The brainstem has traditionally been considered an area of the brain with autonomous control of mostly homeostatic functions such as heart rate, respiration, and the sleep and wakefulness state, which would preclude the necessity to exhibit the high degree of synaptic or cellular mechanisms of plasticity typical of regions of the brain responsible for flexible, executive control, such as the medial prefrontal cortex or the hippocampus. The perception that the brainstem does not share the same degree of flexibility to alter synaptic strength and/or wiring within local circuits makes intuitive sense, as it is not easy to understand how “soft wiring” would be an advantage when considering the importance of faithful and consistent performance of the homeostatic, autonomic functions that are controlled by the brainstem. However, many of the molecular and cellular requirements which underlie strengthening of synapses seen in brain regions involved in higher-level processing are present in brainstem nuclei, and recent research suggest that the view of the brainstem as “hard wired,” with rigid and static connectivity and with unchanging synaptic strength, is outdated. In fact, information from studies within the last decades, including work conducted in our group, leads us to propose that the brainstem can dynamically alter synaptic proteins, and change synaptic connections in response to prenatal or postnatal stimuli, and this would likely alter functionality and output. This article reviews recent research that has provided information resulting in our revision of the view of the brainstem as static and non-changing by using as example recent information gleaned from a brainstem pontine nucleus, the laterodorsal tegmentum (LDT). The LDT has demonstrated mechanisms underlying synaptic plasticity, and plasticity has been exhibited in the postnatal LDT following exposure to drugs of abuse. Further, exposure of the brain during gestation to drugs of abuse results in alterations in development of signaling pathways in the LDT. As the LDT provides a high degree of innervation of mesoaccumbal and mesocortical circuits involved in salience, as well as thalamocortical circuits involved in control of arousal and orientation, changes in synaptic strength would be expected to alter output, which would significantly impact behavioral state, motivated behavior and directed attention. Further, alterations in developmental trajectory within the LDT following prenatal exposure to drugs of abuse would be expected to impact on later life expression of motivation and arousal. The laterodorsal tegmentum (LDT) is located in the pontine central gray, just below the caudal portion of the aqueduct, which connects the third and fourth ventricles. The LDT is a heterogeneous nucleus composed of cholinergic, GABAergic and glutamatergic neurons . The effAlthough outside of the scope of the early work focusing on the role of the LDT in state control, anatomical tract tracing studies showed that the LDT does not just project upstream to the thalamus, where it controls cortical neuronal firing patterns typical of alert wakefulness and REM sleep, or downstream to pontine and medullary nuclei controlling the classic, motor-related physiological sign of REM sleep, motor atonia . Rather,As the VTA was known to be important in goal-directed behavior based on seminal studies from the 1970’s, identification of the projections to the mesoaccumbal pathway had raised speculation that the LDT could be involved in limbic processing of behaviorally-relevant stimuli . InvestiAfter decades of study, the VTA-NAc pathway is now known to play a fundamental role in assignment of reward to stimuli, including reward assigned to drugs of abuse, and further, the idea that DA plays a critical role in this function is widely accepted. In the mid 1970s, findings that DA receptor blockers reduced lever press for intravenous amphetamine self-administration in rats led to the suggestion that DA plays a central role in perception and extent of reward . StudiesFurther work showed that electrical stimulation of VTA neurons enhanced cueing effects of amphetamine , while aSeveral sources of modulation within the VTA have been shown to lead to drug-induced DA rises. Drugs of abuse exhibit a variety of pharmacological mechanisms, and rises in DA can occur via actions in the synaptic cleft, direct excitation of DA cells, and/or via activation of non-DA VTA cells . AlthougRecent development of optogenetic tools allowed further characterization of DA VTA neurons which has shown that they display reward-related firing patterns, and that their activity is sufficient to trigger addiction-related behaviors, the later shown even in the absence of any drug exposure . Taken tin vivo and in vitro approaches  brief increase in DA release; (2) followed by a decreased DA release below baseline levels; and (3) a sustained DA release for several minutes, which was at levels approximately 80% higher than baseline. The blockage of nicotinic receptors (nAChRs) or ionotropic glutamate receptors in the VTA attenuated the first component, while suppression of the second component was achieved by intra-LDT blockage of muscarinic receptors (mAChRs) suggesting an autoregulatory role. Intra-VTA infusions of mAChR blockers were shown to attenuate the third, sustained component . Blockade of LTP unctions . To dateunctions , and corunctions . Postsynunctions , and genunctions . AMPAR a synapse . Drugs o synapse .+2 blockage, these synapses are not functional at resting membrane potential, as glutamate release from presynaptic sites is unable to induce appreciable levels of currents in the postsynaptic neuron upon binding to the NMDAR. These NMDAR-containing synapses which lack functional AMPAR, were termed “silent synapses”  agonist to cholinergic LDT cells elicited calcium release from intracellular calcium stores, and decreased the frequency of inhibitory postsynaptic currents (IPSCs) of LDT neurons, suggesting the functional presence of CB1Rs within the LDT nucleus which plays a role in induction of depolarization-induced suppression of inhibition  offspring, monitored the functionality of the LDT postnatally with implications for a role of the LDT in negative behavioral outcomes seen in those exposed to nicotine in the prenatal period, including a higher susceptibility for development of dependency to drugs of abuse , 2019. OIonic currents resulting from activation of membrane-inserted, ionotropic receptors can be pharmacologically isolated, which allows determination of occurrence of specific neurotransmitter signaling, such as glutamate, in the form of spontaneous and miniature excitatory postsynaptic potentials (sEPSCs and mEPSCs), with mEPSCs amplitude and frequency reflecting effects of action potential-independent glutamate release. While the amplitude of such currents has been correlated with postsynaptic function of glutamate receptors, particularly AMPAR, examination of the frequency by which these currents occurs has been a common initial analysis approach to investigate alterations in the probability of glutamate release from presynaptic terminals. A higher frequency of postsynaptic currents predicts a greater degree of glutamatergic release directed to a neuron, whereas a reduced frequency of EPSCs likely reflects a reduced degree of active glutamatergic input to the recorded neuron.In our work, we have found a reduced frequency of mEPSCs in putative cholinergic LDT neurons associated with PNE treatment, suggesting a lower probability of activity-independent glutamate release from presynaptic terminals within this nucleus . A more The ontological expression of ionotropic glutamate receptor subunits in the rodent brain has been well characterized for both AMPA and NMDA receptors, and identified changes in the balance of these subunits throughout the lifespan suggest they play different functional roles during development, in which their subunit switch represents a key step in the maturation of glutamate signaling . Among ANMDA receptors also show PNE-associated changes in the LDT. Interestingly, the subunits affected are the same ones implicated to play a large role in TBS-induced LTP, and in drug-induced LTP in other brain regions where this has been examined. The GluN2B NMDAR subunit has been suggested to play a central role in brain mechanisms of plasticity associated with several physiological brain functions, including learning and memory . StudiesThe GluN3 subunit plays a vital role in the maturation of NMDARs and supports stabilization of glutamate signaling. Expression of this subunit increases and becomes widespread during the first postnatal days, but declines to very low levels throughout the lifespan . In addiExposure to drugs of abuse has been shown to reactivate plasticity mechanisms seen during development in addiction-associated brain regions, which may play a role in the formation of addiction-related memories  as reactThe intrinsic properties of neurons underlying neuronal excitability have been shown to exhibit plasticity in an activity-dependent fashion . This prIn LDT neurons, PNE was associated with alterations in AMPA and NMDA receptors, reductions in glutamate release and activity-dependent intracellular calcium, changes in intrinsic properties, and reductions in firing excitability, which would certainly impact on output to target regions, such as those within the mesoaccumbal pathway. One prominent hypothesis of drug dependence is the hypodopaminergic hypothesis which posits that those with relatively lower DA tone in the mesoaccumbal circuit exhibit a heightened sensitivity to drug stimuli, and are therefore more vulnerable to the reinforcing properties of drugs of abuse. PNE has been associated with a high liability for development of drugs of abuse, and the enhanced susceptibility for development of dependency to drugs of abuse associated with these individuals has been suggested to be due to hypodopaminergic VTA functioning resulting from the early life nicotine exposure . In suppThe LDT had long been thought to be a hardwired “way station,” playing a role as a faithful effector, passively transmitting information to its targets, without any meaningful alteration of the informational content of signaling received from higher-order brain regions. Review articles focused on synaptic plasticity, including those detailing the neuronal plasticity engendered by drugs of abuse, remain centered on higher order structures involved in learning and memory with little to no mention of mechanisms underlying synaptic plasticity occurring in the brainstem, nor what the functional impact of such an