using mouse models of ZIKV infection have shown that anti-interferon-α/β receptor (IFNAR) monoclonal antibody-treated wild type animals, but also Ifnar1−/− knockout mice, as well as Stat2−/−, or Irf3−/−Irf5−/−Irf7−/− triple knockout mice, can die from ZIKV infections; while opposite was the effect upon the wild-type, Irf3−/−, Irf5−/−, Mavs−/−, Tmem173(STING)−/−, and Mb21d1 (cGAS)−/− mice ,68,69,70Furthermore, in addition to the claimed role of antibodies, it has been demonstrated the role of protecting cross-reactive CD8+ T cells seen in HLA transgenic mice, and as well suggested as tool for future vaccine development . Additiohttps://clinicaltrials.gov/, accessed on 31 August 2021, NCT03110770). All types of vaccines are well described elsewhere [Lately, a massive work has been conducted upon vaccine development. The emergency in the Americas and the severe congenital defects triggered the development of different vaccines that were advanced into clinical trials. Indeed, multiple candidates have shown promising results in both animal models and phase I clinical trials. Until 2019, phase I clinical trials with data on animal experimentation comprised mostly DNA or mRNA vaccines having prM/E proteins as immunogen. One of the DNA-based vaccine, named VRC5283, has showed relevant protection in NHP model, and demonstrated immunogenicity in humans, once evaluated safety and efficacy of a three-dose regimen. It has recently completed the phase II in December 2020  achieved mean titers above the 1/100 threshold leading to virus protection ,79. In tInterestingly, it has recently been shown that tetracycline-inducible vaccinia expressing prM and E protein induced high anti-E IgG titers in vitro; on the other hand, in vivo, mice treated with anti-IFNAR1 and vaccinated with a single dose were protected from ZIKV, with an increase of interferon-gamma (IFN-γ)-secreting splenocytes and the induction of a humoral response .Getting access to human or primate fetal brain samples is extremely difficult, thus in vitro models offer an essential alternative. Nowadays, traditional cell cultures and classical virology/microbiology techniques are still in use for exploring viral pathogenesis and carrying out antiviral research, but they have reached their dusk.ZIKV can replicate within a large range of mammalian cell lines as thoroughly reported . StudiesAs ZIKV infection prevented neurosphere formation, these in vitro models were very useful for discovering the special features of the virus, in contrast to other members of its family. As the neurospheres model highlighted in the past , the useOrganoids play an important role in mimicking a viral disease and researchers need to exploit humanized 3D models for a deeper understanding of human infections. Specific human biological phenomena are not amenable in animal models. In fact, the human brain is more complex than the murine one, in relation to specific resident cell populations, their abundance and metabolism. In fact, it has been hard to describe the effect of the virus during the neurodevelopment in animal models due to the species-specificity of the infection .Organoids represent an improvement in human tissue modeling and accessibility to study many human organs and diseases. They are an extraordinary example of 3D cell culture that differ from simpler spheroid models  because they originate through the proliferation of progenitor cells that self-organize into clusters, which build up the architecture and resemble the functionality of the native tissue . The cerPolarity and morphogens  can determine, polarize and differentiate specific brain-regions. The production of specific forebrain, midbrain or hindbrain organoids has the advantage to be more consistent in terms of cell composition . AdditioAnimal models have been employed to study the effects on the risk populations. While few studies tried to understand the viral effect during pregnancy, others focused on ZIKV infection on adults and neonates, and few others explored its sexual transmission, which is unique within the Flavivirus genus.Whenever a new viral threat emerges, rodents are often the most common animal model taken in consideration but not necessarily a guarantee of a proper human surrogate. For instance, in the context of the last coronaviruses pandemics, SARS-CoV2 was shown to have poor affinity for the mouse homologue angiotensin-converting enzyme 2 (ACE2) that is strongly bound in humans .In ZIKV field, mice models have enormously contributed to the current knowledge about the innate and the adaptive immune response, as well as the virus life cycle, tropism, transmission