Advanced sensors/electrodes and signal processing techniques provide powerful tools to analyze surface electromyographic signals (sEMG) and their features, to decompose sEMG into the constituent motor unit action potential trains, and to identify synergies, neural muscle drive, and EEG–sEMG coherence. However, despite thousands of articles, dozens of textbooks, tutorials, consensus papers, and European and International efforts, the translation of this knowledge into clinical activities and assessment procedures has been very slow, likely because of lack of clinical studies and competent operators in the field. Understanding and using sEMG-based hardware and software tools requires a level of knowledge of signal processing and interpretation concepts that is multidisciplinary and is not provided by most academic curricula in physiotherapy, movement sciences, neurophysiology, rehabilitation, sport, and occupational medicine. The chasm existing between the available knowledge and its clinical applications in this field is discussed as well as the need for new clinical figures. The need for updating the training of physiotherapists, neurophysiology technicians, and clinical technologists is discussed as well as the required competences of trainers and trainees. Indications and examples are suggested and provide a basis for addressing the problem. Two teaching examples are provided in the Supplementary Material. Furthermore, it provides extensive information for medical doctors, physiotherapists, occupational therapists, and movement scientists for functional diagnosis, patient path management, evaluation of patient recovery and progress, and quantification of treatment/training effectiveness. Most clinical studies are limited to few subjects  and look more like “proof of concept” studies. Larger clinical studies require funding and competent operators and both are lacking. This work addresses the need for training clinical operators in the use of sEMG as a measurement tool.The milestone book by Basmajian and De Luca  providedwww.seniam.org, now also available in www.robertomerletti.it/en/emg/material/seniam/). The project involved partners from nine EU countries and provided guidelines about electrodes and their placement, signal processing, modeling techniques, and data reporting. Despite their frequent citation in many papers, and their recent updates . Furthermore, increased quadriceps-hamstring coactivity has been reported during sit-to-stand from a chair and during stair descending, as a result of muscle weakness and joint instability . Similarly, Hodges and Richardson adopted sEMG to describe a transversus abdominis neuromuscular activation delay during upper limb movements in subjects with low back pain, identifying this phenomenon as a marker of inefficient muscular stabilization of the lumbar spine and leading clinicians to develop ad-hoc exercise programs aimed at improving spine functional stability [68].3.33.3.1Functional recovery can occur either through the restoration of motor abilities of affected body structures or by means of adaptive strategies aimed at compensating the impaired body function. Adaptation has been largely described in patients with CNS lesions, but it also occurs after impairments of peripheral body structures. Clinical examination alone may not distinguish between motor recovery of the affected structures and adoption of adaptive strategies involving unaffected structures, especially in patients with complete functional recovery. For example, Temporiti et al. described asymmetries in body weight distribution during quiet standing in patients before and after unilateral hip arthroplasty. These patients relied on the unaffected limb to stabilize the whole body and ensure postural stability . Adaptiv3.4Patients undergoing a motor rehabilitation program are required to perform tailored therapeutic exercises, which are defined after an accurate functional assessment. In this scenario, sEMG is essential to provide such assessment as well as to show the presence of arthrogenic muscle inhibition, improper muscle activation timing, or the use of adaptive strategies . The dethttps://collegedunia.com/courses/physiotherapy/syllabus).In this scenario, the professional(s) dedicated to sEMG in clinical practice (1) must be aware of, and be familiar with, the most updated and appropriate literature in the field and (2) must have a strong interdisciplinary medical-technical knowledge. What kind of education should these professionals receive? In general, the complexity of sEMG data analysis does not allow for the inclusion of the required skills in a 3 year Bachelor program and requires a 4 year curriculum  established the European Higher Education Area to improve student and staff mobility, to make higher education more inclusive, accessible, appealing, and competitive. All participating countries agreed to implement a three-step higher education system consisting of Bachelor (BS), Master (MS), and Doctoral (PhD) levels. Given this organization, sEMG could be taught at the BS, MS, and PhD levels, as well as at postgraduate certified courses. At the BS level, the technical general knowledge of sEMG should be provided at least in basic Neural Engineering and Neuromuscular Physiology courses. The sEMG may be taught more deeply at the MS level to neurophysiology technicians and physiotherapists. The PhD courses (when available) offer a wide range of research programs in neurosciences, biomedical signal processing, and biomechanics and provide a background for teaching. Furthermore, the fundamental concepts of sEMG should be taught during the medical specialization courses in Neurology, Rehabilitation, and Orthopedics. There are other non-university high education programs (e.g. in Germany). Some National/International Scientific Societies provide certificates as well as the possibility to develop professional figures or promote dissemination of knowledge.4.1Infrastructure requirement and curriculum (policy making): sEMG instrumentation should be included in physiotherapy labs (as done in India and many other countries), with standardized teaching in a 3 year BS curriculum. A dedicated subject in undergraduate education and in postgraduate programs is desirable, with proportionate weightage in examination and evaluation. This is a practical approach to avoid the inertia to adopt new technologies that is observed in experienced physiotherapists at later times.Clinical practice: there should be a policy on “Standards of Procedures” to guide and enhance sEMG reporting during clinical assessment and monitoring of neuromuscular disorders. Physiotherapists should contribute to these standards.Research thrust: sEMG research in MS and PhD programs (where available) may be promoted by offering grants, seed money, access to literature, fellowships, and idea incubation centers.Faculty development programs should grant credits to attend sEMG-related workshops, conferences on evidence-based practices, etc. to disseminate sEMG techniques. Career enhancements should provide physiotherapists with promotional avenues in reward of exemplary practice, case studies, innovations, peer-reviewed publications, etc.Despite the current situation , physiothttps://isek.org/isek-jek-tutorials/). Other specific “URLs” dealing with academic curricula are:In summary, the current academic curriculum in physiotherapy is not suitable to produce a clinical figure with proper competence in sEMG and other assessment techniques. Its updating is long due and proper pressure and educational initiatives promoted by the professional societies are necessary. The recent initiatives of the ISEK are a good step in this direction (https://collegedunia.com/courses/physiotherapy/syllabushttps://physio.sgtuniversity.ac.in/syllabus-first-year-bachelor-of-physiotherapy-bpt/https://www.getmyuni.com/bpt-syllabus-subjectshttps://www.amsterdamuas.com/programmes/european-school-of-physiotherapy/programme-structure.4.2https://ec.europa.eu/growth/tools-databases/regprof/professions), this profession is well regulated in three countries across the European Union, notably in Hungary (Klinikai neurofiziológiai szakasszisztens), Italy (Tecnico di neurofisiopatologia), and Portugal (Técnico de neurofisiologia). In other countries, sEMG and EEG technicians are professional figures specifically trained within certificated and specific courses (US and UK). In Italy, the Law Decrees of January 26, 1988 and March 15, 1995 provided a clear legal identification of the neurophysiology technician professional profile within the National Health Service. Accordingly, the neurophysiology technician is a health professional figure who works in the field of neurology and neurosurgery, uses the necessary instruments , and is responsible for their operation and for proper bioelectric signal recordings for diagnosis, assessment, or research work in collaboration with the specialized medical doctor. The neurophysiology technician has direct responsibilities of the application and of the result of the devices used. The required title is the 3-year degree (BS) in “Techniques of Neurophysiopathology.” The modern profile of the Neurophysiology Technicians should be not just an exam executor, but an expert specialist, completely integrated in the neurophysiological pathway [Perform all operations concerning sEMG data acquisition in accordance with established recommendations, consensus, and literature updates, including the proper use of devices.Control the quality of sEMG recordings by detecting and correcting interferences and artifacts.Perform sEMG signal processing and interpretation using commercial or home-developed software.Understand the sEMG physiological and clinical correlates.Perform initial and continuous patient assessments to ensure proper comprehension and compliance with testing methods.Demonstrate the knowledge and abilities required to care for patients who require this monitoring mode.Score sEMG activity and computer-generated results and maintain records to ensure comprehensive clinical information.Comply with national and international documentation rules and submit a copy of the results to the physician.Ascertain that adequate equipment and supplies are available to provide quality patient care, maintain inventories, and update reference materials.Assist in the development and presentation of educational lectures, conferences, and promotional activities and act as point of contact for issues pertaining to sEMG studies.One of the professional figures that would best suit the role of sEMG management is the “neurophysiology technician” . Professionals in the field of neurophysiology perform tests that assist physicians and surgeons in the diagnosis and evaluation of diseases of the brain, peripheral nervous system disorders, and sleep using sophisticated electronic testing equipment. According to the report of the European Commission  was founded in 2009 as the professional association of a new healthcare professional, the Technical Physician (www.nvvtg.nl). See: https://www.tudelft.nl/onderwijs/opleidingen/masters/technical-medicine/msc-technical-medicine.Since 2003, the University of Twente and, since 2014, the Technical University of Delft, the University of Leiden, and the Erasmus University (The Netherlands) offer a BS in Clinical Technology and an MS in Technical Medicine (3 + 3 years). Up to 2022, over 600 students graduated, mostly finding jobs in public hospitals. See: This activity has been presented as a task-shifting initiative, that is “passing the buck” of attaining/enhancing competences, work and responsibilities, from one professional group to another, as a remedy against medical workforce shortages, increasing