alteration would be . FurtherAs environmental experiences occurring in both prenatal and postnatal life have been shown to alter its functionality, it will be important to determine the impact of such alterations on drug dependence and other aroused, attentive, and motivated behaviors associated with prenatal nicotine exposure. Additionally, how these changes impact behaviors for which the LDT is more traditionally known to play a role, such as control of the sleep and wakefulness state and arousal, also warrant investigation. Further, additional studies should be conducted with different technical approaches such as optogenetics, electron microscopy, and characterization of early response genes and expression of exogenous molecules known to play a role in information processing-based synaptic alterations in order to confirm findings of plasticity in the LDT, as well as to determine which specific LDT neuronal subtypes can experience plastic changes. In summary, while arousal can be associated with compulsive or rote behavior, it can also be governed by flexible executive control. While such control is often believed to stem from cortical regions, we now know the brainstem could also play a role. Therefore, future studies should be conducted to provide a full characterization of the alterations in plasticity possible in the brainstem, and specifically within LDT neurons, as well as the underlying mechanisms and the behavioral consequences of plasticity-associated deficits promoted in this neural area by experiences throughout the lifespan if we are to truly understand cognitive-based processes including those leading to drug dependence and motivated and goal-directed arousal.ex vivo electrophysiological recordings. Animals were kept in the cage with their maternal progenitor under 12-hour light/dark cycle with lights on at 7:00, controlled humidity (52–62%), and constant temperature (22 ± 2°C). Ad libitum access to food and water was provided to the moms.Animal procedures were authorized by the Animal Welfare Committee (Danish Ministry of Justice) and in accordance with European directives for the employment of rodents in research. All experimental procedures were conducted under valid permits (2014-15-0201-00031 and 2017-15-0201-01195). Naive NMRI male mice  between 11 and 15 days old were employed for 2HPO4 1.2, CaCl2 2.7, MgSO4 (anhydrous) 1.2, Glucose 10, NaHCO3 26; oxygenated in 95% oxygen/5% carbogen]. Coronal slices containing the LDT (300 μm) were obtained at 0–4°C with a Leica vibratome , heated for 15 min at 37°C, and recovered at room temperature for 1 h prior to recordings. A sagittal cut was performed along the midline of the slice to separate the two hemispheres. Slices were placed in a microscope chamber  constantly perfused with oxygenated ACSF at 33°C with a controlled flow rate of 1 mL/min provided by 4 Head Perfusion pump . Slices were visualized on screen via a CCD camera system  attached to the microscope using Live Acquisition software 2.2.0 .Brain slices were obtained as previously described , 2019. B2 3, HEPES 10, NaGTP 0.3 and Na2ATP 4; EGTA free; 285 mOsm). The liquid junction potential was calculated as +14.7 mV at 20°C as previously described (+ or T-type Ca+2 currents were used to classify LDT neurons. A hyperpolarization voltage step (−85 mV) was followed by 7 depolarization voltage steps . To allow the employment of this protocol, QX-314  was added to the patch solution to block voltage-gated sodium channels. Small, round-shaped neurons with presence of T-type Ca+2 current, which are likely GABAergic neurons, were classified as type I. The medium/big-sized, polygonal-shaped neurons containing either A-type K+ current (type II) or both currents (type III), were likely cholinergic cells based both on previous literature  were fabricated on a horizontal puller (Sutter Instruments P-97) and filled with an internal solution  used for presynaptic stimulation was placed in the ventral portion of the LDT nucleus (100–300 μm from the recorded neuron). The electrode was connected to a Stimulus Isolator A365  with the output parameters controlled by a Pulse Stimulator Master-9 , triggered with TTL pulses generated by Clampex. 5 min after the acquisition of the whole-cell configuration (for equilibration), 10 pulses (0.033 Hz) were delivered to the slice via the stimulation electrode to record evoked excitatory postsynaptic currents (eEPSCs) with a voltage membrane command set at −60 mV, as a baseline. Intensity and duration of pulses were set to evoke EPSCs with amplitudes between 50 and 150 pA. Next, the patched neuron was depolarized to 0 mV, concomitant with the application of a theta burst stimulation (TBS) protocol, as previously described for the hippocampus . Four puThe datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Dyreforsøgstilsynet as mandated by the European Communities Council Directive (86/609/EEC).Both authors did literature searching, data collection and/or interpretation, wrote and edited the manuscript.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.


Myelination is an essential process that consists of the ensheathment of axons by myelin. In the central nervous system (CNS), myelin is synthesized by oligodendrocytes. The proliferation, migration, and differentiation of oligodendrocyte precursor cells constitute a prerequisite before mature oligodendrocytes extend their processes around the axons and progressively generate a multilamellar lipidic sheath. Although myelination is predominately driven by oligodendrocytes, the other glial cells including astrocytes and microglia, also contribute to this process. The present review is an update of the most recent emerging mechanisms involving astrocyte and microglia in myelin production. The contribution of these cells will be first described during developmental myelination that occurs in the early postnatal period and is critical for the proper development of cognition and behavior. Then, we will report the novel findings regarding the beneficial or deleterious effects of astroglia and microglia, which respectively promote or impair the endogenous capacity of oligodendrocyte progenitor cells (OPCs) to induce spontaneous remyelination after myelin loss. Acute delineation of astrocyte and microglia activities and cross-talk should uncover the way towards novel therapeutic perspectives aimed at recovering proper myelination during development or at breaking down the barriers impeding the regeneration of the damaged myelin that occurs in CNS demyelinating diseases. During the last decades, the production of myelin in the central nervous system (CNS) has been the focus of a multitude of studies in the context of development or regeneration. Oligodendrocytes, the cells synthesizing myelin in the CNS, were initially found in hind-jawed fishes  can be detected at the embryonic day (E) 12.5 and 16.5 respectively, thus before the start of OPC maturation and axon myelination taking place during the first three postnatal weeks  pathway . Acting through its receptor PDGFRa, PDGF promotes OPC proliferation and mobility and, also inhibits OPC differentiation towards mature oligodendrocytes, by generating a graded response requiring activation of the phosphoinositide 3-kinase and/or phospholipase C γ pathways. Astrocyte-derived PDGF was thus proposed to drive the clock that times oligodendrocyte development  from both cell types  in the CNS, and secreted via the ATP-binding cassette transporter ABCA1  4 may be supplied to oligodendrocytes possibly importing the molecule through MCT1  in astrocytes, exhibited detachment of adjacent paranodal loops of myelin from the axon, increased nodal gap length, thinning of myelin sheath in the optic nerve and finally decrease in visual acuity. These data led to propose that thrombin-dependent proteolysis of the cell adhesion molecule neurofascin 155 that attaches myelin to the axon, is inhibited by the vesicular release of thrombin protease inhibitors from perinodal astrocytes, which likely involves these cells in myelin remodeling necessary for optimal electrical conduction  p65 subunit via the recruitment of PI-3 kinase to the PDGFRa thus leading to a synergism with PDGF  of the dorsal forebrain. This inhibition was mediated by the blockade of several proinflammatory cytokines including IL-1β, IL-6, TNF-α, and IFN-γ. Also, the observation that activated microglia significantly increased O4+ cells but decreased PDGFRa+ OPCs  model of demyelination formed distinct global expression clusters. Unexpectedly, CD11c+ and CD11c− subpopulations were relatively close together indicating that the major difference was related to developmental age rather than subpopulation phenotype. The identification of gene expression profiles associated with microglia from the different conditions showed that under demyelinating conditions, microglia cells were enriched for immune system genes consistent with their activated state. On the contrary, in the neonatal brain, microglia gene profile included genes involved in nervous system development displaying a neurogenic phenotype. Remarkably, upon a quite total ablation of microglia in the adult brain by using an inducible mouse strain, clusters of highly proliferating microglia were detectable throughout the CNS. Although expressing CD11c and nestin, the proliferating cells did not display a neurogenic gene expression profile confirming that the myelinogenic CD11c+ subset of microglia is unique to neonatal CNS , known to efficiently deplete microglia 1 to P8, displaying a high expression of the activation marker Mac3 and dramatically collapsing at P9. Microglia found in the postnatal subcortical (with an amoeboid shape) and cortical regions (with multiple cellular processes) were found to share their early origin from the same CX3CR1via FGFR1/ERK1/2 signaling activation, which results in the control of MBP expression, myelin formation and conduction velocity  family, the second-largest class of GPCRs playing crucial roles in developmental processes , secrete high levels of CNTF during the remyelination phase while CNTF injection in the spinal cord upregulates the transcripts encoding FGF2, a potent OPC mitogen  was identified, as well, together with an increased ability of activated OPCs to migrate and differentiate compared with non-activated ones. Activated OPCs finally express transcription factors such as TCF4 and Sox2, which serve to maintain OPC in the cell cycle and to prime these cells for further differentiation  versican, aggrecan, neurocan and the dermatan sulfate proteoglycan increase in correlation with astrocytosis. In contrast, these molecules accumulate in foamy