Hydrocephalus constitutes a complex neurological condition of heterogeneous origin characterized by excessive cerebrospinal fluid (CSF) accumulation within the brain ventricles. The condition may dangerously elevate the intracranial pressure (ICP) and cause severe neurological impairments. Pharmacotherapies are currently unavailable and treatment options remain limited to surgical CSF diversion, which follows from our incomplete understanding of the hydrocephalus pathogenesis. Here, we aimed to elucidate the molecular mechanisms underlying development of hydrocephalus in spontaneously hypertensive rats (SHRs), which develop non-obstructive hydrocephalus without the need for surgical induction.Magnetic resonance imaging was employed to delineate brain and CSF volumes in SHRs and control Wistar-Kyoto (WKY) rats. Brain water content was determined from wet and dry brain weights. CSF dynamics related to hydrocephalus formation in SHRs were explored in vivo by quantifying CSF production rates, ICP, and CSF outflow resistance. Associated choroid plexus alterations were elucidated with immunofluorescence, western blotting, and through use of an ex vivo radio-isotope flux assay.+/K+/2Cl− cotransporter NKCC1, a key contributor to choroid plexus CSF secretion. However, neither CSF production rate, ICP, nor CSF outflow resistance appeared elevated in SHRs when compared to WKY rats.SHRs displayed brain water accumulation and enlarged lateral ventricles, in part compensated for by a smaller brain volume. The SHR choroid plexus demonstrated increased phosphorylation of the NaHydrocephalus development in SHRs does not associate with elevated ICP and does not require increased CSF secretion or inefficient CSF drainage. SHR hydrocephalus thus represents a type of hydrocephalus that is not life threatening and that occurs by unknown disturbances to the CSF dynamics.The online version contains supplementary material available at 10.1186/s12987-023-00448-x. Hydrocephalus encompasses a heterogeneous group of neurological conditions characterized by an abnormal accumulation of cerebrospinal fluid (CSF) within the cerebral ventricles. It can occur in presence or absence of changes in intracranial pressure (ICP) . HydroceThe present study, therefore, sought to elucidate whether development of hydrocephalus in SHRs is associated with abnormal CSF dynamics such as an increased CSF secretion or an elevated CSF outflow resistance. Elucidation of the molecular mechanisms underlying hydrocephalus formation in SHRs and delineation of the associated CSF dynamics may provide novel insight into the pathogenesis of hydrocephalus.Nine-week-old male SHRs and WKY rats  were used in the study. Animals were housed in a temperature-controlled room with a 12 h:12 h light-dark cycle and had free access to a standard rodent pellet diet and tap water. Animal experiments performed at University of Copenhagen conformed to the legislations for animal protection and care in the European Community Council Directive (2010/63/EU) and were approved by the Danish Animal Experiments Inspectorate (License no. 2018-15-0201-01595). Animal experiments performed at University of Michigan were approved by the University of Michigan Committee on the Use and Care of Animals and followed the Guide for The Care and Use of Laboratory Animals . The majority of the experiments were conducted with six rats of each type for each experimental paradigm , whereas five rats of each kind (based on earlier results) were deemed sufficient for magnetic resonance imaging, brain water quantification, and isotope flux experiments. For Western blotting, only four rats of each type were available for the experiments.2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 25 NaHCO3, pH adjusted with 95% O2 and 5% CO2). Ex vivo experiments (86Rb+ efflux assays) were conducted in HEPES-containing aCSF  120 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 17 Na-HEPES, adjusted to pH 7.4 with NaOH/HCl). HEPES-aCSF was selected, as continuous gas-equilibration of the HCO3− buffered aCSF is technically challenging with small quantities of isotope-containing test solutions, and earlier experiments revealed similar 86Rb+ efflux rates whether the experiments were conducted in heated and gas-equilibrated aCSF or HEPES-aCSF [In vivo experiments were conducted with heated (37°C) and gas-equilibrated artificial CSF  127 NaCl, 2.5 KCl, 2.5 CaCl− 1 humidified air mixed with 0.1 l min− 1 O2. The ventilation was adjusted to result in 4.5 ± 0.5 kPa blood pCO2 according to exhaled end tidal CO2 measured with a capnograph . Blood samples were collected from anesthetized and mechanically ventilated rats through insertion of a femoral artery catheter during the ICP measurements (described below). The blood gas content was determined with an ABL80 blood gas analyzer (Radiometer).Xylazine and ketamine anesthesia was used for the surgeries . Animal body temperature was maintained at 37 °C by a homeothermic monitoring system (Harvard Apparatus). Rats were tracheostomized and mechanically ventilated with the VentElite system (Harvard Apparatus), inhaling 0.9 l minThe rat brain was isolated immediately following decapitation, and the wet brain weight determined on a pre-weighed porcelain evaporation beaker. The brain was afterwards placed in a pre-heated oven at 100 °C and left to dry for 3 days to a constant mass. The dry brain was weighed, and the brain water content determined using the equation: (wet weight - dry weight)/dry weight.2-weighted 2D rapid acquisition with relaxation enhancement (2D-RARE) for reference spatial planning. The following settings were applied: repetition time (TR) = 10,000 ms, effective echo time (TE) = 60 ms, number of averaging (NA) = 3, RareFactor = 16, slice thickness = 500 μm, in-plane resolution = 137 × 137 μm, 50 coronal slices, total acquisition time (TA) = 8 min. For high resolution CSF volumetry, a 3D constructive interference steady-state sequence (3D-CISS) [Anesthetized rats underwent MRI in a 9.4 Tesla preclinical horizontal bore scanner  equipped with a 240 mT/m gradient coil  at the Preclinical MRI Core Facility, University of Copenhagen. During the scan, anesthesia was maintained at 1-1.5% isoflurane in a 1/1 mixture of air/oxygen. The MR scanner was interfaced to a Bruker Avance III console and controlled by Paravision 6.1 software (Bruker). MRI was performed with an 86 mm-inner-diameter volume resonator and a 4-channel surface quadrature array receiver coil. Animal body temperature was maintained at 37 ± 0.5 °C with a thermostatically controlled waterbed and the respiratory rate closely monitored with an MR compatible monitoring system . The MRI protocol consisted of T3D-CISS) , 27 imag3D-CISS) , 29. For™ with DAPI . Images for fluorescence quantification were obtained with an Olympus fluorescence microscope  using the cellSense Software (v 1.18), while confocal microscopy  was employed to verify antibody presence in the correct choroid plexus membrane. Images were taken with the same contrast settings and exposure times. The primary antibodies used were rabbit anti-NKCC1  and rabbit anti-phosphorylated NKCC1 . The secondary antibody used was Alexa Fluor 594 donkey anti-rabbit . Negative controls were made by omission of the primary antibodies and resulted in absence of any fluorescence (data not shown). Fluorescence quantification was conducted in Image J (v 1.52a) [Brain sections were immunostained with the primary antibodies overnight at 4°C, followed by incubation with the secondary antibody for 2 h at room temperature. Sections were nuclei stained using Fluoroshieldv 1.52a) . For eacChoroid plexus samples were immersed in Western blot sample buffer , sonicated, and the protein content determined with the Bio-Rad protein assay kit (Hercules). Then, 50 µg protein samples were loaded on a 4–20% SDS-PAGE gel. The protein samples were transferred onto a Hybond-C PURE Nitrocellulose membrane (Amersham) and blocked with 5% non-fat milk (LabScientific Inc) at room temperature overnight. After blocking, membranes were incubated with the primary antibodies overnight at 4°C. Membranes were then immunoprobed with the secondary antibodies for 1 h at room temperature. The antigen-antibody complexes were visualized with the ECL chemiluminescence system (Amersham) and exposed to a Kodak X-OMAT film. The relative densities of bands were analyzed with ImageJ (v 1.52a). The primary antibodies were rabbit anti-phosphorylated NKCC1  and mouse β-actin . The second antibodies used as 1:2000 were goat-anti rabbit  and goat-anti mouse . ECL chemiluminescence system (Amersham) and Kodak X-OMAT film (Sigma) were used for final visualization. The raw western blot image is attached as Additional file 1, Fig. 86Rb+  and 3H-mannitol . 3H-mannitol does not enter the choroid plexus epithelial cells and serves as an extracellular marker [86Rb+ activity was corrected for extracellular background using 3H-mannitol. Data are shown as the natural logarithm of the 86Rb+ activity at time each point (AT) normalized to the initial 86Rb+ activity (A0) as a function of time. The slope from linear regression analysis was used to determine the 86Rb+ efflux rate constant (min− 1) [Rats were anesthetized with an intraperitoneal injection of ketamine and xylazine  and euthanized by decapitation. The brains were immediately isolated and immersed in ice-cold HEPES-aCSF for 10 min, followed by separation of the two hemispheres and isolation of the choroid plexus from the lateral ventricles. After acute isolation, the lateral choroid plexus was allowed to recover for 10 min in 37 °C HEPES-aCSF prior to 10 min of incubation in an isotope solution containing: (min− 1) . The phaCSF secretion rates were quantified with the ventriculo-cisternal perfusion technique –36. Anesp = CSF secretion rate (µl/min), ri = infusion rate (µl/min), Ci = fluorescence of inflow solution (a.u.), Co = fluorescence of outflow solution (a.u.), calculated based on stable time intervals from 60 to 90 min.where VAnesthetized and mechanically ventilated rats were placed in a stereotactic frame and a 3.6 mm diameter cranial window was drilled with care not to damage the dura. The epidural cannula  was filled with HEPES-aCSF, connected to a pressure transducer APT300 and transducer amplifier module TAM-A (Hugo Sachs Elektronik), and secured to the skull with dental resin cement . On the contralateral side of the skull, a 0.5 mm burr hole was drilled , and a 4.5 mm brain infusion cannula  was placed into the lateral ventricle. 