impact of technology, as well as a way of lowering healthcare cost by shifting tasks from physicians to less costly personnel . However5measurement, confounding factors, interferences, artifacts, noise ,47;mono-dimensional (1D), bi-dimensional (2D or images), and three-dimensional (3D or movies) signals evolving in time and space ,16,80,81time and frequency representation of a signal: Fourier transform, amplitude and power spectra of a signal ;amplitude and frequency signal features ;signal diffusion in space and crosstalk between signal sources ;muscle fiber conduction velocity and its measurement ,85;decomposition of sEMG into its constituent MUAP trains ,86,87;neural drive to a muscle and its measurement ,87;input impedance, common mode rejection ratio, frequency response of a sEMG amplifier, signal sampling, and A/D conversion ,10;identification of reliable literature and familiarization with trustworthy resources in the field of sEMG; andemphasis on checking equipment and procedures when using sEMG .The need for updating the academic education of rehabilitation clinical operators has been extensively underlined in the literature and in the previous sections. Knowledge of the state of the art, including technology, is fundamental for posing and answering meaningful clinical questions. Many clinical questions were unthinkable before the decomposition of sEMG into its constituent MUAP trains was developed 20 years ago. Physiological knowledge, clinical questions, and technology are strictly inter-related ,43,47. Phttp://youtube.com/neuromechTV. In agreement with Jippes at al. [believe that providing schools that are conducted 100% online could significantly benefit a larger number of students and professionals” [https://www.robertomerletti.it/assets/pdfs/dr_sanjeev_gupta_video_lectures.pdf.The above concepts are strongly interdisciplinary and is difficult to find teachers competent in all of them. In addition, this teaching requires extensive experience . Althougs at al. , these asionals” . Relatedhttps://isek.org/isek-jek-tutorials/).Other Summer Schools in closely related fields have been associated to the International Conferences in Neurorehabilitation ,93,94 anversus in-presence lecturing [Teaching regular academic in-presence lectures on these concepts requires 30–50 hours at the graduate level and 10–15 at the UG level, but this time could be reduced to half by using available online material. With a few exceptions, experienced clinical operators, trained many years ago (end even today) in the therapeutic application of heat, electromagnetic radiation, ultrasound, and electrical stimulation, would not be suitable teachers of the above concepts because of lack of competence. Biomedical engineers would likely lack the perception of the real clinical questions and needs. Neurophysiology technicians lack competence in the field of movement analysis and rehabilitation. The impact of clinical technologists has not yet been tested in this field. Therefore, a considerable effort is needed to combine “Teach the Teachers” initiatives, selection/preparation of good on-line material for home study, and discussion of recent literature in “journal clubs.” Availability of equipment for laboratory exercises, and teachers able to teach how to use this hardware and the related software, is a requirement that today is rarely satisfied. This is particularly the case in countries where Doctoral Degrees in physiotherapy are not available, a fact resulting in a vicious circle [ecturing  is quite5.1Many authors consider that sEMG teaching should take place at the MS level or at postgraduate Master courses. According to other authors, the main category to target for sEMG teaching is students of undergraduate (UG) programs. Many of these students will not pursue further studies and should therefore receive basic notions about sEMG at the UG level. The advantage with UG students is that they may reflect more keenness and curiosity toward sEMG practice. Students show interest in advanced instrumentation and their capacity to grasp technology is generally higher than that of practicing physiotherapists who are more oriented toward managing their patient loads. The natural tendency to resist change in clinical practices is less likely to be exhibited by the UG student community, especially if it is linked with curricular credits.5.1.1A three pronged strategy is necessary to introduce sEMG knowledge and instrumentation in the learner’s mind. The three prongs for such approach are (a) Educate, (b) Mandate, and (c) Reinforce. Educate: a thoughtful combination of appropriate theoretical concepts blended with practical sessions at para-clinical level of UG education is necessary to lay a strong foundation of sEMG practice and timely overlap of sEMG methods with related methods and protocols (e.g. biomechanics and IMU). This is the stage where the matrix of clinical practice is being laid in a budding clinician (learner) mindset. Mandate: the knowledge and application of sEMG should be made mandatory for clinical practice by statutory measures. It should include teaching and evaluation of sEMG knowledge with emphasis on quantitative reporting of muscle and movement features, sEMG clinical practice, student exchange programs (at the centers of excellence in sEMG), and attendance in seminars/conferences on sEMG. It should be made clear from the beginning of UG education that sEMG is an integral part of movement and treatment assessment. Reinforce: This is an unexploited area in sEMG education. Students should be posted as interns at sEMG laboratories as observers with limited but challenging tasks assigned to them. Fellowships, apprenticeships, academic document writing, supervised review of journal articles, and assisting teachers in preparation of notes and presentations are the non-conventional methods of teaching/learning that help the learners to reinforce their sEMG knowledge.5.2The interdisciplinary nature of sEMG requires that sEMG courses should be offered at the second or third year of UG and at the graduate level to students who satisfy the minimal following requirements.Clear concepts of basic physics and mathematics at senior secondary 12th grade standard level.Understanding of basic neuromuscular anatomy and physiology required to apply sEMG theory and practice.Capacity to overcome initially frustrating problems such as electrode placements, signal processing, feature extraction, parameter selection, and noise related issues.Basic understanding to handle electrical instrumentation safely and diligently.Basic understanding of computer software and digital platforms such as MS-Office, MATLAB, and similar.Good knowledge of data banks, such as PubMed, and ability to search and read articles.Minimal requirements for a student entering a sEMG course:Good knowledge and teaching acumen for principles of physics and mathematics related to biomechanics, electrophysiology, and biomedical signals.Sufficient understanding of neuromuscular anatomy and physiology required to weave sEMG concepts into the students.Experience with common clinical situations of movement disorders and use of sEMG to cite examples and relate to the learners quests.Knowledge of common clinical practices of neuromuscular diagnosis and treatment such that an integrated teaching and assessment approach can be formulated.Extraordinary communication and soft skills for the translation of mathematical concepts in a simple and comprehensible form.Theoretical and practical knowledge of biomedical signal processing and MATLAB. Good supply of examples and exercises.Full awareness of the sEMG capabilities and limitations. Familiarity with the literature.Motivating and flexible personality to manage and steer learning goals in a problem-solving attitude suitable for the audience.Ability to organize and manage a journal club.Minimal requirements for an sEMG educator/trainer:These capacities are hard but not impossible to find among post-doctoral students and researchers working in existing research laboratories. The increase of the number of these figures of biomedical/rehabilitation engineers or life scientists with dual education must be promoted.6There is a widespread consensus about (1) the need of reducing the gap between available knowledge in the field of rehabilitation assessment technology and the clinical application of such technology in physiotherapy and related fields, (2) the need to adopt EBP approaches to increase and quantify the quality of planning and evaluation of interventions and the effectiveness of therapies, and (3) the need to increase the competence and technical expertise of clinical operators in these fields. However, consensus on how to address these problems, how to form teachers, and how to disseminate knowledge and improve academic education, training, and competence of professional clinical operators is lacking. Consensus about a new educational curriculum for the next generation of clinicians is particularly urgent, not only because of the evolving technology but also because new technologies are changing our knowledge of physiology ,95.From the single channel recording and simple biofeedback applications of the 1980s, sEMG techniques evolved into the HDsEMG methodology, providing electrophysiological images and movies (similar to those of EEG), and are now addressing the clinical applications of decomposition of sEMG into the constituent motor unit action potential trains, the issue of EEG–EMG coherence, and the study of essential tremor, stroke, and other pathologies of the neuromuscular system. The current state of the art and the promising perspectives require a major updating of the competences of clinical operators in the interdisciplinary fields of biomechanics, biomedical signal processing, and neurophysiopathology. This competence is today available in research laboratories more often affiliated to Schools of Biomedical Engineering than to Schools of Medicine or Schools of Allied  Medical Professions.https://rishii-project.com/rishii-at-a-glance/.There is a strong need for simplification, standardization, and clinical versions of sEMG signal processing approaches and equipment that must be jointly developed by engineers and clinicians and then transferred to manufactures. A policy push to mandate sEMG as integral part of clinical practice in neuromuscular sciences is required to reinforce and update the learner’s knowledge and ensure its reflection in clinical procedures. A more concerted and transdisciplinary approach to aggressively reduce threats to sEMG acceptance should be initiated involving all stakeholders to reverse the current decaying situation. Few schools, training physiotherapists, and (in some countries) neurophysiology technicians teach sEMG technology and its clinical applications. The new figure of “clinical technologist” with strong interdisciplinary competences has been trained, at the BS and MS academic levels, and tested for 15 years in the Netherlands. Its impact in the rehabilitation field is still under evaluation. Discussion about the academic level at which courses on sEMG and other technologies should be offered  is under way and is reflected in this work. Novel applications of sEMG in many fields  are rapidly developing and there is an urgent need to review the academic training of existing or new professional figures able to translate the available sEMG knowledge to these areas. There also is a strong need for internationalization of curricula among Higher Education Institutions, currently promoted by the EU and India project The Supplementary Material provides two examples of support material for UG lectures dealing with the Fourier transform and the HDsEMG.


