Families of organ donors play an important role in the deceased organ donation process. The aim of this study was to gain insight into donor family care by creating an inventory of practice in various European countries. A questionnaire about donor family care and contact between donor families and recipients was developed. Representatives of the organ donor professionals of 15 European countries responded (94%). The donor coordinator plays a key role in care for the donor family. All countries provide information about the donation results to the families, although diminished due to privacy laws. Anonymous written contact between donor families and recipients is possible in almost all countries and direct contact in only a few. Remembrance ceremonies exist in most countries. Half of the respondents thought the aftercare could improve. This first inventory shows that differences exist between countries, depending on the organisation of the donation process, the law and the different role of the professionals. Direct contact between donor families and recipients is rarely supported by the donation organisation. To date there has been limited research about the experience of donor family aftercare and we would urge all donation organisations to consider this as a priority area.  Organ transplantation is a well-accepted medical treatment for organ failure. The main source of donor organs is patients dying in the intensive care unit (ICU), after declaration of brain death , or after withdrawal of life sustaining treatment . In the ICU, “patient-centred care” is the predominant model, which means that individual’s specific health needs and desired health outcomes are the driving force behind all health care decisions and quality measurements . PatientIf the decision is taken that ICU treatment is futile, the end-of life care process will start. Organ donation is an integral part of end-of life care for many patients . The famOrgan donation can be overwhelming and stressful for families for many reasons, including the duration of the process and grief at the death of a loved one . HoweverThe aim of this study is to gain insight into donor family care by creating an inventory of practice in various European countries. We focus on two aspects: the formal communication with the family during and after the donation process and the possibility of contact between donor families and recipients.The Deceased Donation Working Group of ELPAT  developed a questionnaire. The items of the questionnaire were based on our professional experiences with the donation process. The questionnaire contained two parts: communication and care for donor families during and after the donation and contact between donor families and recipients.The first part of the questionnaire contained 16 questions, of which 13 were multiple-choice questions with the ability to provide additional comments, and three open questions. Subjects covered included: guidance of the family during the donation process, information and care provided to families after donation and the provision of remembrance ceremonies for the family.The second part contained eight questions, of which six were multiple-choice and two open questions. Subjects covered included: contact, what kind of contact, possibility and experience of meetings between donor families and recipients.Pilot testing of the questionnaire was done with five organ donor coordinators, and using their comments, minor modifications were made to the questionnaire. For each country, one representative was approached by mail or telephone during early 2019; the study was explained and consent was obtained to participate. Each representative was chosen because of their anticipated in-depth knowledge of their country’s donation process, and their ability to obtain information from a diverse group to reduce heterogeneity and subjectivity in the replies.If there were, according to the representative likely to be regional differences in the country, additional representatives were approached as required.From the 16 approached countries, 15 responded (94%). In three countries , more questionnaires were returned from different regions, to explore regional differences. Respondents were all experts on the donation process in their country, although their specific job titles varied; for example, organ donor coordinators, transplant coordinators or specialist nurse for organ donation. As these names differ for the person who fulfills the similar task of coordinating the donation process, we use for clarity in this article one term for all the above job titles: donor coordinator (DC).The organisation of the donation and the roles of the professionals involved in the donation process differ by country. Communication with and support of the donor family in the ICU was, in most countries, organised in cooperation between the DC, the intensivist and the ICU nurse. The DC coordinates the donation process on the ICU, has contact with the family and coordinates the organ retrieval procedure.All countries provide information to the donor family after the donation procedure. This information is given by letter, by telephone and in some countries face to face at the family home or in the hospital. The information is provided by the DC; only in the four countries where there is no contact between the DC and the family, the intensivist provides the information.Respondents from most countries say the way in which information is provided depends on family wishes. In three countries, a national organisation provides the information . In some countries, the donor family is invited to the hospital a few months after the donation procedure, to evaluate and discuss the donation process.The kind of information that is provided after the donation procedure varies; information about which organs/tissues are transplanted, information about the recipients gender, age or health. In two countries (Hungary and Finland), only a standard “thank you letter” is sent without any information about the recipients. During the last years, the information provided has reduced in many countries (n = 7), e.g., now the information about age, gender, time on the waiting list, and the health of the recipient, is limited. Some countries now only provide the information that the organ is transplanted or not. A reason suggested by participants for less information sharing was data protection legislation. Some stated that this is a pity, because less meaningful letters are sent to the family. Another factor suggested was social media, due to the fear that donor families will search for recipients’ information. This was mentioned for two countries.The representatives from six countries expressed overall satisfaction with the care provided for donor families . The main concern from the other country representatives who were not satisfied was that there was no or not enough aftercare for donor families. Structural organised care for families after donation was missing, were some comments, like: “There is no organised care, donor family care should be implemented.” “More national follow up of donor families is needed.” A lack of structured organised after care was a frequent observation, highlighting there was too much variation per hospital or region. Comments: “There should be more follow up with donor families after they leave the hospital, to learn more about their grieving process and implement the lessons learned.” Information was lacking concerning the effects, positive or negative, of the donation to this grieving process. In some countries the opinion was that donor after care is too variable per region or hospital and should be implemented on a national level, to guarantee donor after care for all donor families. Comments: “smaller hospitals are less experienced; there is a wish to give more support to all families, not only to those who ask for help or information.”We were interested if there was a difference in care for donor families on the ICU and families of regular ICU patients who die in the ICU. From the respondent countries, almost half are of the opinion that the care differs. The general opinion was that more attention is given to donor families during the donation process, especially if there is a DC present to coordinate the donor procedure, or a trained professional to give information and support to the family and who can help them in the beginning of the grieving process. Donor families also receive more care after the donation (aftercare) than families of deceased ICU patients, was the opinion of seven countries. “Donor families are invites to special ceremonies,” “donor families receive ‘thank you letters.’” “Yes, the care is different, donor families are invited to the donor hospital 6 weeks after the donation, to talk about their experience.” Families from regular ICU patients who die don’t have these special ceremonies in general. However, some state the care is equal, also families of non-donors are invited to the hospital for after