We thereby establish the concept of cotransport of water as a missing link in the search for molecular pathways sustaining CSF production and redefine the current model of this pivotal physiological process. Our results provide a rational pharmacological target for pathologies involving disturbed brain fluid dynamics.Cerebrospinal fluid (CSF) production occurs at a rate of 500 ml per day in the adult human. Conventional osmotic forces do not suffice to support such production rate and the molecular mechanisms underlying this fluid production remain elusive. Using ex vivo choroid plexus live imaging and isotope flux in combination with in vivo CSF production determination in mice, we identify a key component in the CSF production machinery. The Na +/K+/2Cl− cotransporter in the choroid plexus contributes substantially to CSF production via its inherent ability to cotransport water.Osmotic forces do not suffice to explain the rate of cerebrospinal fluid (CSF) production. Here, the authors show that the Na Prior to exiting the brain, the CSF travels through the ventricular system and part of it re-enters the brain via the para-vascular route along the large arteries and penetrating arterioles3. The CSF is predominantly produced by the choroid plexus, an epithelial monolayer resting on highly vascularized connective tissue and located at the base of each of the four ventricles7. The molecular mechanisms underlying this choroidal fluid production remain unresolved. Dysregulation of CSF production or clearance may lead to brain water accumulation and raised intracranial pressure, as evident in patients with hydrocephalus. Hydrocephalus most commonly occurs as a consequence of obstructed CSF outflow, and is routinely treated by insertion of a ventriculo-peritoneal shunt diverting the excessive fluid from the ventricles into the peritoneal cavity in the abdomen8. However, in certain choroidal pathologies, such as choroid plexus hyperplasia, choroid plexus papilloma, and posthemorrhagic hydrocephalus, the increased intracranial pressure occurs, at least in part, from CSF overproduction10. The molecular mechanisms underlying the pathologic increase in CSF production remain elusive. Insight into the transport mechanisms underlying brain CSF accumulation could provide a rational therapeutic target to reduce this pathologic brain fluid accumulation.The mammalian brain is bathed in the cerebrospinal fluid (CSF), which is continuously produced at a rate of approximately 500 ml fluid per day in the adult human+/K+-ATPase12) followed by osmotically obliged, passive movement of water, partly via the water channel aquaporin 1 (AQP1) expressed at the luminal membrane of the choroid plexus14. However, several observations suggest that such a simple osmotic model may not be adequate: (1) The CSF production declined by a mere 20% in the AQP1 knock-out mice, partly ascribed to the 80% reduction of central venous blood pressure in these mice15. (2) With the known osmotic water permeability across the choroid plexus, detailed calculations have demonstrated that the osmolarity of the CSF must exceed that of the plasma by as much as 250 mOsm (in contrast to the measured difference in osmolarity of 5−10 mOsm17) in order for the CSF to be produced at the observed rate by simple osmosis18. (3) The choroid plexus has the ability to produce CSF against an oppositely directed osmotic gradient21. Taken together, conventional aquaporin-mediated osmotic water transport does not suffice to sustain the rates of CSF production consistently observed in mammals.The CSF production is generally assumed to take place by transport of osmotically active ions (e.g. sodium by the Na22). The coupling between water translocation and substrate transport takes place within the protein itself in a manner that permits water to be transported independently of, and even against, an osmotic gradient23. Examples of such water-translocating cotransporters are the Na+/K+/2Cl− cotransporter 1 (NKCC1) and the K+/Cl− cotransporters (KCCs)26. Isoforms of these transport proteins have been detected in the choroid plexus epithelium29, although their exact isoform distribution, relative expression, and membrane targeting remain largely unknown, as are their ability to transport water independently of an osmotic gradient in the choroid plexus tissue and their contribution to CSF production in vivo. In the present study, we introduce the water-translocating cotransporter, NKCC1, as the main contributor to CSF formation in the mouse choroid plexus.A number of cotransporter proteins have the inherent ability to cotransport water along with the ions/solutes in the translocation mechanism  or NaCl  P = 0.96, df = 14, q = 0.4) reached comparable levels  completely blocked the K+-mediated choroid plexus volume increase: The tissue responded in a passive manner, as when challenged with mannitol as the osmolyte . We were, however, unsuccessful with all five KCC3 antibodies tested, none of which properly recognized KCC3, either due to poor epitope recognition or lack of KCC3 expression in the oocytes. Western blotting of lysates from mouse choroid plexus demonstrated robust immunoreaction with antibodies targeting NKCC1 and KCC1, while KCC2 and KCC4 were below detection limit in this tissue  compared to the total membrane fraction (Ft) . In contrast, abundance of KCC1 was significantly lower in the biotinylated fraction  and resembled the pattern obtained with E-cadherin, a basolaterally located membrane protein32 . Therefore, NKCC1, not KCC1, is localized to the luminal membrane of choroid plexus, from which it could indeed participate in CSF production.Localization of NKCC1 and KCC1 to the luminal membrane of the choroid plexus is required in order for these to directly transport water into the ventricular lumen. Immunohistochemical staining of mouse choroid plexus verified the localization of NKCC1 at the luminal membrane , and the ion concentrations determined with flame photometry (Na+ and K+) or by colorimetry (Cl−). The obtained ion concentrations  illustrate that the intracellular Na+ and Cl− concentrations are substantially higher than the 5−15 mM range usually observed in most mammalian cells34. Calculation of the Gibbs free energy using the obtained ion concentrations (see Methods) yields ΔG = 448 J mol−1, which indicates an outwardly directed transport of NKCC1 in the lumen-facing membrane of mouse choroid plexus epithelial cells under physiological conditions. To experimentally determine the transport direction of NKCC1 in choroid plexus epithelial cells, we performed wide-field Na+ imaging on ex vivo choroid plexus, loaded with SBFI-AM  , indicating that the observed increase in [Na+]i did not result from unspecific influx of Na+, but was indeed due to blocking of NKCC1 transport activity. The unique transport direction of NKCC1 was verified with efflux experiments with 86Rb+, a congener for K+, from pre-loaded ex vivo choroid plexus. The 86Rb+ efflux decreased approximately 85% after exposure to either bumetanide  or furosemide   Fig. . In chorons Fig. . Upon in.3) Fig. , indicat36). In this experimental approach, aCSF containing fluorescent dye (dextran) is perfused via a cannula through the lateral ventricle of the mouse (0.7 µl min−1) with simultaneous fluid collection by a glass capillary from the cisterna magna at 5-min intervals  was obtained in a set of animals anesthetized with isoflurane rather than ketamine/xylazine (see Methods). The vehicle (DMSO)-containing aCSF delivered to the lateral ventricle was replaced with new DMSO-containing aCSF at the time point marked “solution change”. After 60 min additional sample time, the last two sample points . Based on these results, it is evident that NKCC1 is a substantial contributor to the molecular machinery underlying CSF production in an in vivo experimental setting. While this dilution method reflects CSF production of all origins , delivery of a pharmacologic inhibitor via the cannula placed in one lateral ventricle may not reach the transport mechanisms expressed in the choroid plexus at the base of the contralateral ventricle. To obtain an estimate of the reach of such one-sided drug delivery, Evans blue was infused into one lateral ventricle in a manner mimicking the experimental approach above, prior to isolation of the brain. As evident in Fig. To determine the contribution of NKCC1 to CSF production in vivo, we performed experiments on anesthetized mice placed in a stereotaxic frame during ventriculo-cisternal perfusion  (repeated measures (RM)) followed by Tukey’s multiple comparisons test, P = 0.0018, df = 399, q = 4.9) or furosemide  followed by Tukey’s multiple comparisons test, P = 0.0083, df = 399, q = 4.2) significantly decreased the movement of dye compared to control, indicative of NKCC1-mediated movement of ventricular fluorescence and thus CSF flow. Of note, injection of Evans blue, in a manner and quantity mimicking the experimental approach above, predominantly stained the ipsilateral ventricle . The ventricular dye redistribution is employed as a proxy of CSF production, although diffusion of the dye is expected to contribute to its redistribution. Figure cle Fig. , indicatHere, we have demonstrated, by complementary ex vivo and in vivo experimentation, that the high production rate of CSF is sustained by NKCC1 via its inherent ability to cotransport water along with its directional ion translocation in a manner independent of osmotic driving forces. This unconventional means of fluid secretion underlying CSF production represents a paradigm shift in the field and provides a long-needed rational therapeutic target towards brain pathologies involving disturbances in brain water homeostasis and increased intracranial pressure.27. A slightly reduced CSF production was observed in AQP1−/− mice, although accompanied by a severe drop in venous blood pressure in these animals15. With the general view that CSF originates from the vascular compartment38, taken together with a fivefold higher arterial flow in choroid plexus compared to that of the brain as a whole12, such AQP1-dependent reduction in blood pressure15 is anticipated to contribute to the reduced CSF production observed in AQP1−/− mice. Secondly, it has been assumed that CSF production takes place via conventional osmotic water transport following a ventricular build-up of osmotic particles through activity of choroidal membrane transport mechanisms, such as the Na+/K+-ATPase, cotransporters, and/or ion channels13. Concerted action of these transport mechanisms renders the CSF approximately 5−10 mOsm hyperosmolar relative to plasma17. With the established water permeability of the choroid plexus epithelium39, calculations dictate that the transepithelial osmotic gradient entailed to sustain the