First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping researchers promote themselves alongside their papers. Kazuki Omata is first author on ‘ Kazuki OmataDescribe your scientific journey and your current research focusAfter learning that one million red blood cells are produced per second, I developed an interest in hematology. Through laboratory training in the current laboratory of Professor Takashi Kato, I became even more fascinated by the universality and the diversity of hematopoiesis conserved among vertebrates. The focus of the present work is the progenitor of red blood cells. In the next study, I will investigate the signaling pathway in the red blood cell.Who or what inspired you to become a scientist?I was inspired by Jean-Henri Casimir Fabre's ‘Souvenirs entomologiques’ and developed a fascination for biology. Currently, my research is focused on vertebrates, but I still love to learn about the way all living things live.How would you explain the main finding of your paper?In short, we have established a tool and a method to count the cells that become red blood cells in frogs. There are many differences between humans and frogs, and one of the most important differences is metamorphosis. Frogs undergo dynamic changes from larvae to adults, whereas humans do not. Despite these differences, the systems necessary for survival are similar between frogs and humans, including the blood system. Within the unique biological phenomenon of frog metamorphosis, the mechanism of red blood cell production, which is shared with humans, is not fully understood. Therefore, we developed an antibody that can be used to count frog red blood cells. In this paper, we demonstrated the changes in the number of cells that become red blood cells in frogs during metamorphosis. This finding will highlight the difference in the blood production system between humans and frogs.What are the potential implications of this finding for your field of research?This work has provided insights into how three different axes – individuals, organs, and functions – are controlled through metamorphosis and subsequent maturation. Interestingly, humans undergo hematopoiesis in the liver during the fetal period but lose this ability immediately after birth and the bone marrow becomes the major site of hematopoiesis. The method reported in this paper is expected to provide a means to investigate the mystery of why hematopoietic capacity in the liver is lost in humans while maintained in frogs. In addition, the established antibody against the frog molecule can inhibit signaling molecules critical for erythropoiesis. By using this antibody, there is an opportunity to discover unique biological phenomena that have not been found in mice or humans.Which part of this research project was the most rewarding?One of the important parts is antibody generation. To be honest, this part was mainly done by a co-author who worked in our lab about ten years ago. I was not there when the activity of the antibody was first recognised. In writing the paper using this antibody, I had the opportunity to re-examine the data and refer to my senior colleagues' experimental notes and previous presentations, which reminded me of the importance of experimental records.What do you enjoy most about being an early-career researcher?I think that early-career researchers are able to concentrate and work on a single project. By having the time to think deeply about a question, I feel lucky to be able of the imagine the pure joy of biology.What piece of advice would you give to the next generation of researchers?…I think it is important to proactively learn how to speak in a clear and organised manner, how to create videos, and how to use social media effectively…In the future, there will probably be more opportunities for researchers to explain or present something to people without a scientific background. Therefore, I think it is important to proactively learn how to speak in a clear and organised manner, how to create videos, and how to use social media effectively, even if it is only on a small scale.What's next for you?First and foremost, I will complete my doctoral thesis. After obtaining PhD, I aspire to challenge myself by collaborating with individuals who have an engineering background in a company where I can utilise my background in biology to engage in product development.


It is crucial to maintain the intracranial pressure (ICP) within the physiological range to ensure proper brain function. The ICP may fluctuate during the light-dark phase cycle, complicating diagnosis and treatment choice in patients with pressure-related disorders. Such ICP fluctuations may originate in circadian or sleep-wake cycle-mediated modulation of cerebrospinal fluid (CSF) flow dynamics, which in addition could support diurnal regulation of brain waste clearance.ICP was monitored continuously in patients who underwent placement of an external ventricular drain (EVD) and by telemetric monitoring in experimental rats. CSF was collected via the EVD in patients and the rodent CSF secretion rate determined by in vivo experimentation. Rodent choroid plexus transporter transcripts were quantified with RNAseq and transport activity with ex vivo isotope transport assays.+,K+,2Cl- cotransporter (NKCC1), which is implicated in CSF secretion by this tissue.We demonstrated that ICP increases by 30% in the dark phase in both species, independently of vascular parameters. This increase aligns with elevated CSF collection in patients (12%) and CSF production rate in rats (20%), the latter obtained with the ventriculo-cisternal perfusion assay. The dark-phase increase in CSF secretion in rats was, in part, assigned to increased transport activity of the choroid plexus NaCSF secretion, and thus ICP, increases in the dark phase in humans and rats, irrespective of their diurnal/nocturnal activity preference, in part due to altered choroid plexus transport activity in the rat. Our findings suggest that CSF dynamics are modulated by the circadian rhythm, rather than merely sleep itself.The online version contains supplementary material available at 10.1186/s12987-023-00451-2. Our skull represents a fixed-volume compartment enclosing the brain tissue and the fluids that surround its cells and structures; the cerebrospinal and interstitial fluids (CSF and ISF) as well as the blood that supplies the brain with oxygen and nutrients. The Kellie–Monroe doctrine stipulates that due to the constant intracranial volume, the volumes of its components are in a dynamic equilibrium, meaning that an increase in one component is normally compensated by a decrease in another, in order to ensure a stable intracranial pressure (ICP) , 2. WhenSleep increases the interstitial space in rodent parenchyma and is permissive for cortical CSF tracer influx . The sleThus, we set out to quantify day-night ICP fluctuations and resolve the underlying CSF dynamics in humans and experimental rats. Our results reveal that CSF secretion increases in the dark phase, which may contribute to the elevated ICP observed in this phase. The dark phase increase in CSF dynamics occurred in both species, irrespective of their diurnal/nocturnal activity preference, and our findings thus indicate that CSF dynamics might not be sleep dependent as such, but rather modulated by the circadian rhythm.For the present study, patients who were admitted between 1 August, 2017 and 31 December, 2020 and who underwent placement of an external ventricular drain (EVD) and continuous measurement of ICP, could be included. From this group, 50 patient data sets were retrospectively and randomly selected from a patient group with various diagnoses of acute brain injury. Of these 50 data sets, 25 fulfilled the study criteria that (i) ICP measurements were available during both night and day in the same patient; (ii) CSF was drained against an unchanged counter-pressure (drain height) for at least four consecutive days, and (iii) CSF output measurements were stable (defined by less than 10% daily variation in the CSF collection). One of these patients was subsequently excluded since one ICP measurement was above 100 mmHg, which was considered out of normal patient range and potentially could reflect a detection failure. The patient data set employed for the analysis originated from 24 patients  with the following diagnoses: non-traumatic subarachnoid haemorrhage (n = 12), spontaneous intracerebral haemorrhage (n = 6), and traumatic brain injury (n = 6), who were mainly bedridden during the time of measurements, either in the supine position or with a 30–45° headrest elevation. At admission, the patients scored 9.0 ± 4.5 on the Glasgow Coma scale  and patiad libitum and were randomly assigned to experimental groups. For terminal procedures, rats were anaesthetized with 6 mg/ml xylazine + 60 mg/ml ketamine (ScanVet) in sterile water . When prolonged anesthesia was required (CSF secretion measurements), the animals received bolus ketamine injections at half the initial dose as required to sustain anesthesia. A tracheotomy was performed to control ventilation using the VentElite system (Harvard Apparatus) by 0.9 l/min humidified air mixed with 0.1 l/min O2 adjusted with approximately 3 ml per breath, 80 breath/min, a Positive End-Expiratory Pressure (PEEP) at 2 cm, and 10% sigh for a ~ 400 g rat. Telemetric pressure probe insertion procedures were performed using isoflurane  mixed with 1.8 l/min air / 0.1 l/min O2. Body temperature during all surgeries were maintained at 37°C by a homoeothermic monitoring system (Harvard Apparatus).Male Sprague-Dawley rats 9–11 weeks old, housed in temperature-controlled facilities with 12:12 h light cycle, were employed for the study. The rats had access to water and food Implantation was performed as described previously , 19 usin3−-containing artificial cerebrospinal fluid  120 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 25 NaHCO3, pH adjusted with 95% O2/5% CO2, 307 ± 1 mOsm) containing 1 mg/ml TRITC-dextran , was perfused at 9 µl/min. CSF was sampled at 5 min intervals from cisterna magna with a glass capillary  placed at a 5° angle . The cisterna magna puncture and associated continuous fluid sampling prevents elevation of the ICP during the procedure. The dilution of the infused solution is ascribed to endogenously secreted CSF, irrespective of origin of this fluid [p = ri × (Ci – Co/Co), where Vp = CSF production rate (µl/min), ri = infusion rate (µl/min), Ci = fluorescence of inflow solution, and Co = fluorescence of outflow solution. For the ventriculo-cisternal perfusion assay conducted during the dark phase, the anesthesia and all parts of the experiments were conducted either in the dark or with a red LED lamp for dark adaptive lighting (OcuScience), with the animals’ eyes completely covered following anesthesia, thus ensuring that the animals had minimal exposure to light. The CSF secretion rates were obtained approximately 8 h after initiation of the light/dark phase (zeitgeber time (ZT) 8/20 h).The CSF secretion rate was determined with the ventriculo-cisternal perfusion technique, with a total number of 12 animals used. One animal died during experimentation and was excluded alongside its light-dark phase matching counterpart. An infusion cannula  was stereotaxically placed in the right lateral ventricle of an anesthetized and ventilated rat , through which pre-heated  HCOis fluid : Vp = rith) were isolated in the light phase  and in the dark phase , the latter in the dark with only a red LED lamp for dark adaptive lighting (OcuScience) occasionally lit to ensure that light exposure was minimal during anesthesia and brain isolation. 