route, and possible treatments. Most mice models are generated based on the type I IFN pathway genes impairment making mice susceptible to the Flaviviridae family (as well as to other Arboviruses) infections. In the same way, the dexamethasone-induced immunosuppression is an alternative way to generate murine models for ZIKV research, providing human-like symptoms with multiorgan involvement . StudiesAdditionally, neocortex formation is accounting for the highest human cognitive functions, but given the neocortex’s different developmental pattern and architecture in humans and rodents, the latter are not often appropriate for analyzing a specific time-frame of infection or neural progenitor’s phenotype. Another shortcoming in murine models is a low gyrification index , thus a NHPs have also been used as animal model for studying the virus life cycle and pathogenesis, as ZIKV also has them as hosts and reservoirs. The first animal model described susceptible to ZIKV was in fact rhesus monkey , with histat-2-deficient guinea pig and hamsters, with certain range of advantages, however they cannot resemble many clinical aspects of ZIKV infection and they are relatively unexplored in therapeutic research, to the best of our knowledge [Alternative models are nowledge ,103.Above all, it must be remembered that animal models carry their own microbiota, which composition is different from human’s, influencing the pathogen behavior, and adding interferences in clinical manifestation of the disease. In The first organoid model mimicking brain tissues derived from embryonic stem cells (ESCs) and was used to study ZIKA pathogen-host interaction. A link between MR766 ZIKV strain and TLR3 was identified both in human ESC organoid cerebral model and in murine neurospheres as well . A seconOther types of cellular models were differently useful for ZIKV research. Putative ZIKV receptors (AXL and TYRO3) have been previously identified in 2D cell lines but were not confirmed as positive hits when organoids were used . ZIKV E The 3D systems can also simulate basic defense mechanisms. In fact, trophoblasts isolated from full-term placentas resist infection by diverse viruses, including ZIKV, and transfer this resistance to nonplacental cells through the activity of paracrine effectors, including the constitutive release of type III interferons (IFNs). It has been developed a 3D cell-line–based model of human syncytiotrophoblasts, cells that lie in direct contact with maternal blood, that recapitulate the antiviral properties of primary trophoblasts through the constitutive release of type III IFNs (IFNλ1 and IFNλ2) and become resistant to ZIKV infection . A similAs already reported in 2018 by Majolo et al., the increased use of organoids and other relevant 3D cellular models has followed the clinical and research urgency. By modeling key functional characteristics of the pertinent tissue and allowing replication, organoids are currently an important experimental virology platform . The mosCerebral organoids obtained from iPSCs were used to evaluate a new class of antiviral compounds. The most promising compound, an 8-Oxoguanine DNA Glycosylase (OGG1) inhibitor, interacts with members of the Hsp70 chaperone network, disturbing ZIKV particle release and rescuing ZIKV-induced toxicity . Thanks While a pronounced infection of microglia may be expected, astrocytes are more mysterious and recently focus of further investigations. Among human iPSC-derived cell types, in fact, astrocytes were found preferentially targeted by ZIKV in comparison to neurons, and more prone to oxidative stress, mitochondrial and DNA damage after ZIKV infection .Organelles can be utilized by pathogens and ZIKV specifically reorganizes the endoplasmic reticulum. In particular, ZIKV-infected brain organoids indicated transformation of ER sheets to convoluted membranes, visible by super resolution microscopy . In ordeAll animal models show a certain grade of disparity between gene and protein expression compared to humans, making likely unsuccessful unbiased studies. As for primary human neurons and other cell tissues used to modelling ZIKV infection, ethical permissions are always required and represent a limitation for assembling organoids, which are made of iPSCs or derived from primary biopsies. Organoids derived from brain, an organ that is mainly constituted of non-epithelial cells, requires sophisticated strategies ; those aThe majority of current brain organoids models ZIKV infection lacking the immune and vascular systems . SpecifiCerebral organoids have cell types similar to those found in the human