care.In 11 countries remembrance ceremonies are organised for donor families, only in four countries  was this not the case . The cerWho is responsible for organising the ceremonies differs per country: private organisations, a recipient organisation, local organisations from donor hospitals, or the event can be organised by the transplant foundation, or regional teams. All countries who provide ceremonies, state that they were satisfied with these ceremonies. Comments on the ceremonies: “very helpful for donor families,” “families are happy with the attention,” “well visited meetings,” “important to share experiences and emotions with other families.” According to some countries improvement could be the presence of a professional during the meetings, like a DC, to answer questions and give specific information.In all countries but one (Croatia), written anonymous contact between recipient and donor family is possible, through an intermediate, the DC. In one country (Switzerland) a website was developed, where recipients and donor families can post their thoughts, thanks, experiences, and histories. Here was also a guide/template for a “thank you letter” to be used for transplant patients to a donor family. Initiative for contact is taken more by recipients than by donor families.Four countries responded that a formalised process exists for donor family and recipient(s) to meet face-to-face . However“Is this a good thing”?“Should there be a role for the health care professionals during these meetings?” “Expectations should be well managed”“Is the motivation of the donor family and the recipient the same?”“Meetings don’t feel right, not intent to cooperate as a professional.”“What if there is some pressure felt from the donor family to the recipient”?“Meetings can only be possible if the donor family and recipient find each other through social media.”A few general comments were made, and suggestions to improve the care for donor families. For example, offering the family a conversation with a psychologist, if they are in need for support or to be more active in approaching recipients to send a kind of thank you letter to the donor family.To be able to compare if the information provided to the donor families from the DC changed over the years, we asked participants to send examples of two letters: a recent letter and one from approximately 10 years ago. Letters were received from five countries. In four countries, the letters were changed, and in all of these cases less information was given about the recipients. Information about time on the waiting list, age (only an age period) or health information about the recipient was no longer given in the latest letters. The recent letters were simpler and more straight forward, consisting only of information concerning whether the transplantation of the organ was successful, with no more detailed information about the health process of the recipient. The reason for this change was reported as being stricter privacy legislation in the different countries.This study is the first survey about the care of donor families, during and after the donation process and contact between the donor families and the recipients in 15 European countries. It shows that there is a variability between countries, and in some countries small differences between centres or regions. For example, the role of the DC and the moment this professional is participating in the donation process. Depending on the way donation is organised there are also differences in the communication with and care of the donor family. Communication during the donation process with the family is mostly led by the DC; only in a small number of countries is there no direct contact between the DC and the donor family. Generally, DC contact with the donor family starts after consent for donation, though a minority of DCs have contact before consent. There is some evidence that early contact with the family and involvement of the DC in the request for donation can have a positive effect on the perceived support for the family and the consent rate for donation .The amount of information provided to the family about the outcome of the donation and transplantations depends on the legislation and its interpretation, in a specific country. In several countries this information has become more limited in recent years due to data protection legislation. This means that less information about the recipients can be given, mainly restricted to age range, transplant outcomes and sometimes gender of the recipients. It is possible that concerns about privacy and strict interpretations of data protection legislation may not be justified; if a recipient consents to the processing of more data in order to facilitate higher levels of communication with donor families, this would be permitted by the General Data Protection Regulation. However, different countries may have stricter national laws in place, or transplant professionals may be being given legal advice that takes a very strict interpretation of data minimisation where that is not necessary.Face-to-face meetings between donor family and recipients only take place in a few countries, but there is concern about the impact these meetings can have on both donor families and recipients. There are some studies about the contact between the donor families and transplant recipients , 13. OutThe aftercare given to donor families can be different from the care given to non-donor families on the ICU. This could be influenced by providing more and longer intensive contact with the donor family, more extensive conversations and explanations about the donor procedure by the intensive care staff and coordinator of the donation procedure. In the literature, families who consented to donation felt more supported than families of non-donors . IncreasRemembrance ceremonies exist for donor families in almost all countries, and the impression of the DC is that the ceremonies benefit the donor family, but satisfaction with the ceremonies is not routinely measured. There is attention to the needs of donor families, but a lack of specific information about these needs is also mentioned. No specific studies are performed to measure the impact of ceremonies on the donor family.Data collection was completed before the COVID pandemic, so it had no impact on the results. However, the pandemic may have affected the provision of donor family support, such as public remembrance ceremonies, which are temporarily reduced.Given the results of our research, we can make several recommendations. Donor family aftercare is an essential part of the donor process and should be delivered in a structured way and embedded into the organisational process. In order to establish best practice for a country, research on family views is needed. What services do donor families need? This research can be done using groups, such as donor family advisory group, or interviews with donor families. Research should also be conducted to evaluate letters from a donor family perspective; what information benefits the donor family? Are meetings between recipients and donor families beneficial for both? Those working with donor families and recipients should reflect on this inventory, and comparative practices, and consider whether they are meeting the needs of donor families. Furthermore, organisations can tend toward risk aversion in data protection at the expense of helping families; this tendency should be avoided. Research evaluating the impact of COVID on donor family care should also be performed.This first inventory of 15 European countries about the care provided to donor families during and after donation and contact between donor families and recipients, shows, as expected, that there are differences between the countries. These differences depend on the organisation of donation, the law (and its interpretation) and the different roles of the professionals involved in donation. Donor family aftercare is provided in all countries and some countries provide remembrance ceremonies. In most countries, direct contact between donor families and recipients is not supported by the donation organisation. To date there has been limited research about the experiences and satisfaction of donor family aftercare and we would urge all donation organisations to consider this as a priority area.This is the first multi-country study to compare the way care to donor family and aftercare is provided to donor families and contact between donor families and recipients, which provides valuable insights. Since this is a first inventory, it provides only an initial overview of the different aspects. More research is necessary to explore in depth in how communication and aftercare is given, and the experiences and satisfaction of the donor families, with including possible suggestions for improvement.Belgium, Croatia, Denmark, Finland France, Germany, Hungary, Iceland, the Netherlands, Norway, Slovenia, Spain, Sweden, Switzerland, United Kingdom.