12 animals were used for these experiments. The choroid plexuses were stored in RNAlater (Sigma) at − 80 °C prior to RNA extraction and library preparation (performed by Novogene Company Limited with NEB Next® Ultra™ RNA Library Prep Kit (NEB)) prior to RNA sequencing  on an Illumina NovaSeq 6000 (Illumina). Quality control and adapter removal were done by Novogene. The 150 base paired-end reads were mapped to rat reference genome Rnor_6.0.104 (Rattus norvegicus) using Spliced Transcripts Alignment to a Reference (STAR) RNA-seq aligner (v.2.7.9a) [https://github.com/Sorennorge/MacAulayLab_Nocturnal_rythem_RNAseq.Rat choroid plexus  , 23. The.2.7.9a) . Gene in.2.7.9a) . The cou2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 17 Na-HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.56, 37°C) for 10 min prior to initiation of the experiment. 86Rb+ influx: The experiments were initiated by placing the choroid plexus in HEPES-aCSF containing 1 µCi/ml 86Rb+  and 4 µCi/ml 3H-mannitol  per lateral choroid plexus. The choroid plexus was subsequently swiftly rinsed in cold isotope-free HEPES-aCSF containing 2 mM ouabain (4 °C) and transferred to a scintillation vial and dissolved in 100 µl Solvable . For the light and dark phase influx experiment, 12 animals were used (six either phase), but one dark phase lateral choroid plexus (ouabain) was lost during experimentation. 86Rb+ efflux: The experiments were initiated by placing the choroid plexus in HEPES-aCSF containing 1 µCi/ml 86Rb+ and 4 µCi/ml 3H-mannitol at 37°C for 10 min to allow isotope accumulation in the tissue. The choroid plexus was briefly washed (2 × 5 s) in 37°C HEPES-aCSF prior to transfer  to different HEPES-aCSF solutions (37°C) containing 20 µM bumetanide  or DMSO vehicle (0.05%) for a total of 40 s, choroid plexuses from each animal randomly allocated to each condition. The efflux medium from each of the solutions was transferred into separate scintillation vials, as was the choroid plexus, the latter dissolved in 100 µl Solvable . The 86Rb+ counts were corrected for 3H-mannitol counts (extracellular background), and the natural logarithm of the choroid plexus content At/A0 was plotted against time [86Rb+ efflux rate (min− 1) by linear regression analysis. For the light and dark phase efflux experiment, 12 animals were used (six either phase), but one dark phase lateral choroid plexus (bumetanide) was lost during experimentation. 500 µl of Ultima Gold™ XR scintillation liquid  was added to all scintillation vials and the radioactive content quantified in a Tri-Carb 2900TR Liquid Scintillation Analyzer (Packard).Rat lateral choroid plexuses were isolated in the light phase  and in the dark phase , the latter in the dark with only a red LED lamp for dark adaptive lighting (OcuScience) occasionally lit during anesthesia and brain isolation to ensure that light exposure was minimal. The choroid plexus was placed in HEPES-aCSF (containing (in mM) 120 NaCl, 2.5 KCl, 2.5 CaClr marker ) at 37°Cnst time  to obtait-test was conducted, otherwise either a Mann–Whitney (unpaired), or Wilcoxon (signed rank) (paired) test. RNAseq was evaluated with the Welch test. If more than two parameters were compared (isotope experiments), a one-way ANOVA was employed. Correlation analyses were performed with Pearson’s test (telemetric data) or Spearman’s test (patient data). All statistical tests are indicated in the figure legends.Data were analysed with GraphPad Prism 9.0 . All data are shown as mean ± SD and P < 0.05 was considered statistically significant. All data were tested for normal distribution using the Shapiro–Wilk test and data sets were tested for outliers (Grubbs’ test) when required, as indicated in figure legends. For normally distributed data, a one-sided or a two-tailed 2 = 0.05, P = 0.33).Determination of ICP relies on surgical placement of a pressure probe and hence cannot be readily conducted in healthy individuals. To that end, we enrolled patients admitted to the Neurointensive Unit at Copenhagen University Hospital – Rigshospitalet, Copenhagen, Denmark with conditions see "" sectionTo determine if the nightly elevation in ICP could be a functional consequence of altered CSF dynamics in the dark phase, we took advantage of the CSF collection via the EVD as a proxy read-out of CSF secretion. To ensure stable measurements for each patient, we included only patients with consecutive light and dark measurements with the same EVD resistance  and in the aorta . The av2 = 0.06, P = 0.65) or not  and dark phase (ZT 20 h), with the rats completely shielded from visible light both prior to the anesthesia induction and during the experimental procedure see "" section+-driven Cl−/HCO3− exchanger NCBE  and the electroneutral Na+,HCO3− cotransporter 1 NBCn1  and the dark phase (ZT 20 h), n = 5–6  and the dark phase (ZT 20 h). Quantification of transcripts coding for transporters involved in CSF secretion  Fig. 4AA4A, illu+/K+-ATPase and the NKCC1 [+/K+-ATPase activity was determined with an isotope uptake assay based on the K+ congener 86Rb+, which readily replaces K+ in the transport cycle  phase in anesthetized mice  and whenus awake  or contrus awake . Notablyus awake . HoweverLimitations to this study include employing a patient group rather than healthy control individuals. As ICP measurements and CSF collection are invasive procedures, these can only be conducted upon clinical indications. Thus, it was necessary to employ a patient group, which, in addition, remained near-horizontal for the time of measurements in order to avoid posture-related changes in ICP. One must therefore bear in mind that these processes may be different in healthy individuals. The patient group was characterized by acute brain injury; however, this term covers different pathophysiological processes depending on the underlying specific disease, although increased ICP is part of the final common pathway for all these diseases. Some of these patients may, in addition, suffer from vasogenic edema, which could contribute to the collected volume of CSF, although vasogenic edema is not expected to display diurnal variation. The clinical treatment was generally aimed at controlling ICP, but treatment guidelines did not depend on the time of day; in fact, it is likely that clinical management may have ‘buffered’ natural changes in ICP. Thus, both ICP and cerebral perfusion pressure were zealously monitored, and moderate or marked ICP increases were treated with targeted temperature management , higher doses of sedatives-analgesics, mild hypocapnia, and bolus doses of hypertonic saline. Furthermore, the cerebral perfusion pressure was continuously maintained within a narrow target range by adjustment of vasopressor therapy; thus any spontaneous mild changes in ICP would lead to an adjustment of the noradrenaline infusion rate to obtain a similar change in MAP and thus keep the cerebral perfusion pressure constant. The collected CSF was used as a surrogate marker of the CSF secretion rate; however, although the drainage pathways may well have retained (part of) their capacity in the given setting, they may not depend on time of day. Thus, we believe that day-night fluctuations are well reflected by the collected volumes. Another limitation to the study is the lack of information regarding sleeping patterns and phases, in addition to potential patient medication, which were measured neither in the patient cohort nor in rats. Regardless of sedation, sleep disturbances are prevalent and severe in generally critically ill patients , 51 and irrespective of nocturnal/diurnal activity preferences and bipedal/quadrupedal posture. The elevated ICP therefore does not appear to associate with sleep, but rather dictated by the circadian rhythm. This retained circadian pattern may stem from the highly conserved choroid plexus originating from a common ancestor with a predicted diurnal activity preference [In conclusion, our data demonstrate that CSF secretion is elevated in the dark phase in humans and rodents, in part due to elevated activity of a choroid plexus transporter. The altered CSF flow is suggested to serve as a mediator of the detected dark-phase ICP elevation in both species, eference . With theference –55. It iAdditional file 1: Figure S1. Cerebral perfusion pressure. Figure S2.86Rb+ uptake in excised rat lateral choroid plexus as a function of time. Figure S3. Correlation analysis of the nightly change in paired intracranial pressure and mean arterial pressure in patients  and freely behaving rats. Figure S4.  NKCC1 and Na+/K+-ATPase protein expression in choroid plexus acutely excised in the light and the dark phase.  Figure S5. Quantification of brain water content in brains excised during the lightand the darkphase.


