Increasing evidence has associated positive fluid balance of critically ill patients with poor outcomes. The aim of this study was to explore the pattern of daily fluid balances and their association with outcomes in critically ill children with lower respiratory tract viral infection.A retrospective single-center study was conducted, in children supported with high-flow nasal cannula, non-invasive ventilation, or invasive ventilation. Median (interquartile range) daily fluid balances, cumulative fluid overload (FO) and peak FO variation, indexed as the % of admission body weight, over the first week of Pediatric Intensive Care Unit admission, and their association with the duration of respiratory support were assessed.p = 0.001). Median cumulative FO% was 4.6 (− 0.8 to 11) and peak FO% was 5.7 (1.9–12.4). Daily fluid balances, once patients were stratified according to the respiratory support, were significantly lower in those requiring mechanical ventilation (p = 0.003). No correlation was found between all examined fluid balances and respiratory support duration or oxygen saturation, even after subgroup analysis of patients with invasive mechanical ventilation, or respiratory comorbidities, or bacterial coinfection, or of patients under 1 year old.Overall, 94 patients with a median age of 6.9 (1.9–18) months, and a respiratory support duration of 4 (2–7) days, showed a median (interquartile range) daily fluid balance of 18 (4.5–19.5) ml/kg at day 1, which decreased up to day 3 to 5.9 (− 14 to 24.9) ml/kg and increased to 13 (− 11 to 29.9) ml/kg at day 7 (In a cohort of children with bronchiolitis, fluid balance was not associated with duration of respiratory support or other parameters of pulmonary function.The online version contains supplementary material available at 10.1186/s44158-023-00093-8. Positive fluid balance has been shown to impact respiratory function, with fluid overload (FO) > 10–20% negatively affecting alveolar gas exchange, especially in patients with lung inflammation and in pediatric acute respiratory distress (PARDS) . BronchiThe aim of this study was to explore patterns of daily fluid balances in a cohort of children with bronchiolitis admitted to our PICU. We also assessed the association of daily fluid balance, cumulative FO, and peak FO in the first 7 days of PICU admission with ventilatory support duration and respiratory function. The characteristics of fluid balances were also compared in two different time periods, before and after the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) pandemic.An observational cohort study was conducted including patients with bronchiolitis admitted to Meyer Children’s Hospital PICU from September 2019 to January 2022. We included all patients under 4 years of age with a diagnosis of acute lower respiratory tract viral disease. We excluded patients whose primary admission diagnosis was not due to a respiratory condition and those who were receiving chronic ventilatory support  before PICU admission. The decision to extend the generally accepted age for bronchiolitis  was taken owing to the unusual wave of older children admitted in 2021–2022 with clear clinical symptoms of bronchiolitis and nasal swab viral positivity . The rep2), inspiratory oxygen fraction (FiO2)), the worst daily blood gas analysis results (when available), morning laboratory data , type of ventilatory support , including fraction of inspired oxygen. For those who required ventilatory support for more than 7 days, the last day of ventilatory support was recorded. Duration of respiratory support was registered as the number of days when any support was needed, regardless of the modality. In all cases, the length of PICU stay was registered. Patients were categorized according to the most invasive support they had received during the first week of PICU admission. Isolated viruses were reported for all patients, and further pathogens were reported if coinfections occurred.Demographic information, significant clinical history, admission bronchiolitis severity score (BSS), and pediatric index of mortality 3 (PIM3) were gathered from the electronic clinical chart. Clinical data were collected in the first 7 days of PICU admission: the worst daily vital parameters : all fluid intakes  and fluid outputs  were considered for daily fluid balances. Insensible losses were calculated as 1 ml/kg/h in patients below 10 kg and 0.5 ml/kg/h in others. The cumulative fluid balance was calculated as the sum of daily fluid balances over the first 7 days of PICU admission. Cumulative FO was calculated as the percentage of cumulative fluid balance indexed over admission body weight, and peak FO% as the highest positive fluid balance reached during the first 7 days of stay in PICU, indexed over admission body weight. Diuretics were administered as needed, typically as single boluses, when the clinicians identified a reduced diuresis or an excessively positive fluid balance. This kind of prescription was not standardized.2 0.3–0.6) or full-face mask NIV , and eventually intubation if clinical signs and symptoms (mainly oxygen saturation and respiratory pattern) did not show improvement. Patients could also be centralized from peripheral hospitals, and in such cases, transportation was generally managed with invasive ventilation . In these cases, intubation occurred a few hours before transportation to our unit.The indication and timing for PICU admission in our unit are based on a combined assessment of BSS (in case of a score above 4), and clinical evaluation . Once in PICU, respiratory support was escalated from HFNC (2 l/min/kg and FiO2 0.3–0.5) to helmet CPAP (positive end-expiratory pressure (PEEP) 6–10, FiO2 was also analyzed. Furthermore, we compared patients admitted before and after the SARS-CoV2 pandemic, who apparently had different disease severity, in order to assess if any difference existed in the two populations in terms of fluid balance and associations with respiratory support duration.The primary objective of this study was to assess daily fluid balances in children with bronchiolitis admitted to PICU. Secondary analyses included the association of daily fluid balances, cumulative FO, and peak FO with respiratory support duration. The association of daily fluid balances with worst daily SpOp value of < 0.05 was considered statistically significant. Statistical analysis was performed with the GraphPad Prism 9.0 software package .All data are presented as median (interquartile range). Mann–Whitney test was used to assess the differences between variables. Pearson correlation was applied to verify the association between continuous variables. One-way analysis of variance, two-way analysis of variance, and mixed-effects analysis were applied to analyze the differences over time between continuous variables. Tukey’s multiple comparisons post hoc test was applied to assess differences between groups at single time points. A Overall, 103 patients with lower respiratory tract viral infection were admitted to the PICU in the selected periods. Of these, 6 patients were excluded as they were over 4 years old, and in 3 cases respiratory insufficiency was not the primary PICU admission diagnosis. Ultimately, 94 children were admitted in 2 different time periods: 29 between September 2019 and February 2021 and 65 between September 2021 and February 2022.Median weight, age, isolated pathogens, administration of diuretic therapy, and severity scores are depicted in Table In regard to the highest level of respiratory support during the PICU stay, 9 patients received HFNC, 59 children were assisted by CPAP with helmet or by NIV with mask, and in 26 cases mechanical ventilation was needed .2, SpO2), hemodynamic , laboratory and urine output parameters at admission, and their variation over time are described in Supplementary Table Sr − 0.5, p = 0.002). The median duration of PICU stay was 6 (4–10) days, and hospital stay was 11 (8–17) days. No patient died.Respiratory   ml/kg at day 1; it decreased to 5.9 (− 14–24.9) ml/kg on day 3 and increased thereafter to 13 (− 11–29.9) ml/kg on day 7 (p = 0.003)  Fig. B.p = 0.27 at mixed effect analysis).We did not find any correlation between respiratory support duration and daily fluid balances, cumulative FO, and peak FO Table . The medSimilarly, oxygen saturation was not associated in the first 7 days with daily fluid balances Table .In subgroup analysis, after inclusion of patients requiring invasive mechanical ventilation, or patients with respiratory comorbidities, or patients with bacterial coinfection, or patients under 1 year old, we still could not find any correlation between fluid balance and respiratory support duration (Supplementary Table Sp = 0.0002), as well as weights (p = 0.03) and ages (p = 0.01), whereas PIM3 was higher (p = 0.001) (Table p = 0.72) were not different in the two periods and their correlation with daily fluid balances was absent in both eras   were compared and about a double number of patients were admitted in the second period. Fluid balances over time were significantly lower in the first era  have been admitted with lower respiratory tract viral infection compared to the previous period. The number of these admissions to the PICU was two-fold in 2021–2022 with respect to previous years. This could be due to the “immunity debt” caused by the strict non-pharmacologic interventions against SARS-CoV2, which led to a substantial reduction in typical seasonal viruses among infants and children, who did not develop any kind of immunity against these pathogens , 27–32. 2/FiO2 because of several missing data . The lack of association between fluid balances and respiratory support duration in our study may be due to a low signal, since median FO and median peak FO were below the “recognized” threshold of danger. However, our results may imply that managing fluids within such threshold is safe. Moreover, even the subgroup of patients with the highest positive fluid balances  did not show worse outcomes. In other words, our study might contribute to show that in children with bronchiolitis this threshold (10%) does not affect lung function. Respiratory support duration  may be a suboptimal outcome and can be influenced by attending clinicians’ personal evaluations. However, we estimated that PICU and hospital stay were also influenced by many factors, including logistical requirements . Fluid balance before PICU and hospital admission could not be assessed. This issue should be more deeply investigated, especially in infants, as bronchiolitis could be associated with difficulty in feeding and with dehydration. Fluid administration beyond maintenance fluids did not follow any goal-directed protocol but was administered to optimize suspected hypovolemia . This aspect may significantly affect the amount of input fluids. As a matter of fact, our patients were generally hemodynamically stable with healthy cardiac function and prompt response to fluid boluses. For this reason, our center tended to apply a relatively restrictive fluid management. How to standardize the calculation of inputs and outputs remains unclear. In accordance with Ingelse et al. and Flores-Gonzalez et al., in our study the contribution of enteral feeding to daily input was considered exactly as parenteral input [Our retrospective study has several limitations. We did not attempt to associate the duration of respiratory support with PaOal input , 24, whial input . Fluid bIn a cohort of children with bronchiolitis admitted to PICU for respiratory insufficiency, fluid balance was positive at day 1, it decreased on day 3 and increased thereafter with a median peak FO of about 5%. Fluid balance was not associated with respiratory support duration or other parameters of pulmonary function regardless of age, type of respiratory support, comorbidities, or the presence of bacterial coinfection. Further standardization of fluid balance assessment is needed to ultimately clarify whether fluid management has an impact on outcomes of children with lower respiratory tract viral infection.Additional file 1: Supplementary Table S1. Laboratory, hemodynamic, respiratory variables in the studied population from day 0 to day 7. Supplementary Table S2. Correlation of different subgroups with fluid balance from day 1 to day 7.