Theapparent piezoelectricity of biological materials is not yetfully understood at the molecular level. In particular, dynamic noncovalentinteractions, such as host–guest binding, are not includedin the classical piezoelectric model, which limits the rational designof eco-friendly piezoelectric supramolecular materials. Here, inspiredby the conformation-dependent mechanoresponse of the Piezo channelproteins, we show that guest–host interactions can amplifythe electromechanical response of a conformationally mobile peptidemetal–organic framework (MOF) based on the endogenous carnosinedipeptide, demonstrating a new type of adaptive piezoelectric supramolecularmaterial. Density functional theory (DFT) predictions validated bypiezoresponse force microscopy (PFM) measurements show that directionalalignment of the guest molecules in the host carnosine–zincpeptide MOF channel determines the macroscopic electromechanical properties.We produce stable, robust 1.4 V open-circuit voltage under appliedforce of 25 N with a frequency of 0.1 Hz. Our findings demonstratethat the regulation of host–guest interactions could serveas an efficient method for engineering sustainable peptide-based powergenerators. Owing to the flexible alkyl segments in the β-alanine-histidinepeptide, carnosine-based linkers can adopt a wide range of conformationalstates through low-energy torsional rearrangements, which enablesguest-specific flexible response of the Car_Zn framework; that inturn affects the electromechanical behavior.40 Even though bio-inspired host–guest interactions have nowbeen exploited in materials design,43 studies involving guest-modulatedelectromechanical behavior are relatively underexplored.45 The unambiguous demonstration of guest molecule-directed electromechanicalproperties would offer a means of dynamically modulating piezoelectricresponse, allowing better understanding of piezoelectricity in softmaterials and providing an additional functionality for emerging eco-friendlypiezoelectric devices.51Implementing the designprinciples for the adaptive peptide-basedmetal–organic framework formulated by Rosseinsky and co-workers,scientists have synthesized topologically distinct two- or three-dimensionalpeptide-MOF architectures by changing the type and sequence of aminoacids on peptide ligands.d33 response. Density functional theory (DFT) predictive models wereused to map the piezoelectric tensor of the full set of Car_Zn crystalsand investigate the relationship between microstructure and electromechanicalbehavior , dimethylformamide (DMF), acetone,acetonitrile (MeCN), and ethanol (EtOH). Atomic-level structural analysisof different peptide-MOFs obtained by X-ray crystallography revealedthat the MeCN guest molecule uniquely directs assembly of a triclinicframework in which the polarization is not constrained by symmetry,which endows the crystal with 18 nonzero piezoelectric coefficients,including a measurable longitudinal behavior 1a,valid33 .The powder X-ray diffraction (PXRD) patterns of the Car_Zn MOFs exhibitedhigh crystallinity and were strongly consistent with the simulationdata from the single crystal structure, signifying the same crystallineform . Scanning electron microscopy(SEM) characterizations demonstrated neat prismatic morphologies ofall assembled crystalline architectures , Car_Zn MOFs exhibited anunusual triangular prism morphology measuring tens of micrometersin length, and the morphology could be clearly observed to becomea rectangular prism when the guest was switched to DMF or acetone. Because of the differentsolubility of the carnosine ligand in solvents of different polarity,the nucleation and growth of Car_Zn MOF could be significantly affectedby the choice of solvent52 . As suggested by the morphologicalanalysis of the Car_Zn·(EtOH) single crystal , the anisotropic growth of the (020) surface alongthe [001] direction was suppressed mainly by the addition of polarprotic alcohol, which could form a shell of hydrogen bonded alcoholmolecules around the carnosine linker.33 Furthermore, because the shape of a triangular prism can be approximatedas a truncation of a rectangular prism, in terms of morphology, thesesolvent molecules provide similar crystalline habits for the growthof Car_Zn MOFs. Intriguingly, the addition of MeCN induced a new structurepolymorph with a hexagonal prism crystal morphology, with a lengthup to 60 μm . This uniquemorphology change may be attributed to the solvent template effectwith the induced conformational distortion of the assembly triggeringa phase transition to create the electromechanically active framework.55Car_Zn MOFs were assembled via a zinc nitrate and carnosine reactionin the presence of five different guest molecules under controlledexperimental conditionstectures 1c–g. Witolvent52 S10. As sFigures S13–S16 and Table S1). Under the above-mentioned different solventconditions, the imidazole and carboxylic acid groups of carnosinewere deprotonated to coordinate with Zn(II), forming the Car_Zn framework] complex per asymmetric unit. However, in the Car_Zn·(MeCN)polymorph –NHR′in the chain was confirmed by comparing with the alternative iminolRC(OH)=NR′,56 which did notfit the observed carnosine–Zn crystal packing with coordinatedordered solvent molecules.To further characterize the specific guest molecule-mediatedassemblymechanism at the molecular level, the as-prepared crystals were thoroughlyanalyzed via X-ray crystallography . Although the solvent-filledchannels could still be observed in Car_Zn·(MeCN), the alignmentof the pores changed from the b-axis to the a-axis . In addition,the torsional flexibility of the carnosine ligand allowed the Car_Znframework to be structurally sensitive to the template effect of thespecific guest molecules. In each structure, the carnosine linkerachieved a distinct conformation by adjusting its torsion angles,responding to the size, shape, and hydrogen-bonding characteristicsof the guest molecules in the Car_Zn framework channel , orienting the carnosine amide and amine groups toward the IPA,forming guest–host (IPA–Car) hydrogen bonds  . Those guest–host hydrogenbonds together with the host–host (Car–Car) intermolecularhydrogen bonds  andcarnosine–Zn(II) coordination bonds stabilized the framework. Uniquely with MeCN, the guest molecule did not form hydrogenbond interactions with the carnosine molecules, and the Car_Zn·(MeCN)framework was solely stabilized through the carnosine–Zn(II)coordination bonds and host–host intermolecular hydrogen bonds, with the ethanolguest solvent molecule producing the stiffest crystal with a predictedYoung’s modulus of 27 GPa. With the incorporation of acetoneor DMF, the predicted Young’s modulus of the crystals was reducedto ∼14–17 GPa, while MeCN or IPA solvents produced significantlyweaker crystals with a predicted Young’s modulus of 7–9GPa. The Car_Zn MOF containing guest molecules of EtOH, DMF, and acetonethen showed the lowest piezoelectric strain constant (Tables S4–S8), reflecting their increasedaverage elastic stiffness constants and lower piezoelectric polarization.DMF, acetone, and EtOH guests produce MOFs with predicted d16 = 6.9, 11.2, and 5.5 pC/N, respectively.Thenoncentrosymmetric structure with the directional polar guestmolecule array and hydrogen-bonding networks signifies internal polarization,implying piezoelectric properties.Tables S7 and S8), showing that increased flexibility in the IPA crystalbalances its decrease in charge tensor values. Furthermore, as werevealed in the single crystal diffraction results, Car_Zn·(MeCN)crystallized with significantly altered unit cell parameters and adoptinga lower symmetry unit cell (monoclinic to triclinic), thus allowingfor a complete 18 nonzero component piezoelectric tensor . While its predicted d33 value of 4.7 pC/N is modest, it has six piezoelectricstrain constants between 9.8 and 18.2 pC/N (dmax = d15). In common with mostbiological materials,59 the low dielectric constantsof the peptide-MOF structures permit significant voltage constantoutputs, here as high as 435 mV m/N (g15) (Table S4). These values can be attributedto an overall lower elastic stiffness and |eij| values between 0.15 and 0.33 C/m2.Despite their differing mechanical properties, both alcohol-containingcrystals Car_Zn·(EtOH) and Car_Zn·(IPA) showed a nearlyidentical range of piezoelectric strain constants with maximum valuesof 7.3 and 5.5 pC/N, respectively (Tables S2 and S3), primarily due to the directional alignment of thesmall solvent molecules , asthe carnosine molecules were equally bisected along the b- and c-axes  was found along the acetonitrile MeCNsolvent molecular dipole60  was inverselyproportional to the interion spacing along each axis , which demonstrates how guest molecule-directed supramolecularpacking determines the electromechanical response by stabilizing specificinterion spacing. Thus, the most significant piezoelectric chargeconstants in the Car_Zn·(MeCN) crystal were along the a-axis (Table S4). On the otherhand, in the Car_Zn·(IPA) crystal , the channels were off-center near the corners of thecell, so when a shearing force was applied along the c-axis, it was much easier to push the molecular layers close together, enabling a maximum d36 piezoelectric response of 7.3 pC/N (Table S8). The relatively low charge tensor values in the crystals containingalcohol guest molecules may be due to the juxtaposed orientationsof the two alcohol molecules in each channel, resulting in the alcoholmolecular dipoles canceling out rather than contributing to the piezoelectricresponse. As discussed above, the orientation of the guest moleculesin the channel controls the macroscopic electromechanical propertiesof the MOFs, with the highest piezoelectric responses obtainable fromthe favorable alignment of the crystallized guest molecules (Table S9).The computed electromechanical properties stem from the guestmolecule-mediatedcarnosine packing patterns in the crystals. For Car_Zn·(MeCN),the DFT models predict decrease in the largest elastic stiffness constant was applied to investigate the piezoelectricity of the Car_Zn·(MeCN)crystal. During the PFM measurement of the piezoelectric response,the tip of the PFM was in contact with the single crystal and remainedstationary during the entire measurement process. The piezoelectricresponse of the single crystal was extrapolated from the linear relationshipbetween the applied voltage (in volts) and the resulting deformation(in picometers).61 For the out-of-planeresponse, the resulting deformation was proportional to the effectivelongitudinal piezoelectric coefficient, which can be denoted as dLeff . Likewise, for the in-plane response, the resulting deformationwas proportional to the effective shear piezoelectric coefficient(dSeff) . The linear relationship between the verticaland in-plane piezoelectric response as measured by the photodiodesystem and applied voltage indicated a genuine piezoelectric propertyof the Car_Zn·(MeCN) crystal . The results reveal that the largest measured dLeff of Car_Zn·(MeCN) is 4.7 pm/V andthe largest dSeff is 15.5 pm/V,in very good agreement with the theoretical maximum values predictedby DFT of 4.7 and 18.2 pm/V, respectively  crystal showed a Young’s modulusof 10.97 ± 2.28 GPa along the thickness direction, which ledto a point stiffness of 103.83 ± 26.43 N m crystal 3c,d. Thias dLeff S29a. Lik63 A coin-sized powergenerator was designed and fabricated by tightly sandwiching the Car_Zn·(MeCN)crystals array between two gold-coated silicon dioxide substratesconnected to an external measuring instrument . A constant and stable mechanical force was applied to the generatorthrough the dynamic mechanical test system, and the short-circuitcurrent and open-circuit voltage were measured to characterize thegenerated electrical output signal. The peptide-MOF-based device-generatedthe corresponding periodic current (40.45 ± 2.99 nA) and voltage(1.42 ± 0.063 V) output signals when the generator was periodicallycompressed with 25 N force . Finally, the stability measurement suggested that power generationcould be sustained under a cyclic force (19 N). The output voltageshowed no attenuation over 400 press/release cycles for an hour , indicating the high durability ofthe peptide-based devices.Intrigued by the piezoelectricproperties of the characterizedpeptide-MOF Car_Zn·(MeCN) crystal, we tested the potential ofthe peptide-MOF crystals for use as the active layer in a prototypepiezoelectric energy generator.strument 4a. The ement (64 S33. A coment (64 S33. A coment (64 S33. A coP1)with unconstrained polarization, which created a significant piezoresponseas mapped by using DFT calculations and microscopy. Reminiscent oftransmembrane proteins and diphenylalanine peptide nanotubes, theextensive directionally aligned guest molecules in the narrow channelform a macroscopic dipole that can couple with shear force to generatethe piezoelectric response. As a proof of concept, we demonstratethe utilization of a stable, robust peptide-MOF with useful 1 V outputin a prototype sustainable, eco-friendly power generator. Our findingsillustrate the rational modulation of peptide-MOFs to embed tailoredfunctionalities and pave the way for supramolecular engineering ofpiezoelectric biomaterials for nanotechnology applications throughfurther manipulation and design of internal host–guest interactionsthat confer dramatic changes in materials morphology and improve deviceperformance.In this study, we developeda simple but powerful “guestmolecule-mediation effect” approach for tailor-made designof piezoelectric peptide-MOF crystals. We show the guest moleculeMeCN selectively alters the crystal morphology and acts as a structure-directingagent to lower the symmetry of the unit cell. As a result, unlikethe four other guests we tested, the Car_Zn·(MeCN) MOF crystallizedinto the lowest symmetric system (space group


Spinal cord injury (SCI) is a disabling neurological disorder that causes neural circuit dysfunction. Although various therapies have been applied to improve the neurological outcomes of SCI, little clinical progress has been achieved. Stem cell–based therapy aimed at restoring the lost cells and supporting micromilieu at the site of the injury has become a conceptually attractive option for tissue repair following SCI. Adult human neural stem/progenitor cells (hNS/PCs) were obtained from the epileptic human brain specimens. Induction of SCI was followed by the application of lentiviral vector-mediated green fluorescent protein–labeled hNS/PCs seeded in PuraMatrix peptide hydrogel (PM). The co-application of hNS/PCs and PM at the SCI injury site significantly enhanced cell survival and differentiation, reduced the lesion volume, and improved neurological functions compared to the control groups. Besides, the transplanted hNS/PCs seeded in PM revealed significantly higher migration abilities into the lesion site and the healthy host tissue as well as a greater differentiation into astrocytes and neurons in the vicinity of the lesion as well as in the host tissue. Our data suggest that the transplantation of hNS/PCs seeded in PM could be a promising approach to restore the damaged tissues and improve neurological functions after SCI. Traumatic spinal cord injury (SCI) is a disabling neurological condition that can lead to permanent motor and sensory dysfunctions , 2. DespAs the host micromilieu plays a crucial role in stem cell fate determination, the differentiation, maturation, and migration