When adjusted with donor BMI, pancreas donor risk index, and donor sex, receiving pasireotide translated into OR 0.49 (95% CI: 0.25–0.96 p = 0.037) for CCI ≥ 33.7. Pasireotide was independently associated with lower postoperative morbidity within 90 days of SPK compared to octreotide.In elective pancreatic surgery, somatostatin-analogues pasireotide and octreotide are variably used to reduce postoperative complications, but knowledge on their role in pancreas transplantation is limited. This study compared pasireotide and octreotide for their association with complications after simultaneous pancreas-kidney transplantation (SPK). This retrospective study included consecutive patients undergoing SPK’s from July 2013 to July 2022. Between July 2013 and April 2020, octreotide was administered 0.1 mg s.c. once daily and between May 2020 and July 2022 pasireotide was administered 0.9 mg twice daily, both until third postoperative day. Complications within 90 days postoperatively were collected, and reoperation rate and Comprehensive Complication index (CCI) ≥ 33.7  were used as primary outcomes. Of the 213 patients undergoing SPK, 150 patients received octreotide and 63 pasireotide. Baseline characteristics were comparable. Reoperation rate was 25.3% (  Simultaneous pancreas-kidney transplantation (SPK) offers superior survival over kidney transplantation only (or pancreas after kidney transplantation) to patients with type one diabetes T1D) and end-stage kidney disease (ESKD) D and end. It imprIn elective pancreatic surgery, somatostatin-analogue octreotide has been variably used to prevent pancreatic fistula, but, according to current best evidence, octreotide has no effect on reducing complications, including pancreatic fistula after pancreatic resection . OctreotPasireotide, another somatostatin-analogue with higher somatostatin receptor affinity and longer half-life, reduced the number of clinically significant pancreatic fistulas compared to placebo after pancreatic resections , as wellPerioperative octreotide had been routinely used at our center in all SPK since the beginning of our pancreas transplantation program in 2010. Our institutional policy was recently changed to substitute octreotide with pasireotide as of May 2020.The aim of this study was to compare octreotide and pasireotide for their association with postoperative morbidity after SPK, as well as to assess their association with early postoperative laboratory value trends.This was a retrospective cohort study comparing the association of pasireotide and octreotide with postoperative complications after SPK. Consecutive patients suffering from T1D and ESKD undergoing SPK’s at Helsinki University Hospital, Helsinki, Finland, between 8th July 2013 and 12th July 2022 were included in the study cohort. On 1st of May 2020, our institutional policy was changed from routine perioperative administration of octreotide to pasireotide. Patients undergoing SPK during 8th July 2013 and 30th April 2020 received octreotide 100 ug once daily starting at induction and up to at least 3rd postoperative day (POD). After 1st of May 2020, patients received pasireotide 900 ug twice daily starting at induction and up to the 3rd POD. All the grafts were from donors after brain death (DBD). Immunosuppression, surgical technique, and postoperative care remained similar throughout the study period, and have been described in detail elsewhere . InstituBasic patient and donor demographics were collected. Donor age, donor sex, donor BMI, donor height, donor reason of death, pancreas cold-ischeamia time (CIT), and donor ethnicity were used to calculate pancreas donor risk index (PDRI) for every patient . The PDRAll postoperative complications occurring before the 90th POD were collected retrospectively from electronic patient records and graded according to the Clavien-Dindo classification . The ComValues of postoperative laboratory tests reflecting pancreatic secretions and inflammation—plasma amylase, drain fluid amylase, and C-reactive protein (CRP) - on each morning up to 7th POD, and laboratory test values reflecting graft function—fasting c-peptide levels, estimated glomerular filtration ratio (eGFR) and HbA1c—up to 180th POD were collected. Plasma amylase values are reported as a multiplication of our institutional upper limit of normal (ULN) to allow for better comparability between centers using different assays. Trends of laboratory values stratified by the type of somatostatin were analyzed to assess for possible differences in exocrine/endocrine suppression. Some cases had missing laboratory test values and multiple imputation (with 10 iterations) was performed with basic patient demographics as dummy variables to account for these missing values. Multiply imputing missing values is associated with smaller bias than excluding cases with missing values , followed by pancreas graft associated complications [13/49 (26.5%)], and postoperative ileus [5/49 (10.2%)]. No significant differences were observed for these reasons of reoperation between the groups (The reoperation rate up to 90th POD was 25.3% (e groups . Four (1p = 0.009 (p = 0.015. Other studied outcomes were similar between the groups (The median (IQR) CCI was similar between the groups at both 30th and 90th POD timepoint with little difference between the timepoints demonstrating that most of the severe complications occurred during the first 30 postoperative days . The 90- = 0.009 . In addie groups .n = 80, 37.6%) are presented in p-values over 0.150 in univariable analyses with morbidity, and were omitted from the backwards stepwise logistic regression. According to the final multivariable analysis, the probability for CCI ≥ 33.7 was lower for patients receiving pasireotide when adjusted with the identified risk factors PDRI, donor BMI, and donor sex, 0.49 (95% CI: 0.25–0.96), p = 0.037. VIF -values for somatostatin, PDRI, donor BMI and donor sex in this multivariable model were 1.08, 1.15, 1.11, and 1.03, respectively, showing neglibile multicollinearity between the variables.Pre- and intraoperative risk factors for the morbidity of one reoperation  and spent on average 2 days less in the hospital. The shorter hospital stay could be confounded by an overall trend to shorter hospital stays over the years, as the patients in the pasireotide group were operated later during the study period. In addition, pancreas CIT was statistically significantly shorter in the pasireotide-group, but this finding did not translate into an association with morbidity. This may be explained by the fact that median CIT was relatively short in both cohorts , and previous studies have identified CIT exceeding 12 h to associate with heightened morbidity . When adThere are several limitations to our study. Use of octreotide was introduced at the beginning of our SPK-program in 2010, and it was adapted and modified from other existing protocols. Partly due to the lack of evidence supporting octreotide use and the promising results from the pancreatic surgery RCTs by Allen et al. in 2013 and Tarvainen et al. in 2020 our protocol was changed . This isPasireotide is safe to use for patients receiving SPK transplant and its use was independently associated with reduced severe complications up to 90 days post-transplantation. Further prospective randomized study in larger cohorts is warranted but may be difficult to carry out due to relatively large number of patients required for statistical power.