Medauroidea extradentata using cryo-scanning electron microscopy (cryo-SEM) and white light interferometry (WLI). The secretion was investigated with cryo-SEM, revealing four morphologically distinguishable components. The 3D WLI measurements of the droplet shapes and volumes over time revealed distinctly different evaporation rates for different types of droplets. Our results indicate that the subfunctionalization of the tarsal secretion is facilitated by morphologically distinct components, which are likely a result of different proportions of components within the emulsion. Understanding these components and their functions may aid in gaining insights for developing adaptive and multifunctional biomimetic adhesive systems.Attachment to the substrate is an important phenomenon that determines the survival of many organisms. Most insects utilize wet adhesion to support attachment, which is characterized by fluids that are secreted into the interface between the tarsus and the substrates. Previous research has investigated the composition and function of tarsal secretions of different insect groups, showing that the secretions are likely viscous emulsions that contribute to attachment by generating capillary and viscous adhesion, leveling surface roughness and providing self-cleaning of the adhesive systems. Details of the structural organization of these secretions are, however, largely unknown. Here, we analyzed footprints originating from the arolium and euplantulae of the stick insect  Attachment to the substrate is an important phenomenon influencing the everyday lives of most insects, as it is used to accomplish different tasks, such as locomotion , resistiTo generate attachment, most insect tarsi utilize adhesion supported by lateral shear (friction) . Two typHydra spp.) can achieve temporary adhesion based on their adhesive secretion, which consists mainly of fibers [Besides insects, other animal groups also produce a fluid onto the substrate to support the adhesion process ,15,16. Hf fibers . The fluf fibers .Gromphadorhina portentosa (Blattodea) revealed that the tarsal secretion is produced by exocrine cells into a storage volume underneath the outer part of the endocuticle. It is then released through channels onto the substrate [Locusta migratoria (Orthoptera) is a highly viscous emulsion consisting of lipid droplets in a water-like solution [A histological investigation of the smooth tarsal adhesive organs of ubstrate ,26. The ubstrate ,33,34,35ubstrate , Hymenopubstrate ,38, Coleubstrate ,44,45,46ubstrate ,47, and ubstrate ,34,42,47solution . The ide(I)Coccinella septempunctata and the fly Calliphora vicina indicated different viscosities of 21.8 and 10.9 mPa × s, respectively, showing that the physical properties of the fluid diverge between the species [It can increase the adhesion to a broad range of substrates. Experiments on the attachment performance of Phasmatodea  and Cole species . The pre species ,7. Addit species .(II)Carausius morosus with polystyrene beads and manipulating the amount of adhesive fluid showed that a high amount of fluid led to a faster recovery rate of adhesion than a low fluid amount. Thus, it is an important part of the self-cleaning mechanism of smooth adhesive pads [It contributes to decontamination. Contaminating the adhesive pads of the stick insect ive pads ,31.(III)It can compensate for different surface chemistry of substrates. Chemical analyses of the fluids allow for the interpretation of the interaction with different surfaces. Due to the presence of two phases (water-soluble and lipid-soluble phases), the emulsion should improve the attachment to hydrophilic and hydrophobic surfaces, as it acts as a coupling agent between the pad and substrates with different free surface energies ,28,52,55Experiments investigating the functions of tarsal secretions in several insect groups demonstrated that the fluid could support three main functions as follows:Tettigonia viridissima (Orthoptera) revealed that the fluid within the pad contributes to its viscoelastic behavior and the frictional forces subjected to the substrate [Some of the components of the tarsal secretion resemble those found on the surface of the insect cuticle, potentially helping to reduce the evaporation rate of water through the adhesive pad and assisting in communication ,56. An iubstrate ,58. As hDespite considerable insights into the compositions and functions of tarsal secretions, approaches to investigate the details of fluid depletion in insects are scarce. Particularly, details of the interactions of the tarsal fluids with the substrates and between different components of the fluid on the surface of the attachment pads and on the substrate remain unexplored. Insects with large smooth attachment pads possess large areas that need to be covered by these secretions and are particularly prone to contamination.Medauroidea extradentata. Stick insects are among the largest insects [We therefore analyzed the morphological characteristics of the footprint residues of the stick insect  insects  and they insects ,60,61,62 insects ,63, and  insects ,64,65. T insects ,67. We uMedauroidea extradentata   A becauseThe morphology of arolium and euplantulae represents the most common and least derived setup among phasmids with smooth adhesive microstructures on both attachment pads, without micro-ornamentation  Figure B. IndiviMicroscope slides (76 × 26 mm) and glass coverslips (12 mm)  were used as sampling substrates for investigation using white light interferometry (WLI) and cryo-scanning electron microscopy (cryo-SEM). The glass surfaces were thoroughly cleaned with the following protocol prior to sampling footprints C1: (1) 12 for 20 s. The tarsus was placed on a carefully cleaned glass slide or glass coverslip within a marked area. Glass slides for cryo-SEM investigation were previously sputter-coated with a 20 nm layer of gold–palladium  at −140 °C of the SEM Hitachi S-4800 . The frozen footprints were then observed in the SEM at −120 °C at an accelerating voltage of 3 kV without sputter coating. Subsequently, each sample was sputter-coated with gold–palladium (layer thickness 10 nm) in the preparation chamber at −140 °C and observed again at −120 °C with 3 kV accelerating voltage. Sputter coating was used to enhance the visualization of the surface structure of the footprint components. For some frozen samples, sublimation (freezing-drying) at −80 °C in the prechamber was performed prior to observations. Contrast adjustment and image cropping were performed using the software Photoshop CS6 .The glass slides with the footprints were examined in the white light interferometer New View 6000  and analyzed using the software MetroPro . The glass slides were placed under the WLI and measured at 20.6–22.9 °C room temperature and 61–64.3% ambient humidity. Areas of interest, where enough of the fluid accumulated to form a measurable droplet, were selected with the build-in-mask function C5. The dTo determine the evaporation rate of the droplets, the initial volume (day 0) was considered to be 100%, and the change in volume percentage over minutes (%/min) and days (%/day) was determined  and analyzed using the software TinyTag Explorer 6.0 . For the measurements in the closed glass chamber, 250 measurements at intervals of 30 min were conducted, and for the measurements at the WLI 200, measurements at intervals of 1 min were conducted.We analyzed the appearance of the fluid and solid residuals resulting from the contact of the tarsus with the glass surface. Cryo-SEM enabled us to visualize the components in their frozen state at −120 °C with a high magnification. Immediate freezing with liquid nitrogen after deposition allowed us to investigate the footprints in the condition just after fluid depletion. The micrographs, hence, show a temporary impression of the footprint at the time of its application .M. extradentata is shown with representations of all four components  A to smalA measurement of the change in volume over time (evaporation rate) of 68 fluid droplets from 25 footprints was performed over a period of up to 75 days . The chaThe droplets revealed evaporation rates with different slopes. Based on their evaporation behavior and rates, we distinguished three different types of droplets indicated by different colors in The non-evaporating droplets showed an evaporation rate, which is the change in the droplet volume over the measured days, that was lower than 0.34%/day. In total, 14 droplets were assigned to this type, which showed mean evaporation rates of 0.0531 ± 0.283%/day and 0.0000369 ± 0.000196%/min .The slowly evaporating type included all droplets with an evaporation rate between 0.34%/day and 3.2%/day. The group contained 12 droplets and displayed mean evaporation rates of 1.075 ± 0.479%/day and 0.000747 ± 0.000333%/min .The fast-evaporating type showed a faster volume loss and included all droplets with an evaporation rate higher than 3.2%/day. This type was present in 44 droplets and exhibited mean evaporation rates of 8.629 ± 7.503%/day and 0.00599 ± 0.00521%/min .p < 0.001), whereas there was no significant difference between the evaporation rates of the slowly evaporating and non-evaporating types   = 44, N (slowly evaporating) = 12, N (non-evaporating) = 14).A statistical comparison between the evaporation rates of the three types showed that the evaporation rate of the fast-evaporating type was significantly higher than that of the other types  includiSometimes, an additional imprint with a similar composition as those from the pads appeared between the imprints of the two euplantulae A,D. ThisThe cryo-SEM allowed us to observe the tarsal fluid at a high resolution and thus identify four morphologically distinct structures . The staM. extradentata are readily available, of the composition of the fluids of other insect species allows us to draw assumptions on the composition based on the morphological observations  also showed that it is a two-phase microemulsion, which consists of a volatile hydrophilic phase and a non-volatile hydrophobic phase ,71,72.The fast evaporation rate could be a result of the fraction of the tarsal fluid that has a potentially high volatile content . A slow evaporation rate can be indicative of a droplet type that contains a higher proportion of non-volatile components  and a lower proportion of volatile components. The non-evaporating part of the tarsal liquid might consist of hardening non-volatile components  27,28,3,42,47,73Calliphora vicina and the beetle Coccinella septempuctata with an atomic force microscope over a time span of 60 min [M. extradentata is the presence of a range of different evaporation rates including both fast and slow evaporation rates in different droplets from the same footprint. As Peisker and Gorb measured the evaporation in the first 60 min, and we observed the evaporation for up to 80 days with larger time spans between measurements, a more precise comparison within the same time scale would be necessary for these examples. However, the tarsal secretions from the hairy attachment systems investigated therein seem to consist of more uniform droplets compared to the