brain but those are not organized in the same way. The gyrification of the human neocortex has not been implemented in most organoid protocols, but recently an effort has been done for this purpose , concomiTo note, immune cells are derived from yolk sac and mesoderm while brain organoids are usually derived from neuroectoderm, thus the microglia population is difficult to be displayed  or included in the model ; of courAnother aspect to consider is gender-related differences for ZIKV infection. It seems that a strong response inhibits ZIKV to control the infection in females, while in males, the infection correlates with viral persistence . In lineLast, organoid protocols still lack standardization and the production of organoids is relatively expensive. The complexity associated to brain organoids brings challenges regarding their 3D morphological, molecular and neurophysiological analysis . Being aIt is known that ZIKV is able to cross the blood–brain barrier (BBB), reaching the CNS without damaging it . BrieflyIn addition to vaginal organoids , the devOrganoids can be included in organ-on-chips ,154, as Gyrification of the human brain is connected to the compressive mechanical forces and microfluidics can be useful trying to achieve this particular process. Additionally, bioengineering and bioprinting technology may facilitate the control of spatial and temporal distribution of the organoid cellular system and implement extra cellular-matrix or vascularization  usually By using a personalized medicine approach, genome-wide screens, aiming to find host factors which restrict the viral disease or promote viral replication, would be progressively more applied to organoid cultures . Next geLately, Wang et al. used patient’s cells to generate asparaginyl-tRNA synthetase1 (NARS1)-mutated cortical brain organoids for modeling microcephaly, showing reduced proliferation of radial glial cells and demonstrating the importance of protein translation for brain development .In addition, to overcome missing interregional interactions by region-specific organoids, the researchers are developing (for various organs) assembloids, mainly formed by joining patterned pre-mature or mature region-specific organoids . For insAll these advances may soon be applied to ZIKV research to generate sophisticated datasets and relevant knowledge about the viral pathogenesis and immune evasion.Given the urgent need for ZIKV prophylaxis and treatment, repurposing of approved drugs appeared to be a viable and immediate solution. In fact, already in 2016, chloroquine treatment was shown to partially rescue mouse neurospheres and to protect fetal mice from microcephaly . Many otTo date, no therapeutic treatment is available to treat ZIKV-infected patients and research in therapeutics has focused on many natural compounds for years. Hydroxycholesterol was the first potential antiviral blocking ZIKV entry, attenuating ZIKV-viremia in non-human primates, microcephaly in mice and seen preventing the infection in human cortical organoids . AnotherGinkgo biloba that have raised considerable interest due to inhibiting human cytomegalovirus (HCMV) genome replication and ZIKV infection of normal human astrocytes (NHA) [Other natural compounds have currently provided encouraging results. For example, cavinafungin, a natural compound , targetses (NHA) . Additioes (NHA) . On the es (NHA) .Clearly, all the mentioned compounds need to benefit from brain organoid in vitro analyses to be entitled as truly effective against ZIKV.ZIKV infection constitutes a global harmful threat. The current COVID-19 pandemic has shown that new tools and technologies for the advancement of basic science are required and the organoid platform could participate as pre-clinical platforms for studying all pathogens. In virology, in vivo models are usually made up by specific gene manipulation or applying harmful conditions to animals, while recent advanced in vitro models, definitely cruelty-free, can be directly generated without prior knowledge of the specific responsible genes, obtaining clinical specimen from the patients . ConsideProper integration of disease control measures and effective surveillance programs should be supported by the continuous improvements both in diagnostic methods and research models, such organoids, to guarantee the emerged “one-health” point of view .In conclusions, the application of organoids represents a relevant approach for deciphering pathogenicity mechanisms in humans and evolving valuable predictive models. Human organoids uniquely help researchers in the world collective fight against Zika disease .