macrophages in active plaque centers, suggesting their engulfment together with myelin. In inactive lesions and normal-appearing white matter, proteoglycans are decreased and display undetectable abnormal heterogeneous aggregation, respectively  associated with pro-inflammatory activities and the alternative polarized state (M2) involved in OPC recruitment and differentiation associated with anti-inflammatory activities  as well as a decreased CD14 expression and increased major histocompatibility complex II (MHC II) and stem cell antigen-1 (Sca-1) expressions notably not detected in old mice or Alzheimer’s disease models -34 signals, essential in myeloid cell development. CSF1R activation induces the expression of the chemokine CCL2, itself leading to a dramatic increase of CD11cThe ability of microglia/macrophages to phagocyte myelin debris is critical for promoting OPC recruitment and differentiation into myelinating cells  plays an important role in monocyte/macrophage phagocytosis of myelin as suggested by pioglitazone-induced activation of peroxisome proliferator-activated receptor c, one of the permissive binding partners of RXR, shown to inhibit pro-inflammatory differentiation of MS patient-derived monocytes/macrophages and to enhance myelin phagocytosis , is increased in the cuprizone model of demyelination in both astrocytes and microglia and may be required for myelin clearance and oligodendrocyte repopulation . Although using amphotericin B is precluded by its ability to stimulate TNF secretion and its toxic properties (Doring et al., in vitro and microglia/macrophage activation in vivo (Mei et al., via a yet undetermined direct or indirect effect (Natrajan et al., in vivo together with attenuating microglial responses to inflammatory stimuli by decreasing TNF and nitric oxide secretion and preventing activation of NF-κB (Wang H. et al., Several molecules modulating microglia/macrophages are presently trialed in patients. For instance, the anti-muscarinic molecule Clemastine, modulates macrophage inflammatory responses New molecules are continuously being identified. Among them, a recombinant version of a naturally occurring human IgM, rHIgM22, has been shown to promote remyelination in Theiler’s virus infection and cuprizone animal models and to stimulate myelin phagocytosis in a dose-dependent manner (Zorina et al., Besides the growing number of data supporting the beneficial consequences of promoting the pro-regenerative phenotype of microglia/macrophages, several critical works support the deleterious effects of microglia activated through the classical way. First, activated microglia synthesize a multitude of cytokines, chemokines, cell adhesion glycoproteins or reactive oxygen radicals able to damage axons, myelin, oligodendrocytes (Minghetti and Levi, via a mechanism depending on the presence of astrocytes converted into harmful cells by the inflammatory extracellular vesicle cargo (Lombardi et al., As competent presenters of antigen, microglia activated in a classical way express molecules for antigen presentation such as MHC II and its co-stimulatory factors CD40 and CD86, characterized as classical markers of microglia activation in MS and Alzheimer’s disease patients (Gobin et al., In summary, the majority of experimental evidence reported in this review, points to astrocytes and microglia/macrophages as being crucial in both developmental and repairing oligodendrogenesis and myelination. During development, astrocytes and a unique phenotype of neonatal microglia secrete different patterns of molecules controlling oligodendrocyte development and myelination. Although both cell types shape the number of OPCs with consequences for subsequent myelinogenesis, astrocytes are also implicated in a tight crosstalk between OPCs and axons making them indispensable for myelination. Also, by providing a substantial fraction of lipids incorporated into CNS myelin, astrocytes are required for the proper generation of the myelin sheath. Under pathological and demyelinating conditions, reactive astrocytes and activated microglia/macrophages both exhibit a dual activity inducing detrimental or beneficial effects, the balance of which appears to be critical for tissue regeneration. Besides the secretion of a wide range of molecules, both cell types can phagocyte myelin debris. However, in contrast to the beneficial role of microglia/macrophages-induced phagocytosis, myelin uptake occurring as an early response of astrocytes results in recruitment of immune cells possibly increasing tissue damage in demyelinating conditions driven by inflammation. As a whole, the review indicate that beyond the secretion of a multitude of cues directly controlling diverse facets of OPC biology, astrocytes and microglia/macrophages strongly modify the lesion microenvironment. Further delineation of this crosstalk should open the way towards novel therapeutic approaches aimed at recovering proper developmental myelination and relieving the obstacles on the failure of regeneration of damaged myelin characterizing CNS demyelinating diseases.All authors contributed to the design and documentation of this review article.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.


Because of the interactions of T3 with oxytocinergic and dopaminergic neurones of the hypothalamic paraventricular nucleus, these findings provide new neuroendocrinological insight for possible relations with both attachment and metabolic sequelae of early life stress. Necessary is a mutual integration of all recent advances in the yet separated fields.Attachment is a concept that was developed and researched in developmental psychology in uptake of findings on filial imprinting from ethology. In the present period, however, attachment concepts are increasingly applied to and investigated in animal research, thereby translating back criteria that were established for human infants. It herein appears that findings on filial imprinting are becoming more and more forgotten, whilst basic findings in human infants are not reflected in investigations on attachment in animals. To re-integrate both domains, the present article undertakes the effort in briefly reviewing and recapitulating basic findings in human attachment and recent research on filial imprinting. In specific, replicated were critical roles of the conversion of thyroid prohormone by 2 iodothyronine deiodinase (Dio2) into triiodothyronine (T Octodon degus  are combined basal ganglia and neocortical association areas (homologous to V5 in humans) of the telencephalon, the intermediate medial mesopallium (IMM) that has a central role in imprinting via visual cues  indicated that thyroid hormone 3,5,30-triiodothyronine (T3) determinates onset and timing of the sensitive period, that activation of neuronal metabotropic GABAB receptors in the IMM triggers imprinting projections, and that ionotropic GABAA receptor activation terminates respective dendritic outgrowth towards arcopallium and intermediate hyperpallium apicale (IMHA)  into T3, and both thyroid hormones modulate the onset of the sensitive period, through activation of neuronal thyroid receptors (TR), but without apparent involvement of genomic mechanisms  is a protein complex involved in sensing nutrients and regulating protein synthesis triggered by signalling of insulin and its growth factors. Experience-dependent neuronal plasticity in outgrowth of dendritic spines is triggered by mTORC1, and it was shown that mTORC1 determines pre-synaptic spine morphology  existenNewborns at familial risk for autistic spectrum tested for face-like patterns and biological motion did not exhibit the inborn face preference or predisposition, but, in contrast, showed preferences for non-biological motion and inverted faces, discriminative at ROC 0.85 , who had personally experienced early nanny care in a nobility family environment. He sought to provide an empirical basis for mental “object relation” experiences during psychotherapy. Bowlby  traced hr = 0.27.Since the description of the strange situation procedure, with its discrimination in secure vs. insecure attachment relations (later: four attachment classifications), prospective studies tried to identify the causes for attachment security. Recurrent facial and vocal synchronies  state that feline attachment patterns are possibly based on temperament. This long-standing assumption has now been largely refuted by meta-analysis  effects, which are therefore disputed. Implications of dopamine, serotonin and catecholamines point to general stress diathesis in attachment formation under risk. Possible epigenetic mechanisms, as suggested by findings in ELS studies   that the hypothalamic oxytocin receptor promoter is subject to epigenetic modification by DNA methylation, which regulates 3 .The ectoenzyme CD38/adenosine diphosphate-ribosyl cyclase is a transmembrane receptor confined to the brain, whose activation triggers downstream intracellular calcium signalling pathways, critical for oxytocin release  research has established maternal separation as one central experimental paradigm. ELS research is supportive of cerebral functional and structural alterations, along with long-term metabolic sequelae Lemche , followiOctodon degus  ? Do pets show indications of a development of mental person representations in terms of social cognition? Is feeding the causation of human-like attachment patterning? Is sensitive parenting on the owner side with emotional facial and vocal stimulation the cause of attachment-like bonding behaviours towards the human caregiver? Are epigenetic modifications the key to attachment stability in animals and humans?The reviewed findings from the fields of filial imprinting and early attachment indicated that, despite common starting points, the fields have moved on in separated trajectories. It has become apparent that few research has hitherto attempted to integrate the hypotheses and findings of the other field. Whereas experiments in filial imprinting have aimed at elucidating neuronal plasticity in cognitive and memory domains, a great deal of work on human or rodent attachment was based on the oxytocin hypothesis Insel , the dop3 regulates the sensitive period for imprinting learning and memory consolidation opens a new route for such integration. It is long established that T3 activates the mammalian OXT gene promoter independently of oestrogen  in its hypotheses and experimentation, whilst research in filial imprinting should make use of neuroendocrine findings in attachment research. This integration would also strengthen the study of metabolic consequences of insecure attachment and early life stress.


