Magnetic Resonance Imaging (MRI) has become a valuable imaging modality in ophthalmology, especially for the diagnosis and treatment planning of patients with uveal melanoma, the most common primary intra-ocular tumor. We aim to develop and evaluate the value of silent Zero Echo Time (ZTE) MRI to image patients with ocular tumors at 7Tesla. Therefore, ZTE and different types of magnetization-prepared ZTE , have been developed. After an initial validation with 7 healthy subjects, nine patients with an eye tumor have been evaluated. The ZTE scans were compared to their Cartesian equivalent in terms of contrast, motion-sensitivity, diagnostic quality and patient comfort. All volunteers and especially the patients reported a more comfortable experience during the ZTE scans, which had at least a 10 dB lower sound pressure. The image contrast in the native ZTE was poor, but in the different magnetization-prepared ZTE, the eye lens, cornea and retina were clearly discriminated. Overall the T2-prepared scan yielded the best contrast, especially between tumor and healthy tissue, and proved to be robust against eye motion. Although the intrinsic 3D nature of the ZTE-technique provides an accurate analysis of the tumor morphology, the quality of the ZTE-images is lower than their Cartesian equivalent. In conclusion, the quality of magnetization-prepared ZTE images is sufficient to assess the 3D tumor morphology, but insufficient for more detailed evaluations. As such this technique can be an option for patients who cannot comply with the sound-levels of Cartesian scans, but for other patients the conventional Cartesian scans offer a better image quality. In recent years, MRI has become a valuable imaging modality in ophthalmology due to its ability to non-invasively image parts of the eye which are not accessible by conventional optical techniques –5. One oHowever, one of the main challenges in MRI is motion and in ophthalmology in particular: eye-motion. This results in significant image artifacts, which can mask extrascleral extension of the tumor. Different strategies have been developed to reduce the amount of motion artefacts in ocular MRI scans, which can last several minutes. Among them are cued-blinking protocols, which incorporate regular pauses in the scan during which the subject is allowed to blink,7. TheseZero echo time (ZTE) imaging,9, a 3D In this study we evaluated the use of 3D, ultra-high field, magnetization-prepared ZTE imaging to visualize the eye for two main reasons. First, ZTE sequences have a very low acoustic noise level, which significantly increases patient comfort and decreases the involuntary blink reflexes. Secondly, the inherent isotropic spatial resolution of this 3D sequence supports reformatting in all directions retrospectively, which is advantageous in terms of the highly irregular shape of tumors. Often multiple cuts in different orientations are required to assess the optimal treatment and therefore a isotropic 3D sequence significantly helps to simplify the scanning workflow. After outlining the basic features of the ZTE sequence, aspects of magnetization preparation for eye imaging will be discussed together with results of an initial patient study performed at ultra-high field strength.1+) of the quadrature RF transmit coil , the flip angle is limited to 3°. Signal reception is performed using a dedicated proton eye receive coil (diameter 40 mm), integrated into special goggles to allow visual communication with the patient[The ZTE sequence was integrated into the existing 7T MRI system  software without any hardware changes. In the current implementation ZTE imaging is performed with a short block excitation RF pulse (12.8μs duration), applied in the presence of a read gradient which slightly changes direction each TR . The inddiameter 0 mm, int3 to limit the total scanning time. The basic ZTE imaging parameters were chosen as follows: FOV of (120 mm)3, TR 2.0 ms, 100% angular sampling, resulting in a total scanning time 0:57 minutes for a native ZTE with proton density (PD) contrast. Due to the length of the RF pulse and finite switching time from transmit to receive state on the unmodified MR system, the TE was fixed to 67μs. Extra data sampling at a reduced read gradient strength was used to fill the missing samples in the centre of k-space [1 relaxation. A similar contrast to its Cartesian equivalent was achieved by setting the same inversion time of 1280 ms. Second, T2-weighting was achieved by applying a T2-preparation module[1+ effects. Preliminary evaluations showed that an echo time of 30 ms resulted in sufficient T2-weighting to differentiate the sclera from the surrounding structures. Third, spectral partial inversion recovery (SPIR) was employed for fat suppression, while finally, a saturation recovery contrast (SAT) was used, applying a 90° saturation RF pulse followed by a ZTE sampling block. The sampling time per shot and shot intervals, listed in 3, resolution version of the T2-prepared ZTE sequence was developed. This acquisition time of this scan was approximately 3 minutes, which is similar to its Cartesian equivalent, allowing for a clinically relevant comparison.In a pre-study, different types of magnetization-prepared sequences were evaluated to determine the optimal contrast to discriminate the tumor from the surrounding structures. These scans were performed at an isotropic spatial resolution (1.0 mm) k-space ,15. Aparon module with twoTo compare the sound level of the ZTE scans with their Cartesian equivalent, the souBefore studying the performance of the magnetization prepared ZTE in patients, three in-vivo tests were performed in a small volunteer cohort . Written consent was obtained according to the rules of the institution.3 a TR/TE/FA of 5.6 ms/2.9 ms/7° respectively, and an inversion delay of 1280 ms using low-high k-space sampling for 519 ms. The images were visually compared to the ZTE results by two independent readers , ocular MRI expert with 7 years experience (JWB)).In the first study the basic image quality of the magnetization prepared ZTE was investigated. This helps to judge the basic potential of the ZTE regarding the degree of contrast manipulations compared to selected Cartesian sequences specifically tailored previously for high definition eye tumor imaging,7. In prThe effects of magnetization transfer (MT)  in magne2 prepared ZTE approach was chosen and modified to include the above mentioned 3 seconds blinking time, resulting in a slightly prolonged acquisition time (2:53 min) compared to the basic T2 prepared ZTE approach (1:54 min). This scan was performed twice. In the first run the volunteer was asked to close the eyes for the entire scan and relax. No further instructions were given during the scan. Before the second run, the subject was instructed to follow the cued-blinking paradigm and the visual instructions were enabled. The uninstructed scan was always performed before the instructed scan, as after one scan with visual blink instructions, subjects tend to still synchronize their blinking with the pauses in the MR sound, regardless of the disabled visual cues. The image quality of both runs was compared visually.The third study addresses the sensitivity of ZTE to eye-motion. For Cartesian magnetization prepared imaging, cued blinking approaches are used to freeze potential eye motion. For this purpose, the patient gets visually instructions to focus (eye opened) on a fixation cross on a projection screen during MR data acquisition. Every three seconds, an interval without data sampling is added to the scan during which the fixation cross automatically changes into a red circle by which the subject is signaled to blink shortly. Previous studies ,7,18 hav1 and T2 scans with a cued-blinking paradigm. The ZTE scanning was performed for a maximum-possible total scanning time of 10 minutes, in order to not exceed the maximum time in the magnet constraint.The ZTE protocols described above  were used in a small patient pilot study approved by the local ethical committee, with written consent obtained. Additional ZTE-scanning was performed during a normal eye tumor diagnostic session based on Cartesian scanning, which consisted of FLAIR, T2-weigthed scans were used. In one patient the native ZTE and FLAIR were performed too. In two other patients, the native ZTE scan was evaluated, while in one patient the FLAIR variant was used.First four patients  were scanned with the lower resolution ZTE-scans to assess the optimal contrast for imaging ocular tumors and to confirm volunteer findings. Due to the scanning time constraints the actual ZTE scan selection was randomized among the patients. In all patients the fat-suppressed and T2-prepared ZTE scan, to assess the clinical potential of this ZTE approach for ocular tumors. In these scans and their Cartesian equivalent 3, FOV:40x47x38mm3, scan time: 3 minutes) the tumor thickness, a three-dimensional measure which determines the optimal treatment modality[Subsequently, five additional patients  were scanned with the high-resolution T1 contrast can be seen between the sclera and the vitreous body, due to the relative long T1 of the vitreous body of approximately 6 s[1. The lens, cornea and retina can clearly be discriminated. Fat suppressed SPIR-ZTE 2-prepared ZTE equivalent, 2-prepared ZTE was 82dB. This increase in sound pressure was caused by the fast gradient switching of crusher gradients in the preparation module. The subjective experience of sound level of these magnetization prepared ZTE scans, was however similar to the native ZTE scan, as this gradient switching only produces a very short sound. This can be reduced in future implementations by using more appropriate crusher gradients, which might, however, slightly increase the scan time as these optimized crushers might need more time for a similar amount of signal suppression. For all Cartesian scans, the sound pressure was approximately 93dB, as the sound pressure was predominantly caused by the switching readout gradients. The relatively long duration of these sounds, at least half a second depending on the read-out scheme, made the subjective experience less comfortable than the short clicks, of only a few milliseconds, of the T2-preparation module of the ZTE acquisition.All volunteers and patients reported a much more comfortable experience during the ZTE scans than during their Cartesian equivalents, with much less propensity to blink due to the almost silent nature of the scans. All ZTE scans were approximately 10-20dB quieter than their Cartesian equivalent, 2-prepared ZTE gives the highest contrast. In addition, there is significant contrast between the vitreous body and subretinal fluid. Although the FLAIR is yielding one of the essential contrasts in Cartesian eye imaging, the resulting scanning time for the FLAIR ZTE is unacceptably long. Furthermore, as every read-out starts in the center of k-space, the ZTE FLAIR has some pronounced contrast washout, which is not present in the Cartesian FLAIR due to the low-to-high k-space sampling scheme. The saturation prepared ZTE, which results in a similar contrast to FLAIR ZTE due to the long T1 of the vitreous body, partly resolves these limitations as the magnetization preparation pulses can be repeated every 900 ms instead of every 3 seconds for the FLAIR ZTE. This reduces the scan time from 6 to less than 2 minutes and allows for a short echo-train to minimize the contrast washout. The saturation pulses do, however, suppress most of the MR-signal, which results in a lower SNR.Although native proton-density weighted ZTE MR-images provide insufficient contrast in the human eye, magnetization-prepared ZTE allows for sufficient contrast and flexibility for ocular imaging. The SPIR-prepared ZTE is not particularly useful in measuring tumor size due to lack of contrast, but it could be useful for investigating diseases of the optic nerve since the combination of SPIR and ZTE gives good visualization of the optic nerve at rather high temporal sampling efficiency. The MT-effect, associated with the applications of off-resonant RF pulses, also introduces contrast between the lens and the vitreous body. The TAs the ZTE uses 3D radial FID-sampling, no spatial localized RF-excitation pulses can be used, making the technique intrinsically susceptible to aliasing artefacts stemming from outside the FOV. The use of a local receive coil limits the spatial receive sensitivity to the eye and surrounding tissues, allowing for a FOV of roughly 120 mm without fold-over artifacts. Although for bilateral eye imaging an increased FOV would be needed, this should not necessarily lead to increased scanning times since for both eyes an individual receive coil would be needed, which would allow for the use of under-sampling techniques such as SENSE or GRAPPA. ,24The main advantage of ZTE eye