high CSF production rate would need to be about 50 times greater than that observed18. Thirdly, while the rate of CSF production indeed increases with raised ventricular osmolarity, CSF production continues even when the ventricular fluid is hyposmotic to plasma21. In goat, the in vivo CSF production continued despite opposing osmotic gradients of up to 100 mOsm20.The limitations of a conventional osmotic model for CSF production are apparent from three independent lines of evidence: The minimal effects of genetic deletion of AQP1, the low osmotic water permeability of the epithelium, and the ability of the choroid plexus epithelium to transport water uphill against a transepithelial osmotic gradient. Firstly, discovery of AQP1 in the lumen-facing membrane of choroid plexus initially promoted its implication in CSF production22, rendering the combined solute and water transport near-isotonic. NKCC1 is prominently featured among these and has in complementary cell systems been demonstrated to cotransport water26. Here, we reveal that the mouse choroid plexus displays the ability to transport water against a significant osmotic gradient in a K+-induced, NKCC1-mediated manner. While the high [K+]o strongly favors inward transport by NKCC1, and thus robust cell swelling, the high [Na+]o is, although to a lesser degree, likely to do so as well. However, with the low apparent affinity of NKCC1 for K+40, the inwardly directed transport under conditions of high [Na+]o and low [K+]o and the resulting NKCC1-mediated cell swelling will be limited and thus likely masked by the parallel osmotically induced cell shrinkage. Our data align with an earlier study on salamander choroid plexus, performed with ion-sensitive microelectrodes, which, however, relied on the KCC for movement of water independently of the prevalent osmotic gradient25. It should be emphasized that the NKCC1-mediated component of CSF production is a molecular property within the transport protein and not a result of unstirred layers around the protein or changes in intracellular osmolarity22.Simple osmotically obliged water entry into the ventricle is therefore insufficient to sustain the well-established rate of CSF production and here we propose cotransport of water as the molecular mechanism underlying CSF production. Our research group earlier demonstrated that cotransporters function as unconventional water transporters carrying 300–500 water molecules along with the transported solutes per transport cycle27 and rat41 choroid plexus. We observed KCC1 expression at the mRNA43 and protein level and localized it to the basolateral membrane, while KCC2-4 mRNA and protein expression was negligible or below detection limit in our samples. Lack of KCC2 mRNA and protein in choroid plexus confirms previous studies44, whereas a few reports have assigned KCC3 and KCC4 protein to choroidal tissue29 despite absence or downregulation after birth of mRNA encoding these isoforms (this study and ref. 43). We speculate that the discrepancy might arise from insufficient antibody specificity, by the developmental changes in KCC mRNA expression43, and/or the distinct expression profile of the different choroid plexuses45. We are, nevertheless, left with the conclusion that in adult mouse choroid plexus, NKCC1 is expressed in the membrane facing the ventricular lumen and KCC1 in the basolateral membrane. The remarkably high intracellular concentrations of Na+ (~30 mM) and Cl− (~35 mM) in the mouse choroidal epithelium align with values obtained in rat46 and suffice to promote outwardly directed NKCC1 transport. This atypical NKCC1 transport direction was experimentally verified by complementary techniques, aligns with rat tissue47, and supports the notion that NKCC1 is uniquely well-suited to participate in CSF production via its outwardly directed transport in the choroid plexus epithelium  and revealed robust expression of NKCC1. The observation of NKCC1 localization in the lumen-facing membrane of mouse aligns with reports on human−1 than determined earlier in non-ventilated mice (0.38 ± 0.02 μl min−115) and in our unpublished pilot experiments. Determination of CSF production by the ventriculo-cisternal perfusion method relies on dilution of ventricularly delivered dextran by the newly produced CSF. This method, however, cannot distinguish between CSF formed by the choroid plexus (presumably the majority7) and that entering the ventricles via the ependymal cell layer following its secretion across the capillary wall in the reminder of the brain50. The endothelial expression of NKCC1 is negligible41, and the bumetanide-sensitive fraction of the CSF production is therefore assigned to the highly NKCC1-expressing choroid plexus. Inhibition of NKCC1 (upon intraventricular delivery of inhibitor) decreased CSF production by 50% in mice, the quantitative importance underscored by a similar finding in dogs51. Notably, intravenous delivery of CCC inhibitors consistently fail to affect CSF production rate53, in alignment with the luminal membrane expression of NKCC1 illustrated in summary Fig. Optimal CSF production relies on sustained physiological parameters in the anesthetized animal. We therefore propose that to reliably measure the rate of CSF production and assign quantitative contributions of different transport mechanisms, it is crucial to make every effort to maintain core body temperature and ensure proper artificial ventilation of the animal, and hence physiological values for arterial partial pressure of carbon dioxide and oxygen, arterial pH, heart rate, and blood pressure. Of note, ventilated mice had a significantly higher CSF production rate of 0.66 ± 0.02 μl min+-coupled bicarbonate transporters and the Na+/K+-ATPase also contribute to CSF production14, and may potentially cotransport water. Figure 54. The involvement of bicarbonate transporters has been investigated via inhibition of the carbonic anhydrase by acetazolamide56. As this treatment appears highly vasoconstrictive especially in choroid plexus57, the ensuing decreased choroidal blood flow, which in itself lowers CSF production38, may complicate delineation of the direct role of the bicarbonate transporters. The slightly increased effect of furosemide over that of bumetanide in the ventricular-cisternal perfusion experiment, despite the lack of KCCs in the luminal membrane of the choroid plexus, may be assigned to the low-affinity inhibitory action of furosemide on carbonic anhydrase52 and potentially on chloride/bicarbonate exchange58. The less invasive and rapid whole animal imaging qualitatively confirmed the role of NKCC1 in CSF production.While in larger animal models, it would be technically feasible to place cannulas in both lateral ventricles and apply double lateral perfusion (to determine the full contribution of NKCC1), it is highly likely that other luminally -expressed membrane transport mechanisms such as Na−/− mouse model). While NKCC1−/− mice are viable, they, in addition, display a range of physiological deficits59 and we therefore relied on the well-tested action of bumetanide. With such a specific and effective inhibitor, the advantage of the pharmacological approach is the acuteness of the transporter inhibition, maintenance of normal epithelial ion concentrations, the ability to employ each mouse as its own control, and the guarantee of no developmental effects  trafficked to the opposite choroidal membrane60).Notably, genetic deletion of NKCC1 is predicted to cause severe alterations in choroidal epithelium ion concentrations, and therefore in the driving forces and activity of other choroidal transport mechanisms 2/d2 with 80% power and type 1 error and using SD (standard deviation) = 0.06 from ref. 15 and d (effect size) = 0.11 combined from refs. 51.All procedures involving animal experimentation conformed to European guidelines, complied with all relevant ethical regulations, and were approved by the Danish Animal Experiments Inspectorate with permission no. 2016-15-0201-00944. Adult male B6JBOM (Taconic) or C57BL6J (Janvier Labs) mice were used for the animal experimentation in ages ranging from 8 to 12 weeks. Housed with 12:12 light cycle and access to water and food ad libitum. Sample size for the in vivo studies was chosen using the formula2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 17 Na-HEPES, pH 7.4). The brains were kept in ice-cold aCSF-HEPES for a minimum of 10 min to cool the tissue and ease dissection. Choroid plexus was isolated from the lateral and the fourth ventricles (the latter only employed in expression studies and ex vivo volume live imaging) after removal of the most lateral 2−3 mm of the brain hemispheres followed by separation of the two brain hemispheres.Choroid plexus (CP) was isolated after cervical dislocation, decapitation and rapid removal and placement of the brain in cold artificial CSF solution (aCSF-HEPES) containing . The coverslip was placed in the closed laminar perfusion chamber (Warner instruments) prior to loading with 16.67 µM calcein-AM (Life tech), 8–10 min loading followed by a 6–7 min washout period to remove excess dye. Live imaging was performed at 37 °C on choroid plexus mounted on an inverted Nikon T2000 microscope stage with a ×60 1.4 NA plan Fluor objective placed underneath the coverslip with immersion oil. The tissue was superfused with aCSF-HEPES at a flow rate of 1 ml min−1 and swift solution change was ensured with a PC-16 controller (Bioscience Tools). Hyperosmolar solutions were obtained by addition of 100 mOsm mannitol (100 mM), NaCl (55 mM), or KCl (55 mM). Na+-free aCSF-HEPES , pH 7.4). Osmolarities were determined with an accuracy of 1 mOsm with an osmometer Type 15 (Löser Messtechnik). The fluorophore was excited by light of a wavelength of 495 ± 15 nm delivered by a polychrome IV monochromator (Photonics). The emitted fluorescence of wave lengths 510–535 nm was recorded for 2–10 ms at a frequency of 1–5 s with a 12-bit cooled monochrome CCD (Charge-Coupled Device) camera and analyzed with FEI Live Acquisition software. Each choroid plexus was randomly assigned and all image analyses were blinded to the experimental conditions. Images of mouse choroid plexus were converted into black, and background into white using ImageJ percentile threshold adjustment. The 2D changes in the black to white ratio were used to measure relative changes in the size of the choroid plexus. These values represent an underestimation of the actual 3D volume changes, assuming these are approximately isotropic but with the relative comparisons employed in this study, this limitation does not affect the outcome of our data.Following isolation, the choroid plexus was stored in ice-cold aCSF-HEPES up to 3 h prior to experiments and mounted on glass coverslips coated with Cell-Tak® (BD