5 µl HEPES-aCSF was injected through the epidural ICP cannula to ensure a continuous fluid column between the dura and the probe before starting the baseline ICP recording. After 20 min, heated and gas-equilibrated aCSF was infused at 5, 10, 15 and 20 µl/min for 10 min each. The CSF outflow resistance for each infusion speed was then calculated as :\documeninf is the average ICP during the last 3 min of the infusion given in mmHg, ICPbase is the baseline ICP in mmHg, and Vinf is the infusion speed in µl/min. Rout for each rate of infusion was calculated and averaged across all infusion rates for each rat to determine CSF outflow resistance.Where ICPP < 0.05 was considered statistically significant. All data are presented as mean ± standard deviation and n corresponds to the number of individual animals.Data analysis and statistical tests were conducted in GraphPad Prism . Data were tested for normality with the Shapiro-Wilk test. For normally distributed data, an unpaired two-tailed t-test was conducted. For non-normally distributed data, a Mann-Whitney test was conducted. For more than two groups, an ordinary one-way ANOVA followed by Sidak’s multiple comparisons test was conducted. Statistical tests are indicated in the figure legend. P < 0.05), which amounted to < 1% difference in brain water percentage  in SHRs when compared to that of WKY rats, the difference was not significant (0.23 ± 0.05 min− 1 in SHRs vs. 0.18 ± 0.03 min− 1 in WKY rats, n = 5, P = 0.10, Fig. To determine whether the increased expression of pNKCC1 in the SHR choroid plexus translated to an increased NKCC1 transport activity, the transport rate of NKCC1 was quantified with an ex vivo radio-isotope based transport assay in choroid plexus acutely isolated from SHRs and WKY rats. In this assay, radioactive ime Fig. a. The 86P = 0.75, Fig. P = 0.95, Fig. To resolve whether the ventricular enlargement and excessive brain water accumulation observed in SHRs originated from an increased CSF secretion, possibly mediated by the subtle alterations in NKCC1 activation, we quantified the CSF secretion rate in anesthetized and mechanically ventilated SHRs and WKY rats with the ventriculo-cisternal perfusion technique –36. HereHere, we demonstrate that development of hydrocephalus in SHRs is associated with enlargement of the lateral ventricles, brain water accumulation, and choroid plexus transporter alterations. However, we found no evidence of altered CSF dynamics as evident by similar CSF production rates, ICPs, and CSF outflow resistances in SHRs and their normotensive control counterparts, the WKY rats. The pathophysiological mechanism underlying development of hydrocephalus in SHRs thus remains unresolved.Progressive ventricular enlargement in SHRs has been documented since the 1980s , 11. In 2 levels and cerebral blood volume. Therefore, in this study, the rats were mechanically ventilated during the ICP recordings to overcome potential confounding factors related to uncontrolled breathing. Different methodological approaches thus demonstrate that CSF accumulation in SHRs does not elevate the ICP. Although the CSF accumulation observed in patients with hydrocephalus often translates to a clinically elevated ICP [The CSF accumulation, however, did not translate to an elevated ICP, as quantified through an epidural pressure probe. This finding is in agreement with previous reports , 42, in ated ICP , these tated ICP  and by pated ICP . The latated ICP , 48–50, ated ICP . To our ated ICP –14. The ated ICP . Howeverated ICP , 53, theCSF accumulation might arise from increased CSF secretion. However, no evidence of increased CSF secretion in SHRs was found in the present study, albeit increased levels of phosphorylated NKCC1 were observed in the SHR choroid plexus. Water translocation through NKCC1 normally constitutes ~ 50% of the total CSF secretion in mice, rats, and dogs , 36, 41 The CSF accumulation observed in SHRs could, alternatively, originate from inefficient CSF drainage, thereby promoting accumulation of CSF. To the best of our knowledge, the present study is the first to quantify the CSF outflow resistance in SHRs in an attempt to uncover whether inefficient CSF drainage could underlie development of hydrocephalus in these rats. However, we observed similar CSF outflow resistances in SHRs and WKY rats, which implies no drainage deficiency in SHRs. With the undisturbed CSF secretion rate and CSF drainage capacity, the underlying molecular mechanisms supporting the SHR ventriculomegaly remain unresolved.Analysis of blood gas and electrolyte content in SHRs and WKY rats provided no further clues to the disease pathogenesis, except for lower blood glucose levels in SHRs, which could be indicative of an altered metabolic activity in these rats. Notably, all experiments in the present study were conducted using nine-week-old rats, an age at which hydrocephalus already appears pronounced in SHRs. The pathogenic alterations underlying development of hydrocephalus in SHRs could potentially take place at an earlier age, possibly even prior to the onset of hydrocephalus. Hence, we cannot rule out that the CSF secretion rate or the CSF outflow resistance may be altered at an earlier age in SHRs, which may contribute to the disease pathogenesis. Hydrocephalus development in SHRs may, alternatively, be genetically determined as pharmacologically induced reduction of the SHR blood pressure fails to attenuate the observed ventriculomegaly , or it mIn conclusion, SHRs display ventricular enlargement and brain water accumulation with a striking absence of ICP elevation, possibly due to compensatory brain tissue volume reduction. The increased brain water content did not arise through increased CSF secretion nor through inefficient CSF drainage, and its origin therefore remains unresolved. Future revelation of the molecular mechanisms underlying SHR ventriculomegaly could aid in unravelling the etiology of pathologies such as normal pressure hydrocephalus, in which patients also display ventriculomegaly with no ICP elevation. Despite their normal ICP, these patients often benefit from ventricular shunt placement –62, suggBelow is the link to the electronic supplementary material.Additional File 1: CSF volumes, western blotAdditional File 2: Blood gas analysis


Pm3+) afforded donor radii (rD) that allow for the prediction of Ln-donor distancesregardless of the nature of the rare-earth cation and its oxidationstate. This set of donor radii can be used to rationalize structuraldata and identify particularly weak or strong interactions, whichhas important implications in the understanding of the stability andreactivity of complexes of these metal ions. A few cases of incorrectatom assignments in X-ray structures were also identified using thederived rD values.We present a setof donor radii for the rare-earth cations obtainedfrom the analysis of structural data available in the Cambridge StructuralDatabase (CSD). Theoretical calculations using density functionaltheory (DFT) and wave function approaches (NEVPT2) demonstrate thatthe Ln-donor distances can be broken down into contributions of thecation and the donor atom, with the minimum in electron density (ρ)that defines the position of  critical points correspondingwell with Shannon’s crystal radii (CR). Subsequent linear fitsof the experimental bond distances for all rare earth cations (except  Lanthanide-donor distances can be estimated by using crystalradii and donor radii, which were obtained from the simultaneous analysisof a large number of X-ray structures of lanthanide complexes. Z = 57) to Lu (Z = 71), whose coordination chemistry is dominated by thetrivalent oxidation state.1 The Ln(III)ions are characterized by their high hydration energies3 and their hard character according to Pearson’s classification.4 Thus, stable complexation of the Ln(III) ionsis generally achieved in aqueous media with hard polyaminopolycarboxilateligands. The high coordination numbers adopted by these ions (often8–9)6 make octadentate ligands such as H5DTPA7 and H4DOTA,8 particularly useful for stable complexation inaqueous media  complexes in magneticresonance imaging11 and a DOTA derivativein radiotherapy using the β-emitter 177Lu-radionuclide.12 Some Ln(III) ions are also widely used as luminescenttags for bioanalytical purposes, in particular Eu(III) and Tb(III)complexes, which emit in the visible region of the spectrum.14 Luminescence upconversion has also been recently achieved in molecularLn(III) complexes.16 Furthermore, the magnetic propertiesof Ln(III) molecular complexes are currently receiving a great dealof attention due to their behavior as single-molecule magnets.18The lanthanide series comprises a verycoherent group of elementsof the periodic table from La  currently contains around450 structures of Ln(III) complexes with these families of ligands.The structures reported contain a wide variety of donor groups appendedto the cyclic structure of cyclen or the linear diethylenetriamineunit of H5DTPA. This rich source of structural data providesa unique opportunity to rationalize the coordination chemistry ofthe Ln(III) ions.The applications described above triggered intense researcheffortsdevoted to the preparation of a wide variety of H19 These ionic radiiwere obtained by analyzing the M–O and M–F distancesfrom oxides and fluorides. Refined values of Shannon’s radii,as well as a new set of ionic radii for uncommon combinations of CNsand oxidation states, were obtained recently using machine learning.20 Ionic radii for Ln(III) ions derived from EXAFSdata in solution are in good agreement with Shannon’s radii.21 In the case of transition metal ions, ionicradii are also affected by the electronic spin as high- and low-spinconfigurations are characterized by different radii and covalency.As mentioned previously, the Ln(III) ions are hard Lewis acids accordingto Pearson’s classification, and the Ln(III)-ligand interactionsare essentially determined by electrostatic and steric factors. Thus,the CN and coordination polyhedron in a particular Ln(III) complexare mainly dictated by the nature and topology of the ligand and thesize of the metal ion. The lanthanide series is characterized by asmooth contraction in ionic radii on increasing atomic number, whichamounts to only ∼15%.22 As a result,Ln(III) complexes often form an isostructural series of complexesin which the Ln(III)-donor distances progressively decrease acrossthe series. However, abrupt changes in coordination number and coordinationpolyhedron have also been observed.23 Nevertheless,the Ln(III) ions certainly comprise the most coherent family of metalions across the periodic table in relation to their coordination properties.Recent work suggests that a linear variation of Ln-donor distancesacross the series is more common than a curved dependence.25The ionic radii of the Ln(III) ions were definedby Shannon inhis seminal paper for coordination numbers (CNs) 8 and 9, and in somecases, 6, 7, and 10.26 Indeed, covalence significantly affects interatomic distances, evenin Ln(III) compounds.27 