imaging compared to conventional Cartesian imaging is the increased patient comfort due to its silent nature which all patients preferred. The robustness of the ZTE against eye-motion (due to oversampling the centre of k-space) makes it a robust alternative for cued-blinking paradigms. Claustrophobic patients or very nervous subjects often fail to adhere to the cued-blinking instructions which results in significant motion artefacts, that may mask clinically relevant details in the MR-images. For the3 resolution. Although this resolution is sufficient to show how different contrasts can be achieved in eye ZTE-scanning trough magnetization preparation, the significant partial volume effects hinder clinical interpretation of these images. The sclera could, for example, not always be identified as it is on average only 0.5 mm thick. The ZTE scans with a higher resolution on (0.8mm)3 mitigated this issue up to a certain extent, but some ringing artifacts still mask most fine anatomical details. However, as these scans do show sufficient detail to determine the tumor dimensions, they can be used to determine the optimal treatment strategy for these patients, [Although this pilot study included only a limited number of patients and not all sequences have been tested in all patients due to time constraints, it shows the potential strengths but also the weaknesses of ZTE in eye applications. The initial ZTE scans to determine the optimal contrast used a conservative 1 mmatients,  which maFor additional, more comprehensive, evaluations, such as the screening for scleral invasion of the tumor, an increased resolution compared to the current ZTE protocol will be needed. Given the large FOV needed for the ZTE, such a scan would take at least six minutes to acquire a sufficient number of spokes, if not more, and to achieve a decent SNR. Such long scan times make the scan very susceptible to slow drifts in gazing direction  which bl0 inhomogeneities can for instance influence the T2-preparation module, as off-resonance effects can compromise the performance of the adiabatic RF pulses employed, due to their bandwidth restrictions. Also here further work is needed in redesigning those blocks, especially if these techniques will be used in the planning of proton therapy, since the tantalum markers used as an anatomical reference, will increase local B0 inhomogeneities.Additionally, the specific magnetization preparation blocks used for ZTE need attention with respect to image quality. As the eye is surrounded by air, not only anteriorly but also in the nasal cavities, and bony structures of the globe, local field inhomogeneities remain present, even after localized shimming. These BHowever, as the ZTE acquisition does not need a regular pause for the cued-blinking paradigm, the ZTE protocols can acquire more profiles in the same amount of time compared to the Cartesian acquisition, which can partly compensate its intrinsic lower MR-signal strength at the cost of slight contrast changes.Given the combination of the relatively low SNR of the ZTE-technique and the relatively small anatomy of the eye, these evaluations were performed at 7 Tesla MRI to benefit from the increased SNR of high field MRI. Recent advances in MRI have made high quality ocular MRI feasible at clinical 3 Tesla, although at a slightly lower resolution than at 7 Tesla. Since tMagnetization-prepared ZTE imaging of the eye at 7T provides images with good contrast in both healthy subjects and patients with an intra-ocular tumor. Compared to the conventional Cartesian acquisition strategies, the resolution and image quality of the current ZTE protocol is a limiting factor, while its robustness against eye-motion and increased patient comfort are clearly advantageous. As the current ZTE protocol allows for a basic assessment of the tumor dimensions, this protocol could be advantageous for patients who do not comply with regular scan protocols due the high sound pressure of conventional MR-imaging. However, in other cases, the conventional Cartesian scanning is preferred as the higher image quality / resolution provides more diagnostic value.


Neurofeedback (NFB) enables the voluntary regulation of brain activity, with promising applications to enhance and recover emotion and cognitive processes, and their underlying neurobiology. It remains unclear whether NFB can be used to aid and sustain complex emotions, with ecological validity implications. We provide a technical proof of concept of a novel real-time functional magnetic resonance imaging (rtfMRI) NFB procedure. Using rtfMRI-NFB, we enabled participants to voluntarily enhance their own neural activity while they experienced complex emotions. The rtfMRI-NFB software (FRIEND Engine) was adapted to provide a virtual environment as brain computer interface (BCI) and musical excerpts to induce two emotions (tenderness and anguish), aided by participants' preferred personalized strategies to maximize the intensity of these emotions. Eight participants from two experimental sites performed rtfMRI-NFB on two consecutive days in a counterbalanced design. On one day, rtfMRI-NFB was delivered to participants using a region of interest (ROI) method, while on the other day using a support vector machine (SVM) classifier. Our multimodal VR/NFB approach was technically feasible and robust as a method for real-time measurement of the neural correlates of complex emotional states and their voluntary modulation. Guided by the color changes of the virtual environment BCI during rtfMRI-NFB, participants successfully increased in real time, the activity of the septo-hypothalamic area and the amygdala during the ROI based rtfMRI-NFB, and successfully evoked distributed patterns of brain activity classified as tenderness and anguish during SVM-based rtfMRI-NFB. Offline fMRI analyses confirmed that during tenderness rtfMRI-NFB conditions, participants recruited the septo-hypothalamic area and other regions ascribed to social affiliative emotions . During anguish rtfMRI-NFB conditions, participants recruited the amygdala and other dorsolateral prefrontal and additional regions associated with negative affect. These findings were robust and were demonstrable at the individual subject level, and were reflected in self-reported emotion intensity during rtfMRI-NFB, being observed with both ROI and SVM methods and across the two sites. Our multimodal VR/rtfMRI-NFB protocol provides an engaging tool for brain-based interventions to enhance emotional states in healthy subjects and may find applications in clinical conditions associated with anxiety, stress and impaired empathy among others. Neurofeedback (NFB) is a novel application of brain-computer interfaces that aids real-time voluntarily regulation of brain activity. Mounting evidence shows that NFB has promising effects to enhance behavior, cognitive and emotional processes in normative samples –5. NFB hrtfMRI NFB tools can be used to study the causal mechanisms of complex emotions and to inform evidence-based personalized interventions to enhance and recover aberrant emotional states  in normative and clinical samples. One key practical human challenge of fMRI studies includes participants being distracted and experiencing difficulties to feel valid psychological states in the scanner environment, particularly when trying to sustain complex emotions.Recent studies have combined immersive virtual environments with multiple sensory modalities to deliver psychological/cognitive interventions, and to enhance their effectiveness via engaging and motivating individuals to practice –16.Only two proof of concept studies have combined rt-NFB with virtual environments as brain computer interfaces (BCI). An electroencephalography-based NFB study computed brain activity from about 500 participants collectively, during an interactive game of relaxation and concentration over one night , where iIt remains untested whether rt-NFB platforms integrating multisensory virtual environments can successfully recruit complex emotions and sustain these emotions long and strong enough to probe their underlying neural correlates. Such a platform can advance NFB applications, via (i) increasing the ecological validity of rtfMRI-NFB experiments, and their relevance for the daily experiences of emotions outside of experimental settings, (ii) adapting NFB interfaces to the individual and target population so these are more relatable, engaging and effective in generating and sustaining complex emotions that maximize the success of rtfMRI-NFB interventions –20.This study aims to demonstrate the feasibility of an engaging rtfMRI-NFB interface that can be individually tailored and, specifically, to provide a proof of concept for a rtfMRI-NFB integrating a virtual environment as a BCI and musical stimuli using both local  and distributed  analyses. The FRIEND Engine Framework system  was enhaWe also aimed to validate the functional anatomy of these complex emotions during rtfMRI-NFB. After the real-time data was collected, we ran offline fMRI data analyses to verify the effects of the real-time neurofeedback task on brain activity using standard preprocessing and statistical analysis methods.We hypothesized that participants would voluntary change the color of a virtual environment in the BCI during rtfMRI-NFB using the activity of the following regions: (i) for the tenderness condition, the septo-hypothalamic area (when using ROI-based rtfMRI-NFB method) and other brain areas ascribed to positive affiliative emotions i.e., medial orbitofrontal areas (when using SVM-based rtfMRI-NFB method) , 25–27; We used a single subject, repeated measures design with two identical assessments on two consecutive days, counterbalanced by rtfMRI-NFB method . We recruited eight psychiatrically and neurologically healthy postgraduate research students, free of psychoactive medication and with normal or corrected-to-normal vision. Four participants were recruited from the D'Or Institute for Research and Education (IDOR) in Rio de Janeiro, Brazil . To validate the protocol in a different scanner and institution, we also recruited four participants from the Monash Biomedical Imaging (MBI) at Monash University in Melbourne, Australia (MUHREC CF15/1756 - 2015000893). All volunteers provided written informed consent prior to study participation.https://assetstore.unity.com/packages/3d/environments/autumnal-nature-pack-3649) and displayed a first-person navigation at walking speed through hills and cornfields, with a total duration of 10′8″ . Music excerpts were selected from 20 audio tracks, all normalized using the root mean square feature of Audacity software . The audio tracks were previously rated to have comparable volume, pace, and rhythm. For the rtfMRI-NFB task runs, four excerpts for tenderness and four excerpts for anguish were played.The trial types were displayed via changes in the base color hues of the virtual environment and via specific music excerpts. Music excerpts were fixed for each trial type, and not influenced by current neural/psychological states  and was used by participants to practice tenderness, anguish and neutral states before the MRI. With this practice, participants could learn which music tracks and VR color changes in the BCI corresponded to tenderness, anguish and neutral trials.As shown in Figure http://www.mr-confon.de). The volume of the song excerpts was adjusted for each participant to a level where they could comfortably hear the music while performing rtfMRI-NFB.During the training MRI run for rtfMRI-NFB, participants were instructed to feel the tenderness, anguish and neutral states as intensely as possible. This allowed mapping the brain regions that were most engaged by each individual while experiencing the emotions. We mapped and used the activity in these brain regions for each participant as a source for rtfMRI-NFB. The musical stimuli were delivered with MRI-compatible headphones . The training run allowed mapping which brain regions the participant engaged while experiencing tenderness and anguish. The three subsequent rtfMRI-NFB runs provided participants with continuous feedback (every 2″) on their brain activity in the form of updating the color of the virtual scenario in the BCI. The more participants engaged the target brain regions corresponding to tenderness and anguish states, the more the virtual environment would turn into orange and purple shades, respectively. During neutral rtfMRI-NFB trials, participants were not required to change the color of the virtual environment and this remained at baseline color.a priori brain regions (or network of regions). Participants were instructed to enhance the target emotion as to intensify the color hue of the virtual environment BCI from neutral (baseline scenario hue) to orange  or to purple .rtfMRI-NFB was delivered online and continuously