smooth attachment system of M. extradentata.Similar measurements were conducted by Peisker and Gorb (2011), where they measured the evaporation rates of individual tarsal adhesive secretion droplets of the hairy attachment systems of the fly f 60 min . Within Locusta migratoria [Our microscopy observations and experiments reported above allow us to make predictions about the material properties and behavior of the four tarsal secretion components. We may hypothesize that one part of the tarsal secretion being fluid and solidifying over time in the light microscope is the same component that shows no evaporation and the flake-like appearance in the cryo-SEM. The morphological indications for this hypothesis are the particular layered surface structure of sputtered flakes resembling the structure of dried fluid B,E,F, angratoria . TherefoThe passive self-cleaning mechanism of smooth attachment devices is important for attachment maintenance, as it removes contaminants that would reduce adhesive performance ,31. ContCarausius morosus. They discovered that a high amount of tarsal fluid increases the recovery rate of the adhesion and hypothesized that this is due to the liquid filling the gaps [Clemente and colleagues described the effect of the tarsal secretion for self-cleaning in the stick insect the gaps ,31. We cWe observed that all contaminants were either covered or surrounded by the tarsal secretion B–D, therM. extradentata.Various experiments showed that the tarsal secretion has different effects on the attachment force generation, ranging from implementing viscous and capillary forces ,33 to leMultiple experiments indicate that the action of the capillary and viscous forces is important to generate adhesion in wet contacts ,35,54,75The thick film components display morphological characteristics that can be attributed to those of viscous fluids, such as covering a large surface area A,E and pTherefore, the thick films are likely rather viscous and/or possess a comparably high surface tension, which potentially aids in implementing the viscous force at the tarsus–substrate interface.The droplet components show different morphology than the films, as they form individual roughly round shapes that can accumulate into big complexes. We found droplets in the footprints exhibiting a fast evaporation rate (mean evaporation rate of 8.629 ± 7.503%/day and 0.00599 ± 0.00521%/min) C. Due toFor insects, the adaptation to different free surface energies of the substrates is challenging, as this is a factor that can influence locomotion, which, for example, plants utilize to either repel or capture insects ,79,80. ANano-droplets were found along with the larger droplets E, supporM. extradentata. In these experiments, M. extradentata was able to adhere to a highly hydrophobic surface (Polytetrafluoroethylene (PTFE)) even underwater [The droplet and thick film components show a range of surface structures, which can be smooth, rough, or granular A. Assumiderwater .Since different compositions of the adhesive fluid should respond differently to the free surface energy of the substrate, detailed analyses of the chemical composition are required for a deeper understanding of the functional role of the fluid in the tarsal attachment system of the stick insect.An insect’s adhesive secretion is often mentioned as having a function in leveling the surface roughness in the adhesive contact ,51,81,82M. extradentata can have an influence on the leveling of the substrate asperities is the performance of M. extradentata on substrates with varying roughness, which was reported in previous experiments [As the surfaces of natural substrates have fractal roughness at different hierarchical levels ,84,85, aeriments ,68.The cryo-SEM enabled an examination of the pad fluid in its frozen state immediately after deposition. We identified four morphologically different components that originate from the same tarsal secretion. The measurements of the evaporation rate of individual droplets indicate that the liquid consists of a spectrum of slowly evaporating to fast-evaporating components. These observations suggest that the tarsal fluid is a mixture of volatile and non-volatile components that, working in concert, extend the properties of the adhesive secretion.Parts of the adhesive secretion can harden over time into flakes and thus contaminate the adhesive area of the attachment pads. Contaminations can be glued together via new adhesive fluid and can be passively removed via tarsal movement during locomotion. Due to the presence of morphologically and physically different components, the adhesive fluid can support different phases of attachment, including contact generation, contact maintenance, and contact breakage, for example, by filling varying degrees of roughness and generating capillary and/or viscous forces.Medauroidea extradentata could potentially contribute to the range of functions. These results allow for several possible ideas to be generated for further investigations. A detailed chemical analysis of the adhesive secretion would aid in making a correlation between the morphological features and chemical composition. Histological studies could provide insights into the structure and distribution of the exocrine cells that are involved in the production of the secretion. Studies of the composition of the tarsal fluid of different ecologically specialized taxa can aid in understanding the adaptability of tarsal secretions. These insights could be valuable for the development of novel biomimetic adhesive fluids.With this study, we show how the morphologically and physically diverse tarsal secretion of


To assess the therapeutic response to brolucizumab and aflibercept by deep learning/OCT-based analysis of macular fluid volumes in neovascular age-related macular degeneration.In this post-hoc analysis of two phase III, randomised, multi-centre studies (HAWK/HARRIER), 1078 and 739 treatment-naive eyes receiving brolucizumab or aflibercept according to protocol-specified criteria in HAWK and HARRIER, respectively, were included. Macular fluid on 41,840 OCT scans was localised and quantified using a validated deep learning-based algorithm. Volumes of intraretinal fluid (IRF), subretinal fluid (SRF), pigment epithelial detachment (PED) for all central macular areas  in nanolitres (nL) and best corrected visual acuity (BCVA) change in ETDRS letters were associated using mixed models for repeated measures.p < 0.0001), 1.7 letters (p < 0.001) and 2.5 letters (p < 0.0001), respectively.Baseline IRF volumes decreased by >92% following the first intravitreal injection and consistently remained low during follow-up. Baseline SRF volumes decreased by >74% following the first injection, while PED volume resolved by 68–79% of its baseline volume. Resolution of SRF and PED was dependent on the substance and regimen used. Larger residual post-loading IRF, SRF and PED volumes were all independently associated with progressive vision loss during maintenance, where the differences in mean BCVA change between high and low fluid volume subgroups for IRF, SRF and PED were 3.4 letters (Deep-learning methods allow an accurate assessment of substance and regimen efficacy. Irrespectively, all fluid compartments were found to be important markers of disease activity and were relevant for visual outcomes. The neovascular type of age-related macular degeneration (nAMD) is recognised as one of the most aggressive ocular diseases with rapid progression and extensive morphological alteration associated with irreversible functional loss. Intravitreal administration of antibodies inhibiting vascular endothelial growth factor (VEGF), has introduced a paradigm-shift in the treatment of nAMD, substantially improving the prognosis, and has rapidly become the gold standard in the management of the disease . Yet, deAmong novel pharmacological developments, the humanised, single-chain variable fragment brolucizumab pursues the promise of enhanced efficacy by better target-tissue penetration and superior therapeutic durability . In the Although used as a measure in many trials, CRT is only a rough indicator for fluid recurrence or resolution, originating from the era of time-domain optical coherence tomography (OCT) with only six radial scans. However, spectral-domain OCT (SD-OCT) offers high-resolution raster scanning of the macular area visualising neurosensory layers and pathological biomarkers such as fluid in precise detail . To reliIn this paper, we performed a comprehensive volumetric retinal fluid analysis of the large HAWK and HARRIER trial data set including a total of 41,840 SD-OCT scans. Advanced AI tools provided a detailed quantitative assessment of the therapeutic response in a fully automated, objective and reproducible manner. Most importantly, we investigated the association of fluid volumes in the different compartments with visual outcomes over the maintenance phase to objectively provide evidence about one of the most controversially discussed topics in AMD management: Does fluid volume and location matter and how should the community optimise the management of AMD patients now and in the future when reliable AI tools will be available.AI-based analysis was performed on the SD-OCT scans of 1078 and 739 patients enrolled in the HAWK and HARRIER clinical trials . HAWK and HARRIER were two 96-weeks, prospective, randomised, double-masked, phase 3 multi-centre studies to evaluate the efficacy and safety of intravitreal brolucizumab vs. aflibercept in treatment-naïve patients with neovascular AMD. Patients were randomised 1:1(:1) to receive brolucizumab 6 mg or aflibercept 2 mg (or brolucizumab 3 mg in HAWK only). Both studies adhered to the tenets of the declaration of Helsinki and Good Clinical Practice and all participants provided written informed consent prior to study inclusion. The two core studies were approved by independent Ethics Committees by the sponsor.Aflibercept patients were treated with a conventional q8w regimen throughout the study duration. Brolucizumab patients were treated by q12w, but adjusted to q8w as early as week 16, if disease activity was diagnosed by the masked investigator and supported by protocol guidance based on functional and anatomical characteristics. The maintenance phase in this specific protocol was defined as the post-loading phase i.e. weeks 12–96. Disease activity assessments were conducted by the masked investigator at week 16, week 20 and every 12 weeks thereafter to determine the subsequent dosing interval. There were additional disease activity assessment visits in the HARRIER trial. The trial protocol is illustrated in Fig. 3, comprising a volume of 6 × 6 × 2 mm3, (2) a Spectralis device  consisting of 512 × 49 × 496 voxels with a size of 11.7 × 125.3 × 3.9 µm3, comprising a volume of 6 × 6.1 × 1.9 mm3, or (3) the Topcon T-1000, T-2000, Atlantis DRI  consisting of 512 × 128 × 885 voxels with a size of 11.7 × 46.9 × 2.6 µm3 and a volume of 6 × 6 × 2.3 mm3.Volumetric macula-centred OCT scans were acquired following a standardised imaging schedule using either (1) a Cirrus HD-OCT III device  consisting of 512 × 128 × 1024 voxels with a size of 11.7 × 47.2 × 