As a result, ionicradii often do not provide realistic estimates of the size and shapesof ions.28 In an attempt to circumventthis problem, some authors derived radii from the distribution ofelectron density,31 which is an observable that can be measured experimentallyor calculated using quantum mechanical calculations. Nevertheless,ionic radii remain a very useful tool of widespread use by chemists,material scientists, and mineralogists to rationalize structural data.In the case of covalent compounds, bond lengths could be predictedto a surprisingly good accuracy as the sum of two atomic radii, providingthe bond is not too ionic, the corresponding coordination numbersare not very different and the concerning bonds do not have multiplebond character .34The calculation of Shannon’s effective ionic radiirelieson the assumption that interatomic distances can be approximated asthe sum of cation and anion radii, which change with the coordinationnumber. Some authors, who argued that it is doubtful that an unambiguousseparation of a bond into a cation and an anion part can be reached,have heavily criticized this assumption.4– derivativescontaining amide pendants were found to possess CN 10 in the solidstate, where solvent molecules and counterions complete the metalcoordination environment.36 A DTPA-bis-amide La(III)complex with CN 11 was also reported.37 All the remaining Ln(III) DOTA4– and DTPA5– derivatives show CNs 8 and 9, with a few exceptionsof seven-coordinate complexes. The latter are Tb(III), Dy(III), andEr(III) complexes with cyclen-based ligands that contain bulky phenolatependant arms.39 The relative abundance of CN8 increases across the lanthanide series as the ionic radius of themetal ion decreases, as would be expected. The concept of the coordinationnumber has also been a matter of debate in some cases. For instance,Ln–OH2 distances in some Ln(III) DOTA4– derivatives increase along the second half of the lanthanide seriesas the size of the metal ion contracts,40 which led to estimates of noninteger coordination numbers.41From the perspective of coordination chemistry in aqueousmedia,most Ln(III) complexes display coordination numbers 8 and 9, thoughsome examples of complexes with coordination numbers 7, 10, and even11 have been described 1. Two La4– derivativeswith oxidation states other than +3 have also been also described.Indeed, four structures of eight-coordinate Eu(II) complexes havebeen reported. Of these structures, three of them are cyclen derivativescontaining phosphonate, phosphinate, or amide pendant arms,43 while the last structure is a DTPA5– complex.44 Recently, the Ce(IV) complexes of DOTA4– and the analogue with methylenephosphonate arms (DOTP8–) were also characterized using X-ray diffraction.45A few structures of lanthanide DOTA4– andDTPA5– derivatives to obtain radii for differentdonor atom types. These donor radii were obtained from the crystalradii determined by Shannon for CNs 8 and 9, assuming that the Ln(III)-donordistances can be approximated by the sum of Ln(III) and donor radii.We have chosen the set of expanded Shannon’s radii20 for our study due to its widespread use in coordination chemistry.The donor radii reported here will provide key information to analyzethe structures of Ln(III) complexes, particularly to identify particularlystrong or weak interactions. This may have very important implicationsin understanding not only the stereochemical properties of Ln(III)complexes but also their stability and reactivity. For instance, particularlyweak Ln(III)–Owater interactions, associated withthe coordination of a water ligand at a sterically hindered cappingposition, were correlated to fast water exchange rates,47 an important parameter to be controlled to optimize the efficiencyof MRI contrast agents. Long Ln(III)–O distances involvingcarboxylate groups were also correlated to fast dissociation ratesfollowing the acid-catalyzed mechanism.48 We have also extended our analysis to Y(III) and Sc(III), whichtogether with the lanthanides form the rare earths. The coordinationchemistry of Y(III) is very similar to that of the lanthanides49 and is relevant for the development of radiopharmaceuticalsfor both imaging and therapy.50 The coordinationchemistry of Sc(III) with polyaminocarboxylate ligands is currentlyin its infancy, but interest is growing due to the potential of the 44Sc-radionuclide in PET imaging,51 and its potential as a diagnostic match of β– emitters 177Lu- and 90Y-radionuclides.12In this work, we sought to take advantage of thelarge body ofstructural data reported for Ln(III) DOTA4– andDTPA5– derivatives, although the number of hitsvaries significantly across the lanthanide series. A total of 443structures of DOTA4– and DTPA5– derivatives containing Ln(III), Y(III), or Sc(III) ions have beenanalyzed, among which 83% correspond to DOTA4– derivatives.The number of DTPA5– derivatives reported for Gd(III)(31%) is, however, significantly higher than for other lanthanides,a situation that is reflected in the application of these complexesas MRI contrast agents in clinical practice. A few additional structurescontaining Ln–H and Ln–C bonds have been excluded fromthis study. The lanthanides with the highest number of hits are Eu(21%), Gd (19%), Yb(10%), and Tb (8%), which reflects the numerousinvestigations related to the photophysical properties of Eu(III)and Tb(III) complexes and the development of Gd(III)-based contrastagents. These three lanthanides sit at the center of the lanthanideseries, with Eu and Tb flanking Gd. Thus, the structural data availableare far from being uniformly spread along the series. A significantnumber of hits were also obtained for Dy(7%). However, very few structureswere reported for lanthanides such as Tm  and Sc  and amine N atoms (NA) are the most common donoratoms in the family of complexes analyzed here. The Gd–NA and Gd–OC distances for complexes withCN 9 display fairly good Gaussian distributions centered at 2.667and 2.378 Å, respectively  nine-coordinate complexes, whichreflects the contraction in bond distances as a result of lanthanidecontraction. These shifts of the maxima by ∼0.07 Å areclose to that predicted by the corresponding difference in the (revised)Shannon’s radii (0.065 Å). Decreasing the CN from 9 to8 results in a further shift of the maxima of the Gaussian distributionsto 2.555 and 2.264 Å, which represents a contraction of ∼0.05Å, again close to the variation expected according to the ionicradius (0.057 Å).O atoms of carboxylate groups  complex stability estimated recently.10 The analysis of the electron density at thecritical points of the concerned bonds also suggests that Gd–NA bonds are weaker than Gd–OC bonds.53 It has been also noted that Ln–N bondsare characterized by rather shallow potential energy surfaces, whileLn-OC bonds display potential energy surfaces with deeperenergy minima.54 Indeed, some of the Gd–NA distances shown in 55 and one can reasonably associate these distancesto weakinteractions.The half-width at halfheight of the Gaussian distributions ofGd–N30 Chemical bonds arecharacterized by the presence of  critical points (CPs)at coordinate rc, where the first derivativeof the electron density vanishes (∇ρ(rc) = 0). At this point, the electron density is a minimumalong the internuclear axis and a maximum in the plane perpendicularto that axis. A  CP is situated on the interatomic surfaceseparating the basins of two neighboring atoms.56 Thus, prior to the estimation of donor radii, we analyzedthe electron density along the Ln-donor axes of the selected systems.Theseparation of theLn-donor distances into contributions of the metal ion and the donoratom should ideally reflect the shape of the electron density (ρ)along the bond axis. systems3. For th65 The value of ρ then decreases along theinternuclear axis and reaches a minimum, which corresponds to theposition of the  CPs. 2O)]2–. One can notice that the five Gd–OC distancesare quite similar, while more significant differences are observedfor the three Gd–NA distances. All Gd–OC bonds display very similar positions of the CPs at distancesof ∼1.275 Å from the metal ion. The minimum of the electrondensity deviates significantly from the ionic radius (IR) of Gd(III)(1.107 Å for CN 9).20 However, theposition of the CPs agrees rather well with the crystal radii (CR)of Gd(III) (1.247 Å for CN 9). CR were introduced by Fumi66 and are considered to correspond more closelyto the size of an atom (or ion) in a solid, as pointed out by Shannonin his seminal paper.19 CR differ fromIR by a constant factor, with the former being 0.14 Å longerthan the latter. IR were introduced to comply with the 1.40 Åradius for O2– (CN = 6) proposed by Pauling,67 while CR use a radius of 1.26 Å for theanion. The positions of the critical points for Gd–NA and Gd–OW bonds  are further apart from the Gd(III) ion comparedwith Gd–OC bonds, deviating from the CR by ca. 0.14Å. The values of ρ at the CPs are lower for Gd–NA than Gd–OC bonds, as observed previously.53 It is worth noting that calculations performedat the NEVPT2 level provide nearly identical ρ values alongthe Gd-donor axes .The highest values of ρ are centered on the nuclear positionsof the metal ion and, to a lesser extent, on the donor atom. Thisreflects that most of the electron density sits around the nuclearpositions, which act as attractors in the gradient vector field ∇ρ.C bonds for [Ln(DTPA)(H2O)]2– complexes. This suggeststhat the Ln–OC bonds can be broken down into contributionsof the metal ion radius and the radius of the donor atom. The positionsof the CPs are in very good agreement with those marked by the CR.Similar conclusions can be drawn by analyzing the Ln–NA bonds .Acomparison of the electron densities along the Ln–Oomplexes3b shows 3+ and [La(DOTAM)(CF3SO3)(EtOH)]3+ complexes, in which the La(III) ions display CNs 8 and 10, respectively(AM bonds(OAM = amide oxygen atom) are indeed affected by the CN,with the minima obtained for CN 10 (∼1.371 Å) and 8 (∼1.320Å), correlating well with the corresponding CR .IR andthus CR are defined as specific coordination numbers. Totest whether the shape of ρ across the bond path supports thisassumption, we carried out calculations on the [La(DOTAM)]ectively3c. Calcu4– and DTPA5– derivatives were characterized by using X-ray diffraction.The electron densities calculated for the Eu–OPHOS bonds in [EuIII(DOTP)]5– and [EuII(DOTP)]6– show that the position of theCPs shifts according to what would be expected considering the CRof Eu(II) (1.389 Å) and Eu(III) (1.206 Å) and the averageEu–OPHOS distances .A similar conclusion can be reached by analyzing the [CeIII(DOTA)(H2O)]− and [CeIV(DOTA)(H2O)] systems .The size of a metal ion isobviously affected by its charge. Thecoordination chemistry of the rare earths is largely dominated bythe trivalent oxidation state. However, complexes of Ce(IV) and Eu(II)with DOTAA bonds are characterizedby rather shallow minima, so that the position of the CP and thatmarked by the CR are characterized by similar values of ρ. Wealso note that the coordination geometry is likely to have a minoreffect on the position of the CPs, as evidenced by the results obtainedfor the capped square antiprismatic (SAP) and capped twisted squareantiprismatic (TSAP) isomers of [CeIII(DOTA)(H2O)]− .The results of our calculations support that the Ln-donor bondscan be indeed divided into a contribution from the metal ion, whichcan be approximated by CR, and a contribution from the donor atom.This approach has of course some limitations, as the size of the metalion determined by the electron density changes depending on the natureof the donor atom, as illustrated in 4– and DTPA5– derivativesusing the following expression:dLn–D representsthe Ln3+-donor distance, rD is the radius of a given donor atom, and CRLn is thecrystal radius of the Ln3+ ion. In ouranalysis, we used CR obtained after adding a value of 0.14 Åto the refined Shannon’s radii reported recently. Thus, a plotof dLn–D versus CRLn should provide a linear trend whose intercept corresponds to rD when the slope is fixed to 1. 