ability of hNS/PCs after transplantation is a decisive factor in determining their integration with host tissues . LentiviEpilepsy surgery offers a unique opportunity to achieve hNS/PCs from the resected brain tissues of patients suffering from medically refractory epilepsy . The hNSAll experiments were performed in accordance with the National Institute of Health Guide for the Care and Use and were approved by the Ethics Committee of Shefa Neuroscience Research Center, Tehran, Iran. After receiving informed consent, fresh tissue was obtained from the temporal lobe of two patients with mesial temporal lobe epilepsy during surgical intervention. The past medical history of patients is shown in Table A contusion model of SCI was performed in rats. The subcutaneous application of cyclosporine A (1 mg/100 g body weight/24 h) was begun on day 3 before the cell transplantation and continued for another 2 days [4 green fluorescent protein (GFP)-labeled hNS/PCs treatment, and (iv) PM + hNS/PCs group: SCI rats with 1 × 104 GFP-labeled hNS/PCs + PM. All data assessments were conducted blinded to the group allocation.Thirty-two adult male Wistar rats (220–250 g) were maintained in the animal house of the Shefa Neuroscience Research Center. The animals were housed in controlled condition  with unrestricted access to standard laboratory food and water, and then divided randomly into four treatment groups (8 rats per group): (i) Control group: SCI rats with PBS treatment, (ii) PM group: SCI rats with 0.15% PM treatment, (iii) hNS/PCs group: SCI rats with 1 × 10The resected epileptic brain tissues were washed twice in cold PBS containing 1% penicillin/streptomycin. After dissection of tissues, enzymatic digestion and mechanical dissociation were performed. Briefly, tissue sections were treated with Accutase  for 15 min at room temperature and the suspension was repeatedly pipetting up and down. The enzyme was replaced with an equal volume of the fresh medium to stop the enzymatic reactions. Then, the suspension was centrifuged for 5 min at 110 g and the supernatant discarded and finally dissolved in 1–2 ml of Dulbecco’s modified Eagle’s medium/F12 . The cultures were maintained as neurospheres in uncoated tissue culture flasks in a serum-free medium. For cell culture and expansion, the medium contained DMEM /F12 supplemented with 3% B27 supplement , 0.5% N2 supplement , 10 ng/ml basic fibroblast growth factor , 20 ng/ml epidermal growth factor , 2 μg/ml heparin , 1% penicillin/streptomycin, and 1% glutamine . The culture medium was replaced with a fresh medium every 3 days. The neurospheres were expanded by passaging into fresh medium every 7 days. The expanded neurospheres were visualized by an inverted microscope. The numbers of the neurospheres were analyzed in six randomly selected well from 96-well plates using the ImageJ software.Immunofluorescence assay was done to identify the expression of markers in the isolated cells. After discarding the cell growth medium, fixation was carried out with 4% paraformaldehyde in PBS for 20 min at room temperature. Permeabilization of cells was conducted using 0.2% Triton X-100, and 5% normal goat serum . Then, the cells were exposed to the primary antibody against the glial fibrillary acidic protein (GFAP) , nestin , and Sox2  as markers of adult neural stem/progenitor cells. Subsequently, the cells were incubated with secondary antibodies, including goat anti-rabbit IgG , goat anti-mouse , and rabbit anti-goat  for 2 h at room temperature in the dark. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were photographed using fluorescent microscopy . Samples were incubated with secondary antibodies alone as negative control.Transduction of hNS/PCs with recombinant lentiviral particles was performed to track them after transplantation. The lentiviral transduction process has been described previously . Briefly2 (3.0–2.2 mm) was used to provide equal pressure distribution on the spinal cord.All animals were anesthetized intraperitoneally (i.p.) by ketamine (80 mg/kg) and xylazine (10 mg/kg) during the surgical process and their body temperature was maintained at 37 °C. SCI was performed according to the weight compression method. The lack of feedback to noxious stimulation was considered a symptom of general anesthesia. After that, the back region of the rat was shaved, and the skin was disinfected. After the exposure of the spine by incision, laminectomy was done at the T9 and the T10 segments. Spinal cord compression was applied using 35 g weight placed on the dura for 15 min. The weight with concave shape and area of 6.6 mmAfter the injury, the device was removed and the muscle, fascia, and skin were sutured separately, and then i.p. injection of lactated Ringer’s solution was used to treat dehydration. Gentamicin (6 mg/kg) was given by intramuscular injection for 3 days after surgery. Manual bladder expression was done to remove urine twice daily until recovery.4 GFP-labeled hNS/PCs, or 100 μl of PBS containing 1 × 104 GFP-labeled hNS/PCs + PM was prepared. The solutions  were injected in the center, rostral, and caudal parts of the injury site with a distance of about 3 mm within 5 min using a 26-g Hamilton syringe. The tip of the needle was inserted to a depth of 1 mm for the injection of solutions. After transplantation, all animals survived for 8 weeks.Ten days following induction of SCI, animals were anesthetized as described previously and the lesion site was re-exposed. 100 μl of PBS, 100 μl of PM (0.15%), 100 μl of PBS containing 1 × 10The recovery of the locomotor function was assessed by using the BBB motor performance test.The BBB score was assessed 4 days after the induction of SCI. To standardize the extent and severity of the SCI, rats with hind limb score differences greater than 4 were removed from the study. The treatment was performed on day 10 after the induction of SCI. BBB assessments were done for 8 consecutive weeks after the treatment. BBB scores were measured to categorize the combinations of left and right hind limb movements, trunk stability, stepping and paw position, consistent toe clearance, and tail position. Briefly, each rat was observed and recorded for 4 min in an open field under blind conditions. Left and right hind limb movements were individually scored. BBB scores were evaluated the average of both hind limbs in individual animals.Eight weeks after the treatment, animals were anesthetized (as described previously) and transcardially perfused with 1X PBS containing heparin (pH 7) followed by cold 4% paraformaldehyde. The segments of the spinal cord containing the graft were rapidly dissected out and immediately fixed in 4% paraformaldehyde for 24 h. After tissue processing, tissues were embedded in paraffin and cut at 5-μm thickness.D(A1 + An) + D (A2 + A3 +…+ An−1), where A is the area of the cavity and D is the distance between sections [To assess the lesion volume formed after SCI, a series of sections repeated at 100-μm intervals were dyed with hematoxylin and eosin (H&E) staining. Finally, the images were taken from each sample and then analyzed using the Axio-Vision 4 software. The total lesion volume was calculated by the following formula: 0.5sections .Before immunofluorescent staining, the deparaffinization process was performed to remove the penetrated paraffin from the tissue. The slides were immersed in a sodium citrate buffer at pH 6.0 and heat samples near boiling in a water bath for 10 min. Sections were then permeabilized by treating with 0.25% Triton X-100 , and blocked for 1h by 0.5% BSA  with 5% NGS. Sections were then exposed overnight with the diluted primary antibodies, including mouse anti-nestin  for neural stem/progenitor cells, mouse anti-GFAP  for