Biosciences), prepared according to the manufacturer’s instructions  and 5′-TCTCCTTTAGGCCGAGGGTG -3′ (reverse) to generate an SLC12A4 product of 150 bp; 5′-TGCTCATTGCCGGACTCATT-3′ (forward) and 5′-CCACGTTCTGATCCTGGTCC-3′ (reverse) to generate an SLC12A5 product of 195 bp; 5′-CAGCTGGGGGCTCATACTTC-3′ (forward) and 5′-ACTCCGAAAGATGGCAGCTC-3′ (reverse) to generate an SLC12A6 product of 170 bp; 5′-AGCTCAACGGCGTAGTTCTC-3′ (forward) and 5′-CTGTTCAGCCCTTCCGTCAG-3′ (reverse) to generate an SLC12A7 product of 136 bp; 5′-CCGGGTTCCTATAAATACGGACTG-3′ (forward) and 5′-CAATCTCCACTTTGCCACTGC-3′ (reverse) to generate a Glyceraldehyd-3-phosphate (GAPDH) product of 195 bp; 5′-CTCCGGAAAGGCCAAGACAA-3′ (forward) and 5′-TTTGACGCATTTCCTGCCAAC-3′ (reverse) to generate an H2A family, member Z (H2AFZ) product of 198 bp.Total RNA from mouse choroid plexus in RNAlater  was purified with the RNeasy micro kit (Qiagen) and the RNase-free DNase set (Qiagen), according to the manufacturer’s instruction. A total of 0.5 µg total RNA or 1 µg cRNA was used for reverse transcription using the Omniscript RT mini kit (Qiagen) . cDNA was amplified by quantitative PCR conducted using LightCycler 480 SYBR Green I Master Mix (Roche Applied Sciences). Reactions were carried out in triplicates on a Stratagene Mx3005P QPCR system from Agilent Technologies. The following primer pairs were applied for amplification of targets: 5′-GCAAGACTCCAACTCAGCCAC-3′ (forward) and 5′-ACCTCCATCATCAAAAAGCCACC-3′ (reverse) to generate an SLC12A2 104.3%; SLC12A4 95.1%; SLC12A5 101.2%; SLC12A6 101.8%; SLC12A7 96.1%, GAPDH 100.5%; and H2AFZ 97.6%). GenEx  was used for data analysis including testing for best reference gene combinations. Target genes are normalized to GAPDH and H2AFZT and presented as relative expression.Primers were designed using NCBI’s pick primer software. The optimum concentration for each primer set was determined to 200 nM. The initial melting was performed at 95 °C for 10 min. During the subsequent 40 amplification cycles, the melting temperature was 95 °C (20 s), the primer annealing temperature was 60 °C (22 s), and the elongation temperature was 72 °C (20 s). After completed amplification, melting curves were generated to confirm amplification specificity. Standard curves of 4× serial dilutions of cDNA were made using either reverse transcribed cRNA (target genes) or total RNA from mouse choroid plexus (reference genes) in order to determine the amplification efficiencies for each of the utilized primer-sets (Xenopus laevis were obtained from frogs purchased from Nasco (Fort Atkinson) or purchased from Ecocyte Bioscience (Germany). The oocytes were surgically removed from anesthetized  frogs. The follicular membrane was removed by incubation in Kulori medium  containing 10 mg ml−1 collagenase  and trypsin inhibitor  for 1 h, prior to wash in Kulori medium containing 0.1% bovine serum albumin  and incubated in 100 mM K2HPO4 with 0.1% BSA for 1 h. The oocytes were kept in Kulori medium until experiments. cDNA encoding rNKCC1 was obtained from Professor Kai Kaila  and cDNA encoding mKCC1-4 was obtained from Professor Hans Gerd NothWang  and all sequences were verified prior to use (Eurofins Genomics). cRNA was prepared from linearized plasmids using the mMESSAGE mMACHINE T7 kit (Ambion) and extracted with MEGAclear (Ambion), according to the manufacturer’s instructions, prior to microinjection of 50 ng cRNA per oocyte with a Nanoject microinjector (Drummond Scientific Company). The oocytes were kept at 19 °C for 5 days after which membrane preparations were obtained by homogenization of 20–40 oocytes expressing each construct in 1 ml buffer containing  and pefabloc (0.4 mM), both from Sigma-Aldrich). The supernatant was recovered following 10 min centrifugation at 250 × g and subsequently centrifuged at 14,000 × g for 20 min to obtain the total membrane fraction.Oocytes from 66 , anti-KCC2; 07-432 , anti-KCC3&4 67 , other anti-KCC3 tested; HPA034563 , H00009990-A01 , and sc-19424 . Secondary antibodies; IRDye 800CW donkey anti-goat; P/N 926-32214, IRDye 680RD donkey anti-mouse; P/N 926-68072, IRDye 800CW donkey anti-rabbit; P/N 926-32213, and IRDye 800CW goat anti-rabbit; P/N 926-32211. All from LI-COR Biosciences, 1:10,000-1:15,000.Isolated choroid plexuses  were pooled from each mouse and sonicated on ice  in phosphate-buffered saline (PBS). Western blotting was performed using precast SDS-PAGE gels  and immobilon FL-membranes (Merck Millipore). Both primary and secondary antibodies were diluted in Odyssey blocking buffer (LI-COR Biosciences):PBS-T at 1:1. Signals were detected with an Odyssey CLx imaging system and image analysis was performed using Image Studio 5 (both from LI-COR Biosciences). Full blots with original markers are included as Supplementary Fig. 2, 10 triethanolamine, pH 7.5) was injected to the ventricle and the mouse left on ice for 15 min followed by isolation of choroid plexus. After isolation and quenching to remove excess biotin, the choroid plexus was transferred to a lysis buffer  for 30 min, all according to the manufacturer’s instructions. The samples were sonicated 3 × 10 s at 70%  and centrifuged at 10,000 × g for 5 min at 4 °C. An aliquot was removed  before proceeding with the biotin  fraction purified on NeutrAvidin (Thermo Fisher) columns (Pierce).Mice were anesthetized with isoflurane, placed in the prone position in a stereotaxic frame (Harvard Apparatus), a dorsal midline incision was made over the skull and upper cervical spine to expose the cranium, after which a brain infusion cannula  was placed in the lateral ventricle using the coordinates: 1.0 mm lateral to the midline, 0.5 mm posterior to bregma, and 2.5 mm ventral into the brain. After sacrificing the mouse by cervical dislocation, 1.5 mg EZ-link Sulfo-NHS-SS biotin (Thermo Fisher) in 100 μl biotin buffer  and anti-e-cadherin; 610181 , prior to labeling with secondary antibody; donkey anti-mouse; Alexa555 , donkey anti-rabbit; Alexa488  and nuclear staining with Topro3 . Anti-KCC1 tested; HPA041138 (Atlas Antibodies), anti-KCC1 (kind gift from Professor Thomas J. Jentsch), and ab115607 (Abcam). The digital images were acquired with a DM IRE2 inverted confocal microscope (Leica Microsystems).Sections obtained from tissue from male C57BL/b mice from Taconic (Denmark) were immunostained with anti-NKCC1. 5−10 min later, it was followed by an i.p. injection with ketamine . With intervals of 25−35 min, the mouse was re-dosed with ketamine . Anesthetized mice, after tracheotomy, were placed in a stereotaxic frame and clear CSF collected from cisterna magna. Immediately after decapitation, blood was collected and choroid plexus isolated, weighed, dried at 105 °C, and extracted in 0.1 M HNO3 on a horizontal shaker table 69. The ion concentrations of the choroid plexus were calculated based on previous observations of choroid plexus epithelium containing 79% water and 8.5% blood69. Na+ and K+ content was quantified by flame photometry (Instrument Laboratory 943) while Cl− concentration was quantified by a colorimetric method using QuantiChrom™ Chloride Assay Kit (MEDIBENA Life Science & Diagnostic Solution). Gibbs free energy for NKCC1 was calculated as Eq. 1To obtain enough material, samples from four mice were pooled for each of the four experiments. Mice were anesthetized with xylazine  injection; 1 mg mlR = gas constant (8.314 J mol−1), T = temperature , [X]i = intracellular ion concentration, and [X]o = ion concentration in CSF.where + imaging of choroid plexus bathed in aCSF-HEPES, the epithelial cells were loaded (~20 min) with the membrane-permeable form of SBFI  ester, 200 µM, Teflabs) To this end, the dye was pressure-applied (5 s) directly onto the cells on several positions70. Afterwards, the tissue was perfused with aCSF-HEPES for at least 20 min to allow for de-esterification of the dye before imaging experiments were commenced. Wide-field Na+ imaging was performed utilizing a variable scan digital imaging system  attached to an upright microscope . The microscope was equipped with a ×40/N.A. 0.8 LUMPlanFI water immersion objective (Olympus Deutschland GmbH) and an orca FLASH V2 camera (Hamamatsu Photonics Deutschland GmbH). SBFI was alternatively excited at 340 and 380 nm. Images were obtained at 1 Hz and emission was collected >440 nm. Fluorescence was evaluated in regions of interest (ROIs), representing single cells. Signals were background-corrected as follows: The fluorescence intensities obtained at each excitation wavelength (340/380 nm) were dynamically subtracted frame by frame by the respective fluorescence emissions of a ROI in the field of view, which was free of SBFI-labeling (background). After background subtraction, the fluorescence ratio (F340/F380) was calculated from individual ROIs and analyzed using OriginPro Software (OriginLab Corporation v.9.0). For imaging of cell survival of the choroid plexus after bumetanide treatment, the epithelial cells were loaded with Calcein-AM  as described for SBFI loading. This was done after 10 min of bumetanide treatment, right before the blocker washout (or after 10 min of aCSF as a control). Calcein was excited at 488 nm, emission was collected 510 nm. Each choroid plexus was randomly assigned to each group.For Na2, 1.3 MgSO4, 1 NaH2PO4, 25 NaHCO3, 10 glucose, pH 7.4, equilibrated with 95% O2/5% CO2) but allowed to recover at 37 °C for 5–10 min before beginning of the experiment. Choroidal isotope accumulation was performed by a 10 min incubation in equilibrated (95% O2/5% CO2) aCSF-based isotope medium  and 8 μCi ml−1 3H-mannitol, NET101 (extracellular marker), both from PerkinElmer), followed by 15 s wash prior to incubation in 0.5 ml equilibrated (95% O2/5% CO2) efflux medium , each choroid plexus randomly assigned to each group). 