2O)]− complex, whichcontains a long Er–OH2 distance of 2.732 Å,confirm the presence of a  CP along the Er–O internuclearaxis .Having shown that the Ln-donor distancescan be reasonably separated into metal ion and donor contributions,we sought to estimate radii for different donor groups commonly presentin DOTArD values are obtained from the simultaneous fit of bond distancesobserved for all rare-earth complexes, excluding radioactive Pm. Thus, this approach provides a clear advantage overthe simple analysis of bond distances for a particular Ln3+ ion, Sc3+ or Y3+. Indeed, this approach allowsfor accumulating enough bond distance data for a specific donor atom,even if none or a few values are available for specific Ln3+ ions. Furthermore, the fitted rD valuesare universally valid for all earth rare cations in any oxidationstate, with the only limitation being having reliable CRLn data. This provides a very useful tool to analyze complexes withmetal ions  or oxidationstates (Eu2+ and Ce4+), for which limited structuraldata are currently available.The advantage of this analysis is that the Ln, which varies depending on the rare-earthelement, its oxidation state, and coordination number. The lowestvalue of CRLn corresponds to Sc3+ with CN =8 (1.011 Å), while the upper limit is given by La3+ with CN = 11 (1.459 Å). Linear plots for other donor atomsare shown in Figures S6–S13). Thelinear fits of the data afforded the rD values listed in py)Cl]3+ complexesreported by Wada,69 in which Eu–Cldistances of ∼2.2 Å were reported -1,4,7,10-tetraazacyclododecane, 6– and BF4–, which made us suspect that a F atom was assigned incorrectly toa chloride atom. It is worth noting that terminal Ln–F bondsare significantly shorter than bridging F–Ln–F bonds.64 Thus, the value of rD reported in The linearplot obtained for chloride deserves further comment5. In spi70 using the chloride salt. Addition of KPF6 to an aqueoussolution of the complex afforded single crystals with the formula[Eu(Lpy)(H2O)](PF6)3·2H2O. The [Eu(Lpy)(H2O)]3+ cationcontains a coordinated water molecule with a Eu–OW distance of 2.378(7) Å, rather than a chloride or fluorideanion , which isexpected considering the positive charge of the complex.71 Interestingly, the crystal data can also berefined satisfactorily assuming that the ligand present in the apicalposition is a chloride or a fluoride anion. The refinement of thestructure with these incorrect atom assignments yields very similar R1 and wR2 factors (Table S2), and no major (A type) alerts in the checkcif reports.Thus, most likely, the structures reported by Wada contain a Eu–Frather than a Eu–Cl bond.The extremely short Ln–Cl distances reported by Wada promptedus to prepare and crystallize the complex. Both the ligand and theEu(III) complex were prepared following reported procedures,deanion 6. Thisdd) of amine and carboxylate donor atoms obtained for threerare-earth cations representative of large (La3+), medium(Gd3+), and small (Yb3+) cations. The valuesof Δd display rather good Gaussian distributionswith maxima close to Δd = 0. The fact thatthe maximum of the Gaussian distribution does not drift across thelanthanide series provides confidence in the reliability of our approach.One can notice that Ln–OC bonds are characterizedby narrower distributions than Ln–NA bonds, as mentionedabove. Most of the calculated bond distances fall within the −0.1Å < Δd < 0.1 Å window (<90%).Furthermore, the bonds with the largest deviations are associatedwith unusual coordination features. For instance, the La–OC bonds characterized by Δd = 0.271Å correspond to a dinuclear La3+ complex, with CN11 and carboxylate bridges coordinating through a μ2-η2:η1 mode.72 The La–OC distances with Δd = ∼ 0.17 Å also correspond to bridging carboxylate groupscoordinating in a μ2-η2 mode.73d obtained for all rare-earthcations and nine different donor atoms are shown in x0) and the width at halfheight (Δd1/2). For some donor atoms, the limited number of data preventedaccurate Gaussian fitting. All fitted Gaussian functions show verysmall x0 values, indicating that the Gaussianfunctions are centered at dLn–D ∼ rD + CRLn (Δd ∼ 0). The absolute values of x0 are generally equal to or lower than 0.01 Å, withthe exception of the Ln–Ow bonds, for which x0 = −0.026 Å. The Ln–Ow bonds are also characterized by a rather broad distribution,with Δd1/2 = 0.123 Å  exceeds thosewith negative Δd values. The shape of the histogramsresembles, to a certain extent, that of a potential energy curve associatedwith a bond distance, which is often described by a Morse function.74 This suggests that shortening the Ln-donor distancebeyond the value of x0 provokes a steeperincrease in energy than weakening the Ln-donor interaction. The asymmetryof the histogram is particularly evident in the Ln–OW bonds, for which positive deviations of Δd of up to 0.35 Å are observed  and Er(III) DO3A derivativecomplexes containing a phenanthroline pendant.80 The N atom of the phenanthroline unit at the capping positionis characterized by long Ln–N distances with Δd = +0.185 and 0.197 Å for Eu(III) and Er(III), respectively.Conversely, the pyridyl donor atom at the upper plane of the SAP coordinationpolyhedron is characterized by a short distance (Δd = −0.066 and −0.092 Å for Eu(III) and Er(III),respectively).We note that the histograms are not perfectly symmetrical, showinga hump on the positive side. This implies that the number of distanceswith positive deviations .87A relatively small number of X-raystructures analyzed here containedprotonated carboxylate groups (13 data points), which display ΔrD valuesdo not correlate with the contributions of these donor groups to complexstability. For instance, we have estimated recently that phosphonategroups contribute with Δlog K = 5.0 units tothe stability of a Gd3+ complex, while the contributionof a phosphinate group is only 1.9 log K units.10 However, both donor groups display virtuallyidentical rD values.It is worth noting thatthe rD values reported here allow forthe estimation of a typical Ln-donor distance for any rare-earth complex.This is very useful, as there is no set of radii available for donoratoms that can provide good estimates of lanthanide-donor bond distances.The crystal CR reported by Shannon do not provide good estimates ofLn(III)-donor distances in polyaminopolycarboxylate complexes. Furthermore,Shannon defined different CR for the oxide anion depending on thecoordination number (1.21–1.28 Å).19 The value of 1.21 Å (for CN 2) is 0.08 Å longerthan the donor radius of a carboxylate oxygen atom (1.132 Å).Differences even increase when taking CR for higher coordination numbers.The deviation is significantly larger for a phosphonate or phosphinate(up to 0.1 Å). This is a huge difference, as it roughly correspondsto the distance between the center and the tail of the Gaussian distribution(88 for a N atom of1.55 Å is significantly larger than the donor radius estimatedin our work for an amine donor (1.425 Å) and deviates even morefrom that of a pyridyl donor (1.329 Å). The situation worsensfor neutral O atoms, as the donor radii of amide (1.130 Å) andwater (1.231 Å) differ by 0.1 Å, and the van der Waals radiusof O is much larger (1.52 Å).89 Thus, it isclear that there is currently no reliable set of radii that allowsfor the calculation of Ln-donor distances with reasonable accuracy.The ribution8. The si4DOTA and H5DTPA, the bond distances of complexesbased on the 18-membered macrocycle PYAN , and the availabilityof structures for many of the rare-earths. A plot of the measuredbond distances against the distances calculated using the donor radii shows a reasonably good distributionaround the identity function.In order to test the usefulness of donor radii to predict bondlengths in lanthanide complexes based on platforms other than HThiswork has established a straightforward approach to derivedonor radii for complexes of rare-earth cations, providing a toolto estimate Ln-donor distances (including Y and Sc) regardless ofthe nature of the rare-earth cation and its oxidation state. Our approachrelies on separating the Ln-donor distances into contributions fromthe cation, approximated by the CR, and the donor atom. Theoreticalcalculations provide support for this approach, at least for complexeswith polyaminopoly(carboxylate) ligands used for stable complexationin aqueous media. The rare-earth cations are particularly well-suitedfor the analysis presented here, as they represent the most coherentseries of elements within the periodic table in terms of their chemicalproperties.The work presented in this contribution providesa rich sourceof data that will be very helpful in analyzing the structural dataof rare-earth complexes. For instance, particularly strong or weakinteractions can be easily identified, providing foundations for theinterpretation of thermodynamic and kinetic data. Additionally, theseradii can be very helpful in crystallographic analysis to preventincorrect atom assignments. The approach presented here can be easilyapplied to other donor groups, which were excluded here due to thescarcity of data for these donors in our data set . Historically, IR have served as a very usefultool for geochemists, mineralogists, chemists, and material scientiststo rationalize structural data. However, our results indicate thatCR provide a better estimate of lanthanide ion size than IR. Therefore,we recommend the use of CR for the rationalization of structural dataof Ln(III) compounds and possibly of other metal ions.