via an updated platform of the FRIEND Engine Framework v 0.5 . We defiWe used two different rtfMRI-NFB methods to compute brain activity unknowingly to participants, test the capability of this software and explore whether the patterns of brain activity were more robustly recruited via either SVM based rtfMRI-NFB or ROI based rtfMRI-NFB. Half of the sample was randomly allocated to SVM method on day one and ROI method on day two, and the opposite order was used for the other half. We counterbalanced the presentation of the emotion trial types Figure . The visThe rtfMRI-NFB ROI method computed percentage signal change (PSC) within the 10% most active voxels with an a priori defined ROI, measured across four blocks of the first training NFB run. ROIs included the septo-hypothalamic area when contrasting tenderness versus neutral conditions  and the curr_volROI is the mean of the ROI on the current volume, B is the number of volumes in the previous baseline condition and kROI is the mean of the kth volume (sig(k). The feedback value was used to modulate the color of the virtual environment, so that the higher the percent signal change, the more participants changed the color to orange and purple for the conditions of tenderness and anguish, respectively.where h volume  weightedxt, the projected value was given by xtw+b, where w is a vector containing the hyperplane coefficients and b is a constant . We used a SVM classifier with a linear kernel and a cumulative training, meaning that all brain activity patterns observed during the rtfMRI-NFB task thus far are used at the end of the run to retrain and update the SVM classifier/model to use in the following runs. The projected value of a new observation was used to define the neurofeedback information, in our case, the color tonality of the virtual environment. For a new image volume, composed by real numbers constant . The morThe SVM rtfMRI-NFB method used a feature selection mask that included brain regions implicated in positive affiliative emotions , and that excluded from SVM training and decoding those areas involved in sensorimotor or visuospatial processing .MRI and rtfMRI-NFB data were acquired in the two sites using a 3T Philips Achieva - at the D'Or Institute for Research and Education, in Rio de Janeiro, Brazil (Site 1) - and a 3T Siemens Magnetom Skyra - at the Monash Biomedical Imaging facility and the Brain and Mental Health Imaging laboratory, Monash Institute of Cognitive and Clinical Neurosciences, Monash University in Melbourne, Australia (Site 2).2, slice thickness = 1 mm, 170 slices, slice order ascending). Head motion was minimized via foam padding and straps over the forehead and under the chin. In Site 2 we used an isotropic T1 MP-RAGE scan (with TR/TE = 2.3/2.0 (s), flip angle = 9°, matrix size 256 × 240, FOV = 256 × 240 (mm), slice thickness = 1 mm, 170 slices, slice order descending).Immediately before the rtfMRI-NFB task, we acquired high-resolution anatomical images. In Site 1 we used an isotropic T1-weighted 3D turbo field echo sequence (TR/TE = 7.2/3.4 (s), flip angle = 8°, matrix size 240 × 240, FOV = 240 mm*-weighted EPI (BOLD contrast), with TR/TE = 2,000/22 (ms), matrix = 64 × 64, FOV = 240 mm2, flip angle = 90°, isotropic voxel = 3.75 mm3, 24 slices. Before each fMRI run, we collected five dummy volumes for T1 equilibration. In Site 1 we used an optimized sequence with SENSE factor of 1.5 and dynamic stabilization to enhance temporal signal-to-noise . In both sites fMRI data were acquired with T2to-noise  in brainThe assessment protocol is overviewed in Figure Beck Depression Inventory .Future developments for rtfMRI-NFB platforms incorporating virtual environments as BCI may include providing feedback on different properties of brain functions including but not limited to connectivity and multiple ROIs concurrently (both possible with the Friend Engine platform), and tailoring rtfMRI-NFB tasks with multi-sensory BCIs to the needs of the individual and target population in large samples , to identify the characteristics of those who respond best and least and inform evidence based interventions. Our results warrant further rtfMRI-NFB studies using personalized interfaces in large cohorts to examine the therapeutic potential of rtfMRI-NFB in clinical samples, and its ability to enhance cognitive and emotional wellbeing in normative populations.The datasets used in the current study are available from the corresponding author on reasonable request.VL and BM led the study execution, protocol setup, data analysis, and the writing of all the aspects of the manuscripts from start to completion. RB led the stimulus and neurofeedback setup in the D'Or laboratory in Rio and provided assistance in setting up the neurofeedback platform at Monash University in Melbourne. CS contributed to data collection and led the neurofeedback software setup in the Melbourne site. MY and CT-C advised on the running of the project and revised the manuscript. JM advised on the running of all the aspects of the study, overviewed the experimental protocol setup and the running of the fMRI analyses; and contributed to the writing of the first and other drafts of 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.


Acute pulmonary embolism (PE) is a disease frequently encountered in clinical practice. While the management of haemodynamically stable, low risk patients with acute PE is well established, managing intermediate disease often presents a therapeutic dilemma. In this review, we discuss the various therapeutic options available in this patient group. This includes thrombolysis, surgical embolectomy and catheter directed techniques. We have also explored the role of specialist PE response teams in the management of such patients.  Data from the US suggest a far higher incidence. The Centers for Disease Control and Prevention estimates that there are 60,000 to 100,000 annual deaths from deep vein thrombosis or PEs in the US, and sudden death is felt to be the first symptom in about a quarter of cases3. In Europe, epidemiological models based on data from six European Union countries have estimated the total number of PE events per annum as 95 per 100,0004.Acute pulmonary embolism (PE) is a relatively common disease with a variable clinical presentation. The annual incidence of diagnosed cases in the UK has previously been reported as 34.2 per 100,000 person-years5. This distinction is important as massive PE is associated with increased mortality. In the International Cooperative Pulmonary Embolism Registry (ICOPER), the 90-day mortality rate for patients with acute PE and systolic blood pressure of less than 90 mm Hg at presentation was 52.4% compared with 14.7% in the remainder of the cohort6. Additionally, in the Management Strategy and Prognosis of Pulmonary Embolism Registry of 1001 patients with acute PE, in-hospital mortality was 8.1% for haemodynamically stable patients versus 25% for those presenting with cardiogenic shock and 65% for those requiring cardiopulmonary resuscitation7.An important challenge in the management of acute PE is patient risk stratification. Massive or high-risk PE is characterised by sustained hypotension (systolic blood pressure of less than 90 mm Hg for at least 15 minutes or requiring inotropic support) not attributable to another cause. In contrast, submassive or intermediate-risk PE refers to an acute PE without systemic hypotension but with either right ventricular (RV) dysfunction or myocardial necrosis8, and its relative ease of administration is an added advantage. Typically, such patients subsequently receive parenteral anticoagulants followed by vitamin K antagonists or the newer direct oral anticoagulant (DOAC) agents.Low-molecular-weight heparin is a well-established initial therapy for patients with acute PE who remain haemodynamically stable. It has been found to reduce the incidence of complications and thrombus size compared with unfractionated heparin12. The severity of PE, the type of thrombolytic agent and heparin used, and the dose and duration of administration varied across the trials. Furthermore, the clinical criteria defining haemodynamic instability, shock and haemorrhage were not standardised. Only a few studies focused on clinical outcomes, and most lacked the statistical power to permit definite conclusions.Over the past 50 years, almost 20 randomised studies on thrombolytic therapy in acute PE have been published and their results reviewed in meta-analyses13. The ideal dose of thrombolysis and how it should be administered are also unclear.Major haemorrhage following thrombolytic therapy also remains a concern, and the reported incidence is between 0 and 33% depending on the type and dose of thrombolytic agent used16. However, the role of thrombolysis in the management of haemodynamically stable patients with submassive or intermediate-risk disease is still a matter of debate17.International guidelines recommend thrombolysis for the management of PE patients with haemodynamic instability18.The large, randomised Pulmonary Embolism Thrombolysis (PEITHO) trial compared the composite end point of mortality and haemodynamic collapse in over 1000 intermediate-risk patients who received heparin plus tenecteplase or placebo. Tenecteplase significantly reduced the risk of haemodynamic decompensation within 7 days but also was associated with a 10-fold increase in intracranial haemorrhage (2% versus 0.2%) and a fivefold increase in major haemorrhage (6.3% versus 1.2%). The 2% rate of intracranial haemorrhage may reflect a combination of full-dose tenecteplase plus a simultaneous loading bolus of heparin. Surprisingly, the follow-up results of PEITHO showed that thrombolysis did not affect long-term survival or reduce residual dyspnoea, RV dysfunction, or the incidence of chronic thromboembolic pulmonary hypertension (CTEPH)et al. compared outcomes in patients with acute PE treated with thrombolysis compared with anticoagulation alone19. This included patients with intermediate-risk disease. The use of thrombolysis was associated with lower all-cause mortality and risk of recurrent PE. However, the risk of intracranial haemorrhage was also greater in thrombolysed patients19.The results of various meta-analyses comparing full-dose thrombolysis with standard anticoagulation have been mixed. Chatterjeeet al. found no evidence for a benefit of thrombolytic therapy compared with heparin for the initial treatment of unselected patients with acute PE20. However, there was a suggested benefit for haemodynamically unstable patients which should be weighed up against the statistically significant increased risk of non-major bleeding20. Similarly, Donget al. concluded that outcomes in terms of death rate, recurrent PE and haemorrhagic events were similar in patients who received thrombolytic therapy compared with placebo or heparin21.Conversely, Wanet al. concluded that recanalisation procedures  did not offer a clear advantage in the treatment of PE compared with standard anticoagulation alone22, a number of novel therapeutic modalities have recently been proposed. However, their precise role in clinical practice remains unclear. We have therefore attempted to review some of these and analysed available data relating to their use in a clinical setting. We have focused in particular on submassive or intermediate-risk disease.The development of alternative therapeutic modalities for patients presenting with large-volume PE, with or without haemodynamic compromise, has been the subject of great interest. Whereas JimenezLow-dose thrombolysis has been suggested as a potential treatment strategy for acute PE, particularly in patients presenting with intermediate-risk disease. It remains unclear whether early thrombolysis in this patient group has an impact on clinical symptoms, functional limitation, or CTEPH at long-term follow-up.A small randomised trial of 83 patients suggested that thrombolysis, compared with anticoagulation alone, might improve functional capacity at 3 months. In the PEITHO trial, long-term (at 41.6 ± 15.7 months) clinical follow-up was available for 358 patients with intermediate-risk PE who survived the acute phase; persisting symptoms, mainly dyspnoea, were present in 33% of the patients. However, the degree of functional limitation was mild in the majority of cases regardless of whether the patients had been randomly assigned to thrombolysis or anticoagulation alone. In agreement with the clinical findings, the majority of patients (85% in the tenecteplase arm and 96% in the placebo arm) had a low or intermediate probability—based on the definition of the European Society of Cardiology (ESC) guidelines—of persisting or new-onset PH at echocardiographic follow-up. A standardised diagnostic work-up for CTEPH was not mandated by the trial protocol. Consequently, the findings of this study do