0.2 ml of the efflux medium was collected into scintillation vials every 20 s  and replaced with fresh aCSF. At the end of the experiment, choroid plexus was solubilized at room temperature with 1 ml Solvable  in the leftover efflux medium. The isotope content was determined by liquid scintillation counting with Ultima GoldTM XR scintillation liquid  in a Tri-Carb 2900TR Liquid Scintillation Analyzer (Packard). The choroid plexus 86Rb+ content corrected for 3H-mannitol (extracellular background) was calculated for each time point, and the natural logarithm of the choroid plexus content At/A0 was plotted against time47. Slopes indicating the 86Rb+ efflux rate constants (s−1) were determined from linear regression analysis.Choroid plexus was isolated as above, initially in cold aCSF , each calibrated with respiratory partial pressure of carbon dioxide and arterial oxygen saturation . Ventilation of a ~25 g mouse; approximately 150 μl per breath of a mix of 0.1 l min−1 O2 and 0.9 l min−1 air and approximately 150 breath per min, sight = 10 % increase in tidal volume and Positive End-Expiratory Pressure (PEEP) = 2 cm H2O. The heart rate was continuously monitored . After tracheotomy, 1 ml of warmed 0.9% NaCl was injected subcutaneously to decrease risk of dehydration. The infusion cannula was placed as described in the biotinylation method, and was glued to the scull . The perfusion solution was heated to 37 °C in an inline heater  before entering the infusion cannula. A micropipette held in a 5° position was introduced into the cisterna magna. After observing CSF in the micropipette, infusion of equilibrated (95% O2/5% CO2) aCSF  or 0.03 % w/v Evans blue (314-13-6 Sigma-Aldrich)) and 0.1% DMSO was initiated at a rate of ~ 0.7 μl min−1. Addition of dextran and vehicle (DMSO) increased the osmolarity of the aCSF by approximately 20 mOsm. The final osmolarity of the aCSF employed for the ventricular-cisternal perfusion thus amounted to approximately 313 mOsm, designed to match the mouse plasma osmolarity  in order to limit osmotically induced experimental confounders into the experimental paradigm. An extra set of mice were anaesthetized with isoflurane (0.5–1.5 %) at the time of CSF production rate determination. Anesthesia was induced as above (to facility the tracheometry) but the animal transferred to isoflurane immediately thereafter with no subsequent additional ketamine injections. In this manner, the animal had not been subjected to ketamine/xylazine for at least 2 h and the dominant anesthesia predicted to be isoflurane. After an hour of infusion, the animals anesthetized with the standard ketamine/xylazine described above were exposed to a new aCSF solution containing either 2 mM furosemide, 100 μM bumetanide (expected ventricular concentrations of 1 mM and 50 μM) or the vehicle, DMSO . The solution change took 5–10 min. Outflow was collected in 5-min intervals by introducing a second micropipette into the fixed cisterna magna micropipette and the fluorescent content measured in a microplate photometer . The production rate of CSF was calculated from the equation:Mice were anaesthetized with ketamine and xylazine (as described above) during the experimental procedure and their body temperature monitored and maintained at approximately 37 °C using a homeothermic system (Harvard Apparatus). To obtain near-physiological conditions, the mice were ventilated  after tracheotomy and settings continuously optimized for each animal using a capnograph  and a pulse oximeter (MouseOxVp = CSF production rate (µl min−1), ri = infusion rate (µl min-1), Ci = fluorescence of inflow solution, Co = fluorescence of outflow solution.where Ventricular injections were done as above, except with variation in the ventricular depth: A cannula was placed 2.5 mm into the brain and 2 μl aCSF with vehicle(DMSO)/inhibitor  was injected during 2 s. Within 5 min, the cannula was removed and a new placed 2.2 mm into the brain and the second solution injected. This second cannula contained aCSF with vehicle/inhibitor, as above, in addition to a carboxylate dye . With a lapse of 1 min after injection, the first image was obtained on the Pearl Trilogy Small Animal Imaging System . At the termination of each experiment, a white field image was taken. Due to the swift protocol, these mice were not ventilated. Images were analyzed using Image Studio 5.2 and the region of interest defined as a square, starting at lambda. For test of whether the injected solution reached both ventricles, 0.03% w/v Evans blue  dissolved in aCSF was injected in a manner mimicking the injection of fluorescent dye.Chemicals were freshly dissolved prior to each experimental day. Furosemide  was dissolved directly into the aCSF while a stock solution (100 mM in DMSO) was prepared with bumetanide .t test, one-way or two-way (RM)ANOVA followed by the Tukey’s multiple comparisons post hoc test were employed as indicated in figure legends. Data are obtained from choroid plexus from individual animals and presented as mean ± SEM with significance set at P < 0.05.Data analysis and statistical tests were carried out with GraphPrism 7.0 (GraphPad Software). One-sample We confirm that all relevant data from this study are available from the corresponding author upon request.Supplementary InformationPeer Review File


Handwashing with soap is recognized as a cost-effective intervention to reduce morbidity and mortality associated with enteric and respiratory infections. This study analyzes rural Indonesian households’ hygiene behaviors and attitudes to examine how motivations for handwashing, locations of handwashing space in the household, and handwashing moments are associated with handwashing with soap as potential determinants of the behavior. The analysis was conducted using results from a UNICEF cross-sectional study of 1700 households in six districts across three provinces of Indonesia. A composite measure of handwashing with soap was developed that included self-reported handwashing, a handwashing demonstration, and observed handwashing materials and location of facilities in the home. Prevalence ratios were calculated to analyze associations between handwashing with soap and hypothesized determinants of the behavior. Our results showed that determinants that had a significant association with handwashing with soap included: (1) a desire to smell nice; (2) interpersonal influences; (3) the presence of handwashing places within 10 paces of the kitchen and the toilet; and (4) key handwashing moments when hands felt dirty, including after eating and after cleaning child stools. This study concludes that handwashing with soap may be more effectively promoted through the use of non-health messages. Handwashing with soap at critical moments, such as before eating and after defecation, can prevent infectious diseases by interrupting the transmission of infectious agents. Empirical evidence suggests that handwashing with soap reduces the risk of diarrhea ,2,3, acuGood hygiene is of vital importance in Indonesia due to the lack of basic sanitation in the country. Approximately 20% of the Indonesian population, or 51 million people, are estimated to defecate in open spaces, such as fields, bushes, and beaches . Open deThe Government of Indonesia (GoI) has promoted handwashing with soap as part of the national sanitation program, Sanitasi Total Berbasis Masyarakat (STBM), or the Community Approaches to Total Sanitation and Hygiene . The STBAlthough handwashing may be understood as a simple task, multiple levels of influence or behavioral determinants exist to shape this behavior. Previous research has suggested that adult caregivers, female sex, higher levels of education, high socioeconomic status, place of residence, and access to water and sanitation are associated with handwashing with soap ,12,13. HAccurate measurement of behaviors is foundational for estimating the prevalence of handwashing with soap and assessing hygiene interventions. Yet, the difficulty of measuring handwashing behaviors is well-recognized . EmpiricStructured observations, where researchers directly observe people’s handwashing behaviors for a few hours or more, can collect rich contextual data and objective measures of handwashing practice at critical moments including after defecation . By assuWhile formative research has explored how people may be motivated to wash their hands with soap in Indonesia ,26, quanIn this study, we aim to identify the potential determinants of handwashing with soap, estimated by the use of a composite measure for each household enrolled that includes: (1) self-reported handwashing with soap; (2) an observed handwashing demonstration; and (3) observation of handwashing materials  available to household members. Applying this composite measure, the study aims to assess how motivational drivers, key times that individuals wash their hands, and the presence of a handwashing place in the household are independently, as well as collectively, associated with handwashing with soap. Respondent’s age, sex, education, place of residence, household wealth levels, access to water for household needs, and ownership of a private toilet are included in the analysis. Motivations are included as the psychological determinants of handwashing behaviors. Handwashing moments  and the presence of a handwashing place in the household serve as physical cues to induce handwashing with soap. It is hypothesized that motivations for handwashing, self-reported handwashing moments, and access to handwashing facilities will be significantly associated with handwashing with soap, which may be collectively moderated by socio-demographic characteristics and structural factors.In February 2014, UNICEF commissioned Taylor Nelson Sofres (TNS) Indonesia to conduct a knowledge, attitude, and practice (KAP) survey to establish a baseline of sanitation and hygiene conditions in selected communities at the beginning of STBM implementation. UNICEF specifically selected six districts, which are typically more resource-constrained and have low levels of access to sanitation in Indonesia. Based on the multi-stage cluster sampling method, 2036 households were initially identified and contacted by house-to-house visits. A total of 1786 households had someone at home, and 1700 agreed to participate in the survey, attaining the participation rate of 95.2%. The questionnaire was orally administered in the local language after obtaining verbal informed consent with TNS following international ethical standards (ESOMAR) adherence and having approval from the Government of Indonesia to undertake such surveys in Indonesia. Study participants were from six districts of Indonesia across three provinces, including Alor, Sumba Timur (NTT Province), Luwu Utara, Takalar, Barru (South Sulawesi Province), and Jayapura (Papua Province) .The outcome variable of this study is handwashing with soap, measured using three indicators of handwashing. First, respondents were asked what they usually use to wash their hands without any prompt. Second, the availability of water and soap at a handwashing place in the household was assessed through direct observation. Lastly, the research team asked respondents to show how they usually wash their hands and directly observed the materials used for their handwashing demonstration. Study participants were regarded as performing handwashing with soap in this study if they reported to wash hands with water and soap, if water and soap were observed at the handwashing place, and if use of water and soap was observed during the handwashing demonstration. This study will use the composite measure of handwashing with soap for all the bivariate and multivariate analyses, unless stated otherwise.The exposure variables of interest include psychosocial and structural factors