For better quantification of perfusion with arterial spin labeling (ASL), partial volume correction (PVC) is used to disentangle the signals from gray matter (GM) and white matter within any voxel. Based on physiological considerations, PVC algorithms typically assume zero signal in the cerebrospinal fluid (CSF). Recent measurements, however, have shown that CSF‐ASL signal can exceed 10% of GM signal, even when using recommended ASL labeling parameters. CSF signal is expected to particularly affect PVC results in the choroid plexus. This study aims to measure the impact of CSF signal on PVC perfusion measurements, and to investigate the potential use of PVC to retrieve pure CSF‐ASL signal for blood–CSF barrier characterization. In vivo imaging included six pCASL sequences with variable label duration and post‐labeling delay (PLD), and an eight‐echo 3D‐GRASE readout. A dataset was simulated to estimate the effect of CSF‐PVC with known ground‐truth parameters. Differences between the results of CSF‐PVC and non‐CSF‐PVC were estimated for regions of interest (ROIs) based on GM probability, and a separate ROI isolating the choroid plexus. In vivo, the suitability of PVC‐CSF signal as an estimate of pure CSF was investigated by comparing its time course with the long‐TE CSF signal. Results from both simulation and in vivo data indicated that including the CSF signal in PVC improves quantification of GM CBF by approximately 10%. In simulated data, this improvement was greater for multi‐PLD (model fitting) quantification than for single PLD (~1–5% difference). In the choroid plexus, the difference between CSF‐PVC and non‐CSF‐PVC was much larger, averaging around 30%. Long‐TE (pure) CSF signal could not be estimated from PVC CSF signal as it followed a different time course, indicating the presence of residual macrovascular signal in the PVC. The inclusion of CSF adds value to PVC for more accurate measurements of GM perfusion, and especially for quantification of perfusion in the choroid plexus and study of the glymphatic system. ASL signal in the CSF is non‐zero, and including this in partial volume correction leads to better estimates of GM CBF. Differences in GM signal between CSF‐PVC and non‐CSF‐PVC averaged 10% over all the GM, and 30–40% in the choroid plexus. CSF‐PVC is especially recommended for choroid plexus perfusion measurements. Consequently, any given voxel in an ASL image contains signal from a number of different tissue contributions, introducing bias to the resulting perfusion measurements in any ASL quantification method. For this reason, partial volume correction (PVC) algorithms have been proposed as a means to extract the pure perfusion parameters for gray and white matter tissue, separately.T2 CSF signal, making this sequence time consuming to acquire and infeasible in a clinical setting. Employing a PVC algorithm to retrieve CSF signal without the use of long echo times not only may allow for rapid assessment of the BCSFB in future studies, but also this technique could be applied retrospectively to already acquired data. Such considerations formed the impetus for this study, which has two primary aims:to assess the effect of CSF‐ASL signal on apparent GM cerebral blood flow estimates using PVC algorithms that previously neglected the contribution of CSF;to investigate the use of PVC to isolate the CSF‐ASL signal as an alternative to ultra‐long TE acquisitions.Moreover, the inclusion of CSF contributions in the PVC model may offer the opportunity to isolate the CSF signal and thus quantify water transport across the blood–CSF barrier (BCSFB) without modifications to existing ASL sequences. This would be especially useful as current CSF‐ASL measurements require ultra‐long echo times (TE) to isolate the long‐22.19 Arterial transit times (ATTs) were set to 1.0 and 1.2 s for GM and WM, respectively.T1 of blood was set to 1,650 ms,α, to 0.85. For the purposes of this simulation, the blood–brain partition coefficient, λ, was assumed to be 1, and the equilibrium magnetization of tissue, M0, was set to 1 . Pure WM ASL signal was computed for all label durations (LDs) and post‐labeling delays (PLDs) of the MRI experiment using the Buxton model,T2 of WM set to 60 ms.T1 and T2 of 4300 and 1500 ms, respectively)T2 of 100 ms).Tbl→CSF  centered around the average of 60 s as previously found in healthy volunteers.Tbl→CSF) to high/rapid exchange (short Tbl→CSF). To create simulated ASL signal maps, the pure GM, WM, and CSF signals are multiplied by standard tissue probability maps (TPMs) from SPM12  for the corresponding tissue/fluid type. These maps were then downsampled to a typical ASL resolution, from 1.5 mm isotropic to 3 × 3 × 6 mm3, to create partial volume (PV) maps. Finally, white Gaussian noise with an SNR of 20  was added. All simulations and data analysis were performed in MATLAB R2019b .To test the impact of CSF‐ASL signal on pure GM perfusion measurements, a simulated dataset was created as follows: GM and WM CBF were set to 60 and 20 mL/100 g/min respectively.2.2n ms (n = 0–7). For quantification, an M0 image  was acquired with TR = 10 s and TE = 10 ms to ensure maximum recovery of the longitudinal magnetization of tissue. A high resolution T1‐weighted (T1w) MP‐RAGE image was acquired  for the estimation of tissue PVs. The total length of the scan session was approximately 1 h.Thirteen healthy volunteers  were scanned after providing written informed consent following institutional review board regulations in accordance with the Declaration of Helsinki. All scans were performed on a Philips Achieva XT 3 T system . The multi‐echo pCASL sequence is the same as the one we employed in our previous publication,2.3The image analysis pipeline described below is shown schematically in Figure 2.3.11w image (high resolution) was segmented using SPM (six tissue types) to compute the probabilities for GM, WM, and CSF in every voxel, which were employed as estimates for the PV. Three tissue masks were created from these maps with a tissue probability threshold of more than 30%; these masks were combined into one intracranial mask , to remove any background signal and the skull from the T1w image. This “de‐skulled” image was coregistered to the M0 scan to transform it to the ASL space . Subsequently, the same transformation matrix was applied to the TPMs for GM, WM, and CSF, resulting in PV estimates for each tissue in every voxel in ASL space as input for the PVC algorithm.ASL control and label images were pair‐wise subtracted to extract the ASL signal at all LD/PLDs and TE values. The T2.3.2σ = 2 voxels before any data processing. Since the original PVC‐algorithm does not include a macrovascular component, and neither does the simulated dataset, we opted to exclude macrovascular signal from in vivo data as much as possible by fitting the functionMBuxton from the Buxton perfusion model.M0 values used for this and subsequent analyses were an average over the GM  divided by the blood–brain partition coefficient λ = 0.98 mL/g.1b represents the T1 relaxation time of arterial blood (set to 1650 ms).The in vivo ASL images were smoothed with a Gaussian kernel of 3 × 3 voxels and 2.3.3ri is a sum of contributions from individual tissue types: ∆Mri=∑j=13Pjri·∆mjri, where Pj is a row vector containing the tissue type fractions of this voxel  as taken from the PVs, and ∆mj(ri) is a column vector of the signal intensities for each tissue type. This makes an underdetermined system with three unknowns  and only one measurement. To solve this, we assume that ∆mj is constant over a small kernel of n × n voxels (in plane) centered at ri (represented as ∆mjri¯j) and the equation becomes ∆Mrk=∑j=13Pkj·∆mjri¯j, with ∆Mrk a column vector containing the n2 signal values for all voxels within the kernel, and Pkj an n2 × 3 matrix with the tissue probabilities for the three tissue types in each voxel in the kernel. For a kernel of 5 × 5 voxels in plane, this results in a system of 25 equations for three unknowns, with the solutionThe linear regression PVC algorithm has been described in detail elsewhere.2.3.4To compare the results of PVC with and without the inclusion of CSF, we focused on the effect on GM CBF (as it borders with CSF and is the most relevant and reported ASL perfusion parameter). In the simulation dataset, the error on the GM signal was obtained by computing the relative difference (as a percentage) between the PV‐corrected GM signal and the ground truth. This was done for PVC with (CSF‐PVC) and without CSF (non‐CSF‐PVC), where the CSF signal is assumed to be zero. In the case of the in vivo data, no ground truth is known, so the relative difference between CSF‐PVC and non‐CSF‐PVC was calculated in lieu of the error. The PVC‐CBF was computed using both single PLD and multiple LD/PLD data: for single