not support a role for thrombolysis with the aim of preventing long-term sequelae after intermediate-risk PE, although they are limited by the fact that clinical follow-up was available for only 62% of the study population.23. However, it must be noted that the definition of pulmonary hypertension in the trial does not meet internationally defined diagnostic criteria for this condition. Nevertheless, patients in the treatment arm were found to have a greater reduction in PASP compared with the control group, and although long-term functional outcomes were not formally assessed, these results may be significant in this regard.The Moderate Pulmonary Embolism Treated with Thrombolysis (MOPETT) trial evaluated the role of half-dose  thrombolysis with tissue plasminogen activator (tPA) and anticoagulation versus standard anticoagulation alone in the management of patients with moderate PE. A statistically significant reduction in the development of pulmonary hypertension, defined in the trial as pulmonary artery systolic pressure (PASP) of greater than 40 mm Hg by echocardiography, was reported in the treatment group compared with the control group. There was no significant difference in mortality or bleeding, although the average length of hospital admission was shorter in the treatment groupet al., who demonstrated similar improvements in RV dysfunction and radiological clearance in patients who received half-dose tPA (50 mg/2 hours) compared with those who received the full dose (100 mg/2 hours)24. Bleeding was less common in patients who received the lower-dose regimen, although this difference was not statistically significant24. Similarly, Goldhaberet al. found no differences between a reduced-dose bolus and full-dose tPA with respect to bleeding complications. Additionally, efficacy was similar in the two treatment groups25.The efficacy of low-dose thrombolysis compared with full-dose has been evaluated by Wanget al. described the successful use of low-dose thrombolysis with 25 mg tPA in a high-risk patient who had a cardiac arrest soon after lung resection surgery26. The patient was still alive after 6 months despite developing a mediastinal haematoma that required surgical drainage and blood transfusions26. Low-dose thrombolysis has also been successfully used in the treatment of a patient with submassive PE and right heart thrombus following cardiac surgery27.The role of low-dose thrombolysis in the management of post-operative patients has also been considered. Recent surgical intervention has generally been accepted as a relative contraindication for thrombolysis, particularly for those who have undergone cardiothoracic procedures. ShenFirst successfully performed by Kirschner in 1924, surgical embolectomy fell out of favour following the advent of thrombolytic therapy in the 1970s. However, the risk of bleeding associated with systemic thrombolysis has meant that this modality has re-emerged as a treatment of choice for patients in whom systemic thrombolysis is contraindicated. According to the ESC guidelines, surgical embolectomy should be considered  in such patients and in those in whom thrombolysis has been unsuccessful in achieving haemodynamic stability. This procedure may also be a consideration in patients with acute PE who require surgical excision of the right atrial thrombus or closure of a patent foramen ovale to prevent paradoxical emboli.29. More recently, a multi-centre analysis of over 200 patients by Keelinget al. quoted in-hospital mortality of 11.7%30. This included patients who experienced pre-operative cardiac arrest30.Results from small retrospective analyses have previously shown that surgical embolectomy is an effective treatment modality with 30-day mortality figures of 6 to 8%. This includes critically unwell patients presenting with cardiogenic shock and cardiorespiratory arrest requiring cardiopulmonary resuscitation35.34. Furthermore, surgical embolectomy has been associated with similar intracranial bleeding rates but fewer major bleeding complications compared with thrombolysis.The role of surgical embolectomy in the management of patients in whom thrombolysis is not contraindicated is more controversial, particularly because of the paucity of head-to-head trials comparing the two treatment modalities. Nevertheless, recently published data suggest that short- and long-term mortality rates from the two interventions are comparable36. Additionally, Lehnertet al. demonstrated that surgical embolectomy resulted in a significantly lower amount of residual emboli compared with thrombolysis35.Surgical embolectomy has also been shown to result in a greater difference between pre- and early post-operative systolic pulmonary artery pressure and RV diameter in patients presenting with acute high-risk PE compared with thrombolysisThe growing role of percutaneous interventions in modern medicine has led to an interest in the development of similar strategies for the treatment of PE, especially in patients in whom thrombolysis is contraindicated or not indicated. Various modalities of catheter-directed mechanical thrombectomy and localised low-dose thrombolysis have been developed over the past few years. Although data related to their use are limited at present, it is expected that the next few years will see further progress in this regard.37.The Pulmonary Embolism Response to Fragmentation, Embolectomy, and Catheter Thrombolysis (PERFECT) data study assessed clinical outcomes using changes in mean pulmonary artery pressure and right heart strain in 101 patients who underwent catheter-directed or pharmaco-mechanical thrombectomy and/or catheter-directed thrombolysis for massive or submassive PE. Data from the study suggested improved clinical outcomes in these patients. Additionally, the average dose of thrombolysis was lower than that used for systemic therapy and there were no major bleeding complications38. However, a retrospective comparison of ultrasound-assisted and standard catheter-directed thombolysis by Lianget al. revealed no significant difference in outcomes or complication rates between the two techniques39. More recently, similar results were reported by Schissleret al., who found no significant differences in length of hospital admission, RV dysfunction on follow-up echocardiography and 1-year mortality in patients who received this treatment modality compared with standard anticoagulation alone40. The SEATTLE II trial is thus far the largest study looking at the efficacy and safety of ultrasound-assisted catheter-directed fibrinolysis. One hundred fifty patients with either massive or submassive PE were given 24 mg of tPA, administered by either unilateral or bilateral catheters. The change in RV-to-left ventricle (LV) diameter ratio was used as the primary efficacy outcome. The trial showed a mean reduction of 0.42 along with a statistically significant reduction in mean pulmonary artery systolic pressure. Although one patient had a severe bleed in the form of a groin haematoma resulting in hypotension, there were no intracranial bleeds41. It is worth noting, however, that this was a single-arm trial which involved no head-to-head comparison with other therapeutic modalities. Similar physiological improvements were noted by Kaymazet al., who assessed outcomes in 141 patients who received this modality42. Interestingly, bleeding rates and long- and short-term mortality were not related to age42.The addition of ultrasound assistance to localised catheter-directed thrombolysis is postulated to facilitate the delivery of the thrombolytic agent to the intended target and additionally accelerate fibrinolysis by causing disruption of uncrosslinked fibrin fibres into smaller fibreset al.43. The addition of ultrasound assistance resulted in a significant reduction in RV dilatation compared with anticoagulation alone. No increased risk of bleeding was demonstrated43. A trial comparing the safety and efficacy of this modality with peripheral, low-dose thrombolysis is ongoing44.A randomised controlled trial comparison of additional ultrasound-assisted catheter-directed thrombolysis with anticoagulation versus anticoagulation alone was carried out by Kucher45. Additionally, the Optimum Duration of Acoustic Pulse Thrombolysis Procedure in Acute Pulmonary Embolism (OPTALYSE PE) study is attempting to determine the ideal dose of thrombolytic agent and length of ultrasound procedure in patients with submassive PE46.Recruitment into another trial comparing standard and ultrasound-assisted catheters is already under way, and both short- and long-term outcomes are being compared47. Preliminary results suggest a significant reduction in RV size and a relatively low rate of major adverse events48. However, in the absence of a control group, it is difficult to draw any definitive conclusions regarding its long-term feasibility outside a trial setting. Nevertheless, the FlowTriever system recently received US Food and Drug Administration approval for the treatment of PE in the US and this may lead to a more widespread use of this technology in the future.Like catheter-directed therapy, mechanical embolectomy is used relatively infrequently in clinical practice and data regarding its use have historically been limited to isolated case reports and cohort studies involving small numbers of patients. This treatment modality was recently the subject of the multi-centre FlowTriever Pulmonary Embolectomy (FLARE) Clinical Study trial, which assessed the use of an aspiration catheter in 106 patients with intermediate-risk PE49. However, data from small cohort studies have not been as encouraging. In a meta-analysis of various modern catheter-directed thrombolysis modalities, Kuoet al. found that this technique was associated with the highest complication rates, including five deaths in the total cohort of 6850. Additionally, although Zeniet al. reported good immediate angiographic improvement following the use of rheolytic embolectomy in 17 patients, 10 subsequently required an adjuvant thrombolytic infusion51.Thrombus fragmentation with the injection of saline at high velocity, so-called rheolytic embolectomy, is another form of mechanical embolectomy that has been used in the management of acute PE. The smaller fragments can then be suctioned out of the vessel with a catheter. This technique has been described in various case reports, including in an especially challenging case involving a patient with heparin-induced thrombocytopenia and a recent ischaemic stroke who subsequently presented with an acute PE52. However, despite approval for use of a rotational thrombectomy system in the treatment of peripheral and arteriovenous dialysis fistulae thrombosis in the US, data regarding its role in the management of patients with pulmonary emboli are lacking.The use of a rotational catheter to cause thrombus fragmentation and at least partial clearance of a central embolic occlusion has also been described. This technique can be combined with localised infusion of a thrombolytic agent to improve its effectiveness53. Although initial data suggested that the most common treatment recommended by the team was anticoagulation alone, 11% of patients received systemic or catheter-directed thrombolysis. Additionally, PERT activations increased by 16% every 6 months over the course of the initial 30-month period, highlighting the potentially greater role that such teams are likely to play in the management of acute PE in the future54. The argument for the use of such teams is strengthened by a recent analysis of outcomes for patients treated by activation of the PERT pathway in Kentucky. This showed significantly lower intensive care unit and overall in-hospital length of stay compared with patients treated at the discretion of the attending team alone. However, there was no statistically significant difference in mortality between the two groups55.In 2012, Massachusetts General Hospital introduced the pulmonary embolism response team (PERT). The team is composed of specialists from a number of clinical backgrounds and provides expert multi-disciplinary evaluation and management input for intermediate- and high-risk patients with PE56.A number of other PERT programmes have subsequently been developed in the US and led to the development of the PERT Consortium, which intends to be “the driving force behind increased survival rates and the future of PE treatment”The optimum management of acute PE continues to be a clinical challenge. The early management of intermediate-risk disease, in particular, remains a subject of debate. Although anticoagulation continues to be the mainstay of treatment, head-to-head trials of reperfusion strategies are still needed. PE referral centres and PERTs will facilitate multi-disciplinary decision-making for treatment of higher-risk patients and hopefully improve patient outcomes.