2.0 µmBCVA was measured and OCT acquired monthly at all visits. The OCT scans of all patients were collected centrally by two reading centres (Vienna Reading Center and Duke Reading Center) after certification of study photographers according to a predefined imaging protocol. All OCT images were transferred post-hoc to the Laboratory for Ophthalmic Image Analysis (OPTIMA) at the Medical University of Vienna in a pseudonymised format to conduct the AI-based fluid analysis. The post-hoc analysis presented here was conducted in compliance with the Declaration of Helsinki and approval was obtained by the Ethics Committee at the Medical University of Vienna (EK Nr: 1246/2018).3) of IRF, SRF, and PED were computed within the central 1 mm, 3 mm and 6 mm macular fields. A fully automated three-dimensional OCT segmentation of the three fluid compartments was performed on all of the 41,840  available volumetric scans, using a previously developed and validated deep learning algorithm for IRF and SRF quantification, the Vienna Fluid Monitor  . The algTo test for differences in fluid volumes across the treatment arms and patient subgroups, a mixed model for repeated measures (MMRM) was applied on the full analysis set (FAS). To test for differences across treatment arms, the fixed factors were: baseline volume, age group , treatment, visit, and treatment-visit interaction. To test for differences across OCT devices, the fixed factors were: baseline volume, age group, device, visit, and device-visit interaction. To test for differences across racial subgroups, the fixed factors were: baseline volume, age group, race, visit, and race-visit interaction. Finally, to test for differences across age subgroups, the fixed factors were: baseline volume, age group, visit and age group-visit interaction. Fluid volumes were log(x + 1) transformed to adjust for the skewed non-normal distribution of fluid volumes. The results were back transformed for graphical representation on the original nL scale.High fluid volume subgroup was defined for each fluid type  as patients with the highest 25% quartile of mean post-loading (weeks 12–96) fluid volumes in the central 6 mm. The remaining 75% of patients were classified as Low fluid volume subgroup. Because majority of eyes were dry by the end of the loading dose, an alternative subgroup definition based on the median volume would have utilised cutoff values of <1 nl for IRF and SRF, which would not be representative of High fluid volume subgroup. MMRM was then applied on FAS to model the change of BCVA from week 12, with the following fixed factors: BCVA at the week 12 visit, IRF, SRF, PED volume subgroups and all visit and volume subgroups interactions. The two studies, HAWK and HARRIER, were analysed separately to allow for replication of the findings.To evaluate a treatment agnostic association of BCVA and the amount of the residual post-loading dose fluid, Automated volume quantification provided measurements for 1076 of 1078 patients  in HAWK, and 736 of 739  in HARRIER. This efficacy represents 99.7% of all patients and 98.5% of all OCT scans. A breakdown of the processed OCT scans per device vendor is shown in Table The result of modelling the adjusted mean volumes in all three fluid compartments, for the central 1, 3 and 6 mm for brolucizumab 6 mg and aflibercept 2 mg treatment in both studies is shown in Fig. p < 0.001 for the difference in mean post-baseline SRF) in both studies. This trend was consistent across all macular fields. The fluctuations in SRF volumes in the aflibercept arm were more pronounced than those for IRF in the same group, ranging from a variability of ~1nL in the central 1 mm to ~6nL in the 6 mm area. Similar to SRF, PED resolution with brolucizumab was greater under an identical injection schedule up to week 16 and was maintained throughout the study duration. Statistical analysis revealed that in the central 6 mm the mean post-baseline PED volume obtained with brolucizumab 6 mg was significantly lower than the one obtained with aflibercept 2 mg in HARRIER (p = 0.005), with the same trend in HAWK (p = 0.15). This trend was found consistently across all macular fields.For all treatment arms, the result of modelling of the adjusted mean volumes in all three compartments, in the central 1, 3 and 6 mm, is shown in Fig. The results of modelling the adjusted mean volumes of the three compartments for specific patient subgroups are provided in the supplement. Analyses of the subgroups defined by OCT device  are shown in Figs. High/Low volume subgroups are shown in Fig. High and Low volume subgroups, the difference in mean BCVA change were 3.4 letters for IRF (p < 0.0001), around 1.7 letters for SRF (p < 0.001) and 2.5 letters for PED (p < 0.0001), averaged over both studies and all time points. This finding revealed that in addition to IRF, larger residual SRF and PED volumes were also associated with progressive vision loss during follow-up. Finally, averaging the fluid volume/function correlation over both studies and all visits, we evaluated the mean BCVA change from week 12 for all combinations of fluid volumes thus defining subgroups of differential disease activity  was observed in the subgroup characterised by Low disease activity in all compartments. In contrast, a most substantial visual loss with a mean of −6.5 letters (p < 0.001) was observed in subgroups with High disease activity in all three fluid compartments. Combinations of different high and low volumes ranged in between these two boundaries.The results of modelling the adjusted mean BCVA change over time from week 12 for each fluid compartment in the central 6 mm individually for In contrast to human expert procedures in clinical practice, AI tools can automatically and precisely quantify fluid volumes in different compartments to measure disease activity and therapeutic efficacy. Automated quantification of fluid using AI therefore offers an ideal tool to determine therapeutic efficacy in an objective and reproducible manner, and to help improve the management of nAMD in both clinical trial and real-world settings. In our study, AI-based analysis of fluid volumes from a large set of 41,840 volumetric SD-OCT scans allowed for an individualised yet global conclusion of fluid amounts and function, and confirmed that fluid in all compartments mattered regarding visual outcomes. Lower levels of IRF, SRF and PED during maintenance were all associated with superior visual outcomes. Thus, fluid volumes in all three fluid compartments must be taken into account as markers of disease activity equally and independently relevant for visual function in nAMD, which has major implications for treatment decisions.The accurate quantitative analysis of the therapeutic fluid response based on realistic volumes reveals characteristic mechanisms of action with different substance features. In respect to brolucizumab, this feature design comprises its structure with a lack of fragment crystallisable domains improving bioavailability and small size adding better target-tissue penetration , 18. Sim® label.Brolucizumab as an innovative intravitreal therapeutic biologic format can come at the costs of higher risks, comprehensively discussed in the recent literature. Since first regulatory approval of brolucizumab in September 2019, treating ophthalmologists started to report incidences of vasculitis including occlusive retinal vasculitis (Beovu update for ASRS members). From these reports, the ASRS reviewed clinical data from 25 eyes including 23 eyes with imaging (ASRS Beovu update). 92% of cases were associated with inflammation and 84% of vasculopathy events were reported as occlusive with arterial vessels most commonly affected (Beovu update ASRS). The ReST committee therefore recommended a careful evaluation for any signs of active inflammation prior to brolucizumab administration as brolucizumab and aflibercept are contraindicated per FDA labels in the setting of inflammation. Novartis appointed an independent Safety Review Committee in March 2020 to independently review these post-marketing cases as well as to perform an unmasked post-hoc review of all cases of investigator-reported intraocular inflammation, retinal vascular occlusions and endophthalmitis in HAWK an HARRIER. . The review of the HAWK and HARRIER cases revealed occlusive vasculitis in 2.1% of patients with 1 in 200 individuals beginning brolucizumab therapy losing six or more lines in vision in association with occlusive vasculitis, at the end of study participation. Based on the review of the post-marketing cases, these events have also been added to the BeovuAssociation between fluid and function in nAMD is highly complex and non-linear as the disease is also associated with progressive alteration of neurosensory layers such as the outer nuclear layer, the photoreceptor and the RPE layers. This is consistent with the lack of correlation observed between CRT and BCVA. It is by precise quantification of fluid volumes respective to the individual compartments that the role of fluid increase and decrease can be fully understood and demonstrated in a statistically sound manner. Chakravarthy et al have recently highlighted an interdependency of “fluctuation” in fluid volumes and vision loss . Our groEvaluation of fluid defined by different retinal compartments is a relatively novel approach. In clinical trials, retreatment criteria based on retinal fluid are thoroughly defined and overseen by certified experts in a reading centre setting. Nevertheless, manual, i.e. human determination of fluid in and underneath the retina is demanding even for trained individuals. IRF poses the biggest challenge as it is typically presenting with dozens of variable cystoid spaces embedded more or less distinctly within the neurosensory layers. When 1213 pairs of time-domain and SD-OCT scans from the CATT study were analysed by a professional reading centre, agreement on IRF was only 73% with misleading artefactual interpretation of dark areas as IRF . For detIn the comparative analysis of the HAWK and HARRIER regimen, IRF resolved well with both substances. In general, IRF has been shown to respond rapidly and intensively to anti-VEGF therapy in multiple study settings and different diseases –28. In tThe presence of SRF has been reported to show less association with BCVA changes than IRF. In a post-hoc analysis of the CATT population, eyes presenting with foveal SRF at year two demonstrated better BCVA than eyes with extrafoveal or no SRF . In HARB3 at baseline to 0.23 mm3 at week 8 using a semi-quantitative manual approach. A precise assessment of PED volumes in the current analyses revealed a superior effect on the reduction of PED using brolucizumab. This capacity may be useful as it demonstrates the impact of the substance on the neovascular lesion origin in MNV types 1 and 2, particularly if durability in reducing the biological disease activity is the goal. The fact that fluctuations at the level of the PED still occur during a follow-up as long as 96 weeks, impressively reflects the progressive nature of disease activity within the subretinal fibrovascular complex.A good efficacy of aflibercept in resolving PED