PLD, the data with LD/PLD = 2/2 s was employed with the equation found in Reference prob = 0–10%, the second GMprob = 10–20%, etc. Since we are interested specifically in the interaction between GM and CSF, all voxels that contained more than 30% WM were eliminated from these ROIs.To estimate the size of the effect of CSF signal as a function of GM probability, regions of interest (ROIs) were created from the GM TPM for bins of 10% GM probability: i.e., the first ROI contains all voxels with GMWe also studied the effects of PVC in vivo in the choroid plexus specifically. An ROI was created by delineating the posterior part of the lateral ventricles in three slices where the choroid plexus is located, starting from a CSF mask with a low PV threshold of 30%. This ROI therefore contains both the choroid plexus itself and a margin of CSF that immediately surrounds it.Finally, to determine whether CSF signal estimation from PVC could act as a surrogate for long‐TE acquisitions to isolate pure CSF signal, PVC‐CSF signal was averaged over an ROI of the subarachnoid space for each LD/PLD pair, and similarly the long‐TE signal was averaged over the same ROI . The time courses of these signals were plotted for comparison.33.1The simulated data are shown in Figure Tbl→CSF is shortest. When CSF is included in the PVC (middle), the error on the GM signal is largely resolved. The CSF signal error (right) is large at early time points, and as we move toward longer time points the signal is first strongly affected by noise levels, then becomes more stable, and the error is finally shown to be small and centered around zero.The signal maps from the simulated data were subsequently processed with both non‐CSF‐PVC and CSF‐PVC. The relative error on the resulting GM signal compared with ground truth is shown in Figure prob ROI is shown for a single LD/PLD of 2/2 s (closest to the recommended pCASL settings used in the clinic for perfusion imaging) and all Tbl→CSF values. Without CSF correction , the error consistently exceeds the error when CSF is included , but the difference decreases with increasing GMprob, and for voxels with 90% GM or more the results are similar, with the error approaching zero. CSF‐PVC yielded results that were very similar for all Tbl→CSF values and LDs/PLDs; therefore, we show only one curve instead of several overlapping curves. In Figure Tbl→CSF value of 60 s  and all time points. For the two earliest time points, there is no improvement in using CSF‐PVC, as the low CSF signal only introduces additional error due to the presence of noise in the absence of signal. For LD/PLD = 1.5/1.5 s, the effect of using CSF‐PVC is mixed, as only voxels with less than 50% GM see an improvement in GM signal estimation. For LD/PLD = 2/2 s and higher, the reduction in error from using CSF‐PVC is substantial and reaches up to 100%.To average and compare this error, voxels were binned according to their GM probabilities. These ROIs are shown in Figure Next, CBF was quantified from the PV‐corrected GM signal, first by using the single PLD equation given in the ASL white paper3.2Figure prob . For direct comparison of the effect of CSF‐PVC in the choroid plexus versus in the GM, Figure In Figure In Figure 4In this paper, we have investigated the effect of CSF signal on ASL PVC, used to extract pure perfusion parameters for given tissue types, and compared the real CSF signal time course from long‐TE acquisitions in vivo with the CSF signal extracted using PVC. Our main conclusions are that ASL signal in the CSF may lead to errors in GM‐CBF estimates of approximately 10% when CSF signal is neglected in PVC, and that CSF‐PVC cannot be used as an alternative to long‐TE ASL experiments for measuring water transport across the BCSFB from traditional ASL scans. The origin of these two seemingly conflicting conclusions will be the main topic of this discussion.Tbl→CSF exchange times. These results are to be expected, as when CSF is excluded from PVC the signal originating from this compartment is labeled as either GM or WM signal, and because of the close proximity of GM and CSF in the brain this will largely fall into the GM compartment. This effect will be most severe when more labeled spins are present in the CSF—i.e., for larger CSF PV, longer LD/PLD, and shorter Tbl→CSF (faster exchange of water from blood to CSF). This leads to systematic overestimation of pure GM signal in most GM‐containing voxels, which is largely corrected by the inclusion of CSF in PVC. For the clinical ASL parameters of LD/PLD = 2/2 s, the error is consistently lowered in CSF‐PVC for voxels with 90% GM or less. For earlier time points, this improvement is limited, especially due to the fact that the CSF‐ASL signal becomes small compared with the noise, and for longer time points it is increased. This is particularly visible in Figure Tbl→CSF.First, using simulated and in vivo data, we show that errors on pure perfusion parameters are reduced when CSF is included in the PVC algorithm. In simulated data, the error on the pure GM signal Figures  and 4 isIn vivo, the difference between non‐CSF‐PVC and CSF‐PVC GM signal exceeds 10% in most voxels Figure , which iT2 of CSF. This would also have the added advantage that CSF signal and thus water transport measurements across the BCSFB could be extracted retroactively from existing ASL data, potentially providing more insight into brain clearance mechanisms. To determine this, we compared the time course of the CSF signal as measured in long‐TE ASL  becoming entangled as a result. Nevertheless, both of these contributions should not be attributed to GM when performing CBF quantification, and PVC should benefit from the inclusion of these components. Therefore, the CSF‐PVC approach remains optimal, even though it does not limit its correction to CSF signal. This statement is also supported by simulation studies showing that the CSF‐PVC approach results in improved GM‐CBF measurements in the absence of macrovascular signal, and that in this case the CSF signal is also successfully isolated.Moreover, our results show that the choroid plexus is particularly sensitive to PVC and that the inclusion of CSF signal in this correction will be crucial when measuring perfusion in this small brain region. As there is growing interest in brain clearance mechanisms, and specifically in CBF measurements in the choroid plexus, we believe that the CSF‐PVC method adds value and removes some bias from these measurements. This is true especially when looking at the glymphatic system in the aging brain. While it has been shown that CSF and perivascular space volume increase with age,prob differed between the simulation , which does not allow for large‐scale movement of the ASL signal to spread through the CSF compartment by diffusion or flow. As we have already discussed, the simulation also did not include macrovascular blood signal, while the in vivo data did. This explains the surprising result that the orders of magnitude (~10%) of both the effect of CSF signal in PVC in simulations and the difference between CSF‐PVC and non‐CSF‐PVC in vivo are so similar, when we show that these do not represent the same signal . In reality, our simulation overestimates the effect of CSF signal, because simulated maps assume that CSF‐ASL signal appears everywhere that CSF is present, while in vivo CSF signal only appears where exchange of water from blood to CSF occurs. Therefore, the overestimation of CSF signal in simulations appears to be of the same order of magnitude as the overestimation due to macrovascular contamination in vivo. Additionally, our simulation is a simplified model where only GM, WM, and CSF signals are present, so naturally the PVC algorithm, which also assumes only three compartments, retrieves ground‐truth values with greater accuracy than it would in vivo, where other sources of signal and artefacts may be present. Finally, we noticed that the ROIs based on GMn Figure  and in vn Figure . In the In the scope of this study, we investigated the effects of CSF only on linear regression PVC algorithms; however, other more complex versions of PVC have also been developed—for example, based on information from multi‐PLD acquisitions and statistical models5Including CSF in PVC leads to an improvement in pure GM signal estimation of approximately 10%, with larger differences, reaching up to 40%, observed in the choroid plexus. This improvement carries over to multi‐PLD CBF quantification; however, for single‐PLD CBF competing effects lead to more limited gains from CSF‐PVC. We also show that PVC CSF signal is not a substitute for long‐TE CSF signal, as it suffers from macrovascular blood signal contamination. We would therefore recommend the use of CSF‐PVC, especially when measuring perfusion in the choroid plexus.