The overall mortality is increased compared to the general population and a major cause of death includes malignant diseases, especially oral and esophageal cancers. We here present a case series of four APS-1 patients with oral tongue cancers, an entity not described in detail previously. Scrutiny of history and clinical phenotypes indicate that chronic mucocutaneous candidiasis and smoking are significant risk factors. Preventive measures and early diagnosis are important to successfully manage this potentially fatal disease.Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) or Autoimmune polyendocrine syndrome type-1 (APS-1) (APECED, OMIM  It is clinically defined by the presence of two of the three main components: hypoparathyroidism (HP), primary adrenocortical insufficiency (PAI), and chronic mucocutaneous candidiasis (CMC)  or Autoimmune polyendocrine syndrome type-1 (APS-1)  , 6 due tis (CMC) , severe is (CMC) –9, fulmiis (CMC) , 11.Malignancies are not uncommon in APS-1, and squamous cell carcinoma (SCC) of the oral or esophageal mucosa is the most common entity , 5. In aOral malignancies typically develop in middle-aged or older individuals, often in the fifth or sixth decade of life, and there is a well-established association between both smoking and heavy drinking, and SCC –15. OtheRecently, we have come across several APS-1 patients diagnosed with oral tongue cancers, which seems to be a distinct entity associated with APS-1. Here we highlight their clinical presentation, diagnosis, treatment, and follow up. We also briefly discuss the biological aspects of oral malignancies in the context of APS-1.AIRE-mutations are summarized in Table Basic APS-1 characteristics of the patients including AIRE sequencing. She has smoked regularly from the age of 14 years (currently 1–4 cigarettes a day), but only consumed 4–5 units of alcohol per year.This Finnish female patient (born 1967) was diagnosed with HP at the age of 18 months. She presented with CMC in the mouth and esophagus from the age of three years; regular antifungal medication had not been used. The APS-1 diagnosis was established in early childhood by At the age of 37 years endoscopic esophagus dilation was performed because of stenosis. At the age of 45 years she presented with a 1 cm ulceration on the right side of the tongue. Histology revealed locally invasive SCC of World Health Organization (WHO) Grade 1 without positive neck nodes . A radical resection was performed. No postoperative radiotherapy was given. She is now disease free after an uneventful five-years follow up.AIRE sequencing. Renal transplantation was performed at the age of 24 years because a tubulointerstitial nephritis causing end-stage renal failure. She presented with particularly severe CMC infections from the age of 40 years. The yeast was fluconazole and itraconazole resistant, but amphotericin B sensitive, and she received local treatment with this medication. She has never been a regular smoker and reported current alcohol use of about four units per week.This Finnish female patient (born 1965) was diagnosed with HP at the age of two years and has had oral CMC since the age of 10 years. The APS-1 diagnosis was made based on clinical manifestations and confirmed by in situ of the right side of the tongue and a radical surgical resection was performed. However, a local recurrence of SCC  occurred one year after the initial treatment and a hemiglossectomy with a radial forearm free-flap reconstruction was performed  with 5 mm clinical margins. PDT was delivered as an alternative postoperative treatment option due to the history of recurrent multifocal SCC, assuming that repeated surgical resections and conventional radiotherapy would have caused a higher risk for further impaired oral function. During a 16-year follow up, two granulomatotic gingival lesions have been resected 13 years after the primary diagnosis; histology showed mild dysplasia, gingivitis and fungal infection.At the age of 30 years she was diagnosed with carcinoma AIRE sequencing. His gastrointestinal manifestations have been treated with mycophenolate mofetil and tacrolimus with a good response. The patient has also been diagnosed with asplenism. He neither smokes nor uses alcohol.This Norwegian male patient (born 1996) presented with autoimmune hepatitis and severe malabsorption during the first year of life. He also had recurrent CMC infections in early childhood. PAI was diagnosed at 11 years of age. The APS-1 diagnosis was confirmed by At the age of 21 years he developed severe glossitis and pain in the tongue Figure , and strThis male patient with APS-1 (born 1970) was the son of Persian Jews who were first cousins. He presented, at the age of three years, with alopecia areata, which advanced during the next 4 years to alopecia totalis. HP was diagnosed at the age of five. During the following years additional diseases developed including PAI, vitiligo, bilateral cataract, keratitis, pernicious anemia, hepatitis, and asplenism. He had CMC since childhood and had numerous episodes of oral and oesophageal candidiasis which was treated with nystatin, ketoconazole and fluconazole. There was no history of smoking or alcohol consumption.Staphylococcal aureus septicaemia.At the age of 38 years, a 2 cm mass was observed on the right side of the tongue. Biopsy revealed SCC. Partial glossectomy with selective neck dissection was performed and postoperative radiotherapy to the primary site and to the neck was delivered. Unfortunately, two years later, the tongue tumor recurred. Chemotherapy was initiated, but without effect on tumor growth and the patient died a few months later due to We here report four APS-1 patients with oral tongue SCC at a relatively young age treated with radical surgical resection alone or in combination with chemo-radiotherapy, or PDT in one case. All patients had severe CMC since childhood; some in combination with other well-known risk factors for oral malignancies such as smoking and alcohol use. Apart from early onset, the clinical presentation and histology of the tumors was similar to other patients with oral malignancies. Based on our findings, a prevalence of oral tongue malignancies in the entire APS-1 cohort can be estimated to about 1–2 percent. However, our case series highlights the importance of aggressive CMC treatment and regular follow up examination of the oral mucosa in APS-1 patients.Candida albicans despite the relief of symptoms found in response to treatment , which is a self-antigen in melanocytes and a cancer antigen in melanomas (Aire-deficient thymic stroma was sufficient to confer more effective immune rejection of melanoma in otherwise Aire wild-type hosts (AIRE probably has functions beyond thymic negative selection of T cells and may play a role in the development of malignancies. This also underpins the value of APS-1 as a powerful model disease for studying immunological mechanisms in both autoimmunity and malignancies.All our patients described here had typical APS-1 manifestations and disease-causing survival . Moreove in mice . Howeverpression , 28. Anoelanomas . This leelanomas . In addipe hosts . Taken tOral manifestations in APS-1 should be properly investigated to reveal initial signs of oral malignancies including tongue carcinomas. This malignancy seems to be a hitherto undescribed distinct entity associated with APS-1. CMC should be aggressively treated, and the risk factors reduced, to avoid development of oral malignancies in these patients. A regular investigation of the oral cavity is recommended as part of the annual follow up.Written informed consent has been obtained from all patients alive and from the next of kin of the deceased patient for publication of the case report and accompanying images.All authors contributed in the clinical characterization of the patients and in writing and critically reviewing the manuscript. D-EC described the histologic pictures.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.