has also been shown in previous studies . Even inHigh/Low fluid volume subgroups was provided in a treatment agnostic manner. Optimal BCVA outcomes are achieved at low fluid volumes in all categories and higher volumes are associated with the poorest visual function. Nevertheless, larger residual SRF and PED volumes were also associated with vision loss during maintenance. Low IRF in combination with a residual presence of SRF and/or PED is still combined with good BCVA maintenance and the deficit range is limited to two letters. However, resistant IRF at higher levels may still be accompanied by good vision, if SRF and PED volumes have resolved. BCVA declines further, if the PED volume is increasing. A correlation of an expanding PED and increasing IRF has been shown previously as a common mechanism for progressive visual loss in the VIEW studies and may support the value of PED reduction to prevent functional decline due to accumulation IRF [The most revealing aspect of this paper is the accurate correlation of macular function and fluid resolution enabled by the large dimension and precision of volumetric fluid determination in HAWK & HARRIER. The progressive loss in BCVA during follow-up in the higher volume quartile was clearly confirmed for all fluid types. To set this observation into an adequate proportion with clinical relevance, a modelling of the adjusted mean BCVA change over time from week 12 for each fluid compartment for individual tion IRF .An obvious limitation of this study and the generalisation of our findings is its retrospective nature and the focus on imaging data obtained from an a priori standardised clinical trial setting. AI-based fluid analysis is a novel achievement and has only recently become available for use in large dimensions. Even from large clinical studies only few data sets have so far undergone systematic volumetric fluid quantification , 28, 37.In conclusion, automated quantification of retinal fluid volumes based on deep learning in two large randomised controlled trials allowed to evaluate pathognomonic resolution patterns for different retinal compartments and to objectively compare the therapeutic efficacy of pharmacological substances. It is conceivable that such advanced diagnostic methods determining fluid resolution in a reliable quantitative manner, over time and in different disease phenotypes will provide “fingerprint-like“ characteristics for drugs, regimen and modes of administration e.g. implants and strategies such as retinal gene therapy. Moreover, only small advances in clinical outcomes were achieved between different pharmacological developments, yet an accurate therapeutic guidance by an automated and reliable fluid volume measurement may improve the benefit in a more economic and personalised manner. Also, retinal fluid is just one of a multitude of biomarkers which can be analysed and measured by automated algorithms. Many other relevant features are being explored such as hyperreflective foci, photoreceptor integrity, neurosensory layer atrophy etc. Regarding the current anti-VEGF-based therapy of exudative macular disease, a quantitative correlation of fluid volumes with BCVA values offers clinically most relevant insight into the impact of morphological features on neurosensory function as well as prognostic outcomes of intervention. The results of such accurate and objective analyses represent the base of identifying appropriate parameters prospectively guiding disease management in the real world and detecting promising therapeutic targets for innovative treatment strategies in future clinical trials.Intraretinal fluid is associated with worse visual acuity outcomes in neovascular age-related macular degeneration. Tolerance of SRF might not have an impact on visual acuity outcomes.Eyes with high volumes of IRF, SRF and pigment epithelium detachments have worse visual acuity outcomes compared with eyes with low fluid volumes in the maintenance phase. High fluid volumes in all compartments should be treated to optimise anti-VEGF therapy in neovascular age-related macular degeneration.Supplemental Figure legendsSupplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplemental Figure 5Supplemental Figure 6Supplemental Figure 7Supplemental Figure 8Supplemental Figure 9Supplemental Figure 10Supplemental Figure 11Supplemental Table 1


Its scope has also expanded beyond the palliative treatment of advanced malignancies to neoadjuvant, adjuvant and consolidative use with curative intent.During the course of tumour evolution, cancer cells typically accumulate somatic sequence alterations, a fraction of which code for mutation‐associated neoantigens (MANA) that are presented on human leukocyte antigen (HLA) molecules to T cells, eliciting immune recognition in the context of mutant ‘nonself’ neopeptides. Immune checkpoint blockade unleashes a potent immune response against MANAs, that is amplified in the context of a high neoantigen burden. Conceptually, a higher neoantigen burden confers a higher degree of tumour foreignness to the immune system and clinically this is reflected in therapeutic benefit with ICB for patients with TMB‐high tumours. To this end, TMB has been utilised as a proxy for tumour foreignness however has been inconsistently associated with ICB response.In studying the evolving lung cancer genomes under the selective pressure of immunotherapy, we identified loss of MANAs, via copy number loss or subclonal elimination, as one of the key genomic mechanisms underlying the emergence of acquired resistance.We hypothesised that mutations within the overall TMB fall within two categories: that of ‘loss‐prone’ and that of ‘persistent’ mutations Figure .9 PersWe set out to explore this hypothesis by computational analyses of ∼10 000 tumour samples from the cancer genome atlas (TCGA), and identified an extensive variation in the abundance of only‐copy and multicopy mutations between and within tumour types. On average, we found persistent mutations to be a small minority (10%) of the somatic mutations per tumour. Importantly, when we evaluated the differential reclassification of cancers across 33 tumour types, we found an average reclassification rate of 33% in TMB‐high/low versus persistent TMB‐high/low groups. These findings suggest that tumours are differentially classified based on their persistent tumour mutation burden (pTMB). To explore the clinical significance of these findings, we analysed 524 ICB‐treated tumour samples including head and neck cancer, mesothelioma, nonsmall cell lung cancer and melanoma. In all cohorts analysed, pTMB distinguished responding from nonresponding tumours better than TMB or the number of loss‐prone mutations.A key question that arose from these studies was whether persistent mutation burden can be computed from targeted next‐generation sequencing that is widely adopted in clinical practice. To assess the feasibility and clinical value of pTMB in routine cancer care, we performed in silico simulations, where we restricted the TMB and pTMB definitions to genomic regions queried by clinical grade targeted next‐generation sequencing and found that, similar to whole exome sequencing data, pTMB defined based on targeted NGS outperformed TMB in distinguishing responding from nonresponding tumours in the context of ICB. These findings, while requiring further validation, are encouraging with respect to the immediate clinical utility of persistent mutations. Historically, we have been focusing on the density of sequence alterations derived from tumour molecular profiling, ignoring the structural genomic landscape that can be reliably derived from targeted next‐generation sequencing. By integrating sequence and structural landscapes and considering sequence mutations in the context of the background copy number of the locus evaluated, we can maximally leverage next‐generation sequence data and tailor the tumour mutation burden to the subset of alterations most likely to drive immunologic tumour control.In thinking about the clinical implementation of pTMB, there are several critical steps needed to bring consensus on the clinical significance of varying pTMB results in cancers of different lineages. Dichotomised biomarkers, such TMB ‘high’ or ‘low’, are simple and accessible, but it is increasingly clear that their universal predictive ability is limited. Conversely, overly granular metrics allow for nuance, but may not be practical for routine cancer care. While pTMB provides a composite measure for the number of hard‐to‐lose mutations in a tumour, only‐copy or multicopy subclasses may carry differential weight in predicting ICB response, potentially driven by the dominant copy number state of the tumour evaluated. For example, in the setting of a melanoma tumour, multicopy mutations may be most informative, whereas single‐copy alterations would be more predictive in ICB‐treated mesotheliomas. Understanding the relative importance of only‐copy and multicopy mutations across cancer lineages will help to further improve the clinical utility of pTMB. Furthermore, lineage‐specific pTMB thresholds, and stability of such thresholds require additional studies and validation.As pTMB represents one piece in the jigsaw puzzle of tumour‐immune system interactions, it may fit within an integrative composite predictive tool together with tumour‐extrinsic features. Furthermore, placing these observations in the context of other measures of neoantigen quality,In the clinical setting, there are several scenarios in which pTMB may be particularly informative. One possibility is in situations where other biomarkers have conflicting results. For example, if a nonsmall cell lung cancer tumour is found to have high TMB but low PD‐L1, the pTMB result may support decision‐making: is there a strong chance of response to ICB monotherapy, or is combination with chemotherapy required? pTMB levels may also be informative for TMB‐high tumours which are less likely to have a treatment response at the standard ‘high’ cutoff (>10 muts/Mb), such as in breast cancer.In conclusion, the expanding clinical uptake of ICB has not been matched by the development of accurate predictors of response. To this end, measurement of pTMB represents a promising and innovative approach to predicting response to ICB compared to the overall tumour mutation burden. Focusing on the subset of mutations within the overall TMB most likely to drive sustained immunologic tumour control can extend the promise of cancer immunotherapy and improve patient outcomes.V.A. receives research funding to Johns Hopkins University from Astra Zeneca, Personal Genome Diagnostics and Delfi Diagnostics, has received research funding from Johns Hopkins University from Bristol‐Myers Squibb in the past 5 years and is an advisory board member for Neogenomics. V.A. is an inventor on patent applications  submitted by Johns Hopkins University related to cancer genomic analyses, ctDNA therapeutic response monitoring and immunogenomic features of response to immunotherapy that have been licensed to one or more entities. Under the terms of these license agreements, the university and inventors are entitled to fees and royalty distributions.