To compare the choroidal thickness in eyes with acute non-arteritic anterior ischemic optic neuropathy (NAION) with healthy contralateral eye and normal controls.Thirty-eight eyes with NAION, thirty-eight unaffected fellow eyes, and seventy-four eyes from 37 healthy, age- and sex-matched subjects were included in this prospective comparative case-control study. Choroidal thickness was measured by enhanced depth imaging (EDI) of spectral domain optical coherence tomography (SD-OCT). Peripapillary choroidal thickness (PCT) was measured at 1000 and 1500 PP = 0.42). The mean PCT at 1500 P = 0.007 and P = 0.048). The mean PCT at 1000 and 1500 P = 0.027 and P = 0.035, respectively). The mean of SFCT was significantly thicker in NAION compared to control eyes (P = 0.032); however, there was no significant difference between uninvolved fellow and control eyes (P = 0.248). The mean of PCT at 1000 Thicker choroidal thickness in acute NAION and uninvolved fellow eyes compared to normal eyes suggests a primary choroidal role in NAION pathophysiology. EDI-OCT provides in vivo cross-sectional image of the choroidal layer. Since choroid is located adjacent to optic nerve and shares interconnected blood supply with optic disc, investigators have started to look at its possible role in two of the most common optic neuropathies, that is, non-arteritic anterior ischemic optic neuropathy (NAION) and glaucoma.,7,8,9 NAION is the most common acute optic neuropathy and, as the name implies, it is presumed to be secondary to the vascular insufficiency within optic disc space, but the nature of the vasculopathy and its pathophysiologic mechanism is not definitely known yet.Although the role of choroid in several sight-threatening ocular conditions such as polypoidal choroidal vasculopathy or choroidal neovascularizationEvaluating choroidal thickness in acute NAION may help identify choroidal role in NAION and clarify if the choroidal thickness changes in NAION is a predisposing factor for this condition or the consequence of acute optic neuropathy.The purpose of this case-control study was to evaluate the peripapillary choroidal thickness (PCT) and subfoveal choroidal thickness (SFCT) in eyes with acute NAION and compare them with uninvolved fellow eyes and eyes of healthy controls.This prospective comparative case-control study was carried out at Torfeh Hospital as a university-based tertiary eye center. The study protocol was approved by the Ethics Committee and Review Board of Shahid Beheshti University of Medical Sciences. All investigations were carried out in accordance with the Declaration of Helsinki. Patients with acute NAION and less than 14 days of clinical presentation were included from January 2014 to June 2015.The diagnosis of NAION was based on the presence of a history of sudden painless visual loss associated with a relative afferent pupillary defect that was not attributed to any other ocular, neurologic, or systemic disease, optic disc swelling detected by biomicroscopic examination at the time of presentation and confirmed by peripapillary retinal nerve fiber layer thickness measurement using OCT, visual field testing compatible with ischemic optic neuropathy, and resolution of disc swelling and appearance of optic atrophy within two months.The exclusion criteria were clinical symptoms of giant cell arteritis, high erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) level, any history or clinical evidence of retinal or neurologic disease, previous retinal procedure , intraocular pressure (IOP) All OCT measurements were performed using Heidelberg spectral domain OCT  by the same experienced operator. For each eye, three OCT images were acquired.PCT was measured using horizontal and vertical raster EDI-OCT scans centered at optic nerve head (ONH) [Figure 1]. SFCT was determined using horizontal raster EDI-OCT scans centered at the fovea. Macular OCT scan: 25 horizontal optical coherent tomographic sections were obtained in a 20Choroidal thickness was measured along the perpendicular distance from posterior border of retinal pigment epithelium to anterior scleral border  using the OCT manual measurement tool. For peripapillary choroid, we measured the choroidal thickness at 1000 analysis was used to compare the groups. Furthermore, adjusted results for hypertension and diabetes were obtained using another GEE model. To consider the multiple comparisons, issue in pairwise comparisons, we used the Bonferroni method. The correlation of variables was obtained by the Pearson method. All statistical analyses were performed by SPSS software . All tests were two-sided and PTo present data, we used mean, standard deviation, median, and range. To assess normal distribution of quantitative data, we used Kolmogorov–Smirnov test and Q–Q plot. To compare the results between groups at baseline, we used P = 0.95). The mean interval between the initiation of symptoms and OCT acquisition was 6.7 P = 0.64) and mean axial length (P = 0.87). Affected cases were more likely to have diabetes mellitus (DM), but the prevalence of systemic hypertension was comparable in both affected and healthy individuals [Table 1].Among 46 enrolled eyes, 38 eyes with acute NAION were included for final analysis. Eight eyes were excluded because of poor-quality OCT and inability to determine choroidal–scleral junction. Thirty-eight uninvolved fellow eyes and seventy-four eyes of 37 healthy, age- and sex-matched subjects were included as the control group. The mean age of NAION patients and control subjects were not significantly different (62 Intraobserver and interobserver reproducibility of PCT and SFCT are shown in Table 2. There was excellent reproducibility in the intraobserver and interobserver tests (ranging from 0.934 to 0.992 for the intraobserver and from 0.874 to 0.961 for the interobserver reproducibility).PP = 0.042, respectively). Moreover, PCT1000 was significantly thicker in involved eyes than uninvolved fellow eyes (P = 0.025) [Table 3].The mean PCT at 1000 P = 0.007 and P = 0.048, respectively). In addition, PCT1500 was significantly thicker in the eyes with NAION than the uninvolved fellow eyes (P = 0.035) [Table 3].The mean PCT at 1500 P = 0.038), however, there was no statistically significant difference between the fellow and control eyes (P = 0.248) [Table 4].The mean SFCT was 275 PPP = 1.000).CMT was significantly higher in the NAION than the uninvolved and control eyes (PP = 0.006). There was no statistically significant difference among the NAION, uninvolved fellow, and control eyes regarding the Gc-IPL thickness in the inner macular area [Table 4].Central Gc-IPL thickness was significantly thicker in the eyes with NAION eyes than the uninvolved fellow and control eyes (The differences persisted once adjusted for DM and systemic hypertension [Table 5].In the present study, we found significantly thicker choroid in both acute NAION and unaffected fellow eyes compared to controls, both in peripapillary and subfoveal areas.,9 This study is the first one that measured PCT in acute phase. By adjusting the contrast and resolution scale of the OCT device, we were able to measure choroidal thickness despite edema and disc swelling.All previous studies evaluated the PCT in the chronic phase of NAION.,15Although the exact mechanism of NAION is yet to be understood, ischemia and the so called “compartment syndrome” in optic disc space are the main determinants for the loss of visual function. Therefore, since the choroid mainly supplies – to a limited extent – more anterior optic nerve head regions, it is less likely that choroid has a substantial role in the pathogenesis of NAION. However, several studies reported changes in choroidal thickness in NAION,8,9],98,9] which is consistent with the findings of the current study.Whatever the mechanism of vascular insufficiency in NAION, persistent hypoperfusion is required for the development of ischemic optic neuropathy. The paraoptic branches of the short posterior ciliary arteries are responsible for ONH blood supply and the infarction in NAION is suggested to be predominantly located in the retrolaminar portion of the ONH. Our finding also matches the finding of Nagia et al that observed increased thickness of the PCT, especially 1000 to 1500  Surprisingly, the mean PCT in the affected eyes in our study was lower than the aforementioned studies with chronic NAION. Although one should be cautious to draw any conclusion from these cross-sectional studies, chronic cases were expected to have thinner choroid compared to the eyes at the acute stage if choroidal thickening was secondary to ischemic damage. However, longitudinal studies are needed to evaluate the changes of choroidal thickness following the ischemic damage.Fard et al evaluated the PCT in chronic NAION and reported increased thickness in both affected and unaffected fellow eyes compared to normal controls. further reinforces the primary role of choroid in the development of NAION.The lack of consistent choroidal filling defect in angiographic studies Increased PCT may be a component of crowded disc morphology as suggested by Fard et al. They hypothesized that the thicker choroid may strain the optic nerve axon and its vessel within the narrow central cup and further restrict the crowded optic nerve space.Crowded disc is known as a predisposing factor for NAION, although the mechanism by which it contributes to ischemia is unclear.,9 The NAION progresses through asymptomatic “incipient NAION” to full blown ischemic neuropathy with characteristic clinical presentation in more than two-third of cases,19. By further contributing to “compartment syndrome” in already constricted scleral canal, increased choroidal thickness may have a role in the development of symptomatic axonal damage. In that sense, increased choroidal thickness acts as a “catalyzer” for the progression of ischemic optic neuropathy.Thicker peripapillary choroid in NAION eyes may be a structurally predisposing factor or may be a compensatory mechanism due to ischemia. Thicker choroid, both in acute NAION and uninvolved fellow eyes, rationally suggests the role of choroid as a predisposing factor rather than a compensatory mechanism. Moreover, if increased choroidal thickness was a compensatory factor, it must be decreased after the acute phase, but as mentioned earlier, the result of previous studies in the chronic phase also showed thicker choroid. Thicker choroid may also be a sign of local auto-regulation dysfunction of the choroidal vessel.Moreover, Beijing Eye Study showed no association between SFCT and optic disc size in normal subjects, therefore choroidal thickness could affect the incidence and progression of NAION independent of optic disc size.,9 Garcia-Basterra et al reported thinner PCT in NAION and unaffected fellow eyes compared to normal subjects. The differing results may be because they did not match the refractive state of control subjects and NAION patients, and also they evaluated PCT in chronic phase of AION.In contrast to our study and others, reported no difference between sub-macular choroidal thickness in acute and chronic NAION eyes and control subjects, they included subjects with optic disc edema that had