IFNL4) gene, thus preventing the expression of a functional IFN-λ4 protein. While probing the expression of IFN-λ4 in human peripheral blood mononuclear cells (PBMCs), using a monoclonal antibody that binds to the C-terminus of IFN-λ4, surprisingly, we observed that PBMCs obtained from TT/TT genotype individuals could also express proteins that reacted with the IFN-λ4-specific antibody. We confirmed that these products did not emanate from the IFNL4 paralog, IF1IC2 gene. Using cell lines and overexpressing human IFNL4 gene constructs, we obtained evidence from Western blots to show that the TT allele could express a protein that reacted with the IFN-λ4 C-terminal-specific antibody. It had a molecular weight similar if not identical to IFN-λ4 expressed from the ΔG allele. Furthermore, the same start and stop codons used by the ΔG allele were used to express the novel isoform from the TT allele suggesting that a restoration of the ORF had occurred in the body of the mRNA. However, this TT allele isoform did not induce any IFN-stimulated gene expression. Our data do not support a ribosomal frameshift that leads to the expression of this new isoform, implying that an alternate splicing event may be responsible. An N-terminal-specific monoclonal antibody did not react with the novel protein isoform suggesting that the alternate splicing event likely occurs beyond exon 2. The new isoform is glycosylated similar to the functional IFN-λ4 and is also secreted. Furthermore, we show that the ΔG allele can also potentially express a similarly frameshifted isoform. The splicing event that leads to the generation of these novel isoforms and their functional significance remains to be elucidated.The TT allele of the dinucleotide variant rs368234815 (TT/ΔG) abolishes the open reading frame (ORF) created by the ancestral ΔG allele of the human interferon lambda 4 ( IFNL4 by Prokunina-Olsson and others showed that the TT allele abolishes the ORF, but can express inactive isoforms such as p124 and p143 that are unrelated to IFN-λ4  is a newly discovered type III IFN that has been associated with hepatitis C virus and other infections  homozygous individual was cloned in pcDNA3.1+ (Invitrogen) by using primers listed in IF1IC2, a 1.5 kb gene [from start to stop codon (including the premature stop codon) with all exons and introns] was similarly cloned from human genomic DNA by using the primers listed in A full-length (exons and introns only) 1.4 kb All clones were confirmed by Sanger sequencing of the plasmid DNA. The wild-type (wt) IFN-λ4 cDNA clone, pIFNL4-Halo, was a gift from Ludmila Prokunina-Olsson (NIH), while the codon optimized IFN-λ4 cDNA was commercially synthesized . The TT mutation-addition was introduced into the wt cDNA clones using SDM. Green fluorescent protein  was cloned in either pCDNA+ vector with or without the ATG start codon. GFP without the ATG start codon was also cloned immediately before the TGA stop codon of TT (in pCDNA+) along with a GGGS linker sequence to express a fusion product with the protein translated from the TT allele.Human genomic DNA was extracted from whole blood using a QIAamp Blood DNA Mini Kit (Qiagen). PCR was used to amplify a 1.4 kb amplicon from the genomic DNA, using the primers listed in 2. Cells were seeded into 6-well [for Western blot (WB)] or 12-well (for qPCR) plates and allowed to reach a 70%–80% confluency (∼24 h). Cells were transfected with pcDNA3.1 or pIF1IC2, TT or ΔG, or the cDNA constructs with Lipofectamine LTX and Plus (Invitrogen). Unless mentioned otherwise, 2 μg/well of the constructs was transfected for WB experiments. Twelve hours (for qPCR) or 24 h (for WB) post-transfection, cells were collected and processed for WB/qPCR.HEK293, C33A  , and Huh7.5  cells were maintained in Dulbecco's modified Eagle's medium (Himedia) in addition to 10% FBS and penicillin/streptomycin  at 37°C and 5% COFor fluorescence experiments, HEK293 cells seeded in clear bottom white plates  were transfected with 100 ng of the different constructs (in pCDNA+) at near confluency and the fluorescence intensity was read after 24 h in the EnSight multimode plate reader (PerkinElmer) using Kaleido data acquisition and analysis program.2. After 5 h, cells were either treated or not for 24 h with phorbol 12-myristate 13-acetate , polyI:C , and lipopolysaccharides . After 24 h, cells were collected and processed for WB.The NIBMG Bioethics Committee approved this study with the certificate No. NIBMG/2022/1/0018. Five milliliters of blood was drawn from volunteers after obtaining informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll density gradient method (Sigma). Isolated PBMCs were plated or not (fresh PBMCs) in 6-well plates with RPMI Medium-1640 (Gibco) along with 10% FBS and incubated at 37°C and 5% COIFNL3 gene : MX1, OAS1, ISG15, and IFITM3 were measured using SYBR Green after amplifying the cDNA using the primers described previously . cDNA was synthesized from 1 μg of RNA using the Verso cDNA Synthesis Kit (Thermo Scientific). qPCR was performed in the QuantStudio 5 (Applied Biosystems) using custom-made isoform-specific IFNL4 TaqMan probes and primers as described previously for the p179, p131, and p107 isoforms, and for the eviously . GAPDH wThe proteins were run on 15% SDS-PAGE gels, and WB analysis of human recombinant IFN-λ4 was carried out using the rabbit anti-IFN-λ4  or mouse anti-IFN-λ4  or anti-HA . Following overnight treatment with the primary antibody, the blots were developed with a secondary HRP-linked goat anti-rabbit antibody  or goat anti-mouse HRP-conjugated secondary antibody . For beta-actin (Santa Cruz Biotechnology), primary antibodies at 1:10,000 dilution were used. The blots were developed and imaged in ChemiDoc MP imaging system (Bio-Rad).Deglycosylation was done by using PNGase F . For deglycosylation, 9 μL of cell lysate along with 1 μL of 10 × denaturing buffer was heated at 100°C for 10 min. After denaturation, 2 μL of glyco buffer, 2 μL of 10% NP-40, 1 μL of PNGase F, and 5 μL of deionized water were added. This mixture was incubated at 37°C overnight and then proceeded with WB.Trichloroacetic acid was added to the clarified media in 1:4 ratio and incubated at 4°C for 30 min. The sample was centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was decanted, and the precipitated protein pellet was washed thrice with 200 μL of cold acetone by centrifugation at 14,000 rpm for 5 min at 4°C. Pellet was dried at 95°C for 5 min. Lysis buffer was added to the pellet and proceeded to WB.In the beginning of this study, we were interested to look for the expression of IFN-λ4 in human PBMCs. For the initial experiment, we identified two individuals who were homozygous for the major (TT) and minor (ΔG) alleles  at rs368After incubation, the cells were lysed and probed in WB with a rabbit monoclonal antibody (RAB) that is raised against the C-terminus of IFN-λ4, and that reacts only against the human IFN-λ4 protein by recognizing amino acids 128–152  and of IFNL3 in both freshly isolated PBMCs (without plating) and PBMCs that were cultured and treated, as in IFNL4 isoforms were used  above, we subjected the fresh PBMC lysates from all three genotype individuals to WBs using a mouse monoclonal antibody (MAB) that binds to the N-terminus of IFN-λ4  above, when we queried, we saw that  Premzl,  has spec Premzl, , leading Premzl,  that couIF1IC2 gene in a pcDNA3.1+ (Invitrogen) expression vector; similarly, we constructed another clone, pIFNL4 (TT) (referred to as TT hereafter), that contained the full-length IFNL4 gene  obtained from a TT/TT homozygous individual. We used two different well-characterized  construct to the ΔG allele (referred to as ΔG hereafter) and repeated the experiment along with the TT allele construct . Only thWe simultaneously ran products from a PBMC sample obtained from a TT/TT individual along with the products from overexpressed TT or ΔG constructs in HEK293 cells and saw that at least some of the bands expressed from the ΔG construct moved at similar positions to that from the TT/TT PBMC sample . We alsoInterestingly, these HMW products reacted with the MAB, similar to the results that we saw from PBMC experiments  and S3B.IFN-λ4 is known to be heavily glycosylated (Chen et al., IFNL4 so that the GFP could be expressed only as a fusion protein along with the product of the TT allele (The results show that the novel TT allele isoform cannot express an RAB reacting protein if the start codon is mutated, and it also produces an HA tag suggesting the translation of an in-frame protein beyond exons 4 and 5 (T allele  bottom. T allele .Furthermore, the novel isoform is secreted more efficiently than the products of the ΔG allele in C33A cells, but is secreted to a similar extent in Huh7.5 cells .IFNL4 TT allele that has a restored reading frame beyond the dinucleotide DNA polymorphism rs368234815. Restoration of the reading frame could be due to a ribosomal frameshift, or it could be due to a hitherto unreported splicing event in the body of the mRNA that leads to the reading frame restoration. To address both possibilities, we introduced the TT mutation-addition to the ΔG allele in a p179 expressing cDNA construct. We used two different cDNA constructs, one the wt and another a codon optimized version. However, no RAB reactive products were produced in either of the TT allele carrying versions of the overexpressing cDNA constructs (The above results clearly indicate that a novel protein is produced from the nstructs . This woTo understand the origin of the novel TT allele isoform, we introduced frame changing mutations next to the start codon and subjected the overexpressed products to WBs using the RAB . These i+” protein from the TT allele also raises the possibility of a similar event happening with the ΔG allele (isoform referred to as p123+; However, our results from a + 1 frame-shifted ΔG allele construct (IFNL4 gene regardless of the alleles present at rs368234815, leading to the generation of novel transcripts. PCR and qPCR products generated with cDNA from TT and ΔG allele overexpressed cells were indistinguishable between the two alleles (This hypothesis could be tested to see if an RAB reacting product is generated from onstruct ; indeed,onstruct . These r alleles ; sequenc alleles , table; IFNL4 gene, were identified by RNA-seq and the subsequent molecular analysis (Prokunina-Olsson et al., At least 10 mRNA isoforms, 5 each from the ΔG and TT alleles of human We began this study with an aim to examine the expression of IFN-λ4 in human PBMCs . Our obsIFNL4 mRNA is the most expressed IFN mRNA in bone marrow stem cells of healthy individuals (Gadalla et al., A recent report has shown that