Respiratory syncytial virus (RSV) can cause severe disease in older adults (≥65 years) or those with comorbidities. Understanding the risk of hospitalization after RSV diagnosis through current real-world data analyses is limited by estimates combining cases identified in inpatient and outpatient settings. To address this gap and to better understand opportunities for intervention to prevent such hospitalizations, we quantified the crude 28-day risk of all-cause hospitalizations following RSV infections initially managed in an outpatient setting.This retrospective observational study identified outpatient RSV episodes (clinics and emergency departments [ED]) for RSV between 01Sep2016-31Oct2022 using Optum’s de-identified Integrated Claims-Clinical data set, which contains linked person-level claims and electronic health record (EHR) data. RSV diagnosis was defined using positive test results  and ICD-10-CM diagnosis codes. Comorbidities were identified using diagnosis, procedure, and medication codes. Only the first eligible episode (index date) for each study-defined RSV season (01Oct–30Sep) was included . Episodes hospitalized on the index date or with an RSV-related hospitalization 14 days prior to the index date were excluded. Crude risk of all-cause hospitalization was assessed over a 28-day follow-up period.Study schematic for the retrospective observational cohort study designThe schematic depicts how eligible RSV outpatient episodes were identified and the inclusion/exclusion criteria that were applied to generate the final sample.Of 2,792 included episodes, 62% occurred among females, 55% occurred among patients aged ≥65, and 21% received ED care on the index date. Between 2016-2018, the 28-day risk of hospitalization ranged from 6-9% and declined to 3% in 2019/20 . The risk of hospitalization also varied by age or the presence of comorbidities (Table 1).This table summarizes the crude 28-day risk of all-cause hospitalization for adults overall and stratified by clinical conditions and age groups.This figure displays the crude 28-day risk of all-cause hospitalization following an outpatient episode of RSV, stratified by season and displayed for adults with high-risk conditions.A meaningful risk of all-cause hospitalization following outpatient RSV episodes was observed using a US integrated database that captures a patient’s longitudinal healthcare journey. Risk of hospitalization varied across seasons and was influenced by age and comorbidities. Results include diagnosed cases, and RSV infections may have been underreported due to infrequent standard-of-care testing.Suzanne Landi, PhD, Pfizer, Inc: Employment|Pfizer, Inc: Stocks/Bonds Diana Garofalo, PhD MPH, Pfizer: Stocks/Bonds Amie Scott, MPH, Pfizer, Inc: Employee|Pfizer, Inc: Stocks/Bonds Jill McCarthy, PhD, Pfizer: Contractor Margaret Tawadrous, MD, MS, Pfizer: Full time employee|Pfizer: Full-time employee|Pfizer: Full-time employee|Pfizer: Full -time employee|Pfizer: Ownership Interest|Pfizer: Ownership Interest|Pfizer: Ownership Interest|Pfizer: Ownership Interest|Pfizer: Stocks/Bonds|Pfizer: Stocks/Bonds|Pfizer: Stocks/Bonds|Pfizer: Stocks/Bonds Glenn Pixton, MS, Abbott: Stocks/Bonds|Abbvie: Stocks/Bonds|Pfizer: employee|Pfizer: Stocks/Bonds|Viatris: Stocks/Bonds Niki Alami, MD, Pfizer: Employee|Pfizer: Stocks/Bonds Scott P. Kelly, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Joshua T. Swan, PharmD, MPH, BCPS, FCCM, CareDx: Grant/Research Support|Genentech: Grant/Research Support|Grifols Share Services North America: Grant/Research Support|Heron Therapeutics: Grant/Research Support|Kedrion Biopharma: Advisor/Consultant|Kedrion Biopharma: Grant/Research Support|Pacira Pharmaceuticals: Grant/Research Support|Pfizer: Grant/Research Support|Pfizer: Employee|Pfizer: Stocks/Bonds|VigiLanz Corporation: Grant/Research Support


Xenopus embryos with immature functional muscles excrete archenteron fluid via a tissue-level mechanism that opens the blastopore through a gating mechanism that is unclear. Using microelectrodes, we show that the archenteron has a constant fluid pressure and as development progress the blastopore pressure resistance decreases. Combining physical perturbations and imaging analyses, we found that the pushing force exerted by the circumblastoporal collars (CBCs) at the slit periphery regulates pressure resistance. We show that apical constriction at the blastopore dorsoventral ends contributes to this pushing force, and relaxation of ventral constriction causes fluid excretion. These results indicate that actomyosin contraction mediates temporal control of tissue-level blastopore opening and fluid excretion in early Xenopus embryos.Fluid uptake and efflux play roles in early embryogenesis as well as in adult homeostasis. Multicellular organisms have two main pathways for fluid movement: cellular-level, such as transcellular and paracellular pathways, and tissue-level, involving muscle contraction. Interestingly, early   •In Xenopus embryos, fluid excretion from the blastopore is temporally regulated•Apical constriction at the dorsoventral ends drives the pushing force•The pushing force maintains blastopore closure against archenteron fluid pressure•The release of the ventral constriction causes opening and fluid excretion Cellular physiology; Developmental biology; Embryology In adult organisms, extracellular fluid is transported through pores and tubes at a microscopic cellular level and at a macroscopic tissue level.,,,Extracellular fluid also plays an important role in early developmental processes.Xenopus laevis embryos, which have no functional muscle,,,,,,,,,,Interestingly, early To characterize the blastopore gating mechanism, in this study we used microelectrodes to measure archenteron fluid pressure. We demonstrate that archenteron fluid pressure remains constant, but the upper limit of blastopore pressure resistance decreases as development progress. Furthermore, by combining physical perturbation experiments and imaging analysis, we found that the upper limit of pressure resistance is regulated by the force with which the CBCs push against each other. Focusing on actomyosin contraction to mediate this pushing force, we detected the phosphorylation of myosin light chains (pMLC) at the blastopore ventral and dorsal ends during the mid-neurula stage. However, in the late neurula stage just before fluid excretion, pMLC signals at the ventral end of the blastopore were attenuated. Injection of an inhibitor of actomyosin contraction into the ventral end artificially causes blastopore opening and fluid excretion, even in the mid-neurula stage. These results demonstrate that actomyosin contraction in the ventral end of the blastopore defines the upper limit of pressure resistance and that the decrease in pressure resistance opens the blastopore to allow fluid excretion. Our findings show that even early embryos with immature functional muscles can regulate blastopore opening via actomyosin-mediated apical constriction.Xenopus laevis, extracellular fluid in the blastocoel moves into the archenteron. The archenteron fluid is then excreted from the embryo through the blastopore, a process called “collapse of the archenteron” that occurs via a mechanism that has not been fully characterized . We saw the excretion of archenteron fluid from the embryo after stage 20 whether or not the vitelline membrane was present  and without (lower) the vitelline membrane during archenteron fluid excretion. The time at which the fluid excreted was set to t = 0 min. Green, cells; magenta, extracellular fluid. Second part: Time-lapse image of the blastopore during archenteron fluid excretion. The vitelline membrane was removed prior to time-lapse imaging. The time at which the fluid excreted was set to t = 0 min. Green, cells; magenta, extracellular fluid.Xenopus embryos, the unidirectional fluid flow induced by rotating cilia present in the gastrocoel roof plate (GRP) plays a central role in the determination of the left-right axis between stages 15 and 19.We next artificially removed the archenteron fluid before stage 20 and examined the effects on development. When the anterior part of the archenteron was punctured with a glass needle at stage 14 (before excretion) or stage 20 (just before excretion), most of the fluid was excreted from the opening, and strong archenteron contraction was observed, as in stage 24 embryos  E-1J. In Extracellular fluid is directionally excreted from the blastopore to the medium . As suchTime-lapse image of fluorescent beads ejected from a perforation in the archenteron.Xenopus embryos, suggesting that changes in concentrations during measurement must be considered.This system measures pressure via the electrical resistance of the fluid such that the salt concentration of the fluid flowing into the microelectrode affects the measured value. Previous reports indicate that the salt concentration of archenteron fluid is higher than that of 0.1x Barth’s medium used for Using Arc-buffer, we examined whether the archenteron pressure (Parc) increases due to developmental progression by measuring the pressure at three stages: stage 18 (before excretion), stage 20-21 (just before excretion), and the stage during excretion  A. IntrigTime-lapse image of a lateral view of an embryo. A microelectrode was inserted into the embryo having fluorescently labeled archenteron fluid. The time at which the microelectrode was inserted into the archenteron was set to t = 0.We next hypothesized that the upper limit of pressure resistance (Pres), at which the blastopore maintained closure against the hydrostatic pressure of the archenteron, may decrease due to the progression of developmental stages and could fall below the fluid pressure in the archenteron, resulting in fluid excretion. To examine the upper limit of blastopore pressure resistance, we first inserted a pressure-measuring microelectrode into the archenteron and analyzed fluid pressure (Parc) . ThereafTime-lapse image of Arc-PR-buffer being injected into the archenteron while measuring fluid pressure. The time at which the Arc-PR-buffer was injected into the archenteron was set to t = 0.We next focused on the regulatory mechanism of blastopore pressure resistance. The blastopore is formed at the posterior end of the embryo, where mesoderm invagination occurs, and has a slit-like gap extending in the dorsoventral direction A. The slTime-lapse images of the lateral view of a normal (upper) and aluminum foil-inserted (lower) embryo. The time at which the fluid was excreted was set to t = 0. Green, cells; magenta, extracellular fluid.Time-lapse image of the blastopore with the glass bead inserted. The time when the glass bead was removed was set to t = 0 s.The closure of the blastopore shortly after bead removal K-3N suggWe next analyzed the stage-dependent shape changes of the CBC using the CBC thickness and the contact surface length as an index. The CBC thickness was decreased at stage 20-21 relative to that at stage 18  up to a total volume of 400 nL. Second part: Tungsten wire was used to measure force required to open the blastopore at stage 18 (upper) and stage 20-21 (lower). The archenteron fluid was pre-labeled with phenol red.These results show that the upper limit of pressure resistance of the blastopore is mainly controlled by the pushing force of CBCs and that a decrease in pushing force may open the blastopores to allow fluid excretion.,Xenopus embryos, bottle cells during gastrulationWe next focused on the mechanism to generate pushing force, which resists the positive pressure of the archenteron fluid and maintains blastopore closure. During development, actomyosin, which comprises actin fibers and