Approximately 55-85% of women worldwide have experienced postpartum blues (PPB) during 6-9 weeks after delivery without receiving the counseling program; more than 20% of them have developed into postpartum depression. Study objectives were to evaluate the effect of the Self-EAR program to improve the postpartum blues scores and serum allopregnanolone level among newly blues mothers.During June 2015 to May 2016, the randomized controlled trial was conducted among 76 Nulliparous blues mothers who were screened with Stein’s postpartum blues scores ≥ 3. All participants were randomly assigned either to the intervention group (Self-EAR program) and the control group (standard postpartum care program). The Self-EAR program was transformed into audio files which were installed in an MP3 digital device before providing it to the intervention group in order to be implemented at home three times per day for four weeks. Participants were assessed at baseline, 1-month, 2-month and 3-month follow-up for serum allopregnanolone level. Data were analyzed by using descriptive statistic, chi-square test, t-test, and repeated measure analysis of variance.After the 3-month follow-up, the results revealed positive effects of the Self-EAR program on postpartum blues scores  and serum allopregnanolone concertation . The participants in the intervention group had experienced significantly lower postpartum blues scores; on the other hand, they had significantly higher serum allopregnanolone level when compared with the control group.The findings suggested that the Self-EAR program was effective to improve postpartum blues scores and allopregnanolone level among newly postpartum blues mothers. Usually after childbirth, most women experienced face fluctuated emotion or mood swings, including teary eyes, Irritability, sleep disturbance, lack of concentration, and absent-minded within the first week until 6-9 weeks postpartum.6In the physical change of PPB, many studies of hormone during postpartum periods were contradicting reports, for example, progesterone, estradiol, and cortisol.A randomized controlled trial was conducted in rural Thailand from June 2015 to May 2016. Participants were given an adequate verbal explanation of the trial; they were asked to sign written informed consent for participation. The study protocol was approved by the Ethics Review Committee for Research Involving Human Research Subjects, Health Science Group, Chulalongkorn University (no.122/2015). The inclusion criteria were as follows, willingness, nulliparous mothers who were screened by Stein’s postpartum blues questionnaire ≥ 3 and screened by Edinburgh Perinatal Depression Scale (EPDS) < 13; maternal aged 20 to 35 years. Those who had complications from medical and obstetrical complication; psychosis disorder; on antipsychotic medication, unable to understand and read Thai; and unaltered accommodation after three months of childbirth were excluded. Participants were randomly assigned to the intervention and the control groups by using simple random sampling (SRS). The mean delivery conducted in hospital was 1,753 cases per year. Around 80 participants were eligible from 150 women were admitted to the postpartum unit. Hence, 40 women were allocated in each group by using SRS. There was one dropout participant in the intervention group and three dropout participants in the control group with the reason of moving to another province. The team was trained by the expert in laboratories to operate Allopregnanolone ELISA kits. Stein’s postpartum blues questionnaireThe Self-EAR program is based on the self-empowerment, the self-affirmation, and relaxation techniques. The Self-Empowerment techniques proceeded the three aspects approach including Self-Control, Self-Motivation, and Self-reinforcement. The Self-Affirmation techniques used to guide behavior and decisions, especially to cope with a negative thinking by repeating affirmations to oneself every day and every time. The relaxation techniques practiced by using the progressive muscle relaxation. In the pre-research phase, a focus group discussion was used to develop the 10-minute MP3 audio files by brainstorming ideas from 11 postpartum blues mothers and four nursing instructors. The Self-EAR program was transformed into audio files which was installed in an MP3 digital device before providing it to the intervention group to be implemented at home three times per day for 4 weeks. The control group received the regular and routine standard postpartum care program.The demographic characteristics of participants at baseline are shown in Comparison of mean postpartum blues scores presented in Comparison of mean allopregnanolone serum level is presented in The participants in the intervention group had a significant difference in postpartum blues scores (p=<0.05) and serum allopregnanolone level (p=<0.05) when compared with the control group. Findings showed only postpartum blues scores had statistically significant differences in both group measurements and the interaction effect between measurements depending on groups, whereas serum allopregnanolone level yielded statistically significant differences only in the interaction effect between measurements depending on groups (p=0.001) .The mean difference of postpartum blues scores between the intervention and the control groups was lowest at 1-month, 2-month, and 3-month follow up . The mean difference of serum allopregnanolone level between the intervention and control groups was highest at 1-month and 3-month follow up . .This research study demonstrates the effectiveness of the Self-EAR program that integrated three techniques uniquely to new mothers who had faced with the blues after childbirth. Both postpartum blues scores and allopregnanolone serum level in the newly blues mothers were improved and sustained to 3-month follow-up. The Self-EAR program decreased postpartum blues scores by increasing allopregnanolone serum level. Previous researchers, who conducted progressive muscle relaxation among pregnant women, discovered an improvement in well-being outcomes such as reduced pain level, perceived stress, and promote quality of life.The findings suggested that the Self-EAR program can be applied to decrease postpartum blues scores and increase allopregnanolone level among newly postpartum blues mothers.


Microbial metabolites have a profound effect on the development of type 1 diabetes (T1D). The cross-talk between the gut microbiota, the nervous system, and immune system is necessary to establish and maintain immune and gut tolerance. As quoted by Hippocrates, “All disease begins in the gut.” Although this has been recognized for 2,000 years, the connection between the gut and autoimmune T1D is not yet well-understood. Here, we outline new advances supported by our research and others that have contributed to elucidate the impact of microbial metabolites on the physiology of the pancreas and the gut through their remarkable effect on the immune and nervous system. Among many of the mechanisms involved in the gut–beta-cell–immune cross-talk, glial fibrillary acidic protein (GFAP)-expressing cells are critical players in the development of invasive insulitis. Besides, this review reveals a novel mechanism for microbial metabolites by stimulating IL-22, an essential cytokine for gut homeostasis and beta-cell survival. The close connections between the gut and the pancreas are highlighted through our review as microbial metabolites recirculate through the whole body and intimately react with the nervous system, which controls essential disorders associated with diabetes. As such, we discuss the mechanisms of action of microbial metabolites or short-chain fatty acids (SCFAs), IL-22, and GFAP on beta-cells, gut epithelial cells, neurons, and glial cells via metabolite sensing receptors or through epigenetic effects. The fine-tuned gut–neuro–immune network may be profoundly affected by SCFA deficiency related to dysbiosis and diet alterations at very early stages of the initiation of the disease. Thus, dampening the initial immune response or preventing the perpetuation of the immune response by maintaining the integrity of the gut is among the alternative approaches to prevent T1D. Type 1 diabetes (T1D) is a chronic autoimmune disease in which T cells destroy the insulin-producing beta-cells of the pancreas . The betOne example is the nervous system, which is also critical for the pancreas to function . Both thBeta-cells are involved in late T cell priming, suggesting that they are not required during the induction of T1D , 21. So,Exploring further this idea, Slattery's group has recently shown that ablation of autoantigen presentation in GFAP-expressing cell types reduced the development of invasive insulitis in NOD mice . We can Mycobacterium avium subspecies paratuberculosis, MAP Hsp65, which has a high degree of homology with human GAD65, suggesting that cross-reactivity between MAP Hsp65 and GAD65 potentially could be a mechanism of triggering TID  but also increased the level of serum IL-22, an important cytokine that maintains a healthy commensal microbiota, gut epithelial integrity, and mucosal immunity and ameliorates metabolic disease  in NOD mice and patients with T1D –55, simiActivation of IL-22 through microbial SCFAs contribute to the maintenance of gut homeostasis by the close connection between the intestinal-resident innate lymphoid cell 3 (ILC3) and EGCs. IL-22 is expressed by ILC3, which lies close to EGCs , but its+ neurons and acetylcholine-esterase-stained fibers in the myenteric ganglia. For example, Escherichia coli promoted expression of MHC II molecules on EGCs and significantly induced S100B protein overexpression and nitric oxide (NO) release from EGC, which was counteracted by pre-treatment with TLR and S100B inhibitors  expression on human EGCs . Expresshibitors . As suchhibitors . S100B ihibitors , 73. Thehibitors .During chronic tissue inflammation, significantly increased expression of GFAP on glial cells after stimulation with LPS and pro-inflammatory cytokines has been shown , similarOne study has shown that SCFA butyrate can induce increasing excitatory choline acetyltransferase (ChAT) neurons through the butyrate transporter monocarboxylate transporter (MCT), which is expressed by enteric neurons . Howeverin vitro  , 89. Forhe liver . Aside fhe liver . TGR5 alhe liver , implicahe liver .Among the described effects that SCFAs have on modulating the immune system, beta-cell biology, and gut homeostasis, we have uncovered a novel role for SCFAs by modulating the ENS in the gut, central for the control and prevention of T1D. Overall, an immune response to antigens presented not only by GFAP-expressing pSCs in the pancreas but also by GFAP-expressing EGCs in the gut is a novel finding involved in the initiation of the autoimmune process. Could it trigger antigen-experienced autoreactive cells to move up the gut and reach the ductal and beta-cells, and break the GFAP-expressing neuronal mantle of the islets? This is an unexplored field and requires further research. Given the close location and connection between the gut and the pancreas and their intrinsic dependence from the nervous system, this fine-tuned immuno–neuro-islet cross-talk may be profoundly affected by perturbed gut homeostasis at very early stages of the initiation of the T1D. Dampening the initial immune response or preventing the perpetuation of the islet-specific immune response by maintaining the integrity of the gut is among the possible therapeutic approaches to reprogram T1D , 14. ThuEM developed the conceptual idea, wrote and edited the manuscript. AJ 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.