occurred in a time period of less than one month as an acute NAION, but we included patients with initial symptoms occurring less than 14 days; they also measured choroidal thickness in farther points from the foveal center. Interestingly, Schuster et al reported thinner SFCT in the affected and non-affected fellow eyes of patients with NAION. Although the authors considered the enrolled cases as acute NAION, the duration of the disease that was considered acute is not reported in their paper, so we cannot confidently compare our findings with their results. Besides, they used one horizontal line passing through fovea and CT was measured on 1 point on this line. We argue that measuring choroidal thickness at 1 selected point, which is performed manually, could be affected by irregularities of choroidal-scleral boundary. In addition, not all factors affecting the choroidal thickness measurement were adjusted in that study.Gonal et al,25,26 Since macular OCT is not routinely obtained in NAION, the exact incidence of macular edema is unknown in these eyes but it is estimated to range from 10 to 50%. There is no longitudinal study that has evaluated the course of macular edema after the NAION and its effect on the final visual acuity.The thickening of central macular region in our study is in line with previous reports that have shown subretinal fluid accumulation in recent onset NAION. reported that parafoveal Gc-IPL thinning present in chronic NAION eyes was associated with worse vision compared to glaucoma patients in which central vision is preserved and has no Gc-IPL thinning in this area. Parks et al reported the initial thinning of Gc-IP layer in the macula occurring 46.1  We measured Gc-IPL thickness in the period of less than two weeks from initial symptoms, which showed thickening only in the affected eyes. The ischemia induced axoplasmic flow stasis results in axonal swelling and is considered an early event in the “sequence of events” in NAION. In fact, this early axonal swelling is the stage where timely interventions such as anti-inflammatory and neuroprotective agents proved to be promising.,31,32In the present study, we found that the Gc-IP layer in the central 1 mm was significantly thicker in NAION eyes compared to uninvolved fellow eyes and control eyes, while in all quadrants of 1 to 3 mm annulus, there was no significant difference among NAION, uninvolved fellow, and control eyes in terms of Gc-IP layer thickness. The thickening of Gc-IPL in the central 1 mm may be due to intracellular edema due to greater ischemic stress on central fovea in the early phase of NAION and contributes to vision deterioration. Fard et alThis study has several limitations. The sample size was relatively small but is comparable to previous studies on this subject and necessitates further longitudinal studies to clarify whether choroidal thickening precedes NAION or it occurs following the ischemic incidence.,9 Furthermore, recent endeavors to quantify choroidal thickness with automated segmentation have shown excellent consistency with manual labeling. Choroidal thickness has not been adjusted for IOP in our study and it may have caused some random errors, although the induced bias may be far from significance.Although anterior scleral border was delineated manually, intra- and interobserver reproducibility were excellent, which is in line with the high reproducibility reported in previous studies.In summary, we showed that the boundaries of the choroid in acute NAION could be delineated by excellent intra- and interobserver reproducibility. Moreover, our finding of increased choroidal thickness in both eyes of the affected patients compared to controls lends support to the theory of primary choroidal role in the development of NAION.Nil.There are no conflicts of interest.


Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab  was higher than in the blood  from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity ( The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients ,5. HowevOne remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms.Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People’s Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People’s Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit  on an automatic workstation Smart 32 (Da’an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT–PCR) reagent  was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s).p value = 0.0001). In the anal swab cohort  distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort . Patient 2 (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings.In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group  was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry , which mUnfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, wIn summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.


Purpose: To evaluate the incidence of choroidal neovascularization (CNV) in high myopic patients who experienced recent visual loss and to describe the characteristics of CNV that develop as a complication of pathologic myopia and were detected by fluorescein angiography (FA).Material and methods: The fluorescein angiograms of 89 patients with high myopia, who registered a recent decrease of visual acuity were reviewed. The incidence of CNV was evaluated and its angiofluorographic appearance described. Results: 172 eyes of 89 patients  were analyzed. Among 89 patients , in 58 cases (~65%), the angiofluorography revealed the presence of a neovascular membrane, and 8 cases presented bilateral CNV. That made a total of 66 eyes. The majority of CNVs were classic (56%), 20% had an occult pattern and 24% were cicatricial. The location was predominantly subfoveal.Conclusions: Considering that the main cause for visual loss in myopic patients is the new-onset myopic CNV, fluorescein angiography should be performed in cases with recent visual loss. When CNV is suspected, FA can demonstrate the presence of myopic CNV, bringing information regarding the type, area, and activity of the CNV and helping exclude other disorders. Due to its location, predominantly sub- or juxtafoveal and to the extended retinal lesions associated with pathologic myopia, the visual prognosis of these patients is poor despite new active treatments. These eyes develop retinal degenerative changes and complications  in the posterior pole and/ or in the fundus periphery . These changes give the eye fundus a characteristic aspect (myopic fundus). The myopic maculopathy is the cause for the loss of the best-corrected visual acuity of these patients and was also used to define pathologic myopia (PM) [4].Although the definition of high myopia or pathologic  myopia is not uniform [5]. The findings range from the early appearance of a tessellated fundus to the development of diffuse atrophy and lacquer cracks, followed by progression to patchy atrophy. The areas of patchy atrophy have the tendency to enlarge and fuse. CNV usually develops adjacent to the area of patchy atrophy or lacquer cracks .The exact nature of the relation between the level of severity of refractive error associated with specific pathology is still unknown. There is no clearly stated correlation between the severity of refractive error and the specific myopic pathology. Although myopic CNV was reported to occur in eyes without typical myopic degenerative fundus changes and even at lower degrees of myopia, the natural course of the myopic maculopathy was described to follow a progressive pattern . Recently, a new classification system was proposed by Ruiz-Medrano et al., based on three key factors: atrophy, traction, and neovascularization [8].Based on long-term clinical observations and considering the progression pattern of the macular changes, Ohno-Matsui et al. formulated a standardized, reproducible classification system for myopic maculopathy defining 5 categories: “no myopic retinal lesions” (category 0), “tessellated fundus only” (category 1), “diffuse chorioretinal atrophy” (category 2), “patchy chorioretinal atrophy” (category 3), and “macular atrophy” (category 4). Three additional features were added to these categories and were included as “plus signs:” (1) lacquer cracks, (2) myopic CNV, and (3) Fuchs spot. PM is defined as myopic maculopathy category 2 or above, or the presence of staphyloma . In patients younger than 50 years old, myopia is the main cause for CNV, accounting for more than 60% of the cases [2]. The development of the myopic CNV is accompanied by the following acute symptoms: decrease in visual acuity, metamorphopsia and central or paracentral scotoma. On clinical examination, a greyish, flat, small lesion is noticed, usually situated sub- or parafoveally, with or without hemorrhage. Typically, the subretinal membrane is less than 1 disc diameter in size and is located between the neurosensory retina and retinal pigment epithelium (RPE) (type 2), whereas the CNV secondary to age related macular degeneration (AMD) is usually in the sub-RPE space (type 1) .The choroidal neovascularization (CNV) was reported to occur in 5-10% of the eyes with high myopia [9].Ohno-Matsui et al. studied the incidence and predisposing findings for choroidal neovascularization (CNV) in a large series of highly myopic patients. They concluded that 1/ 10 of highly myopic eyes with myopic fundus changes in the macula can develop myopic CNV within 3 years and that 30% of these patients will develop CNV in the second eye within the next 8 years after CNV development in the first eye. The risk of developing myopic CNV was higher in eyes with patchy atrophy or lacquer cracks around the macula; during the follow-up period, these patients developed myopic CNV in 20% and, respectively, about 30% of the cases [10]. Chorioretinal atrophy developing around myopic CNV is the main cause of long-term visual decrease, being influenced by CNV size and patients age .The natural course of CNV is towards regression and scarring, resulting a fibrous pigmented scar (Fuchs’ spot). Chorioretinal atrophy usually develops around the regressed CNV, accounting for the extremely poor long-term visual outcome of the myopic CNV. Yoshida et al. showed that the visual acuity of almost all of the patients dropped to 20/ 200 or less within 5 to 10 years after the onset of CNV . The progression pattern of the myopic maculopathy was demonstrated in long-term clinical studies  and the gradual visual impairment was shown to be caused by the development of CNV or CNV-related macular atrophy and enlargement of macular atrophy. Although anti-VEGF-treatment seemed to be effective in improving and maintaining visual acuity in patients with myopic CNV at 1-year-follow-up , the visual acuity gain decreases in most cases in time and deteriorates irreversibly due to advanced myopic chorioretinal atrophy [21-23].The prognostic of CNV associated with myopia without treatment is not very favorable. Factors generally associated with poor visual prognosis are subfoveal localization, large CNV, lower baseline best-corrected visual acuity and age over 40 [24].Sudden vision loss in a high myopia could be due to development of CNV, but these patients have often already poor visual acuity due to