myosin, causes the apical constriction of epithelial cells upon myosin light chain (pMLC) phosphorylation. Coordinate apical constriction of multiple cells drives large forces that induce changes in tissue shape.We next investigated whether apical constriction was observed in the pMLC-positive tissue. When cell shape was analyzed by fluorescent immunostaining with an anti-E-cadherin antibody, apical surface constriction was detected in the ventral side of the blastopore at stage 18 J. HoweveTo investigate the relationship between actomyosin activity and the blastopore gating mechanism, we examined whether suppression of ventral actomyosin activity at stage 18 (before excretion) causes blastopore opening. Y27632 inhibits Rho-kinase, which is involved in myosin phosphorylation, and in turn inhibits apical constriction.Time-lapse images of a lateral view of mock- (upper) and Y27632-injected (lower) embryos. The time at which the mock or Y27632 injection into the ventral end of the blastopore occurred was set to t = 0. Green, cells; magenta, extracellular fluid.We next examined the effect of Y27632 injection on pMLC using immunostaining. Following Y27632 injection into the blastopore ventral region at stage 18, pMLC signals in the ventral region disappeared, and the effects on the dorsal region were insignificant K and S7JThese results suggest that the release of ventral apical constriction causes blastopore opening; however the possibility that the increase in fluid pressure in the archenteron associated with Y27632 injection causes blastopore opening cannot be ruled out. To evaluate this possibility, Y27632 was injected into the ventral side of the blastopore and the fluid pressure in the archenteron was measured through to blastopore opening. At stage 18, embryos injected with Y27632 showed no change in fluid pressure (approximately 10 Pa) before and after blastopore opening Q and 6S.Xenopus embryos by actomyosin-dependent apical constriction at the dorsoventral ends that generates a pushing force between CBCs  causes defects in laterality. The left-right axis is determined by the leftward flow of fluid driven by ciliary motility in the archenteron. Cilia in the gastrocoel roof plate (GRP) are tilted in the posterior direction and rotate clockwise. Thus, the rightward flow decelerates due to viscous friction and the leftward flow is dominant.On the other hand, no significant effects on left-right axis formation were observed in the early excretion associated with the injection of Y27632. Since the period of fluid excretion induced by Y27632 was brief, a sufficient amount of fluid would have remained in the archenteron. Consistent with this idea, a sufficient amount of fluid excretion was observed in the collapse of the archenteron in Y27632-injected embryos. In addition, Y27632 was injected at the late neurula stage (stage 18), which may have been too late to affect ciliogenesis.Together, our results show that fluid is retained in the archenteron until the left-right axis is determined and that the timing of fluid excretion is temporally regulated.,,In this study, pressure measurements in the archenteron using microelectrodes revealed that a decrease in the upper limit of the blastopore pressure resistance causes blastopore opening. Recently, direct or indirect methods were attempted to measure pressure in the embryonic cavity to elucidate the role of fluid pressure in development.Pressure measurement using microelectrodes can be improved to address the possibility that fluid could leak from the insertion site due to high fluid pressure or debris that clogs the needle tip during prolonged measurements. Further improvements in instruments to measure pressure are expected to provide a better understanding of dynamic pressure changes during embryogenesis.Xenopus embryos excrete from a localized tissue, the blastopore. We measured the upper limit of pressure resistance of the blastopore by combining pressurization with buffer injection and pressure measurement using microelectrodes, and found that a decrease in pressure resistance induces blastopore opening. Measurements of pushing force using a tungsten wire also supported a decrease in pressure resistance. These results indicate that the primary factor causing fluid excretion is an increase in fluid pressure in the blastocoel in mouse embryos and a decrease in the pressure resistance of the blastopore in Xenopus embryos, suggesting that they regulate excretion via different mechanisms. Furthermore, there is a difference between mice and Xenopus: random ejection at the cellular level and localized excretion at the tissue level. One possible explanation for this difference in randomness and localization may be differences in the cavity wall structure. The mouse embryo blastocoel wall has a single-layer epithelium, and thus transient rupture of epithelial TJs would allow efficient fluid drainage.Xenopus embryo archenteron walls have thick tissue comprising endothelium, mesenchyme, and epithelium. Thus, fluid excretion due to epithelial collapse may be more difficult in Xenopus than in mice. In addition, during the late neurula stage, various tissues such as neural tube,Xenopus embryos through the excretion of fluid only through the blastopore. Consistent with these considerations, in fluid drainage in the inner ear of zebrafish that occurs repeatedly during the formation of the semicircular canal associated with cavity wall deformation, is mediated in a localized area, the endolymphatic sac.,In the mouse blastocoel cavity, fluid drains due to the partial disruption of TJs in random regions of dividing cells.In this study, we revealed that actomyosin contraction at both dorsoventral ends of the blastopore drives the pushing force and that relief of ventral contraction induces the collapse of the archenteron. To effectively close the blastopore by the contraction of the dorsoventral ends, the CBC must have a certain degree of stiffness in order to transmit the pushing force from both ends to the center . Therefore, this softening of the CBC could trigger the opening of the blastopore. We used a tungsten wire to measure the force required to open the blastopore and found that the force was reduced in later stages M and 4N.,Previous studies reported that actomyosin contraction is important to decrease the IMZ circumference.,,Recently, various methods involving light were developed to control actomyosin contraction and relaxation. In actomyosin-dependent apical constriction, Ras homolog family member A (RhoA) localized to the apical surface of the cell is activated by Rho guanine nucleotide exchange factor (RhoGEF), after which active RhoA activates Rho kinase (ROCK). Further, active ROCK phosphorylates MLC and inhibits pMLC dephosphorylation by myosin light chain phosphatase (MLCP) to cause myosin-mediated apical constriction.The molecular mechanism by which actomyosin contraction at the ventral end is resolved during the late neurula stage remains elusive. In the convergent extension movement of the neural tube, the Wnt/PCP pathway regulates actomyosin contraction as an upstream signal.In summary, this study revealed that apical constriction at the dorsal and ventral ends contributes to maintaining closure of the blastopore, and that collapse of the archenteron is caused by the relaxation of contractility at the ventral end of the blastopore. Additional features of the design principles of the blastopore remain to be characterized, and identification of upstream factors is needed. However, our results show that tissue-level opening of the blastopore and fluid excretion can be temporally controlled via actomyosin contraction even in early embryos that have immature functional muscles. As morphogenesis mediated by actomyosin contraction is universally observed in embryogenesis, we could speculate that actomyosin contraction plays an important role in controlling fluid transport in embryos of other animals as well.There are several limitations in the present study. First, the molecular mechanisms of blastopore opening remain to be defined. Identification of upstream signals involved in actomyosin contraction would enhance our understanding of the gating mechanism at the molecular level. Second, the spatiotemporal resolution of actomyosin contraction analysis in the blastopore needs to be further improved using optogenetics and other techniques. Since Y27632 diffuses through tissues, the possibility that Y27632 affects other tissues besides the CBC cannot be completely ruled out. Third, we cannot ignore the possibility that the excision and fixation of embryos may have had secondary effects such as tissue deformation. Time-lapse imaging deep within the live embryos using X-ray image analysissoichiro.kato@riken.jp).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Soichiro Kato . Ovulation was induced by injecting mature females with 600 units of human chorionic gonadotropin . Spawned eggs were fertilized in vitro by mixing with sperm suspended in 1.3x Barth’s medium 2, 1.066 mM MgSO4, 3.12 mM NaHCO3).Adult To label embryonic cells, 10 kDa Alexa Fluor 488-dextran (Alexa 488-dex)  or 10 kDa Alexa Fluor 647-dextran (Alexa 647-dex)   was injected into the two blastomeres at the 2-cell stage. At stage 14, archenteron fluid was visualized by injecting 10 kDa Alexa Fluor 568-dextran (Alexa 568-dex)  . Time-lapse images were acquired using an MVX-10 stereomicroscope  equipped with an EM-CCD camera , a BZ-X710 fluorescence microscope  with a 2× objective , an IX83 microscope (Olympus) equipped with a spinning disk unit , or an EM-CCD camera , and 20× objective . Fluorescence images for fixed embryos were acquired using an SP8 confocal microscope  with a 10× , 20× , or 40×  objective.To examine fluid excretion, Alexa 568-dex was injected into the archenteron of embryos with (VM+) or without the vitelline membrane (VM-) at stage 13–14. The vitelline membrane was removed with forceps at stage 14. Time-lapse images were acquired using an MVX-10 stereomicroscope (Olympus) equipped with an EM-CCD camera . Embryos without the vitelline membrane that had excreted fluid were immediately fixed in trichloroacetic acid solution (TCA) containing 1.85% formaldehyde, after which the developmental stage was analyzed. For embryos with the vitelline membrane, the membrane was removed and the embryos were cultured at 12 °C for 10 min prior to fixation.For early removal of archenteron fluid, the anteroventral archenteron wall was perforated with a glass needle and the hole was maintained for 30 min by repeated insertion of the needle. Although most of the archenteron fluid was excreted, the ventral side of the embryos was gently pushed to ensure complete removal of the fluid. Embryos were cultured until stage 45 and laterality was analyzed by the direction of heart tube and gut coiling.To examine archenteron pressure, 1 μm fluorescent beads  diluted in LCMR (1/200) supplemented with 1% skim-milk  were injected into the archenteron at stage 18. Then, the archenteron wall was perforated with a glass needle and movies were acquired using an MVX-10 stereomicroscope (Olympus) equipped with an EM-CCD camera .To measure archenteron hydrostatic pressure, analog signals from a Micropressure system  were converted to digital signals with an AD converter  and the data was then analyzed with Labchart software . Microelectrodes were made by stretching a glass capillary  with a needle puller (PC-10) and adjusting the tip inner diameter to 7.4 ± 0.8 μm. If the tip diameter is too large, buffer will leak from the needle, and needles having too small tip diameters can become clogged with cellular debris that compromises pressure measurements. The probe tip was coated