that may influence handwashing behaviors. A psychosocial factor examined in this study is people’s motivations for handwashing. Respondents were asked what motivates them to wash their hands in an open-ended question without any prompt or any limit to the number of responses that they can give. Each response was recorded as one of the nine reasons derived from literature search on sanitation and hygiene in Indonesia , and indicator variables  were constructed for each motivational factor.This study also assessed seven handwashing opportunities or moments, which consist of before cooking, before eating, after eating, before feeding a child, after cleaning child stools, after defecation, and after work. As with motivations for handwashing, respondents were asked when they usually wash their hands without prompt, and indicator variables were made for each handwashing opportunity . For a structural factor, access to handwashing facilities in the household was directly observed and recorded with the following categories: within 10 paces of the toilet facility, within 10 paces of the kitchen, elsewhere in the household, outside of the yard, and no specific place.Control variables for this study consist of respondents’ age, sex, education, district, household size, access to water and sanitation, and wealth quintile. As a proxy measure of socioeconomic status, the wealth quintile was developed with 12 variables that represent household assets and characteristics  by conducting the principal component analysis .This study employed STATA 13 to perform univariate, bivariate, and multivariate analyses . BivariaDue to the issue of multicollinearity , Alor and Sumba Timur districts were both included as the reference group of the district variable in the multivariate analysis. This study also examined if the presence of a handwashing place near the kitchen and the toilet is associated with handwashing with soap before cooking and after defecation, respectively. Neither of the hypothesized interaction effects was statistically significant, and thus, multivariate models only assessed the main effects of independent variables.The descriptive characteristics of the study participants are summarized in The top three self-reported reasons or motivations for handwashing were to be clean (89.6%), to prevent the spread of disease (52.9%), and to get rid of dirt/smell/sticky things (37.5%). Approximately 60% of households had access to a handwashing place within 10 paces of the toilet or the kitchen, and 19% had a handwashing station in other locations around the household. However, 21.3% did not have a specific location for handwashing. Lastly, almost 90% of respondents reported to wash hands before eating, with only 56.6% of people reporting to wash hands after eating and 52.1% after defecation. Only 7.6% and 13.4% of respondents reported to wash hands before feeding a child and after cleaning child stools, respectively. These results suggested that many respondents do not wash hands at all of the critical moments including before cooking, before eating, before feeding a child, after cleaning child stools, and after defecation.Major differences were observed for the prevalence of handwashing with soap between the individual measures of handwashing and the composite measure . While sThe prevalence of handwashing with soap at critical moments was very low. Based on the composite measure, only 26.7% and 52.0% of respondents were estimated to wash hands with soap after defecation and before eating, respectively. The prevalence was even lower before feeding children (4.0%) and after cleaning child stools (6.9%), and only 1.8% of respondents were estimated to wash hands with soap at these four critical moments.The proportion of older adults who washed hands with soap (49.4%) was lower than that of young adults (56.3%) and middle-aged adults (57.0%) See . The estThe respondent’s education, the district the household was located in, household wealth, and access to water and sanitation were significantly associated with the behavior of handwashing with soap, and the calculated prevalence is presented in The majority of respondents who had access to a handwashing place within 10 paces of the kitchen and the toilet facility was estimated to wash their hands with soap. Compared to respondents without a specific location for handwashing in the household, the prevalence of handwashing with soap was on average 24% and 37% higher among those respondents who had a handwashing place within 10 paces of the kitchen and the toilet, respectively.The estimated prevalence of handwashing with soap by self-reported handwashing moments ranged from 39.3% before cooking to 63.0% after eating. Five handwashing moments, including before cooking, before eating, after eating, after defecation, and after work were significantly associated with handwashing with soap. The direction of associations, however, was not uniform. Reporting to wash hands before eating and after eating were associated with a higher prevalence of handwashing with soap than those who did not report to wash hands at these respective moments. The other handwashing moments , however, were negatively associated.The results of nested GLM regression analysis are presented in In Model 2, motivations of good smell and interpersonal influence were positively associated with handwashing with soap while negative associations were found for the aesthetic reason  and the descriptive norm . By holding other variables constant, respondents who washed their hands for good smell and interpersonal influence  were associated with having 26.2% and 26.0% higher prevalence of handwashing with soap than other respondents without these motivations. Having a motivation to remove dirt, smell, or sticky materials on their hands was associated with 20.6% lower prevalence of handwashing with soap than those respondents without this motivation.In Model 3, respondents who have a handwashing place within 10 paces of the kitchen and the toilet were associated with 28.2% and 46.7% higher prevalence of handwashing with soap, respectively, than those respondents who do not have a specific handwashing place. Thus, the presence of handwashing stations in the household may be a key structural determinant or enabling environment of handwashing with soap. By adding the handwashing place to the baseline specification, the association between self-reported ownership of a private toilet and handwashing with soap was no longer statistically significant. This result suggests that the presence of handwashing stations may mediate the independent effect of the sanitation variable on handwashing with soap, highlighting the need for promoting handwashing stations around the household sanitation facility or the kitchen to facilitate handwashing with soap.In Model 4, four handwashing moments, including before cooking, before eating, after eating, and before feeding children, were significantly associated with handwashing with soap when holding other variables constant. The prevalence of handwashing with soap among respondents who reported to wash hands before cooking was 26.0% lower than that of respondents who did not report to wash hands at this handwashing moment. This finding suggests that people do not often use soap for their handwashing before cooking, and consequently food preparation may remain an important pathway for food contamination. Respondents who reported to wash their hands at the other three handwashing moments had a higher prevalence of handwashing than the respondents who did not report to wash hands at these moments. In this model, household size lost its significant association with handwashing with soap. This may be a reason that the independent effect of household size is significant in the previous regression models, which do not assess the association of handwashing moments with handwashing behaviors. The analysis suggests that in Model 4, self-reported handwashing moments may mediate the association between household size and handwashing with soap.Most of the variables maintain their significant association with handwashing with soap in Model 5, except for a few self-reported motivations and handwashing moments. In this model, cleanliness, good smell, aesthetic motivations, religious reasons, and interpersonal influence have significant associations with handwashing with soap while the descriptive norm is no longer significant. Self-reported handwashing moments that include before cooking, after eating, and after cleaning child stools were significantly associated with the outcome.This study explored potential determinants of handwashing with soap in six districts across three provinces of Indonesia. The results suggested that structural factors, namely household wealth levels, district of residence, and presence of handwashing infrastructure within 10 paces of the kitchen and the toilet, are associated with greater handwashing with soap in the context of this study. Motivations for handwashing, such as good smell and interpersonal influence, were also identified as factors associated with handwashing with soap. The analysis further highlighted that handwashing with soap is more common at certain handwashing moments, mainly after eating and after cleaning child stools, than other critical times, such as after defecation or before food preparation.The estimated prevalence of handwashing with soap by the composite measure was lower than that of individual measures of handwashing in this study. Although this study cannot assess the validity of the composite measure by comparing the estimated prevalence with that of structured observations, a more conservative estimate of handwashing behavior was obtained. The findings in this study can be interpreted to indicate that the combination of multiple indicators, rather than one indicator, may be more useful for household surveys and reduce the risk of overestimating handwashing behaviors. The Demographic and Health Surveys (DHS) and the Multiple Indicator Cluster Survey (MICS) only collect data on the presence of soap and water for handwashing in the household as the proxy measure of handwashing behaviors. Handwashing measurement in these large household surveys could potentially be strengthened by including the direct observation of handwashing demonstration and developing a composite measure to estimate the prevalence of handwashing with soap in low- and middle-income countries.Descriptive analysis revealed that Alor district has the lowest prevalence of handwashing with soap, which could have been induced by limited access to water in this district. A further analysis of the data found that only 46.2% of households in Alor were estimated to have access to water throughout the year, compared to at least 87% or higher in other