The cerebrospinal fluid (CSF) production rate in humans is not clearly defined but is estimated to be 18–24 ml/h :377–387  (Ekstedt J Neurol Neurosurg Psychiatry 41(4):345–353 , and the internal software of LiquoGuard7 was used to measure PRcsf. Statistical analysis used SPSS  by paired :345–353 ), assumin=41): PRcsf of 79ml/h±20SD (p<0.0001). Post-surgical CSF leak (n=26): PRcsf of 90ml/h±20SD (p<0.0001). Subarachnoid haemorrhage (n=34): PRcsf of 143ml/h±9SD (p<0.0001). Intracerebral haemorrhage (n=22): PRcsf of 137ml/h±20SD (p<0.0001). Spinal lesions (n=7): PRcsf of 130ml/h±20SD (p<0.0032). Pituitary adenomas (n=10): PRcsf of 29 ml/h±9SD (p<0.049). Idiopathic intracranial hypertension (n=15): PRcsf of 86ml/h±10SD (p<0.0001). Decompensated long-standing overt ventriculomegaly (n=4): PRcsf of 65ml/h±10SD (p<0.0001). Cerebral infection (n=5): PRcsf of 90ml/h±20SD (p<0.0001).PRcsf was calculated in 164 patients. Suspected normal pressure hydrocephalus (Net CSF production rate may be higher than expected in many conditions, as measured with new device LiquoGuard7 through the study of net flow rate, which may have implications for clinical decisions on CSF diversion. The conventional understanding of CSF production and circulation does not explain the findings of this study. More extensive studies are needed to validate this technique. Historically cerebrospinal fluid (CSF) is believed to be formed at a rate of 18 – 24 ml/h, renewed approximately 3–5 times a day, maintaining a total CSF volume of 90–150mls and a supine ICP between 7 and 15mmHg [Trevisi G, Frassanito P, Di Rocco C (2014)Hydrocephalus is thought to be predominantly due to obstruction in CSF flow and only rarely due to CSF overproduction by choroid plexus hypertrophy, infection or tumour , 12. StiRecent studies employing PC-MRI, radioactive water and biological dyes have challenged the traditional views of CSF hydrodynamics. A significant volume of CSF is formed by the choroid plexus and the remaining amount by the brain parenchyma, ependyma and the blood capillaries constituting the blood-brain barrier (BBB) , 15, 17.+/Cl− co-transporter, Na+/K+ ATPase and glucose transporter GLUT1 [CSF production by the choroid plexus is mediated through apical and basolateral membrane transporters such as aquaporin 1 (AQP1), Ker GLUT1 . At the er GLUT1 .AQP1 and AQP4 are both associated with normal CSF hydrodynamics but are also implicated in several pathologies. AQP4 is associated with the development and resorption of cytogenic and vasogenic cerebral oedema respectively , 24. It Experiments studying the role of transporters in CSF production found > 20% reduction in CSF production in AQP1 null mice, and 7–10 fold decreased CSF production in AQP4 null mice , althougPreviously CSF was thought to maintain a unidirectional bulk flow driven by the hydrostatic gradient generated by the choroid plexus and the arachnoid villi. PC-MRI has revealed an additional pulsatile flow in synchrony with the cardiac and respiratory cycles , 17, 20.Similarly, studies into the impact of barbiturate anaesthesia on CSF flow revealed that PRcsf is approximately 6.5 times less in an anaesthetised animal as compared to an awake animal . StudiesOver the decades, several techniques have been employed to calculate PRcsf. Since CSF is simultaneously secreted and absorbed, a production rate was estimated based on the absorption rate of CSF, calculated from choroid plexus arterial blood flow and haematocrit value . PressurWe measured PRcsf and intracranial pressure (ICP) in patients with various neurosurgical pathologies, using LiquoGuard7, an automated device certified for simultaneous monitoring of pressure and precise drainage of CSF . It was The LiquoGuard pump is already in clinical use for CSF drainage and early mobilisation of patients , ICP monIn 1977 and 1978 Ekstedt published his studies of CSF hydrodynamics in humans, employing constant pressure infusion based on similar techniques used by Davson et al. for animal studies in 1970 , 14. By Electron microscopic examination of the hydrostatic bulk flow through the arachnoid villi exhibited a pressure-dependant one-way vacuolation of pores . AbsorptTherefore, decreasing CSF pressure below cerebral venous pressure halts CSF absorption into the venous circulation. Under such circumstances, CSF collected through external drainage devices represents the basal CSF production.Transporters at the BBB and the blood-CSF barrier create an osmotic gradient which contributes to CSF production and absorption at these barriers , 15. EveA PRcsf study was performed on all patients in our centre who required CSF drainage through an extra-ventricular drain (EVD) or a lumbar drain (LD), as part of their ongoing clinical management from September 2021 onwards.EVD  was inserted through Kocher’s or Keen’s point depending on individual cases. LD  was inserted between L3/L4 lumbar vertebrae. The drainage catheters in all cases were attached to LiquoGuard7. The external tubing of LiquoGuard7 apparatus was primed with 0.9% saline to prevent wastage of CSF.The LiquoGuard7 pump has a pressure transducer, which measures the CSF pressure relative to that reference point. According to published studies in a completely supine individual the external auditory meatus (EAM) correlates to the anatomical brain centre and provides a suitable zero level for CSF pressure measurement .For this study the ICP transducer was applied on the skin of the patients at the level of the EAM with an electrocardiogram sticker provided with the LiquoGuard7 drainage set Fig. .Fig. 1LiThe patients were requested to lie in a flat, supine position. Only patients who were able to safely lie flat for the duration of the study were included. In a flat, supine position the CSF pressure is uniform in all the fluid compartments of the central nervous system, allowing for accurate CSF pressure measurement by the external ICP sensor applied at the level of the EAM  Fig. 2)2).Fig. 2The LiquoGuard pump allows for two modes of drainage: volume-led drainage and pressure-led drainage. For this study, pressure-led drainage was employed. This mode allows for CSF removal and collection in the external collecting bag as soon as the ICP exceeds the set pressure limit. For the purpose of the study the pressure limit of the machine was set at 0mmHg, which is sufficiently low to prevent CSF absorption into the venous circulation.With CSF pressure set at 0 mmHg, the volume of the fluid which was collected into the external collection bag attached to the LiquoGuard7 was set at 150ml/h, which is the upper limit of the LiquoGuard7. This also represented a short-coming of the current study, as it prevents from recording PRcsf above 150ml/hour.During initial studies, it was established that in most supine patient with the above mentioned test parameters, net CSF flow rates stabilised to a constant value within 4 to 15 min. What this means is that in a strictly supine individual connected to a LiquoGuard7 with the external transducer at the level of EAM, when the CSF pressure is set at 0mmHg and the upper limit of volume to be drained is set at 150ml/h, the LiquoGuard7 machine attempts to remove as much CSF as it can, as much as 150ml/h, to try to bring the ICP to 0. Since the patient is strictly supine this ICP is representative of the CSF pressure of the entire central nervous system (CNS) in a neutral position. Hence, during initial studies it was observed that with the above mentioned test parameters the LiquoGuard7 drained out CSF at high flow rates in an attempt to bring CSF pressure to 0, and that this flow rate progressively decreased with each minute as ICP decreased, until the CSF pressure reached a value of 0 and the flow rate stopped decreasing any further but instead remained the same from that time-point onwards. CSF pressure increased above 0 within seconds reflecting release of CSF in the CNS, and was almost instantly brought back to 0 by LiquoGuard7 via draining the released CSF, the quantity of which could be calculated by the internal software of LiquoGuard7 pump and remained constantly stable  with programmable valves in situ. All patients with a particular diagnosis had similar results, regardless of age, gender or co-morbidities. A brief summary of each neurosurgical disorder examined is given below  and 4 decompensated long-standing overt ventriculomegaly (LOVA) patients via extended lumbar drainage through LiquoGuard7 Table .Table 2Dp<0.0001). Of these, two NPH patients with previous spinal pathology had average net PRcsf of 69–92ml/h. At a 6-month follow-up all 22 patients had received a programmable VPS and exhibited a Glasgow outcome scale (GOS) of 5 with improved neurological, psychological and motor status.Twenty-two NPH patients demonstrated an average net PRcsf of 55–82 ml/h (p=2.13). With clinical investigations these patients failed the diagnosis of NPH and were non-responsive to extended lumbar drainage. At a 6-month follow-up these patients had a GOS of 4 with ongoing cognitive and motor deficit.Seventeen suspected NPH patients had an average net PRcsf of 22–32ml/h (p=2.13), but showed subjective and objective symptomatic improvement with ELD. At a 6-month follow-up these patients had received a programmable VPS and exhibited a Glasgow outcome scale (GOS) of 5 with progressive improvement in neurological, psychological and motor function.Two NPH patients were observed to have an average net PRcsf of 22–32ml/h  displayed an average net PRcsf of 83–94ml/h (p<0.0001)  stent insertion. Their net PRcsf post-stenting was calculated to be average 130–150ml/h . Eleven patients eventually received CSF diversion and had a 6-month GOS of 5.p<0.0001) (Table Patients with spontaneous or post-operative CSF leakage had an average net PRcsf of 75–110ml/ (1) Table .Table 4DAt six-month follow-up, 19 out of the studied 26 patients had received permanent CSF diversion and were symptom-free with GOS 5.p<0.0001). At 6-month follow-up four patients had received a shunt and had a GOS of 5 .Seven of the patients with SAH had a pre-existing programmable shunt. The average net PRcsf in 19 SAH patients and 13 ICH patients was above 120ml/h, with the mode of the cohort being 150ml/h  . At 6-month follow-up these patients had GOS of 5 and had returned to their baseline with no further intervention (Table Pituitary adenoma patients requiring CSF drainage through a LD demonstrated average net PRcsf of 26–36ml/h (on Table .Table 8DIn these preliminary observations, several trends of raised CSF production associated with clinical need for early CSF shunting have been noted. These trends are in-keeping with similar incidental observations reported by others over the decades, in which CSF production rate was calculated by different methods without specifying the CSF pressure or the posture of the patient. Yet, the results observed in each of the mentioned pathology are comparable to those calculated in this study.In two patients who underwent lumbar drainage following the removal of EVD, the PRcsf remained the same and did not vary between cranial and lumbar sites of CSF removal. This can be explained by the supine positioning of the patient during measurements, which makes CSF pressure uniform throughout all fluid compartments of the central nervous system.Similarly, multiple CSF production rates calculated in the same patient 1 month apart yielded the same reading. This effect has also been reported by Casey et al. in a patient who was observed to produce four times the ‘normal’ amount of CSF months after suffering from a cerebral infection . These fAquaporin upregulation takes place in two phases. In an acute phase, aquaporin membrane channel permeability increases within seconds to minutes. In the second phase, long-term upregulation is maintained by changes in the rate of aquaporin mRNA synthesis within a few hours to several days . This exIt is observed that relatively low or normal net PRcsf is a predictor of poor outcome in haemorrhagic stroke. Failure to maintain reflex hypersecretion of CSF seems to be associated with poor prognosis. CSF production rate may act as a prognostic tool in cerebral haemorrhage and other neurosurgical pathologies. This will be considered in depth in subsequent publication.Stenting in IIH led to increase in net CSF flow rate. This may be because stenting increases the blood flow through the SSS, decreases the venous pressure and may lead to a change in the hydrostatic gradient resulting from increased secretion of CSF and increased absorption.CSF production rate also appears to be a possible diagnostic tool in several common neurosurgical conditions such as NPH. In the studied cohort, patients who did not exhibit raised PRcsf were lumbar drainage non-responsive. This is significant since predicting shunt responsiveness in the context of NPH is still a clinical challenge . BradleyA history of previous spinal disease was noticed to be associated with increased net CSF flow rate in most patient groups.Age, gender and non-spinal co-morbidities did not have any impact on the net PRcsf of any group, a fact also noted by Ekstedt during his experiments , 14.It was also observed that at 6-month follow-up several patients with raised net PRcsf showed clinical improvement following the insertion of shunt. This is in keeping with previous studies exploring the role of intervention in the improvement of symptoms in patients in various diseases such as NPH and CSF leakage , 27.Based on the individual net PRcsf observation of post-surgical spinal CSF leak patients, tailored CSF drainage of volume 50–150ml/h, continuously for a week, was offered to several patients. The individually personalised CSF drainage led to rapid recovery, prevention of significant complications and shorter hospital stay of the patients. This will be discussed in detail in subsequent publication.Although the principle of this study was based on Ekstedt’s experiments on CSF hydrodynamics, LiquoGuard7 provides several technical advantages . The tecThis study helps provide a new understanding of the association of CSF production rate in these pathologies and to form a clinical decision support guide allowing for earlier decisions on CSF diversion, to provide optimal treatment, potentially preventing brain and spinal cord injury. This represents a paradigm shift in our understanding of hydrocephalus, which has generally been understood as obstruction of flow or absorption of CSF rather than an increase in CSF production. It may also open the possibility of new pharmacological targets for disease management, since increased production is more likely to be amenable to modification through a molecular approach than obstruction which has to be overcome by physical means.Net CSF production rate may be higher than expected in many conditions, as measured with new device LiquoGuard7 through the study of net flow rate, which may have implications for clinical decisions on CSF diversion. The conventional understanding of CSF production and circulation does not explain the findings of this study. More extensive studies are needed to validate this technique and to define its use as a diagnostic and prognostic method.