posterior staphyloma or chorioretinal atrophy. In the presence of large chorioretinal atrophy and other findings that are common in myopic maculopathy , the clinical examination may not be able to reveal an obvious CNV in a high myopia which presents with sudden loss of vision .It is very important to diagnose a CNV, as the visual deterioration can be influenced by anti VEGF treatment [8]. These diagnoses should be identified, as they require different therapeutic approaches from CNV [5].Vision loss in a myopic patient can be due to several pathologies and complications of high myopia, including myopic traction maculopathy, epiretinal membrane, vitreomacular traction and myopic full-thickness or lamellar macular hole, retinal hemorrhage due to new lacquer crack formation and macular exudative changes associated with a dome-shaped macula or a staphyloma . Different studies evaluated the contribution of FFA and SD-OCT in diagnosis of myopic CNV, finding that FFA has better repeatability and reproducibility to make the diagnosis of myopic CNV compared with SD-OCT. Some concluded that SD-OCT is a better tool to rule out the presence of myopic CNV [24]. OCT and now OCTA are non-invasive, easy repeatable imaging tools that aid in an early diagnosis and are important for follow-up of such eyes. OCT alone cannot differentiate myopic CNV from subretinal bleeding due to new lacquer crack formation, which could lead to unnecessary treatment for subretinal bleeding without CNV by anti-VEGF therapy [5]. Leveziel et al. found that exudative features of myopic CNV are more obvious on FA than on SD OCT, suggesting that fluorescein angiography should be performed when new-onset myopic CNV is suspected [25].Fluorescein angiography (FA), indocyanine green angiography, spectral domain optical coherence tomography (SD-OCT), and optical coherence tomography angiography (OCTA) are available diagnostic tools, helpful in making a correct diagnosis .In most of the cases, the angiogram revealed a classic, well defined CNV (well-defined hyperfluorescence in the early phase), sometimes with a hypofluorescent halo, small in size (less than 1/ 2 optic disc diameter), situated sub- or juxtafoveal, presenting minor to moderate exudation in the late phases .It is rare for choroidal neovascular membranes in myopic eyes to have a poorly defined aspect, but these findings have been described . This aspect of CNV occurring in older patients, comparative to those seen in younger patients, may suggest an overlap between myopic CNV and age-related macular degeneration [25].Larger CNVs can occur especially in older patients (≥ 55 years) .The natural course of CNV is towards regression and scarring, resulting in a fibrous pigmented scar (Fuchs’ spot). The aspect of cicatricial CNV on the angiogram presents hyperfluorescence due to staining, but no exudation. Following regression of the CNV, chorioretinal atrophy forms around the scar, being responsible for the poor long-term visual outcome , these being the most common predisposing findings for the development of CNV . A subretinal bleeding is not always associated with choroidal neovascularization in pathologic myopia and can be a sign of new lacquer crack formation .The ruptures of the RPE-Bruch membrane and choriocapillaris complex (lacquer cracks) appear in the posterior pole as fine yellow radial lesions, hyperfluorescent on the angiogram. Lacquer cracks are usually a sign for the progression of the myopic maculopathy and predispose high myopes to visual loss . Our study confirmed this observation . This may confirm the hypothesis that estrogen may play a role in the mechanism of development of CNV [2].It seems that women are more often affected, epidemiologic studies suggesting that myopic CNV is more common in females [2]. In our group, the mean age was 52 with only 37  patients under 50 years of age, suggesting that the visual threatening complications of the myopic maculopathy might develop at an older age. Several studies confirmed that aging is associated with more advanced forms of myopic maculopathy and worse BCVA .Unlike the CNV in age-related macular degeneration, more than 50% of affected myopic patients had a presenting age of 50 or less in most studies [9]. In our study, 14% of the cases were bilateral at presentation (8 among 58 patients).Reviewed articles state that approximately 30% of the myopic patients with CNV will develop CNV in the second eye within 8 years . Long-term visual outcome of myopic CNV is poor, despite new active treatments . Future therapies should focus on preventing subretinal fibrosis, a characteristic feature of the scarring stage in myopic CNV, which is responsible for the irreversible visual loss [8].Although predisposing lesions for CNV  are well known and easily detected, no prophylactic treatment can be applied [FA enables the understanding of the physio-pathological process in eyes with myopic changes and is useful in detecting the presence and type of myopic CNV, it provides information about its extent, location, and activity, and helps exclude other disorders. It gives an extensive description of the myopic fundus and should be performed as baseline diagnostic test in myopic patients with recent visual decrease. In high myopic eyes, the angiofluorographic detection of a CNV is sometimes difficult due to the multitude of associated lesions and its “low-exudative” characteristics. The information provided by FA should be completed with an OCT imaging. Imaging in high myopic eyes is challenging, as each imaging modality has its limitations; a multimodal approach is important for image interpretation, accurate diagnosis and progression of the disease monitoring. A correct assessment of all fundus changes is necessary before any surgical intervention, whether refractive or cataract surgery, in order to evaluate the visual prognosis.Conflict of InterestThe authors state no conflict of interest.Informed ConsentInformed consent has been obtained from all individuals included in this study.Authorization for the use of human subjectsThe research related to human use complies with all the relevant national regulations, institutional policies, is in accordance with the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of the Department of Ophthalmology, “Carol Davila” University of Medicine and Pharmacy, Bucharest.AcknowledgementsNone.Sources of FundingNone.DisclosuresNone.


Plants growing in any particular geographical location are exposed to variable and diverse environmental conditions throughout their lifespan. The multifactorial environmental pressure resulted into evolution of plant adaptation and survival strategies requiring ability to integrate multiple signals that combine to yield specific responses. These adaptive responses enable plants to maintain their growth and development while acquiring tolerance to a variety of environmental conditions. An essential signaling cascade that incorporates a wide range of exogenous as well as endogenous stimuli is multistep phosphorelay (MSP). MSP mediates the signaling of essential plant hormones that balance growth, development, and environmental adaptation. Nevertheless, the mechanisms by which specific signals are recognized by a commonly-occurring pathway are not yet clearly understood. Here we summarize our knowledge on the latest model of multistep phosphorelay signaling in plants and the molecular mechanisms underlying the integration of multiple inputs including both hormonal  and environmental (light and temperature) signals into a common pathway. We provide an overview of abiotic stress responses mediated via MSP signaling that are both hormone-dependent and independent. We highlight the mutual interactions of key players such as sensor kinases of various substrate specificities including their downstream targets. These constitute a tightly interconnected signaling network, enabling timely adaptation by the plant to an ever-changing environment. Finally, we propose possible future directions in stress-oriented research on MSP signaling and highlight its potential importance for targeted crop breeding. Under natural conditions, plants are need to continuously maintain a balance between growth and defense or adaptive responses that are dictated by the severity, duration, and developmental timing at which any particular stress occurs . The essETHYLENE RESPONSE FACTOR 1 (ERF1) was gradually induced by drought stress treatment over 12 h (reaching a maximum after 1 h), and this plays a positive role in drought, salt, and heat stress tolerance via stress-specific gene regulation  action have revealed that cytokinin modulates levels of stress-related genes/proteins . On the gulation .Arabidopsis thaliana).All these findings (and many others described below) show how important plant hormone signaling (including the MSP) and metabolism are during early stress responses. In the next few sections, we attempt to summarize recent observations on the biological relevance of MSP-mediated stress adaptation strategies in plants (mostly in RESPONSE REGULATORS (A-ARRs), the cytokinin primary-response genes, which also function as negative feedback regulators of the signaling pathway (APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene family, mediate a large fraction of the transcriptional response to cytokinins, affecting a set of cytokinin-responsive genes that largely overlaps with targets of type-B ARRs , located both at the plasma membrane and the endoplasmic reticulum . Recentl pathway . In tande-B ARRs . For morcis-acting GCC box and DRE elements depending on whether the stress conditions are abiotic or biotic  and ETHYLENE RESPONSE SENSOR 1 (ERS1) with a functional HK domain, and the HK-like sensor Ser/Thr kinases ETR2, ERS2, and ETHYLENE INSENSITIVE 4 (EIN4), that make up subfamily 2 . Ethylenarr2 mutant is less sensitive to treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) probably due to a positive effect of ARR2 on the expression of ERF1, an ethylene-responsive member of the ERF/AP2 transcription factor family  and the UV-B RESISTANCE 8 (UVR8) family proteins affect plant behavior in response to both quantity and quality of light. The individual signaling branches mediating responses to a wide range of wavelengths as well as their interplay has been extensively studied e.g., . Since lCOP1–SPA E3 ubiquitin ligase  and far-red (FR) light. Phytochromes are synthesized as light-insensitive apoproteins and the functional, i.e., light photoswitchable holoproteins (phys) are assembled following binding with the light-sensitive, linear tetrapyrrole chromophore cofactor phytochromobilin, that is imported to the cytosol from plastids . Upon abn ligase . PIFs con ligase . COP1 inn ligase . More deOne of the first pieces of evidence for direct crosstalk between MSP and light signaling was presented by CKI1 was found to be under the control of phyA, probably via PHY-INTERACTING FACTOR 1 (PIF1) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). Plants defective in phyA and HEME OXYGENASE1 (HO1), whose product is involved in the production of phytochromobilin, show misregulation of CKI1 expression, associated with attenuation of MSP responsiveness to cytokinins and