districts. In focus group discussions conducted by TNS, a female participant from Alor also stated that “I will not let my kids wash their hands unless we have enough water, since getting water is difficult and our priority is for drinking and cooking” . AdequatThe drivers that form hygiene habits or change individual and community-level hygiene behaviors are not fully understood. In this study, disease prevention did not appear to be a primary driver for handwashing with soap. By recognizing that handwashing may not be always facilitated by health-related motivations, previous research has explored the potential roles of emotional drivers  and sociocultural factors  for handwashing promotion ,19. DisgInterpersonal influence was also identified as an influential factor for handwashing promotion. A qualitative study with the same study population revealed that local authorities, community health workers, and religious leaders are influential sources of information on sanitation and hygiene in this study context . AccordiHandwashing moments appeared to provide varying levels of physical cues to induce handwashing with soap. The results suggested that handwashing with soap was more commonly practiced after eating and less commonly practiced before cooking. While the exact reasons for limited handwashing with soap before cooking remain unclear in this study, people may be washing hands with soap inconsistently or wash hands with water only. This finding is in accordance with earlier formative research in Serang district of Indonesia, which reported that new mothers infrequently and inconsistently practiced handwashing at critical moments, including before cooking . While hThere are some important limitations to this study. First, self-reported data could be influenced by social desirability bias . The stuTo generate a research agenda for the future, we conducted a post hoc correlation analysis to assess the association between self-reported motivations for handwashing and three handwashing moments—after defecation, before eating, and after cleaning child stools. This analysis revealed that motivations for disease prevention, cleanliness, good smell, interpersonal influence, and descriptive norms were positively associated with self-reported handwashing after defecation. Cleanliness and good smell were positively associated with handwashing before eating while a negative association was found for the aesthetic motivation. Lastly, disease prevention and aesthetic motivations were positively associated with handwashing after cleaning child stools. Accordingly, motivational drivers differently function to influence people’s handwashing practices at each critical handwashing moment. To promote handwashing with soap at critical moments more effectively, future research can further examine what motivations are associated with people’s handwashing practices at each handwashing moment.This study identified some of the potential determinants of handwashing with soap by highlighting the substantial effects of structural and psychosocial factors. Physical proximity to handwashing stations has been shown to facilitate handwashing behaviors. Future interventions that send the message of good smell and comfort associated with handwashing may be also effective to promote the practice of handwashing with soap. More efforts are necessary to understand which key drivers for handwashing could effectively increase the practice of handwashing with soap at each critical moment, along with increased operational guidance on how to change his personal hygiene behavior at scale and maintain the behavior as a habit.Handwashing with soap is a cost-effective strategy to reduce the global burden of disease, yet this hygiene behavior has not been fully recognized as a global health priority. While water and sanitation have been included in the Millennium Development Goals (MDGs) and the Sustainable Development Goals (SDGs), the progress on hygiene promotion has not been sufficiently tracked globally. While the evidence base of handwashing measurement is not complete, this study revealed a potential utility of composite measures to monitor and evaluate the progress of handwashing promotion. Developing credible indicators to monitor handwashing behaviors can be the first step to further highlight the importance of hygiene behaviors in global health.


For the majority of scholars concerned with the ethics of living organ donation, inflicting moderate harms on competent volunteers in order to save the lives or increase the life chances of others is held to be justifiable provided certain conditions are met. These conditions tend to include one, or more commonly, some combination of the following: (1) The living donor provides valid consent to donation. (2) Living donation produces an overall positive balance of harm–benefit for donors and recipients which cannot be obtained in a less harmful manner. (3) Donation is not liable to cause significant and long-term morbidity to, or the death of, the donor. This paper critically examines the suggestion that these criteria are not sufficient to offer a general account of justified living organ donation in the context of competent volunteers and that key to justified living organ donation is that donors receive sufficient benefits from their donation that these outweigh the harms they suffer. However, although this view—termed here ‘The Donor Benefit Standard’—directs welcome attention to the many and complex motives which may underlie living organ donation, this paper ultimately concludes that given the threats this position poses to individual autonomy and the lives of those in need of organ transplants ‘The Donor Benefit Standard’ should ultimately be rejected. The living donation of organs has been permitted since the dawn of the transplantation era when organ retrieval techniques, tissue preservation procedures, and immunosuppressive medications were not sufficiently developed to allow for the safe transplantation of organs from deceased donors. At this time, in the absence of retrieval from living donors, those in need of organ transplants would almost certainly die. Thus, transplant surgeons viewed living donation as a temporary and uncomfortable necessity, expressing the hope that in the future the need to resort to living donors would be made obsolete as “organs obtained solely from cadavers could be used with a high expectation of survival”  only where organs are freely given and where donation is expected (and necessary) to produce, an all-things-considered benefit for those holding a stake in the donation, a threshold should be placed on the extent to which living organ donors may be harmed as a result of their donation. Where this threshold should lie is a matter of debate but it is generally held that it requires, at the very least, that donation not be certain, or highly likely, to cause death.Although considerations of harm–benefit and valid consent have always received, and continue to receive, significant attention in the ethics and policy literature regarding living donation, comparatively little philosophical attention has been paid to the suggestion that limits should be placed on the extent to which living organ donors may be harmed as a result of their donation. Yet, despite this, few but the most committed of libertarian and utilitarian scholars, are likely to condone Yet, why should a threshold be placed on the harms that living organ donors are permitted to endure? Why should an altruist not be permitted to forfeit his life and donate his organs and tissues to needy strangers, a husband be proscribed from sacrificing himself in order to donate his heart to his much-loved wife, or a father be forbidden from risking death and accepting a life on dialysis in order to donate a second kidney to his daughter? Unlike requirements of informed consent and the production of a favorable balance of harm to benefit which may be seen straightforwardly to reflect the value of respect for individual autonomy and consequentialist moral reasoning, the imposition of a donor harm threshold may be motivated by a number of different considerations.The weakest versions of the donor harm threshold may, for example, be justified via appeal to a corollary of the dead donor rule in deceased organ donation. This rule, often underpinned by the view that the deliberate/foreseen killing of innocents is never permissible, requires that “a human being must be dead before life prolonging organs  can be procured for transplant or other purposes.”  optimal consequences—leads to the requirement that instances of living organ donation should be permitted only where they produce (and are necessary to produce) an overall positive balance of harm to benefit for those holding a stake in the donation, it may also be used to justify a moderate version of the donor harm threshold. After all, although consequentialist moral reasoning can,  lottery  – where call into question or, more strongly, invalidate donor consent. The strong claim that certain and grave harm may invalidate donor consent is, of course, hard to justify. Even on the most demanding accounts of autonomy, where autonomous action is perceived as synonymous with rational action, few would be willing to hold that acts of extreme self-sacrifice are necessarily irrational. Yet, whilst this is so, it does seem that the presence of significant risks of grave harm may well call the validity of donor consent into question. That is, their presence may act as a ‘red flag’, suggesting that it is prudent to reconsider how clearly information regarding possible risks has been conveyed and the extent to which their consent is truly autonomous. For, whilst many are willing to suffer moderate harm for the sake of advancing the interests/welfare of another  very few are willing to sacrifice their very lives for this purpose.Similarly, just as respect for autonomy motivates the requirement that the retrieval of organs from the living should be permitted only where a donor provides his valid consent to donation, so too may it motivate the requirement that harms to donors fall below some acceptable threshold. It may be suggested, for example, that instances of living donation where the harms to donors are both certain and grave always involves the possibility of serious harm, the donor’s physician should consider this practice justified only when it is anticipated that there will be benefits for the donor that exceed the risks to the donor; benefit for the recipient alone won’t do.  for the recipient’s sake alone. Many others, however, are purely self-regarding (aimed at securing benefits and avoiding or mitigating harms for themselves), or self-interestedly other-regarding (aimed at securing benefits for others because the donor’s