astrocytes, and rabbit anti-doublecortin  for immature neurons at 4 °C and rinsed in TBS three-time before the application of secondary antibody. Alexa Fluor 647 goat anti-mouse  and Alexa Fluor 647-goat anti-rabbit  were applied for 60–90 min at room temperature. In negative controls, the primary antibodies were omitted. The slides were then washed with TBS and nuclei were stained with DAPI . The sections mounted with glycerol buffer under coverslips. The percentages of nestin, GFAP, and doublecortin positive cells (GFP-positive cells) per image field were calculated relative to the total number of GFP-positive cells in hNS/PCs and hNS/PCs + PM groups.t-test was carried out to compare one variable between two groups. The P values of less than 0.05 were considered significant.Statistical analyses of the present study were conducted with SPSS version 22. All data are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) test was used to analyze differences among all groups, and post hoc tests were performed with Tukey’s test. An independent nd and 3rd passage . The application of hNS/PCs led to a significant improvement of functional recovery after 7 weeks of the treatment compared to the PM group (P < 0.05). Moreover, treatment with hNS/PCs + PM has demonstrated a significantly higher BBB score after 1 week of the treatment compared to the PM group (P < 0.05). The application of hNS/PCs + PM has shown a significantly higher BBB score on week 5 after the treatment compared to the hNS/PCs group , hNS/PCs (0.58 ± 0.04 mm3), and hNS/PCs + PM (0.42 ± 0.01 mm3) groups compared to the PBS group . Besides, we observed a significant decrease in the lesion volume in the hNS/PCs + PM group compared to the PM groups  compared to the hNS/PCs group (72.9 ± 4.1%, P < 0.05; Figs. P < 0.05; Fig. To explore the migratory ability of transplanted cells and their capacity to differentiate into neuroglial cells in vivo, the expressions of nestin, GFAP, and doublecortin in intralesional, perilesional, and extralesional sites were assessed in the transplanted cells. The mean percentage of GFP-nestin, GFP-doublecortin, and GFP-GFAP positive cells in the perilesional and extralesional regions was significantly higher in the hNS/PCs + PM group compared to the hNS/PCs group (The long-term GFP expression in hNS/PCs using lentiviral vectors allowed us to track the cells within the injured spinal cord and healthy host tissues as well as to monitor hNS/PCs behavior following transplantation. Our data indicate the efficacy of hNS/PCs derived from human epileptic mesial temporal lobe tissues combined with PM in facilitating SCI repair and improvement of neurological functions. Furthermore, the data demonstrated the multilineage differentiation capacity of hNS/PCs and the ability to migrate within the lesion site as well as in the healthy host tissues 8 weeks after transplantation. The peptide hydrogel PM significantly promotes the differentiation and migration of hNS/PCs.The strong expressions of nestin, GFAP, and Sox2 in neurosphere-derived single cells confirmed the potential of harvesting neural stem/progenitor cells from the adult human brain. These cells proliferated as neurospheres in vitro and differentiated into neural and glial lineage in vivo. In keeping with our results, several investigations have shown that adult neural stem cells can proliferate and differentiate into various cell lineages, including astrocytes and neurons –28. In aAdult neural stem cells are promising alternatives to current cell replacement therapies for neurodegenerative disorders , 46. hNSSurgical treatment of intractable epilepsy provides a promising adult human neural stem cell source, which its transplantation with a 3D peptide hydrogel promoted functional recovery, reduced lesion volume, and enhanced cell migration and differentiation ability at the SCI lesion site in rats. Further investigations are warranted to understand the mechanisms involved in the observed functional outcome improvement; particularly, the main factors mediated the integration of hNS/PCs into existing neural circuits in the spinal cord.


Macrophage colony-stimulating factor 1 (M-CSF) is known to play a critical role during fracture repair e.g. by recruiting stem cells to the fracture site and impacting hard callus formation by stimulating osteoclastogenesis. The aim of this experiment was to study the impact of systemic M-CSF application and its effect on bony healing in a mouse model of femoral osteotomy. Doing so, we studied 61 wild type (wt) mice  which were divided into three groups: (1) femoral osteotomy, (2) femoral osteotomy + stabilization with external fixator and (3) femoral osteotomy + stabilization with external fixator + systemic M-CSF application. Further, 12 op/op mice underwent femoral osteotomy and served as proof of concept. After being sacrificed at 28 days bony bridging was evaluated ex vivo with µCT, histological and biomechanical testing. Systemic M-CSF application impacted osteoclasts numbers, which were almost as low as found in op/op mice. Regarding callus size, the application of M-CSF in wt mice resulted in significantly larger calluses compared to wt mice without systemic M-CSF treatment. We further observed an anabolic effect of M-CSF application resulting in increased trabecular thickness compared to wt animals without additional M-CSF application. Systemic M-CSF application did not alter biomechanical properties in WT mice. The impact of M-CSF application in a mouse model of femoral osteotomy was oppositional to what we were expecting. While M-CSF application had a distinct anabolic effect on callus size as well as trabecular thickness, this on bottom line did not improve biomechanical properties. We hypothesize that in addition to the well-recognized negative effects of M-CSF on osteoclast numbers this seems to further downstream cause a lack of feedback on osteoblasts. Ultimately, continuous M-CSF application in the absence of co-stimulatory signals (e.g. RANKL) might overstimulate the hematopoietic linage in favor of tissue macrophages instead of osteoclasts. Osteoblasts, which derive from mesenchymal stem cells (MSC), are responsible for the production of bone extracellular matrix and are able to mineralize into bone matrix. On the contrary, osteoclasts originate from the monocyte/macrophage/hematopoietic lineage and are responsible for bone resorption3. Osteoblasts produce soluble Macrophage colony-stimulating factor 1 (sM-CSF) and membrane-bound M-CSF (mM-CSF) which binds to the colony stimulating factor 1 (CSF) receptor of pre-osteoclasts stimulating osteoclastogenesis4. Thus, M-CSF regulates the resorptive activity of tissue specific macrophages and osteoclasts6. Ultimately, M-CSF seems to play a crucial role in osteoblast-mediated osteoclast production within the bone7. Nevertheless, current evidence is conflicting whether exogenous M-CSF acts anabolic or negatively impacts bone formation: some authors suggest that M-CSF signaling restricts osteoclast formation and thereby protects bone12 whilst others suggest M-CSF to be anabolic by promoting osteoclastogenesis14.Fracture repair requires equilibrium of cellular and molecular mechanisms aiming towards bone formation especially osteoblasts and osteoclasts and their respective effector molecules16.Besides M-CSF the co-stimulatory molecules Receptor activator of nuclear factor kappa-Β ligand (RANKL) and osteoprotegerin (OPG) contribute to the M-CSF/RANKL/OPG axis, which is key for bidirectional signaling between osteoblasts and osteoclasts. The crucial role of M-CSF has been documented in in vitro studies, as human osteoclast precursor cells treated with M-CSF increased their proliferation irrespective of additional RANKL