PRRT2 mutations, the most common genetic etiology of PKD, could cause epilepsy syndromes as well. Standing in the twilight zone between MDs and epilepsy, the pathogenesis of PKD is unclear. Gamma oscillations arise from the inhibitory interneurons which are crucial in the thalamocortical circuits. The role of synchronized gamma oscillations in sensory gating is an important mechanism of automatic cortical inhibition. The patterns of gamma oscillations have been used to characterize neurophysiological features of many neurological diseases, including epilepsy and MDs. This study was aimed to investigate the features of gamma synchronizations in PKD. In the paired-pulse electrical-stimulation task, we recorded the magnetoencephalographic data with distributed source modeling and time-frequency analysis in 19 patients of newly-diagnosed PKD without receiving pharmacotherapy and 18 healthy controls. In combination with the magnetic resonance imaging, the source of gamma oscillations was localized in the primary somatosensory cortex. Somatosensory evoked fields of PKD patients had a reduced peak frequency (p < 0.001 for the first and the second response) and a prolonged peak latency , indicating the synchronization of gamma oscillation is significantly attenuated. The power ratio between two responses was much higher in the PKD group (p = 0.013), indicating the incompetence of activity suppression. Aberrant gamma synchronizations revealed the defective sensory gating of the somatosensory area contributes the pathogenesis of PKD. Our findings documented disinhibited cortical function is a pathomechanism common to PKD and epilepsy, thus rationalized the clinical overlaps of these two diseases and the therapeutic effect of antiepileptic agents for PKD. There is a greater reduction of the peak gamma frequency in PRRT2-related PKD than the non-PRRT PKD group . Loss-of-function PRRT2 mutations could lead to synaptic dysfunction. The disinhibiton change on neurophysiology reflected the impacts of PRRT2 mutations on human neurophysiology.Paroxysmal kinesigenic dyskinesia (PKD) is conventionally regarded as a movement disorder (MD) and characterized by episodic hyperkinesia by sudden movements. However, patients of PKD often have sensory aura and respond excellently to antiepileptic agents.  Magnetoencephalography (MEG) is a powerful tool because it is not only competent in localizing and measuring the cortical neuronal activities, but also able to show the temporal dynamic changes and the reaction synchronicity between different functional areas . Brain oPRRT2 mutations are the most common genetic etiology of PKD, which could cause epilepsy syndromes as well  is a group of diseases characterized by episodic involuntary movements triggered by sudden movements, like initiation of standing, walking, or running. Attacks may consist of dystonic, choreatic, and mixed symptoms. PKD is conventionally regarded as a movement disorder involving substantia nigra and basal ganglia , 11. How as well –23. Stan as well , 25 and  as well , which p as well . This lo as well . To clarPRRT2 mutation, and 18 healthy controls (HC). Distinguished from previous MEG studies for PKD, all our patients were evaluated before receiving any pharmacotherapy so that the medication effects could be avoided. In addition, this study was based on a relative homogenous phenotype as all of the recruited patients presented by paroxysmal dystonia but no chorea. This is also the first study to explore the impacts of PRRT2 mutations on human neurophysiology.This study was aimed to explore the features of gamma synchronizations in PKD with the attempt to address the alternations of brain sensory and motor networks of the disease. We totally recruited 19 patients of newly-diagnosed PKD, 5 with a pathogenic This work was carried out with informed consents obtained from the participants and the ethics approval from the institutional review board of Taipei Veterans General Hospital (IRB 2015-08-001B).This study recruited nineteen patients newly diagnosed as PKD according to the criteria proposed by Bruno et al. . All patPRRT2 gene (NM_145239.2) by Sanger sequencing. The detected mutations were validated via repeated sequencing of sense and antisense strands of the amplicons. The pathogenicity determination took references of the following public genome dababase: the Exome Aggregation Consortium Sequencing Project genome database , dbSNP (https://www.ncbi.nlm.nih.gov/snp/), 1000 Genomes Project (www.1000genomes.org), the National Heart, Lung, and Blood Institute Exome Sequencing Variant database (http://evs.gs.washington.edu/EVS/) and Taiwan Biobank (https://taiwanview.twbiobank.org.tw).Genomic DNA was extracted from the white blood cells in the peripheral blood with standard protocols. All 19 PKD patients received mutation analysis of the coding exons and flanking introns of the Median nerve stimulation was eperformed to provoke somatosensory evoked potentials (SEPs) and fields, which is a standard investigating paradigm using multichannel MEG simultaneous recordings , 30.p > 0.05).Each subject received median-nerve electrical stimulation at the right wrist by an electrical stimulator (Konstant-Strom Stimulator) using the paired-pulse paradigm, which means two identical electrical pulses were delivered with short time interval between them. Each pulse of the paired stimulations was constant-current square-wave with the width of 0.2 ms, inter-stimulus interval (ISI) of 500 ms and inter-pair interval of 8 s, as reported in our previous studies , 31. TheSomatosensory evoked fields (SEFs) for paired electrical stimulation were recorded by using a whole-scalp 306-channel MEG , which is composed of 102 identical triple sensor elements. Each sensor element consisted of two orthogonal planar gradiometers and one magnetometer. In the following analysis, the neuromagnetic activities from magnetometers were discarded for low signal-to-noise ratio ] or MEG artifacts (> 3000 fT/cm) were automatically excluded from further analysis. At least one hundred artifact-free data epochs, in which included the first and second SEF responses to the paired stimulations, were recorded for further analyses.Somatosensory gamma oscillations were evaluated using the SEF responses and transformed into time-frequency domain using Morlet wavelet analysis. A set of wavelets was used with frequencies ranging from 1 to 100 Hz in 1 Hz steps. The default wavelet was set at 1 Hz and 3 seconds for central frequency and time resolution, respectively. Time-frequency spectral power of the SEF responses was averaged across all epochs. At each time-frequency bin was normalized as the percentage change of spectral power relative to the mean power in a reference period between −500 and −100 ms. In the present study, we extracted the prominent channel with the largest SEF responses and calculated the peak power, frequency and latency of gamma oscillation following the first and second electric stimulation at the frequency range over 30 ~ 100 Hz.http://brainvisa.info). The inverse operator of minimum norm estimates analysis was calculated with noise covariance derived from baseline period (−500 ~ −100 ms) and regularization parameter (λ2 = 0.33). Finally, the cortical source activities were obtained and were further analyzed to map the oscillatory activities onto the cortical surface and MRI using the time-frequency power analysis at specific frequency range as mentioned above. The MEG data analysis was performed with Brainstorm (http://neuroimage.usc.edu/brainstorm).To localize the gamma oscillation, the SEF responses were analyzed with a distributed source modeling using weighted minimum norm estimates analysis. The forward model was calculated from the MRI-derived surface model of each participant's brain to describe the signal pattern generated by a unit dipole at each allowed location on the surface. Brain MRIs were acquired by using a 3T MR system (Siemens Magnetom Tim Trio). The surface model was reconstructed from the T1-weighted structural volumetric images , the mean AOE, age of onset (AON) and disease duration (DD) had no significant difference in patients with or without a pathogenic PRRT2 mutation. Although the peak gamma frequencies in both PRRT2-related PKD and non-PRRT2 PKD were significantly lower than HC, it was noticeable that the peak gamma frequency had a greater reduction in PRRT2-related PKD as compared with non-PRRT2 PKD . Moreover, using logistic regression analysis, significant relationships were observed between a PRRT2 mutation and peak gamma frequency . The differences of the peak latency  means that the affected individuals with dyskinesia have slower gamma oscillation responses, and again, reflected the aberrant synchronization of gamma oscillations. The higher power ratio in the PKD group further indicates the incompetence of activity suppression. All together, these findings indicate that somatosensory sensory gating was defective in PKD.The involvement of primary somatosensory area in current study is in line with several functional MRI studies for PKD –35. As tOur group has previously identified decreased postmovement inhibition of the motor cortex and altered theta synchronization in the somatosensory areas between both hemispheres in patient with PKD , 26, 37.The disinhibitory deviation of neuronal networks in PKD could be supported by evidence at the molecular level. Gamma oscillations represent a specific frequency code of GABA (gamma-aminobutyric acid)-ergic inhibitory interneurons which are crucial in the sensorimotor integration in the corticocortical and thalamocortical circuits , 39. GamPRRT2 mutation had a much more decreased peak gamma frequency. PRRT2 is a component of the AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) complex and believed to be involved in synaptic transmission of diverse signaling molecules, including glutamate  in all participants concerning the hemispheric asymmetry in human somatosensory cortical activation . TherefoPRRT2 mutation because they can beneficial substantially by treatment targeting to GABA deregulation and synaptic dysfunction. Our study highlighted a key role of the primary somatosensory cortex in the pathogenesis of PKD. The concept may apply to dystonia and other MDs and provide a potential targets for modulation in the future.This study identified that aberrant gamma synchronization, representing the attenuation of automatic cortical inhibition on sensory gating, is a neurophysiological feature of PKD. Interrupted sensory gaiting, together with reduced postmovement inhibition identified by our previous works, documents the nature of PKD a network disorder which involves not only motor functions but also the sensory processing pathways. The link of gamma oscillations with GABAergic inhibitory neurotransmission discloses a common pathogenesis underlying PKD and epilepsy and makes the rationale of pharmacotherapy by antiepileptic drugs. Our results also stress the importance to identify the PKD patients with a Y-TL contributed to the conception and design of the study, acquisition of data, analysis and interpretation of data, drafting of the article and revision of its content. Y-CC, S-YK, C-CC, H-YY, D-JY, K-KL, W-TC, Y-YL, R-SC, K-YJ, S-FL, Y-TW, and P-SW contributed to acquisition of data, drafting of the article, and revision its content. F-JH contributed to the conception and design of the study, acquisition of data, analysis and interpretation of data, drafting of the article and revision its content, and final approval of the version to be submitted.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.