welfare interests are intimately bound up with the wellbeing of their intended recipient). Some benefits sought are dependent on the outcome of the transplant for the recipient, especially in cases of the directed donation of organs to emotionally related recipients where the donor has direct and vicarious welfare interests in their intended recipient’s survival  sufficient to outweigh the harms they suffer.Would the donor benefit standard, if implemented, lead to a reduction in the number of organs available for transplantation? Glannon and Ross suggest, in a short piece criticising this and other aspects of Spital’s work that a reduction in the number of organs made available for transplantation would be the likely result  and possibly financial (if payment is involved). Whether these potential benefits compensate for the potential harms is a contested matter. Baylis , p. 533necessarily provide sufficient reason to abandon it. For, as Spital correctly notes in a response to Glannon and Ross—if the donor benefit standard overcomes the shortcomings associated with other standards for justified living organ donation, and does not fall prey to serious criticisms on other grounds - that the donor benefit standard will reduce the number of organs available for transplantation would only prove a decisive reason to abandon it if it can be shown that there is “another ethically acceptable method for determining the suitability of volunteers that [yields] more donors”  when compared to existing and less restrictive standards is, of course, regrettable. It does not, however, In his papers Spital suggests that if accepted and implemented the donor benefit standard would provide a number of clear advantages when compared to currently existing criteria for justified living organ donation. These can be summarised as follows:the only standard for justified living organ donation that is “consistent with the clear benefit standard used to determine the acceptability of incompetents as donors” . However, difficulties aside, in cases where it can be shown that retrieval is in the interests of the ‘donor’—and provided adequately and stringently policed safeguards are implemented to protect against exploitation and abuse—retrieval will be to the advantage of both ‘donor’ and recipient.Thus, it is conventionally held that, in these rare cases, organ retrieval may be justified in the absence of capacity to consent where it can be shown: firstly, that there are no alternative competent sources of donor organs; and secondly, that despite the harms of retrieval, the donor is likely to receive sufficient psychological and emotional benefits that they outweigh the harms inflicted. When, how, and indeed key role in legal cases where persons lacking capacity have been proposed as organ and tissue ‘donors’ since the 1950s. In the US, for example, in the case of Masden v Harrison kidney donation from a 19-year old (who was under the age of majority at the time) to his identical twin brother was permitted by a Massachusetts court as a result of testimony from a psychiatrist that “grave emotional impact may be visited upon [the donor] if the defendants refuse to perform this operation”, and thus that the operation was necessary for the “continued good health and future wellbeing of [the donor]”  and communication skills (which makes it difficult to determine what is truly in their interests). As such, it is generally considered safer to attribute to them only self-regarding interests in the context of living organ donation, this will not be the case for most adults. For, their interests do not, except perhaps in cases of those with severe antisocial personality disorders, reduce merely to those of direct and indirect/vicarious interests in their own wellbeing. They also, and importantly, tend to include interests in the interests of others. This is because mature human beings are not, despite the picture painted by many, essentially or ideally independent creatures who are disengaged from others and characterised by self-interest. They are instead socially embedded entities who are “intimately related to other people, groups, institutions and histories” and are thus “motivated by interests and reasons that can only be fully defined with reference to other people and things”  he may also have interests in B’s wellbeing for reasons that fail to regard his own welfare, but regard instead the emotional, social, and moral relationships in which he stands to other human beings. These may include both:With this in mind, we can note that just as a competent person (A) can possess an interest in the wellbeing of another (B) for purely self-regarding reasons  B’s interests are in his personal interest.because A will be either directly or vicariously harmed should he be barred from donating but also, and importantly, because as a result of the relation in which he stands to B  A has purely other regarding interests in B’s wellbeing. That is, an individual may possess an interest in donating an organ to another not simply as a means to securing his own future wellbeing but also because as a result of both the moral and emotional relationships that obtain between individuals, he possesses an interest in donating an organ for the recipient’s own sake.Extended to the case of organ donation, A may have an interest in donating an organ to B both her emotional distress and/or because she desires to be viewed by others as ‘good’ or ‘virtuous’ etc.Of course, whilst the distinction between self-regarding and other/ideal-regarding interests might be clear in theory this will not always be the case in practice. Loving or purely other-regarding interests are “commonly intertwined with and reinforced by… self-regarding interests” as “most of the things we desire for their own sakes we also desire as a means to other things” Feinberg , p. 72. Given this, it seems that the extension of the donor benefit standard from cases of ‘donation’ involving minors and those with intellectual disabilities to cases of donation from autonomous adults is inconsistent with the motivation which underlies its acceptance in the former case. For, whilst when applied to cases of ‘donation’ involving minors and the intellectually disabled, the standard will, more often than not, result in outcomes that can be said to be in the interests of the ‘donor’, this will not be so when it is applied to competent volunteers. As shown above, the donor benefit standard’s sole focus on welfare interests discounts a vast swathe of human interests which may arise from the emotional bonds characterising normal and loving human relationships as well as a number of less common interests and values, such as that of selfless concern for the interests of others motivated by altruism and other strongly held moral commitments and ideals which may be frustrated should the candidate donor’s offer be refused.interests of living donors, many of whom do seek benefits from donation, it seems odd and more than a little paternalistic to suggest that in cases where candidate donors are primarily motivated by prosocial and other-regarding values, donation may be justified only where a physician is convinced that a donor will attain undesired  benefits.Similarly, that the donor benefit standard discounts purely other-regarding interests and values such as altruism and solidarity casts serious doubt on Spital’s claim that the donor benefit standard “accommodates and considers individual values” Spital , p. 106.and in order truly to accommodate and consider the individual values of mature human beings, proponents of the donor benefit standard  should adjust their focus from benefits to that of ‘interests’ more broadly construed. This, however, would require that transplant physicians be permitted to retrieve organs from living donors in cases both where they are convinced that donation serves the welfare interests of donors  and/or where donation results in net donor harm but forwards the other-regarding interests of donors.Importantly then, in order to prove truly consistent with existing moral standards for the treatment of incompetent donors interests of candidate donors - where interests are more broadly construed and include both self-regarding and other regarding interests—such criticisms can be avoided. Despite this, however, it is suggested in this section that an additional criticism of the donor benefit standard may not be so easily solved by emendation.As seen in the preceding subsection, the donor benefit standard is neither consistent with existing standards for the retrieval of organs from persons lacking capacity to consent to their donation nor able to truly accommodate and consider the individual values of competent organ donors. Provided, however, its proponents are willing to expand their account such that donation is justified where it can be shown to serve the fulfil her role she must believe that donation provides benefits for her patient that offset the harms of donation and thus that living organ donation absent this requirement poses a clear and problematic conflict of interest for the physician.This criticism attaches to the claim at the heart of the donor benefit standard: the suggestion that for a physician adequately to seems in good company when he makes this claim. The suggestion that a doctor’s role should primarily be that of his patient’s advocate and thus that he should always endeavour to act in their best interests has historically played, and continues to play, a central role in medical ethics. It is, through “images of the physician at the bedside, dedicated to his present patient… consecrated in art and literature”  which does not require that the living organ donor receives benefits from their donation outweighing the harms they suffer is insufficient.However, this paper claims that although the donor benefit standard directs welcome attention to the interests of living organ donors whose motives for donation are not generally wholly other-regarding, crystallizing the importance of ensuring clear and candid discussions among donors and physicians regarding their motives, there is scant reason to think that a blanket imposition of the donor benefit standard would improve on currently existing standards for justified living organ donation. For, analysis shows both that the benefits commonly claimed by proponents of the donor benefit standard are illusory or flawed and that its imposition would pose significant threats to the autonomy of candidate donors and the lives of those who may benefit from organ transplants. Given this, it is concluded that wherever the donor harm threshold is placed  in living organ donation, it should not be so strong as to require that donors receive sufficient benefits that they outweigh the harms of donation.