application. Hence, the authors suggested M-CSF signaling to be an independent mediator in an effort to target osteoclastogenesis, which is in line with the current literature20.The substantial role of M-CSF in bone resorption has been nicely illustrated in vivo in the context of op/op mice. op/op mice are homozygous for the recessive osteopetrosis spontaneous mutation, causing a complete absence of a functional M-CSF. Thus, op/op mice present with obliterated osteoclast numbers, macrophages as well as monocytes resulting in generalized osteopetrosis. Interestingly, treatment with M-CSF in op/op mice has been reported to enhance the local angiogenesis and support normalization of the macrophage-, osteoclast- and osteoblast population. Doing so, op/op mice were reported to be partially or completely cured after treatment with systemic M-CSF treatment21.However, only few data on the role of M-CSF with regard to fracture healing exist so far. We documented the significance of M-CSF during human fracture healing in 113 patients, who were treated for long bone fractures. M-CSF levels were found to be significantly elevated in the fracture hematoma at the time of injury as well as in patient’s serum over the entire observation period with peak levels of M-CSF at week 1 and 2 after injury emphasizing the active involvement of this molecule in the fracture healing process from the early inflammation phase on. Serum concentration of M-CSF stayed elevated during the entire healing period indicating its importance during the fracture healing process22. Doing so, M-CSF application increased bone mass by 2.6 times at 4 weeks and 2.1 times at 8 weeks after operation compared to controls. Moreover, animals treated with M-CSF displayed higher osteoclasts numbers compared to controls linking M-CSF application and osteoclast numbers in vitro.We then transferred these observations into a rabbit model to study the impact of systemic M-CSF application in of critical size bone defects treated with conventional plating23. Alexander et al. studied the impact of CSF-1 administration in a mouse tibial injury model and its impact on the number on macrophages. The authors observed a significant increase in the number of resident macrophages (i.e. osteomacs) at the site of injury, which was a hole drilled through the entire diameter of the proximal tibia that healed by intramembranous bone formation8.The very opposite was studied by Shantz et al., who treated tibial shaft fractures in mice with a PLX3397, which is a CSF-R1 inhibitor. Doing so, the authors found decreased numbers of macrophages in the fracture callus as well as distinct differences with respect to the animals’ age. Interestingly, PLX3397 treatment caused larger calluses and increased bone formation in elderly animalsGiven the significance of M-CSF in the context of bony healing M-CSF might open a possible therapeutic window for anabolic treatment in an effort to support bony union in a murine fracture model. We hypothesized that systemic M-CSF application enhances endochondral fracture healing through the stimulation of osteoclastogenesis and reinforces the biomechanical properties in a mouse model of femoral osteotomy.25.This animal project was approved by the Animal Ethics Committee of the Medical University of Vienna (TV 132/11). All animal care was carried out according to the Principles of Laboratory Animal Care  as well as in accordance to the ARRIVE guidlines. Eighteen weeks old female heterozygote osteopetrosis mice (op/op)  as well as corresponding 18-week-old female wild type (WT) mice  were obtained. Op/op mice are characterized by an inactivating mutation in the CSF-1 gene resulting in the absence of biologically active M-CSF. Consequently, op/op mice have impaired mononuclear phagocyte development characterized by a deficiency of osteoclasts resulting in severe osteopetrosisAnimals were anesthetized with 0.1 ml/10 g of a combination of ketamine 0.5 ml + 0.15 ml Rompun + 0.1 ml Dormicum in 5 ml NaCl which we administered subcutaneously. As for post-operative pain management animals received Buprenorphin 0.1 mg/kg s.c. at the end of the surgery and Piritramid 15 mg/250 ml in drinking water ad libitum until post-operative day 5.www.risystem.com). Twenty randomly assigned WT mice were implanted a subcutaneous, osmotic mini-pump , which was placed aseptically subcutaneously through a skin incision adjacent to the abdominal wall and was left in place upon sacrifice. The mini-pumps were used as continuous drug delivery system, delivering their content  which was reconstituted in 100 μg/mL in sterile phosphate-buffered saline) at a rate of 24 mg/day over a period of 4 weeks. The remaining WT mice as well as the op/op mice did not receive systemic M-CSF treatment nor a sham-implantation of a pump.All animals received an unilateral osteotomy on their right femur. A transverse osteotomy was created in the mid-diaphysis with a 0.66 mm Gigli saw. The osteotomy was stabilized with an external fixator  treated with osteotomy and external fixator (“group 1”) (2) treated with osteotomy + external fixator + systemic M-CSF application (“group 2”) or were (3) treated with osteotomy only (“group 3”). Further we studied a group of twelve op/op mice, which were treated with osteotomy only and are further referred to as “group 4” . Further, histological and biomechanical parameters were assessed.All femurs were scanned using µCT . The X-ray tube was operated at 70 kVp and 114lA with an integration time set to 300 ms. Scans were performed at an isotropic, nominal, and spatial resolution of 10 lm (high-resolution mode). A threshold of 220 was used. Parameters of interest were assessed, connectivity density (Conn.D.), bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp), respectively. The region of the former osteotomy gap was defined as the region of interest.The collected specimens were fixed in 4% formaldehyde decalcified in Tris–EDTA solution, dehydrated and embedded in paraffin. For histology, the paraffin specimens were cut into 4 μm serial longitudinal tissue sections and stained with hematoxylin and eosin. In order to determine osteoclast parameters, the sections were stained for tartrate resistant acid phosphatase  and counterstained with hemalum Fig. . HistoloThe specimens were subjected to a three-point bending test with a universal testing machine  using a 20 N test load. The femora were placed at their lateral surface on the lower supports at a width of 8 mm in between. Before actual testing a small stabilizing preload (0.5 N) was applied at a speed of 0.155 mm/s. Fracture force to failure and modulus of elasticity were calculated from the load curves.Data are presented as the median ± interquartile range, comparisons were made using a Mann–Whitney-U-Test. Statistical analysis was performed using SPSS version 25.0 . P < 0.05 was considered to indicate a statistically significant difference.2 in op/op mice, which were significantly lower than in the corresponding group of WT mice  tended to have the largest callus compared to WT mice . This was an interesting observation regarding the relevance of M-CSF   showed lower numbers of osteoclasts/mm2 than WT mice in group 1 , this difference was found to be of borderline significance (P = 0.057). Despite the application of M-CSF, which was expected to increase the number of osteoclasts, the actual number of osteoclasts/mm2 in group 2 was almost as low as in group 4, i.e. the M-CSF depleted op/op mice.After we had documented that M-CSF is clinically relevant we reached out to study the effect of M-CSF application in the context of fracture healing in WT mice. Interestingly, studying WT mice treated with external fixator, which received M-CSF treatment (group 2) presented