Intracerebral hemorrhage (ICH) enhances neurogenesis in the subventricular zone (SVZ); however, the mechanism is not fully understood. We investigated the role of brain-derived neurotrophic factor (BDNF) in post-ICH neurogenesis in a rodent model and in patients with ICH using cerebrospinal fluid (CSF).A rat model of ICH was constructed via stereotaxic injection of collagenase into the left striatum. Patients with ICH receiving an external ventricular drain were prospectively enrolled. CSF was collected from rats and patients at different post-ICH times. Primary cultured rat neural stem cells (NSCs) were treated with CSF with or without BDNF-neutralized antibody. Immunohistochemistry and immunocytochemistry were used to detect NSC proliferation and differentiation. The BDNF concentration in CSF was quantified using enzyme-linked immunosorbent assays (ELISA).In the rat model of ICH, the percentage of proliferating NSCs and neuroblasts in SVZ was elevated in bilateral hemispheres. The cultured rat NSCs treated with CSF from both rats and patients showed an increased capacity for proliferation and differentiation toward neuroblasts. BDNF concentration was higher in CSF collected from rats and patients with ICH than in controls. Blocking BDNF decreased the above-noted promotion of proliferation and differentiation of cultured NSCs by CSF treatment. In patients with ICH, the BDNF concentration in CSF and the neurogenesis-promoting capacity of post-ICH CSF correlated positively with ICH volume.BDNF in CSF contributes to post-ICH neurogenesis, including NSC proliferation and differentiation toward neuroblasts in a rat model and patients with ICH. The adult mammalian brain comprises a population of neural stem cells (NSCs) in the subventricular zone (SVZ) adjacent to the lateral ventricles wall . After iNotably, the NSCs and ependymal cells at the SVZ form a pinwheel structure with an apical process in direct contact with cerebrospinal fluid (CSF), which regulates NSCs behavior through specific growth factors and signals . Our preIn addition, brain-derived neurotrophic factor (BDNF), a member of the neurotrophic factor family, participate in the survival, differentiation, and synaptic plasticity of neurons . BDNF suAdult Sprague–Dawley (SD) rats aged 8 weeks (280–300 g) were subjected to induction of ICH. Using a microinjector pump, 0.2 U of bacterial collagenase  in 1 μL saline was infused into the left striatum adjacent to the SVZ  at a constant rate of 0.1 μL/min, leaving the needle in place for an additional 10 min after injection. In sham-operated controls, 1 μL saline was injected using the same procedures. Subsequently, the scalp was closed with sutures, and the rats were placed in a cage under an infrared heating lamp until recovery from anesthesia.g RCF for 10 min at 4°C to avoid blood contamination and were then aliquoted and stored at −80°C. The animal experiment protocol was approved by the Institutional Animal Care and Use Committee at National Taiwan University (IACUC-20180009) and Taipei Medical University (LAC-2019-0236).For collection of rat CSF samples, the cisterna magna was punctured using a U-100 insulin syringe with 28G × 1/2-inch needle  and 0.1–0.2 ml of CSF was then gently aspirated. CSF samples were centrifuged at 800 A total of 15 patients who had been diagnosed with supra- or infratentorial ICH with intraventricular hemorrhage and had undergone external ventricular drain surgery between August 2017 and April 2020 were prospectively included. Among the 15 patients, 7 patients whose CSF samples could not be obtained at least 7 days after ICH were excluded and CSF samples from 8 patients were analyzed. The ICH volume was measured from initial computed tomography (CT) imaging. The functional outcome was evaluated at post-ICH 3 months using the modified Rankin Scale (mRS). CSF was also collected from eight patients with normal pressure hydrocephalus (NPH) to serve as controls. The human study was approved by the institutional review board of Taipei Medical University (TMU-JIRB N201703066) and the included patients all provided signed informed consent.Cortical NSCs were obtained from pregnant SD rats at the gestational age of 17 days according to our previously described protocol . Briefly2O2 in 0.2% Triton X100 in PBS for 15 min, and non-specific binding was blocked using blocking solution containing 3% normal donkey serum and 3% bovine serum albumin (BSA) in PBS for 1 h. Dual-staining was performed using anti-Ki67  and anti-nestin  antibodies to label proliferating NSCs, and using anti-doublecortin (DCX)  and anti-glial fibrillary acidic protein (GFAP)  antibodies to study NSC differentiation. After incubation with the above primary antibodies overnight at 4°C, samples were incubated with Alexa Fluor® 594 and 488  secondary antibodies. Nuclei were counterstained with DAPI  and the slides were imaged using the fluorescence microscope .Rats at post-ICH days 1, 3, 5, and 7 were perfused transcardially with cold phosphate-buffered saline (PBS) followed by 10% phosphate buffered formalin. Brains were then removed and placed in 10% phosphate buffered formalin overnight at 4°C, 15% sucrose for 1 day, then 30% sucrose containing 0.1% sodium azide, and stored at 4°C until sectioning was processed. Tissue blocks were embedded in OCT Tissue-Tek  and cut into a series of 14 μm-thick coronal slices. Brain sections were permeabilized with 0.2% Triton X100 in PBS at room temperature for 15 min, fixed with 10% MeOH and 3% HImmunoreactivity was quantified at the anterior dorsolateral region of SVZ, where the most active neurogenesis occurs at different post-ICH times . The perCultured NSCs were fixed with ice-cold methanol for 20 min, permeabilized with PBS containing 0.25% Triton X-100 (PBST) for 10 min, blocked with 1% BSA in PBST for 30 min at room temperature, and double stained with primary antibodies  overnight at 4°C. After washing, cells were incubated with secondary antibodies  and counterstained with DAPI (GeneTex). Images were captured using the fluorescence microscope . For each condition, Ki67-positive, nestin-positive and DCX-positive cells were counted and divided by the DAPI-positive cells and calculated at least three neurospheres per coverslip. GFAP expression was determined by a GFAP-positive area divided from the whole area of at least three neurospheres per coverslip. All values were then averaged to obtain a final immunoactivity level for each condition.Enzyme-linked immunosorbent assay (ELISA) kits  were used to detect total BDNF protein concentration in CSF per manufacturer’s instructions. All samples were tested in duplicate, and the optical density was read at 450 nm on a microplate reader .post-hoc analysis using Tukey’s test. Correlations between ICH volume and BDNF concentration in CSF and the neurogenesis-promoting capacity of post-ICH CSF were examined using simple linear regression analysis. All statistical analyses were performed using the GraphPad Prism software . All tests of significance were 2-tailed with a threshold for significance of p < 0.05.Data are presented as mean ± standard error of the mean (SEM). Statistical analysis comparing mean differences was performed using analysis of variance (ANOVA) followed by The results of brain sections in SVZ from rats at post-ICH days 1, 3, 5, and 7 were analyzed and compared to those from sham controls. The proliferating cells in the SVZ were detected with Ki67 and DAPI double staining. Compared to all cells in the SVZ, the percentage of proliferating cells in the SVZ lesional side was relatively increased on post-ICH days 5 and 7 . While tIn addition, the cell density of neuroblasts (DCX immunoreactive cells) in SVZ and astrocytes (GFAP immunoreactive cells) in the perihematomal region were analyzed. In the lesional side, the number of neuroblasts at SVZ and astrocytes in perihematomal region were both increased on post-ICH day 7 and day 5, respectively . The farin vitro. Under treatment with CSF collected from rats at post-ICH days 3, 5, and 7, Ki67 and nestin double immunoreactive cells increased in neurospheres compared with those in the control group, indicating that post-ICH CSF can stimulate proliferation of cultured NSCs  (P = 0.007)  . Besides= 0.007) .To confirm the contribution of BDNF on neurogenesis, the promoting capacity of post-ICH CSF on proliferation and differentiation of NSCs was examined to determine whether it would be eliminated by BDNF neutralizing antibody. IgG isotype antibody was used as the control. Under treatment with post-ICH CSF of rats, BDNF neutralizing antibody reduced the percentage of proliferating NSCs and neuroblasts in neurospheres, while IgG isotype antibody showed no similar reduction of treatment effects . In addin = 5), thalamus (n = 2) and putamen (n = 1), with mean hematoma volume 10.1 ± 7.8 ml. Most patients (87.5%) showed unfavorable outcomes at post-ICH 3 months (mRS > 2). The mean BDNF concentration in CSF, collected from patients at post-ICH day 3, was 41.3 ± 14.8 pg/ml.Eight patients with ICH  who were required to receive an external ventricular drain were enrolled . The ICHR2 = 0.53, P = 0.041)    , BDNF concentration in CSF , and neurogenesis promoting capacity of CSF  did not correlate with functional outcomes evaluated by mRS at post-ICH 3 months.Attempts to correlate the ICH volume with BDNF concentration and neurogenesis promoting capacity of CSF revealed that the initial ICH volume correlated positively with BDNF concentration in CSF collected from patients at post-ICH day 3 (= 0.041) . After f= 0.014)  and diff= 0.012) , but was= 0.046) . TherefoAlthough post-ICH neurogenesis is well known from studies using rodent models , the mecIn line with prior studies , the preBrain-derived neurotrophic factor exhibits strong neurotrophic factor signals, which induces neurogenesis in adult SVZ via tyrosine kinase B receptors or p75 neurotrophin receptors . CulturePrior studies emphasizing human post-stroke neurogenesis remain rare, possibly because non-invasive techniques to analyze neurogenesis are still limited. The present study used post-ICH CSF from patients to indirectly investigate the features of post-ICH neurogenesis. We demonstrated that CSF samples collected from patients with larger ICH volume have higher BDNF concentration and higher capacity to promote NSC proliferation and differentiate toward neuroblasts rather than astrocytes. For the first time, the association between ICH volume and growth factor concentration as well as neurogenesis capacity in the brain was shown indirectly. Upon acute stroke, neurons and non-neuronal cells such as astrocytes, microglia, ependymal cells, and endothelial cells produce a substantial amount of BDNF . Notablyin vivo detection of cerebral neurogenesis is still highly limited, the method provided in the present study may remain an alternative for future investigation of post-stroke neurogenesis. Fifth, the mean concentration of BDNF in the CSF of rats (post-ICH day 7) was 2.86 times higher than in patients’ CSF (post-ICH day 3)  . HoweverH day 3) . FinallyCerebrospinal fluid samples from a rat model and patients with ICH demonstrated that acute ICH induced BDNF production, which contributes to post-ICH neurogenesis in both NSC proliferation and differentiation toward neuroblasts. Results provide evidence for future BDNF-based therapy in patients with ICH via effects of augmenting endogenous neurogenesis.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Institutional Review Board of Taipei Medical University (TMU-JIRB N201703066). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at National Taiwan University (IACUC-20180009) and Taipei Medical University (LAC-2019-0236).T-CL and L-KT contributed to the conception and design of the study. T-CL, Y-AC, T-HY, AH, Y-HH, and L-KT organized the database. T-CL, Y-CT, C-CW, Y-HC, K-YC, and L-KT performed the statistical analysis. T-CL wrote the first draft of the manuscript. L-KT wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


We have commonly observed prominent cerebral veins on susceptibility‐weighted angiography (SWAN) in acute meningoencephalitis. This study aimed to investigate the clinical significance of these findings.Cerebral veins on SWAN of 98 patients with acute meningoencephalitis diagnosed from February 2016 through October 2020 were classified into three groups according to the degree of venous prominence . Clinical variables and laboratory findings were compared between these groups. The influence of variables on the prediction of prominent cerebral veins was measured by random forest (RF) and gradient boosting machine (GBM).p = .01) and CSF protein level increased . The etiology of meningoencephalitis, neurological symptoms, and increased intracranial pressure (ICP) signs differed between groups (p < .05). RF and GBM demonstrated that CSF protein level was the variable with the highest power to predict the prominent cerebral vein .As cerebral veins became more prominent, cerebrospinal fluid (CSF) glucose level decreased (69.61 ± 29.05 vs. 59.72 ± 22.57 vs. 48.36 ± 20.29 mg/dL, The presence of prominent cerebral veins on SWAN in acute meningoencephalitis was significantly associated with a low CSF glucose level and a high CSF protein level, as well as ICP. Thus, the visual grade of the cerebral veins on SWAN may be utilized for the management of patients with acute meningoencephalitis. The presence of prominent cerebral veins on SWAN in acute meningoencephalitis was significantly associated with a low CSF glucose level and a high CSF protein level, as well as increased ICP. Thus, the visual grade of the cerebral veins on SWAN may be utilized for the management of patients with acute meningoencephalitis. We retrospectively searched electronic medical records and MR images between February 2016 and October 2020 and identified 132 patients with diagnoses of meningitis or encephalitis. We selected patients who had undergone a CSF study and MRI and were discharged with a diagnosis of meningitis or encephalitis. Ultimately, 98 patients were enrolled in the study. CSF studies and MRI were performed with an average gap of 1.73 days between MRI and CSF analysis . Clinical data included age; sex; hypertension (HTN); diabetes mellitus (DM); smoking history; CSF analysis; neurological symptoms such as seizures, confusion, or aphasia; signs of increased intracranial pressure (ICP) such as a high CSF pressure, unconsciousness, abnormal pupillary dilatation, uncontrolled headache with nausea or vomiting, irregular breathing, and papilledema; duration of symptoms; duration of hospital admission; intensive care unit (ICU) care; and etiology of meningoencephalitis.2.2MRI studies were performed using a GE 3T Discovery MR750 . Our standardized protocol included T2‐weighted imaging, diffusion‐weighted imaging, FLAIR, susceptibility‐weighted angiography (SWAN), and subsequent contrast‐enhanced T1‐weighted imaging with FLAIR. The sequence parameters for SWAN were as follows: repetition time (TR) = 31 ms; echo time (TE) = 3 echoes centered around 23 ms; flip angle = 10°; slice thickness = 2 mm; intersection gap = 0 mm; field of view = 210 mm; matrix number = 320 × 224 and bandwidth = 62.50 kHz.To enhance the visibility of veins and other sources of susceptibility, minimum intensity projection (mIP) of SWAN was acquired with a thickness of 8 mm in the axial plane. mIPs of SWAN sequences of all patients were assessed for the presence of prominent cerebral veins by two independent reviewers who were unaware of the clinical details. We used the three‐point score to evaluate the prominence of cerebral veins: Grade 0 (mild)—superficial and deep parenchymal veins were rarely seen; Grade 1 (moderate)—subependymal veins, deep middle cerebral vein, and basal vein of Rosenthal were clearly seen. However, deep medullary veins were rarely seen; Grade 2 (prominent)—parenchymal veins, deep medullary veins and subependymal vein were clearly seen , blood tests , CSF/serum glucose ratio, CSF/serum protein ratio, neurological symptoms , ICP signs, duration of symptoms, duration of hospital admission, ICU care, and etiology of meningoencephalitis were compared among the three groups with different cerebral vein grades on mIP of SWAN. A one‐way analysis of variance (ANOVA) with a post hoc Tukey's test was performed for continuous variables, while the chi‐square test or the Fisher's exact test was used for categorical variables.p < .05. Interrater reliability was assessed using the intra‐class correlation coefficient (ICC) with a two‐way random model of absolute agreement. All data analyses were performed using R, version 3.5.3.For further analysis, the patients were dichotomized into two groups: patients with prominent cerebral veins and those without prominent cerebral veins . To measure the influence of variables for the prediction of a prominent cerebral vein, the variable importance scores of each clinical variable were calculated using two popular ensemble methods: random forest (RF) and gradient boosting machine (GBM) Breiman, . These e33.1p < .05). As the cerebral veins became more prominent, the CSF glucose level and CSF/serum glucose ratio decreased. Post hoc analysis revealed that the CSF glucose level in patients with prominent cerebral veins was significantly lower than that in patients with mild cerebral veins  and moderate cerebral veins . Patients with mild cerebral veins were more likely to have neurological symptoms than those with moderate cerebral veins , and the variable with the second highest prediction power was CSF glucose . The importance scores of the other variables are summarized in Table 4In the present study, we found that cerebral venous prominence was associated with CSF glucose and protein levels. A prominent cerebral vein on SWAN indicated lower CSF glucose and higher CSF protein levels in patients with acute meningoencephalitis. Moreover, it was significantly associated with ICP. In contrast, when a cerebral vein is rarely seen, it may imply that patients are likely to have neurological symptoms.Prominent cerebral veins on ESI have previously been described in ischemic regions of the brain in patients with stroke   is noted on basal cistern (A) and prepontine cistern (B) on FLAIR.Click here for additional data file.


Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove, and that Ca2+ dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements involved in groove opening and of how lipids reorganize outside the closed groove remain unknown. Using cryogenic electron microscopy, we directly visualize how lipids associate at the closed groove of Ca2+-bound nhTMEM16 in nanodiscs. Functional experiments pinpoint the lipid-protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two π-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryoEM structure determination.Activation of Ca To date, three families of integral membrane proteins have been identified as lipid scramblases, the Ca2+-activated TMEM16s 7, the caspase-dependent XKRs 8, and the ATG9 proteins involved in autophagosome formation 11. Additionally, several other proteins and single-helix membrane spanning peptides have been reported to mediate lipid scrambling 16. These scramblases share broadly conserved functional characteristics, such as mediating rapid and poorly selective lipid transport 7, while displaying a remarkable structural diversity, as each family adopts a different fold 22. Thus, it is not known whether these diverse scramblases function according to a conserved mechanism.Activation of phospholipid scramblases disrupts the plasma membrane asymmetry, and results in the exposure of the negatively charged lipids phosphatidylserine (PS), an important signaling molecule. Thus, lipid scrambling plays a key role in a variety of physiological processes, ranging from recognition of apoptotic cells by macrophages and blood coagulation, to viral entry, membrane fusion and repair 23. In several dimeric TMEM16 scramblases, Ca2+ binding favors opening of a transmembrane hydrophilic groove which has been proposed to serve as the translocation pathway for lipid headgroups 28. The lipid groove is formed by TM3–7 helices of each TMEM16 protomer, and its interior is lined by hydrophilic residues 7  imaging of TMEM16 scramblases in nanodiscs showed these proteins induce a pronounced thinning of the membrane near the groove region, a distortion that is more pronounced when the groove opens 2+-bound human TMEM16K scramblase crystalizes with an open groove but has a closed groove in single particle cryoEM imaging conditions 18, and the fungal nhTMEM16 adopts conformations with an open, closed, and intermediate-closed groove with roughly equal probability when reconstituted in MSP2N2 nanodiscs but is only in an open groove state when imaged in detergent micelles with cryoEM and x-ray crystallography 21. In contrast, other homologues, such as murine TMEM16F and fungal afTMEM16 are less sensitive to the environment, as they exclusively adopt a single conformation in detergent and in nanodisc environments 30. Thus, the extent to which choices of detergent, lipid and/or nanodisc scaffold protein influence the conformation of TMEM16 scramblases is poorly understood.One important factor to consider when interpreting cryoEM imaging experiments is the potential role of the environment in determining the conformation and state distribution of proteins. For example, the Ca2+-bound closed nhTMEM16 reveals how lipids arrange near the groove, thus showing how the scramblase thins the membrane outside a closed groove. Mutating residues that coordinate outer leaflet lipids near the closed groove impairs scrambling only in the absence of Ca2+, suggesting these interactions play a specific role in closed-groove scrambling. Structural analysis of the transition of nhTMEM16 from closed to open state suggests that the E313-R432 salt bridge could be involved in groove opening. Disruption of this interaction by the R432A mutation trapped nhTMEM16 in a closed-groove conformation, suggesting the salt bridge is critical for groove opening. In sum, our work provides insights into how lipids are scrambled outside a closed groove, how the groove opens, and how these processes are regulated by the lipid environment.Here, we used cryoEM to image the fungal nhTMEM16 scramblase in lipid nanodiscs formed from different scaffold proteins and found this to affect both the conformations adopted by nhTMEM16 and their distribution. In the smaller MSP1E3 nanodiscs the scramblase preferentially adopts a closed conformation, whereas in the larger MSP2N2 nanodiscs nhTMEM16 predominantly adopts open and intermediate-open conformations. The 2.64 Å resolution structure of Ca21. We imaged the scramblase in the absence and presence of Ca2+ and in nanodiscs formed from a 7:3 mixture of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-Dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG) lipids. We used the MSP1E3 and/or MSP2N2 scaffold proteins that differ in their average diameter by ~3 nm 32. In 0 Ca2+ and in MSP1E3 nanodiscs, we obtained a single class of particles, which yielded a map with average resolution of 2.93 Å corresponding to a conformation with a closed hydrophilic groove and no density in the Ca2+ binding sites (2+ sites and position of the cytosolic domains (see below). In contrast, the conformational distribution of nhTMEM16 changes drastically when reconstituted in 0.5 mM Ca2+ in MSP1E3 or MSP2N2 nanodiscs. We found that in MSP2N2 nanodiscs the majority of the particles (~69.2%) adopt an open groove conformation, ~17.3% are in an intermediate-open state (see below), and no class with a closed groove could be detected   .2+-free conformation determined in MSP1E3 DOPC/DOPG nanodiscs the Ca2+ binding sites are empty , TM6 is bent, the groove is closed, and the cytosolic domains move away from each other by ~4 Å . This rearrangement resembles that seen in afTMEM16 30. In the previously reported putative apo nhTMEM16 structure  21 these rearrangements were not observed, likely due to the presence of an unassigned density in the Ca2+ binding sites in this map . This suggests that partial ligand binding to these sites is sufficient to induce a rearrangement in the cytosolic domains. The Ca2+-bound open conformation determined in MSP1E3 DOPC/DOPG nanodisc is nearly identical to the previously determined open structure of nhTMEM16 determined in the larger MSP2N2 POPC/POPG discs (PDB: 6QM9) 21 . The open conformation in MSP2N2 DOPC/DOPG nanodiscs has a slightly more opened groove than that in POPC/POPG lipids because of a lateral displacement of the TM4 by ~1.5 Å . Finally, the intermediate conformation seen in the MSP2N2 DOPC/DOPG nanodiscs differs from the previously reported intermediate-closed one, as the TM4 is shifted further away from TM6 by ~2 Å and accompanied with a counterclockwise rotation of the extracellular portion of TM4, such that there is minimal contact between TM4 and TM6 , consistent with the idea this region undergoes minimal rearrangements during groove opening.The cryoEM map of Ca2+-bound and Ca2+-free closed conformations are nearly identical in this region , indicating that protein-lipid interactions are likely unchanged in the two conformations. Thus, if the observed thinning is important for closed groove scrambling, then we expect these residues to play similar roles in the Ca2+-free and Ca2+-bound closed states.The arrangement of the 10 lipids near the pathway (P1-P10) reveals the structural basis of membrane thinning at a closed groove  and thei2+, when scrambling is primarily mediated by the open groove, the three constructs have WT-like rate constants   and P3-P4 lipids (Y327A/F330A/Y439A) or of the IL P6-P10 lipids (M348A/G349A/M353A/K357A) –C. In thonstants . In cont(p<10−7) . The scr(p<10−7) . Thus, t2+-free closed, to Ca2+-bound closed, intermediate, and open states    –I. As Can 0 Ca2+ , when th(p<10−5) –H, very 0−5)  . Extensive classification results in four dimer classes: three are symmetrical and differ only in the occupancy of the Ca2+ binding sites and in the conformation of the intracellular portion of TM6 below 444P . These results suggest that the Ca2+-dependent straightening of TM6 might be important for closed groove scrambling.One key assumption of the closed groove scrambling model is that the structural differences between the apo-closed and Ca(p<10−8) –C. Cryo-27, where we showed that this and several other mutations, such as Y439A, greatly impair activity. The key difference between our current and past experiments is the membrane composition, a DOPC/DOPG mixture here and a POPE/POPG/Egg PC mixture then. To test if changes in lipid headgroups or acyl chain saturation underlie this difference we reconstituted WT and mutant nhTMEM16 in liposomes formed from DOPC/DOPG, POPE/POPG, DOPE/DOPG and POPC/POPG lipid mixtures. Consistent with previous reports 34, WT nhTMEM16 is active in all compositions, with a ~3-fold reduction in POPE/POPG membranes compared to the other conditions (m) of POPE membranes is close to room temperature, whereas those of all other tested compositions is < 0 °C 37. This raises the possibility that scrambling might be sensitive to the formation of local gel-like microdomains with reduced fluidity.Finally, we note that our results on R432A nhTMEM16 contrast with our previous report nditions  and 6C–Dnditions  and 6C–Dnditions –D. Thus,2+ dependent activation of TMEM16 scramblases plays a critical role in a variety of cellular signaling pathways 6. Despite extensive structural and functional investigations, the structural basis of their Ca2+-dependent activation and lipid transport mechanisms remain poorly understood. For example, TMEM16 activation is thought to entail opening of a hydrophilic groove following Ca2+ binding to a site formed by residues in the TM6-TM8 helices. However, it is not clear how Ca2+ binding facilitates the displacement of TM4 to open the groove. Further, while an open groove facilitates scrambling, the closed groove has also been proposed to be scrambling competent 30. However, the structural basis of lipid scrambling outside a closed groove remain unknown.The Ca2+-bound closed nhTMEM16 reconstituted into lipid nanodiscs allowed us to directly visualize how lipid molecules interact with the closed groove of the scramblase . These results suggest that membrane thinning is a general and evolutionary conserved mechanism for lipid scrambling by the TMEM16s. When the groove is closed, thinning is less pronounced and scrambling is slower than when the groove is open, rationalizing the Ca2+ dependence of this process.Our high-resolution structures of Cacan open –E. These21 we can provide a detailed description of the molecular transitions underlying the Ca2+ dependent activation of nhTMEM16 (Supplementary Movie 2). To this end, we make two plausible assumptions: that the previously reported apo conformation corresponds to a partially bound state and, that the 7 observed conformations represent intermediates along the transition from apo closed to Ca2+ bound open. The relative stability of these conformations might be differentially affected by the membrane environment, which would result in slightly different opening trajectories. Nonetheless, we hypothesize that these states are visited -albeit briefly- during opening. Starting in the apo conformation the groove is closed, TM6 is bent, and the cytosolic domains are apart (PDBID: 8TPM)  . Binding of both Ca2+ ions favors the compete straightening of TM6 and the formation of the intracellular π-helix turn, which allows the acidic side chain of E452 on TM6 to come within coordination distance of the bound ion (PDBID: 8TOI)  , F. PartD: 8TOI) , I, whicD: 8TOI) , O. The  at A444 . These r at A444 , J. The nhTMEM16 –F. This 2+ sites, empty in our structure but partially occupied in the previous 21 one . This suggests that the membrane environment might modulate TMEM16 activity by affecting ion binding, as both structures were obtained in similar Ca2+ buffering conditions. Structural differences are also observed in the conformations adopted in the MSP2N2 DOPC/DOPG nanodiscs compared to those seen in MSP2N2 POPC/POPG 21, as both the open and intermediate groove conformations in DO lipids are more open than those seen in PO lipids (2+-free conditions are nominally identical (21, and the lipid composition is the same in our MSP1E3 and MSP2N2 reconstitutions (Our results reveal that the environment plays a critical role in determining the structures of nhTMEM16 conformations as well as the population distribution among them. Indeed, subtle changes in lipid composition, from PO to DO lipids, and/or nanodisc scaffold proteins, from MSP1E3 to MSP2N2, can affect ion binding, protein conformations, and their distributions. Specifically, in MSP1E3 DOPC/DOPG nanodiscs nhTMEM16 can adopt an apo conformation (O lipids  leading O lipids , the Ca2dentical  21, and itutions . Finallyitutions , where titutions . This ra43. Our findings challenge the straightforward assumption that the particle distribution is a faithful reporter of the conformational landscape of the protein. The lack of correspondence between protein activity, particle distribution, and conformation confounds the correlation between the free-energy landscape of a protein, cryoEM particle counts, and activity. Rather, our results show that these distributions also reflect the contributions of the environment, and that even subtle changes in detergent, scaffold protein, or lipid constituents of nanodiscs can induce dramatic shifts in the preferred conformations adopted by the reconstituted protein. These effects should be evaluated for each protein system under consideration, as for example whereas the conformational landscape of WT nhTMEM16 is highly sensitive to the environment, that of its close homologues afTMEM16 or mTMEM16F is not 30, and even the single point mutant R432A nhTMEM16 exclusively adopts only one conformation in different nanodisc scaffolds (44, suggesting these considerations are generally applicable to diverse types of membrane proteins.Recently, much work has been devoted to extracting free energy landscapes of proteins from the analysis of particle distributions in cryoEM imaging experiments caffolds –D. Inter