Zebrafish testis has become a powerful model for reproductive biology of teleostean fishes and other vertebrates and encompasses multiple applications in applied and basic research. Many studies have focused on 2D images, which is time consuming and implies extrapolation of results. Three-dimensional imaging of whole organs recently became an important challenge to better understand their architecture and allow cell enumeration. Several protocols have thus been developed to enhance sample transparency, a limiting step for imaging large biological samples. However, none of these methods has been applied to the zebrafish testis. We tested five clearing protocols to determine if some of them could be applied with only small modifications to the testis. We compared clearing efficiency at both macroscopic and microscopic levels. CUBIC and PACT were suitable for an efficient transparency, an optimal optical penetration, the GFP fluorescence preservation and avoiding meaningful tissue deformation. Finally, we succeeded in whole testis 3D capture at a cellular resolution with both CUBIC and PACT, which will be valuable in a standard workflow to investigate the 3D architecture of the testis and its cellular content. This paves the way for further development of high content phenotyping studies in several fields including development, genetic or toxicology. Testis has long been studied to better understand mechanisms of spermatogenesis, puberty or sterility disorders that are key issues in fundamental research, aquaculture and medical science. In the field of reproductive biology, the zebrafish has become a useful model, by means of many modern molecular tools . The zebrafish model also benefits of the recent advance in genome editing with the CRSIPR/Cas9 technology, which will eventually lead to the production of a high number of mutant lines to be analyzed123D-fluorescence imaging has made significant progress with the development of cutting-edge microscopy technologies. Nonetheless, imaging of large biological samples remains greatly hindered by the natural opacity of tissues. To overcome this limitation, several protocols have been developed that enhance tissue transparency by reducing light scattering, reviewed by Richardson and Lichtman56784We tested the clearing efficiency of RIMS, SeeDB, 3DISCO, CUBIC and PACT methods on the adult zebrafish testis. We evaluated the sample transparency, the optical depth penetration and the tissue deformation. Nuclear staining with propidium iodide (PI) was chosen to test the imaging efficiency. Assessment of the preservation of the GFP fluorescence was performed by using a zebrafish transgenic line that express GFP in Sertoli cellsi.e. testes incubated in PBS, To assess the clearing efficiency of RIMS, SeeDB, 3DISCO, CUBIC and PACT protocols on zebrafish testes, samples were observed at a macroscopic level. The apparent transparency of all cleared samples was compared to that of non-cleared testes (Tg(gsdf:GFP) encodes the GFP protein driven by the promoter of the zebrafish GSDF gene. Testes were cleared using the different methods and the fluorescence was observed afterward with a fluorescence macroscope  imaging on CUBIC-cleared samples, with or without laser compensation. XZ orthoslices of the stacks revealed a deeper signal recovery with 2-photon microscopy without laser compensation, as compared with confocal microscopy. The imaging depth was similar with 2-photon and confocal microscopy with laser compensation . The meaIn order to get insight into the effect of clearing on testis integrity, and based on the assumption that nuclear size modifications reflect cellular size changes, we measured nuclear sizes after clearing . For eacTg(gsdf:GFP) was cleared following the CUBIC method and imaged by 2-photon microscopy. A total volume of 5.787 mm × 2.494 mm × 0.703 mm was acquired  was importantly reduced in testis with 3DISCO and SeeDB, although it was properly preserved with RIMS, CUBIC and PACT. Given the chemical treatments that were applied to testis samples, it is not surprising that the fluorescence was somewhat impaired, if not totally suppressed. The fluorescence of GFP and variants not only depends on the pH and the temperature, but also on the protein secondary structure19et al.thy1 mouse transgenic lines that are well known for their strong fluorescence, as reviewed by Silvestri et al.tert-butanol (adjusted to a basic pH) during the dehydration steptert-butanol and a diphenylether-BABB mix instead of the dibenzylether used in the original 3DISCO81125The native GFP fluorescence of the transgenic line In most cases, the size of tissues is used to evaluate deformations after clearing, as it was successfully done for whole mouse brain or mouse brain slices1649711Although the macroscopic clearing efficiency was the most powerful with 3DISCO in our hands, this solvent-based protocol is based on the use of toxic chemical reagents and results in very hard samples that tend to break, thus complicating the sample manipulation. In addition, we were not able to get resolved images by 2-photon microscopy, although the strong sample shrinkage should have made them easier to acquire in terms of time and data volume. One of the main advantages of CUBIC over PACT is its easiness of achievement. Other studies clearly indicate that CUBIC is an efficient clearing method for various mouse organs202711309i.e. 700 μm) at a cellular resolution by using 2-photon microscopy, which shows the great efficiency of these methods on the zebrafish testis. It is well demonstrated, mainly on brain, that PACT-clearing and imaging with 2-photon/confocal microscopes allows imaging with a high resolution in depth up to more than 1 mmUsing CUBIC and PACT, and optimal imaging conditions, we successfully attempted to image whole testis thickness i.e. 00 μm at Overall, our work allowed for the first time the 3D imaging of whole zebrafish testis. The comparison of several clearing protocols showed that CUBIC and PACT are the most appropriate among the large number of protocols available in the literature. In combination with 2-photon microscopy, they can be applied to investigate testis 3D architecture and cellular content. Application of these protocols on testis of other species, including mammal, will be of utmost interest. This study is also the first step toward the development of high content phenotyping workflows of abnormal zebrafish testes for diagnosis, drug discovery or mutant screening.All experimental procedures used in this study followed the recommendations of the French and European Union regulation on animal welfare and were approved by the INRA LPGP-Animal Care and Use Committee (N°Z-2015-30-VT and Z-2015-127-VT-MF).Tg(gsdf:GFP) zebrafish line were raised at 28 °C until adulthood (aged from 4 to 5 months) in the INRA-LPGP fish facilityAdults of the TM  was dissolved in 5 ml of PBS 0,02 M +0,05% sodium azide6.66 g of HistodenzSeeDB protocol was conducted as described by Ke and collaboratorsSamples were cleared by a simplified 3DISCO protocolProtocol was conducted as described previouslyFixed testes were subjected to clearing following CLARITY protocol7i.e. in CUBIC2 and RIMS respectively). Whole testes were incubated in PI  diluted at 10 μg/ml in PBS +0.1% (v/v) tween +0,05% (w/v) sodium azide at room temperature for 2 days. Samples were then washed overnight in PBS +0.1% (v/v) tween +0,05% (w/v) sodium azide at room temperature.Nuclear staining was performed before clearing for RIMS, SeeDB and 3DISCO, and after clearing for CUBIC and PACT. With these latter, the nuclear staining was performed before incubation in the refractive index solution . The VibMic enables accessibility of deep structures of large whole-mount specimens by the combination of a vibratome (VT1200S) with a SP8 confocal microscope allowing successive mechanic sectioning and block-face imaging. Imaging was performed with a Leica SP8 two-photon/confocal hybrid microscope with a 25X/0,95NA HCX IRAPO water immersion objective . For confocal microscopy, GFP and PI were excited by 488 nm and 552 nm lasers respectively and fluorescence was detected by PMT. For 2-photon microscopy simultaneous excitation of PI and GFP was performed at 850 nm with a Chameleon Vision II laser (Coherent) and fluorescence was detected by NDD HyD detectors (Leica microsystems) with BP 525/50 and BP585/40 filters respectively.Mounting and imaging were performed at the end of the clearing protocol for all conditions. Samples were mounted under #0 coverslips  with 5 spacers of 120 μm each in their respective final clearing solution. To assess the deepness of the PI staining, testes were embedded in 4% low gelling temperature agarose (A9414.Sigma-Aldrich) and examined using the VibMic combination system .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.