with silicon  prior to measurement. A probe filled with 3 M NaCl solution equipped with a dial gauge  was connected to a micromanipulator . Probes were calibrated based on changes in hydrostatic pressure by submerging the needle tip to depths of 0 mm and 20 mm. Changes in water depth during pressure measurement were corrected using a calibration curve of the dial gauge value and water pressure changes.g, 1 min), the collected supernatant was diluted 10-fold and used to fill a glass capillary . Ag wires  coated with AgCl were inserted into the capillary and electrical resistance of fluid in the capillary was measured using a digital multimeter  . To create an Arc-buffer having the same electrical resistance as the archenteron, 0.1x Barth’s medium containing various concentrations of NaCl (8.8–100 mM) was diluted 10-fold and the electrical resistance was measured using the same method (n = 5). A calibration curve generated from 0.1x Barth’s medium containing various concentrations of NaCl as a function of electrical resistance was used to determine the Arc-buffer composition. To measure the upper limit of pressure resistance, Arc-PR-buffer  in Arc-buffer), was injected into the archenteron. When water pressure at the same depth was measured with a microelectrode using Arc-buffer or archenteron fluid from stage 18 or 20–21, no significant differences were observed.To examine electrical resistance at stage 18 or 20–21, archenteron fluid was collected from 10 embryos. After debris was removed by centrifugation  into the archenteron were cultured in 0.1x Barth’s medium or Arc-buffer. There was no difference in the timing of fluid excretion for either buffer.To measure archenteron pressure, embryos from which the vitelline membrane was removed at stage 14 were injected with 10 kDa Alexa 568-dex  into the archenteron and then placed laterally (measurement of pressure and pressure resistance) or posteriorly (measurement of pressure after injection of Y27632) in the hollow of an agarose plate filled with Arc-buffer.To measure blastopore pressure resistance, the pressure probe was first inserted into the archenteron to measure fluid pressure. The needle for injecting buffer was then inserted and Arc-PR-buffer was injected continuously until fluid leaked from the blastopore. The pressure in the archenteron at which fluid leaked out was defined as the upper limit of pressure resistance of the blastopore. Buffer was injected into the archenteron at three different injection rates  and no significant difference in Pres was observed among the different rates. Time-lapse images were acquired using an SZX-7 stereomicroscope (Olympus) equipped with a digital camera .Embryos were fixed in a solution of 2.5% glutaraldehyde and 2% paraformaldehyde in 50 mM HEPES buffer, pH7.4, at 4 °C overnight. After primary fixation, the samples were washed three times with 50 mM HEPES buffer (pH 7.4) for 10 min and postfixed with 2% osmium tetroxide solution for 3 h at 4 °C. Then, the samples were washed three times with 50 mM HEPES buffer (pH7.4) and stained with 0.25% aqueous uranyl acetate for 2 h at room temperature. After staining, the samples were washed three times with distilled water for 10 min at room temperature, dehydrated with a graded ethanol series  for 15 min at room temperature, bathed in propylene oxide for 15 min at room temperature, and embedded in epoxy resin  for 48 h at 60 °C. Ultrathin sections were stained with 2% uranyl acetate followed by lead stain solution and analyzed by TEM .To visualize embryonic cells, 10 kDa Alexa 488-dex  was injected into two blastomeres at the 2-cell stage. At stage 13–14, archenteron fluid was labeled by injecting 10 kDa Alexa 568-dex  into the archenteron. A 5 μm-thick, ∼100 μm-wide strip of aluminum foil  coated with 0.1% BSA was inserted into the blastopore of stage 14 embryos with the vitelline membrane removed. To confirm proper insertion, embryos were fixed with 2% TCA and 9.25% formaldehyde and then cleared with benzyl alcohol/benzyl benzoate (BABB) solution. Fluorescence images were acquired using an SP8 confocal microscope with a 10× objective (Leica).Time-lapse images were acquired using a BZ-X710 fluorescence microscope (Keyence) with a 2× objective (Nikon). To prevent flow of culture medium, high-viscosity 0.1x Barth’s medium containing 3% methyl cellulose  was used.A glass bead (approximately 130 μm diameter) made by heating the tip of a glass needle with a gas torch until a bead formed was inserted into the blastopore. Time-lapse images were acquired using an SZX-16 stereomicroscope (Olympus) equipped with a digital camera . Blastopore closure after removing the glass bead was analyzed in ImageJ using the shading of the hole as an indicator.To visualize embryonic cells, 10 kDa Alexa 488-dex  was injected into two blastomeres at the 2-cell stage. To release the CBC pushing force, the tissue around the blastopore was excised, leaving the dorsal side intact. Then, the ventral side of the blastopore slit was cut and embryos were fixed immediately (within 1 min) in 2% TCA and 1.85% formaldehyde. Fixed embryos were dehydrated in methanol and cleared with BABB solution. Fluorescence images were acquired using an SP8 confocal microscope with a 10× objective (Leica).To examine the CBC shape without incision, 2% TCA and 1.85% formaldehyde were continuously injected into the archenteron at the excretion stage. Fixed and cleared embryos were analyzed using an SP8 confocal microscope with a 10× objective (Leica).To analyze the shape of the CBC with the ventral end of the blastopore resected, an optical coronal section corresponding to the center of the blastopore slit was obtained from embryos cleared with BABB. The fluorescence images were then binarized using ImageJ to obtain the outline of the CBC. For control embryos and embryos having ventral ends that were not cut, the outline was obtained manually. The direction perpendicular and parallel to the slit was set as the x axis and y axis, respectively. The length with the longest y axis distance was defined as the thickness of the CBC F.h and y were obtained from the following equation.x and y indicate the x and y coordinates of the respective points.For curvature analysis, the point on the outline that fell midpoint between the maximum and minimum values of the y axis was set to A. Further, points on the outline ±32 μm away from point A in the y axis direction were set as B (+32 μm) and C (- 32 μm). Curvature was calculated according to the following equation:The length of the contact surface of the slit was defined as the length of a straight line connecting the endpoints of the contact surface determined using ImageJ.To make a force measurement probe, a tungsten wire  was inserted into the tip of a bent glass needle and fixed with UV resin . To calibrate the force measurement probe, the tungsten wire was placed horizontally and 20 nL silicon oil  was repeatedly added to the tip of the wire up to a volume of 400 nL. The probe tip displacement was obtained using an SZX-7 stereomicroscope  equipped with a digital camera  and the values were used to make a force-displacement curve.To measure the force required to open the blastopore, embryos with archenteron fluid labeled with phenol red solution were placed in the hollow of an agarose plate with the posterior side facing up. The pulling probe and measurement probe were inserted separately on both sides of the slit, and only the pulling probe was moved horizontally until the blastopore opened. The opening of the blastopore was detected by leakage of archenteron fluid labeled with phenol red. Time-lapse images were acquired using an SZX-16 stereomicroscope (Olympus) equipped with a digital camera  and the force required to open the blastopore was calculated.To measure CBC stiffness, embryos were placed in the hollow of an agarose plate with the posterior side facing up. The pulling and measurement probes were both inserted into one side of the slit, and only the pulling probe was moved to stretch the CBC in the direction of the slit long axis. Time-lapse images were acquired using an SZX-7 stereomicroscope (Olympus) equipped with a digital camera  and the force required to stretch the CBC was calculated.To visualize cells, 10 kDa Alexa 647-dex  was injected into the two blastomeres at the 2-cell stage. At stage 14, 10 kDa Alexa 568-dex  was injected into the archenteron, after which the vitelline membrane was removed. Embryos were then fixed at the desired stage in 2% TCA for 20 min followed by washing three times in PBS-T buffer (0.3% polyoxyethylene (10) octylphenyl ether in PBS). Thereafter, the peripheral tissue of the blastopore was excised and blocked with blocking buffer (PBS-T buffer containing 10% FBS). Fragmented tissues were then incubated overnight at 4 °C with anti-phosphorylated myosin light chain  and anti-E-cadherin  as primary antibodies that were diluted in Can Get Signal B solution . Tissues were then washed with PBS-T (3 × 30 min) and incubated with Alexa Fluor 488 anti-rabbit IgG  or Alexa Fluor 546 anti-mouse IgG  as secondary antibodies diluted in Can Get Signal B solution. After washing tissues with PBS-T buffer, tissues were dehydrated in methanol and cleared with BABB solution. Fluorescent images were acquired using an SP8 confocal microscope with a 40× objective (Leica).To analyze apical constriction in the mediolateral direction, serial optical transverse sections were acquired using an SP8 confocal microscope with a 40× objective (Leica). To analyze apical constriction in the anteroposterior direction, sagittal sections were reconstructed using serial optical transverse sections. The apical width and apical-basal length of cells in the ventral end of the blastopore were measured and the ratio between them was calculated.For time-lapse imaging of embryos injected with Y27632, 10 kDa Alexa 647-dex was injected into two blastomeres at the 2-cell stage to label embryonic cells. At stage 14, 10 kDa Alexa 568-dex was injected into the archenteron, and the vitelline membrane was removed. At stage 18, 1 nL Y27632 solution  and Alexa Fluor 488 hydrazide ) or Mock solution (LCMR containing 12.5 ng Alexa Fluor 488 hydrazide) was injected into the CBC tissue at the blastopore ventral region. Time-lapse images were acquired 1 min after injection of Y27632 or Mock solution using an MVX-10 stereomicroscope (Olympus) equipped with an EM-CCD camera . Embryos that excreted archenteron fluid within 10 min of injection were counted and further cultured until stage 20 to assess developmental abnormalities.To examine the effect of Y27632 injection on myosin phosphorylation, 10 kDa Alexa 647-dex was injected into two blastomeres at the 2-cell stage. After injection of Y27632 solution or Mock solution without Alexa Fluor 488 hydrazide, embryos were fixed and used for immunofluorescence staining with anti-pMLC antibody.To assess the effect of Y27632 injection on archenteron hydrostatic pressure, archenteron fluid was first labeled with 10 nL phenol red solution. Embryos were then set in the hollow of agarose plate with the blastopore facing upward. Y27632 or mock solution was injected while measuring pressure. Time-lapse images were acquired using an SZX-7 stereomicroscope equipped with a digital camera (ILCE-QX1). Blastopore opening was detected by excretion of phenol red-labeled fluid.No statistical methods were used to predetermine sample size. Each experiment was performed at least three times. All statistical analyses were performed using GraphPad Prism 8 software . All data were evaluated for normal distribution and equal distribution by normality test and F-test, respectively. For parametric data, an unpaired t test F or ordi