occurrence of cytokinin-related phenotypes  controls the activity of (MSP) signaling pathway by transcriptional regulation of the constitutively active HK, thus controlling MSP sensitivity to its primary signal, cytokinin  that do not mediate hormone responses  kinase . Under uproteins . The abiproteins .2+ levels  and cytoskeletal rearrangement  . SnRK2s pression .Since ABA interacts tightly with MSP signaling during the abiotic stress response, the mutual crosstalk might occur at several levels including regulation of phosphorylation of individual signaling components as well as regulation of dynamic chromatin state . These iAltogether, the MSP pathway is not just the cytokinin signaling transduction system, but it acts as a signaling hub, integrating both intrinsic  regulation with that from environmental signals, including light, osmoregulation, and temperature . The impStress, in general, affects the metabolism of cytokinins, ABA, and ethylene, which in turn interact with specific kinases to regulate many stress-related biological processes ranging from regulation of shoot growth under stress to stomata movement. Drought results in the reduction of cytokinin content by modulating cytokinin metabolism and stimulating ABA and ethylene contents in a wide range of terrestrial plants e.g., . Even th2 uptake and water loss by transpiration  which is responsible for removing sodium ions from root xylem. Cytokinins were also shown to regulate salt stress−induced expression of the type−A response regulator ARR5 predominantly via ARR1 and ARR12, revealing the action of particular MSP components in the roots to control sodium accumulation in the shoots  and the type-A ARR genes ARR5, ARR7, and ARR15. This ethylene-mediated repression was supposed to be mediated by direct binding of EIN3 to the promoters of type-A ARRs, thus potentially representing another mechanistic link between canonical ethylene signaling and MSP during plant desiccation , NAC DOMAIN CONTAINING PROTEIN (ANAC), and DRE-BINDING PROTEIN 2A (DREB2A) transcription factors and their downstream genes may explain the increased stress tolerance of AHK1 overexpressing plants  is an important physiological adaptation trait of plants to drought. The recent findings of Furthermore, various types of abiotic stresses including cold, osmotic stress, salt, and drought differentially regulate the expression of several individual MSP components . As mentetr1-6 and etr1-7 mutants have a higher germination rate than wild-type seeds in the dark or following FR illumination (that inhibits seed germination in the WT) indicating a negative role for ETR1 in the control of germination  that further activate downstream ABA-unresponsive and ABA-activated SnRK2s . Fast reTemperature is another fundamental environmental factor, with 10–30°C being optimum for the growth of most higher plants . TemperaArabidopsis -mediated cold acclimation pathway. Based on the hypersensitive response of ahk2, ahk3, and arr7 mutants, as well as the insensitivity of ARR7 overexpression line to ABA, the authors propose that the MSP-mediated cold response is mediated via negative regulation of ABA signaling by AHK/ARRs, thus following the general scheme of negative cytokinin/ABA interaction as in the drought response described above  regulatory mechanisms, mediating the AHK2/3-dependent cold response , , , 2017. Rresponse .Metaphorically, sensor kinases serve as guards at the gate to the cell or nucleus, enabling the perception of various environmental signals. Besides HKs, acting as an integral part of canonical MSP signaling, other sensor kinases integrated into MSP signaling are phytochromes  similar to phyB mutants, demonstrating that circadian light input is modulated by the interaction of phyB with ARR3 and ARR4. Considering the absence of any correlation between sensitivity to cytokinins in higher-order arr mutants and the period phenotype, as well as the distinct (concentration-dependent) effects of exogenous cytokinins, ARR3 and ARR4 seem to control both the length of the period (independent of phyB) and the phase of the period (through phyB) independently of cytokinins (ARRs expression responds to temperature fluctuations independently of cytokinin levels (see above), we may say that type-A ARRs could represent an intersection for light and temperature signaling to fine-tune photomorphogenesis under adverse environmental conditions  in the control over context-dependent chromatin accessibility , the welThe conclusions and implications we summarize above point toward a number of important questions. How is the combination of individual inputs translated into the final MSP signaling output? How is signaling fidelity maintained? In other words, how does the plant recognize and discern the particular origin of the signal, given that a number of sensory kinases recognizing the different type of signals result in phosphorylation of AHPs, and act as a signaling hub within the pathway? Is there any variability in the sensitivity of MSP signaling to individual types of MSP inputs associated with ecotype-specific adaptation to the site of its origin? And finally, what are the mechanisms acting downstream of MSP signaling in the stress response, either independently or in concert with other signaling pathways?Future studies aimed at potential modification of the MSP signaling output at the level of the regulated genes and characterization of the underlying molecular mechanisms will be necessary to answer some of these questions. Studying the existing pool of natural genetic variability in MSP signaling affecting the responsiveness of the pathway to various inputs might provide important evidence on the potential role of MSP signaling and its downstream targets. They would also go a long way in shedding light on the ability of plants to successfully face environmental stresses which are after all an everyday reality of plant life. In combination with tools for targeted genome editing of plant genomes that have become available recently , this tyJS, KN, and JH performed the literature search. KN and JS created the figures. JS, KN, RV, and JH wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Numerous changes in maternal physiology occur during pregnancy that are critical in controlling and maintaining the maternal metabolic adaptations and fetal development. The placenta is the key source through which the fetus receives nutrients, blood, and oxygen for growth. The human placenta releases several molecules into maternal circulation that include hormones, proteins, RNA, and DNA throughout the course of pregnancy. Additionally, extracellular vesicles (EVs) originating from the placenta have been found in the maternal circulation.Methods: In this review, we discuss the role of EVs in maternal-fetal communication during pregnancy.Results: EVs originating from the placenta can be divided into 3 categories based on their size and/or origin: exosomes (50 to 150 nm), microvesicles , and apoptotic bodies or syncytial nuclear aggregates (>1 μm). The cellular microenvironment—such as oxygen tension and glucose concentration—have been found to control EV release from the placenta and their bioactivity on target cells. Furthermore, maternal EVs can stimulate cytokine release from endothelial cells and are involved in several physiologic and pathologic events in pregnancy.Conclusion: Exosomes provide a way to identify the function and metabolic state of cell origin through their ability to reflect the microenvironment that they are released from. Further understanding of how EVs regulate key events in pregnancy may help elucidate how maternal-fetal communication is established in both normal and pathologic conditions. The placenta releases many different molecules that can alter maternal physiology to accommodate fetal requirements during gestation. The placenta may also influence the physiology of the mother via extracellular vesicles (EVs).6 EVs encapsulate a diverse cargo of proteins, lipids, and nucleic acids that are released into the maternal circulation and are subsequently taken up by cells of the maternal immune and vascular systems, thereby modulating the overall maternal physiologic system to adapt to pregnancy-induced changes.4 In complicated pregnancies, this mode of cell signaling plays a role in the manifestation of physical symptoms of disease states, as the release of the EVs is dependent on the microenvironment to which they are exposed.7EVs are increasingly released into the maternal circulation as pregnancy progresses, in both healthy and pathologic pregnancies  or preeclampsia [PE]) .4-6 EVs 11 but this review focuses on EVs during gestation, with emphasis on the trafficking of placental vesicles into maternal circulation to regulate immune and metabolic adaptations in pregnancy.Several recent (2014 through 2020) reviews on EVs during pregnancy have been published,12 EVs are lipid bilayer structures measuring 50 nm to several μm that are released from a multitude of different cell types, including trophoblasts,13 erythrocytes,14 and endothelial cells,15 into the extracellular environment. Within their lipid bilayer membrane, EVs contain RNA, DNA, and proteins, which are hypothesized to modulate the bioactivity of specifically targeted cells.16 Studies published in 2014 and 2018 have demonstrated that EVs may affect cellular adaptations to physiologic changes during gestation, including immune responses, migration, and invasion of placental cells.18Investigation of the potential role of EVs in intercellular communication has been an area of growing research.12 Exosomes are a specific type of EV (50 to 150 nm) that arise from the inward budding of the plasma membrane, forming multivesicular bodies that internalize to form intraluminal vesicles and are then released into the extracellular environment as exosomes. As a result of their mode of formation, exosomes are enriched with intracellular proteins such as CD63, CD9, and CD81. Exosomes are distinguished from other EV cells by their distinct cup-shaped morphology and a buoyant density in a sucrose gradient, ranging from 1.13 to 1.19 g/mL. Compared to exosomes, microvesicles are different in size and originate in a different manner. Microvesicles can be as small as exosomes or as large as a few μm in size and are derived from plasma membrane secretion through direct budding of the membrane itself in response to cellular activation or in response to stress.19 Similarly, apoptotic bodies differ from exosomes in size (>1 μm) and through their formation via membrane blebbing, membrane protrusion, and formation of distinct bodies.The different types of EVs—exosomes, microvesicles, and apoptotic bodies—can be categorized on the basis of their size, surface markers, and content.Regardless of their origin and size, EVs play important roles in cell communication and act as key regulators in disease states and healthy conditions. In the context of pregnancy, the placenta releases a wide range of EVs that are identified in the maternal circulation in both normal pregnancies and pregnancies with complications.5 Placental exosomes are hypothesized to regulate the establishment of maternal-fetal circulation via remodeling of spiral