The short-term effects of botulinum toxin type A (BoNT-A) treatment in stroke patients with upper extremity are well established. This study examined the association between the recovery of motor function of the upper extremity with subjective physical symptoms in outpatients receiving long-term BoNT-A and occupational therapy following stroke. We also investigated the expectations of patients who elected to continue treatment. Forty-seven patients (23 men and 24 women) aged 61 years received BoNT-A treatment more than 20 times. The subjective physical status was analyzed by using the visual analogue scale score through an eight-item questionnaire. Recovery of motor function in the upper extremity was detected by calculating the change (delta) in Fugl–Mayer Assessment (FMA), and ordinal logistic modeling analysis was used to determine the association between the delta-FMA score and the subjective level of agreement for each item. When the ordinal logistic modeling fit was statistically significant, results were interpreted as having logistic probability. The logistic curves discriminating one point (strongly disagree) from five points (strongly agree) were fit in a stepwise fashion. This study suggests that patients receiving long-term BoNT-A treatment and occupational therapy experienced an increased upper extremity mitigation and decreased insomnia after injection, regardless of the recovery of motor function. Patients with post-stroke hemiplegia often suffer from spasticity ,2. SeverThe efficacy of BoNT-A is attenuated in three to four months following administration ,9. ThereTo effectively achieve rehabilitation in hemiplegic patients, it is necessary to understand the factors related to the motor skills of patients . In partIn this study, we aimed to determine the association between the recovery in motor function of the upper extremity and muscle relaxation after treatment with subjective symptoms of pain in long-term outpatient treatment of BoNT-A and aid of rehabilitation after stroke. We also investigated the expectations for subjective physical status of patients who continued for treatments.Baseline characteristics of the 47 selected patients are shown in As a result of the subjective physical survey, the patients indicated that they felt both injection and exercise therapy were beneficial . The logistic curves discriminating one point (strongly disagree with the question) from five points (strongly agree with the question) showed a stepwise fashion fit  to verify our findings. Second, selection bias could be limiting the effects of this study because the patients included were only those who received BoNT-A treatment more than 20 times. Therefore, it is likely that those who had significantly recovered upper extremity motor function during the course of treatment and those who stopped treatment due to deterioration in physical condition were not included in this survey. The magnitude of this bias is unknown. It is necessary to investigate whether similar results can be obtained for patients with a short period of treatment. Third, the study was likely underpowered based on the sample size calculations that were performed, which could contribute to increased type 2 errors. It is also difficult to predict if similar results can be obtained. To fully and scientifically examine the physical effects of BoNT-A treatment, it will also be necessary to recruit and reanalyze patients who comply with long-term monitoring, as this would reduce type 2 errors in future studies. Finally, the data we obtained from patients were limited and confounding variables may have been present, these include the use of sleeping pills, analgesics, or other medications taken for symptom management. Given these limitations, the apparent next step would involve the use of study data to explain BoNT-A treatment to patients and thus allow them to feel safe and receive continuous treatment. However, patients receiving this treatment should be monitored, and improvements in treatment compliance should be verified.This study suggests that patients who received BoNT-A treatment more than 20 times and continued occupational therapy for at least five years experienced increased upper extremity mitigation and decreased insomnia after BoNT-A injection, even without the recovery of motor function.This was a retrospective, longitudinal study. Patients underwent BoNT-A treatment at least 20 times at Tokyo Jikei University Hospital from November 2010 to March 2018. The written approval of consent to receive BoNT-A treatment was obtained from all 47 patients enrolled in the study. This study was approved by the ethics committee of the Tokyo Jikei University School of Medicine . The registry was carried out according to the principles of the declaration of Helsinki.Motor function of the upper extremity in patients with post-stroke hemiplegia and their subjective physical status were examined after BoNT-A treatment. Assessment of motor function of the upper extremity was performed using the Fugl–Mayer assessment (FMA) score . In addiThe inclusion criteria were (1) patients with post-stroke hemiplegia who were continuing BoNT-A treatment and occupational therapy, (2) patients who consented to BoNT-A treatment, and (3) patients who had at least 20 BoNT-A injections . Patient2R (0.60) values in G*Power 3.1 software [A sample size of 53 was derived by logistic regression z-tests insertion of power (0.80), α (0.05), and software , 47 patiPatients received injections of BoNT-A OnabotulinumtoxinA . One vial of BoNT-A (100 U) was diluted with 2 mL saline. Selection of muscles and injected doses were determined by the physician based on clinical assessment. Treated upper extremities were injected with BoNT-A (0–60 U) into the selected muscles at up to four sites .FMA is a comprehensive evaluation index for assessing motor function of hemiplegic patients after stroke, including detecting sensorimotor impairments . The FMASubjective physical survey items were created by physicians, occupational therapists, and clinical psychologists . AnswersTo analyze recovery of motor function in the upper extremity, delta-FMA was calculated from the initial and last FMA scores obtained during BoNT-A treatment.p) of multiple dichotomous responses (Equation (1)) to a linear model (Equation (2)):x is the explanatory variable, iβ is the partial regression coefficient, and e is the residual between actual and predicted data. Therefore, for multilevel ordinal responses, the cumulative probability is calculated at each level to generate a simple regression. In this study, the probability of five levels of subjective agreement was evaluated in association with delta-FMA score. All statistical analyses were performed using the R 3.5.2 software .The association between the delta-FMA score and each item from the subjective physical status questionnaire was determined by ordinal logistic modeling analysis . The pri


ELOVL4 cause three different neurological diseases in humans. Heterozygous inheritance of one set of autosomal dominant ELOVL4 mutations that leads to truncation of the ELOVL4 protein causes Stargardt-like macular dystrophy (STGD3), an aggressive juvenile-onset retinal degeneration. Heterozygous inheritance of a different set of autosomal dominant ELOVL4 mutations that leads to a full-length protein with single amino acid substitutions causes spinocerebellar ataxia 34 (SCA34), a late-onset neurodegenerative disease characterized by gait ataxia and cerebellar atrophy. Homozygous inheritance of a different set of ELOVL4 mutations causes a more severe disease with infantile onset characterized by seizures, spasticity, intellectual disability, ichthyosis, and premature death. ELOVL4 is expressed widely in the CNS and is found primarily in neurons. ELOVL4 is expressed in cell-specific patterns within different regions of the CNS that are likely to be related to disease symptoms. In the retina, ELOVL4 is expressed exclusively in photoreceptors and produces VLC-PUFA that are incorporated into phosphatidylcholine and enriched in the light sensitive membrane disks of the photoreceptor outer segments. VLC-PUFA are enzymatically converted into “elovanoid” compounds that appear to provide paracrine signals that promote photoreceptor and neuronal survival. In the brain, the main ELOVL4 products are VLC-SFA that are incorporated into sphingolipids and enriched in synaptic vesicles, where they regulate kinetics of presynaptic neurotransmitter release. Understanding the function of ELOVL4 and its VLC-SFA and VLC-PUFA products will advance our understanding of basic mechanisms in neural signaling and has potential for developing novel therapies for seizure and neurodegenerative diseases.Elongation of Very Long chain fatty acids-4 (ELOVL4) protein is a member of the ELOVL family of fatty acid elongases that is collectively responsible for catalyzing formation of long chain fatty acids. ELOVL4 is the only family member that catalyzes production of Very Long Chain Saturated Fatty Acids (VLC-SFA) and Very Long Chain Polyunsaturated Fatty Acids (VLC-PUFA) with chain lengths ≥28 carbons. ELOVL4 and its VLC-SFA and VLC-PUFA products are emerging as important regulators of synaptic signaling and neuronal survival in the central nervous system (CNS). Distinct sets of mutations in  Lipids are critical biochemical components of the central nervous system (CNS) that are essential for proper CNS function. The lipid composition of the brain is unique and exceedingly diverse . AberranELOVL4 refers to the human gene, Elovl4 to the non-human mammalian gene, and elovl4 to non-mammalian gene. ELOVL4 refers to the protein in all species.Recent studies have revealed novel functions for Very Long Chain-Fatty Acids (VLC-FA), defined by a chain length of 28 or more carbons, in neural signaling . This reThe ELOVL family of enzymes in mammals is comprised of seven members that all reside in the endoplasmic reticulum (ER) and are thought to form a multimeric complex . TogetheFatty acid elongation occurs by cycling through a four step process , with two carbon atoms added through each cycle. Members of the ELOVL family catalyze the first step, a condensation reaction between a fatty acyl-CoA and malonyl-CoA . The secComplexing of ELOVL proteins is important to their function . ELOVL4 The ELOVL family of elongases is involved in elongation of many different lipid species. Importantly, some ELOVL family elongases show distinct substrate selectivity and mediate very specific elongation reactions, while other members of the family show broader substrate selectivity and can show functional redundancy . Thus, tELOVL4 mediates elongation of long chain PUFA and SFA to form VLC-PUFA and VLC-SFA of 28 carbon chain length, respectively . ELOVL4 cis double bonds, lead to locally disordered phospholipid packing in the membrane, increased fluidity, and potentially affect membrane curvature   . Part of (> C30) , and are (> C30) . In the  (> C30) , which a (> C30) . Conditi (> C30) . VLC-PUF (> C30) .In the CNS, ELOVL4 produces both VLC-SFA and VLC-PUFA in a region-specific manner. In the brain, the main ELOVL4 products are VLC-SFA, which are incorporated in sphingolipids that are enriched in synaptic vesicles and regulate presynaptic release . In retiELOVL1, -4, -5, and -7 are associated with neurological disease. Importantly, the diseases caused by these mutations have some shared characteristics that suggest fatty acid elongation in general is particularly critical to the health of the CNS. No disease causing mutations in ELOVL2, -3, or -6 have been reported to date. The synthetic activities and functions of each ELOVL family member are summarized below.The mammalian ELOVL family of elongases has a number of important functions in the body and, as a group, are particularly important to CNS function, the epidermal water barrier, systemic metabolic functions, and also are likely to be important to fertility. Mutations affecting Elovl1 in mice causes disruption of the lamellae in the stratum corneum of the skin and disrupts the water barrier of the skin leading to perinatal lethality , spinocerebellar ataxia 34 (SCA34), and a neuro-ichthyotic syndrome. All of these diseases have profound effects on the CNS and are discussed in more detail in several sections below.ELOVL4 elongates long-chain PUFA and long-chain SFA of 24 carbon length to VLC-PUFA and VLC-SFA  in humans, which is characterized by gait ataxia, nystagmus, anosmia, and cerebellar atrophy , but their expression levels differ across brain regions . ELOVL2 in situ hybridization  cause SCA38, which is characterized by gait ataxia, dysarthria, abnormal eye movements, and cerebellar atrophy with onset in the third or fourth decade , spinocerebellar ataxia-34 (SCA34) with or without erythrokeratodermia variabilis (EKV), and a severe neuro-ichthyotic syndrome. Each disease is discussed below.Several disease-causing mutations in the human  to date . MutatioELOVL4 cause autosomal dominant STGD3, an aggressive degeneration striking the macular region of the retina with juvenile onset and rapid progression , a late-onset degenerative disease of the cerebellum that may present with or without erythrokeratodermia variabilis   . The gaiof SCA34 .ELOVL4 gene cause a severe neuro-ichthyotic syndrome  effectively eliminate the enzymatic function of ELOVL4 and, thus, lead to SVs that lack VLC-SFA. The absence of the van der Waals forces generated by the VLC-SFA within the SV lipid bilayer would therefore be expected to increase the probability of spontaneous release events by removing a barrier to fusion. In this case, the precise timing of synaptic release would be compromised, leading to bursting activity and seizures, as seen in the hippocampus of knock-in mice homozygous for the 5bp deletion STGD3 ELOVL4 mutation  system. Although PIPs are a minor fraction of the membrane lipids, they are essential for proper targeting of vesicular traffic and for synaptic endocytosis . The cytablished . WhetherAlthough there is abundant information on ER trafficking from yeast and other single cell organisms , the det2 in SV  in neuronal axons has been known for nearly half a century . The axoEpilepsy patients are prescribed a ketogenic diet – a fat-rich diet that is low in sugars – when all anti-convulsive drugs fail to reduce seizures . The cliThere is tremendous interest in nutritional interventions to prevent cognitive impairment in the elderly, including the potential of fatty acids to prevent or slow cognitive impairment . Change Recent studies reveal novel functional roles for ELOVL4 and its VLC-FA products in the CNS, including retina and brain, in health and disease. VLC-PUFA play vital functions in the CNS as precursors of compounds that serve newly recognized roles in homeostatic signaling and regulation of neuronal survival. Most recently it was shown that VLC-SFA have essential functions in synaptic transmission, with disruption of VLC-SFA synthesis leading to seizures and neurodegeneration both in patients lacking ELOVL4 and in mouse models of ELOVL4 mutations. A better understanding of VLC-SFA and VLC-PUFA metabolism and their functions in the CNS holds great promise for development of new therapeutic avenues for treatment of seizures and neurodegenerative diseases.FD, RA, JF, and DS contributed to the writing and editing of the manuscript.RA has a United States Patent  entitled “Very Long Chain Polyunsaturated Fatty Acids, Methods of Production, and Uses.” The remaining 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.