arteries. Upon implantation, the blastocyst adheres to the endothelium of the placenta, and the trophoblastic cells of the fetus differentiate into an inner cytotrophoblast and an outer syncytiotrophoblast layer. As the blastocyst continues to grow, the cytotrophoblast layer forms a layer of multinucleated syncytiotrophoblasts. Within this layer, the cytotrophoblast releases proteolytic enzymes, while the syncytiotrophoblast layer extends finger-like projections into the endometrium and replaces the endothelial cells of the uterine spiral arteries, establishing a connection between the growing embryo and maternal blood. This normal physiologic process is thought to be mediated at least in part by cell-to-cell communication via EVs and is complete by 10 weeks of gestation.Exosomes are released from different organs, including the placenta, in both normal and pathologic conditions.20 Whereas HLA-G is expressed only in extravillous trophoblasts, HLA-G+-EVs have been detected in pregnancy.21 An analysis of EVs released from extravillous trophoblasts using Swan71 cell line isolation showed that the EVs release exosomes.22 The findings of this study strongly indicate that extravillous trophoblasts not only release microvesicles but specifically exosomes.Evidence of EV release by extravillous trophoblasts has been demonstrated through the detection of soluble proteins such as human leukocyte antigen G (HLA-G).24 In a comparison of pregnant and nonpregnant women, the concentration of exosomes was approximately 50-fold greater in pregnant women.25 The increased concentration may be attributable in part to the contribution of placental-specific exosomes that have been identified in maternal circulation as early as approximately 6 weeks of gestational age.9 Specifically, placental-alkaline phosphatase-positive (PLAP+)-EVs were detected in the maternal circulation throughout pregnancy.5 The PLAP+-EVs can be isolated from the plasma of pregnant women and increase during the first 12 weeks of gestation.5 The increased presence of these EVs may be because of the changes and adaptations in maternal physiology.The amount and type of EVs released are affected by the microenvironment.26 In pregnancies with increased stress, such as those complicated by GDM6 and PE,27 more EVs are released. For example, the circulation in women affected by PE contains approximately 40% greater number of EVs compared to healthy, pregnant women.28 Furthermore, studies have demonstrated that changes in the microenvironment can alter the secretion of EVs from cytotrophoblast cells and in turn affect vesicle content and bioactivity.24 Exosomes released from the placenta have been shown to decrease insulin sensitivity and glucose uptake in skeletal muscles, contributing to the pathophysiology of GDM.29 Also, exosomes released from other types of cells can affect placental function and are involved in regulating the physiologic and pathologic mechanisms in pregnancy. For example, exosomes released from adipocytes can mediate changes in placental metabolic status and contribute to GDM.30 Hence, the cross-communication between different organs and the placenta via exosomes is crucial in mediating the maternal metabolic changes in pregnancy.31Changes in the cellular microenvironment, such as hypoxia or increased glucose concentration, also affect EV release. These different microenvironments can also affect vesicle content and the bioactivity of target cells.27 Furthermore, studies have found that states of abnormal placentation, such as PE, have increased concentrations of circulating placental-derived EVs.32 All of these findings suggest that EVs have a significant role during pregnancy. The 43 Future studies are required to develop a proper understanding of the mechanisms of exosome release and their effect on the target cells, which could give insight into their role as markers for the diagnosis of pregnancy complications.As previously stated, the role of exosomes in the remodeling of spiral arteries from a high-resistance, low-capacitance system to a low-resistance, high-capacitance system has been studied.44 As stated previously, exosomes correlationally increase with increasing gestational age,6 and several studies have explored the potential role of placental-derived exosomes in regulating maternal immune response during pregnancy.48In normal physiology, EVs from the placenta balance both immunosuppressive and proinflammatory cytokines to support fetal establishment and curtail rejection. Because a fetus is antigenically unique from the mother, maternal immune responses against the growing organism must at least in some part be avoided by inhibiting maternal T lymphocyte (T cell) and natural killer (NK) cell activation.49 Throughout pregnancy, proinflammatory and anti-inflammatory stages allow for the development, maturation, and parturition of the fetus.49 Placental EVs are believed to have a role in modulating these proinflammation and anti-inflammation states by modulating cytokine release.20 A method through which this regulation occurs could be via the expression of UL16 binding protein 1-5 (ULBP1-5) and major histocompatibility complex (MHC) class I chain-related gene protein (MIC) on the surface of placenta-derived exosomes.48 The interaction with these ligands causes the selective and dose-dependent downregulation of receptor NKG2D, which is present in NK cells, CDC8+, and gamma delta T cells, without affecting the lytic pathway through perforin.48 Exosomes also have a role in expression of components of the B7 family of ligands including B7-H3, which causes downregulation of T cell activation.20 HLA-G5 isoform incorporation within exosomes is another important aspect of exosome involvement, as this class of molecules has been shown to protect fetal tissue from maternal immune cell attack.20More specifically, immune adaptations in pregnancy are believed to be largely attributable to the expression and production of different cytokines that are thought to be regulated by EVs.48 For instance, syncytin-1 is present in placental EV cells. In normal pregnancies, syncytin-1 suppresses the production of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), inflammatory regulators that have links to early pregnancy loss and PE.51 In a related manner, maternal EVs can also be proinflammatory, causing a systemic inflammatory state and resulting in pathologic pregnancies. Studies have shown that placental EVs can induce release of proinflammatory cytokines, including TNF-α, macrophage inflammatory protein (MIP)-1α, interleukin (IL)-1α, -6, -8, and -1β from endothelial cells and activate macrophages to release proinflammatory IL-1β.52 The activation of phagocytic cells, including macrophages and monocytes, is particularly important, as they regulate maternal immune response to maintain a normal pregnancy and also protect against infection.53 Similarly, monocyte internalization of EVs trigger their migration and the production of IL-1β, IL-6, granulocyte colony stimulating factor (G-CSF), and TNF-α.53More specifically, placental EVs inhibit maternal immunity and promote fetal survival through the expression of specific immunoregulatory molecules.54 Because of the compounded effect of placental cytokine release and also cytokine release by adipose tissue, the inflammatory pathways are heightened.55 Increased body mass index in pregnancy has been shown to be associated with elevated levels of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and increased activation of the p38 mitogen-activated protein kinases (MAPK) and signal transducer and activator of transcription 3 (STAT3) inflammatory pathways.56 These proinflammatory states may modify placental function and in turn have fetal effects.Alterations in the levels of inflammatory cytokines in pregnancy result in metabolic disorders such as GDM and PE. The amount of cytokine release is particularly evident in obese pregnant women.59; however, the molecules specifically responsible for these effects are still not well understood. The content of EVs reflects the cellular microenvironment and the physiologic response to stresses in the microenvironment and thus may be an important aspect in further understanding the physiologic mechanisms in pregnancy. Current data suggest that exosomes in maternal circulation may contribute to development of a proinflammatory environment by transferring specific cargo (protein and microRNAs [miRNAs]) to target cells.The outcome of aberrant regulation of inflammatory pathways has been established61 Furthermore, cell-to-cell signaling from mother to fetus in complicated pregnancies helps protect the fetus via compensatory mechanisms.Once established, maternal-fetal circulation is a bidirectional system that nonselectively allows for the exchange between mother and fetus of nutrients, oxygen, wastes, and cytokines necessary for fetal growth. Because of the bidirectional passage, pathologic changes affecting maternal adaptations may also complicate the fetus. On the other hand, cellular trafficking from the fetal to maternal compartment leads to fetal microchimerism, and an appropriate maternal immune response to this phenomenon is key for the proper maintenance of pregnancy.63; however, Miranda et al examined the contribution of placental exosomes to the total exosome concentration in fetal circulation and found that the concentration of placental exosomes in maternal and fetal circulation is decreased in conditions such as fetal growth restriction and small for gestational age.64 Cleys et al studied the umbilical cord blood and serum in pregnant sheep and showed the presence of placental EVs.65 A comparison between miRNAs of these vesicles isolated from the umbilical cord blood and maternal serum demonstrated a difference in the miRNA content. Further evaluation by bioinformatics analysis revealed that the miRNA in fetal circulation is important for embryonic development, and in states with abnormal stressors on maternal development, the expression profile of exosomes is also altered.62 The proteins that were expressed differently caused different outcomes for regulation of cellular processes, including complement and coagulation cascades, enzyme regulator activity, and extracellular regulation. Identifying placental-specific exosomes in fetal circulation and isolating their specific roles in both healthy and pathologic pregnancies are important areas of research and need further investigation.Exosomes have been isolated from fetal cord blood in several studiesEVs represent a mechanism of maternal-fetal interaction during gestation. Exosomes provide a way to identify the function and metabolic state of cell origin through their ability to reflect the microenvironment that they are released from and the metabolic state of their cell of origin. Studies have demonstrated that EVs are released from the placenta into the maternal circulation and have a wide range of functions to regulate immunologic responses to pregnancy and to establish the maternal vascular function. Further understanding of how EVs regulate key events in pregnancy may help elucidate how maternal-fetal communication is established in both normal and pathologic conditions.