with less osteoclasts/mm2 was decreased in WT mice treated with M-CSF, M-CSF application had a pronounced impact on trabecular thickness  hat a significantly lower mean distance between trabeculae in terms of trabecular separation compared to mice with osteosynthesis with external fixator (group 2) , WT mice without any stabilization of their femur osteotomy showed the lowest E modulus compared to those mice, which had additional fixation. This corresponds to an overall beneficial impact on fracture healing by fracture stabilization  and 24 months (“old”). The authors showed that old mice (24 months) display delayed fracture healing with significantly less bone and more cartilage compared to young animals26. Further, the callus size was decreased in old animals as compared to young animals. Therefore, a potential age cut-off regarding the beneficial effects of PLX3397 should be evaluated, as well as for M-CSF. Given the impact of age Shantz et al. postulate that the inhibition of macrophages by blocking the M-CSF receptor c-fms might be a potential therapeutic target in elderly animals to enhance fracture healing in this specific subgroup23. Clearly, the significance of age in the context of osteoclast as well as macrophage activity needs more attention in an effort to clarify the impact on callus size and quality.Further, systemic M-CSF application had a pronounced impact on callus size as well as bone microstructure: callus size in animals treated with M-CSF was significantly larger compared to WT animals without treatment Fig. B. This i37. However, the fact that administration of M-CSF led to a reduction of the number of osteoclasts was unexpected. It is tempting to speculate that this finding might be caused by a stimulatory impact on the osteoblastic MSC niche i.e. a direct stimulatory effect of M-CSF on osteoblasts resulting in increased trabecular thickness. But to date only cells of the monocyte/macrophage/hematopoetic lineage are recognized to display a CSF-receptor, which makes a direct stimulatory effect of M-CSF on osteoblasts rather unlikely. Rather, the lack of osteoclasts in WT mice treated with systemic M-CSF is potentially responsible for missing inhibitory feedback mechanisms further downstream. Physiologically, regular numbers of osteoclasts would control and limit osteoblast activity. Accordingly, the missing feedback of osteoclasts mimics a pro-osteoblastic effect, which imitates a shift towards the MSC lineage. As shown in Fig. In our study M-CSF had a notable impact on fracture healing with respect to trabecular thickness although total bone volume (BV/TV) was not impacted. These results are in line with previously reported data on M-CSF administration and new bone formation in a rabbit modelM-CSF expression by osteoblasts is required for pre-osteoclasts to differentiate into osteoclasts, but M-CSF on its own is obviously unable to enhance this process. While in vitro data suggests that M-CSF is able to stimulate osteoclast development irrespective of additional co-stimulatory signals, e.g. RANKL, this might not be the case in vivo. Most recent data suggest that continuous M-CSF application in the absence of co-stimulatory signals such as RANKL might overstimulate the hematopoietic linage in favor of tissue macrophages instead of osteoclasts. Accordingly, there might be a small therapeutic window of M-CSF concentration where osteoclast activity is just perfectly orchestrated or optimized Fig. . But beyThe tight coupling between bone formation and resorption represents a thin line when aiming to influence this process. The suppression of osteoclasts might have had a positive impact on the trabecular thickness while the net effect regarding bone volume (BVTV) was zero. Ultimately, a co-stimulation of M-CSF together with RANKL might be required to ultimately support cell differentiation into osteoclasts instead of tissue macrophages or osteomacs.38. As we used C57BL/6 mice as control, potential effects of the background mouse stains in B6C3Fe a/a-CSF1 mice need to be taken into account, as they could potentially have affected fracture healing as well. Another limitation is the fact that our assessment focused exclusively on post-operative day 28 instead of assessing multiple time points which would provide a more detailed picture of e.g. oc numbers during the course of fracture healing. In future studies the time course should be assessed in more detail by e.g. assessment of monocyte recruitment to the fracture site via F4/80 staining at pre-defined time points.The following limitations of our analyses need to be acknowledged. In an effort to demonstrate the significant role of M-CSF, we initially aimed to treat op/op mice with M-CSF to overcome their osteoclast depletion. Despite a meticulous surgical technique we failed to mount external fixators given the animals severe osteopetrosis. This forced us to modify the study design to which is presented here instead of comparing three groups that were all treated with external fixation Fig. . Of noteUltimately, our results contribute to the deeper understanding of the crucial importance of osteoclasts in the setting of op/op mice and potential obstacles that are associated with using op/op mice in models of fracture healing. Concomitantly our results stress the crucially important fine-tuning between bone resorption by osteoclasts and bone formation by osteoblasts in an effort to support fracture healing in the future.


IntroductionIn late 2019, a novel coronavirus was identified as the pathogen responsible for a cluster of pneumonia cases in Wuhan, a city in the Hubei Province of China. Elevated cardiac troponin is a marker of myocardial injury, which is commonly seen in hospitalized patients with COVID-19 due to unclear reasons. The frequency of elevated troponin levels in patients with COVID-19 is variable and is reported in up to 7-36% of patients. The troponin level may be associated with the severity of COVID-19, and mild cases of COVID-19 tend to have a normal troponin level. This study aims to determine the frequency of patients with COVID-19 who had elevated troponin levels on presentation to the ED and determine the factors associated with elevated troponin levels. Additionally, the study aims to identify the association of elevated troponin and the outcome of COVID-19.MethodologyA retrospective study wherein the factors associated with elevated troponin levels in COVID-19 pneumonia were evaluated. The study was conducted in King Fahd Hospital of the Imam Abdulrahman Bin Faisal University. The Hospital Information System was used to identify all visits to the ED from March 2020 to November 2020 for patients who tested positive for SARS-CoV-2. In addition, a structured data collection form was used to collect data from the electronic health records. The data collection was conducted by emergency medicine physicians who were given a detailed explanation of the purpose of the study and had training and supervision by the principal investigator.ResultsP < 0.01). In total, 36 (76.6%) patients with elevated troponin levels required admission to the ICU compared with 58 (45.0%) patients with normal troponin levels (P < 0.01). Multivariable binary logistic regression analysis was used to identify the predictors of elevated troponin levels on presentation. The model revealed that being admitted in the ICU was the single independent predictor (P = 0.02).The study involved 214 patients who presented to the ED and had positive results on the SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) test and had troponin-I levels measured. Patients with elevated troponin levels were more likely to require supplementary oxygen compared with those with normal troponin levels (88.0 vs. 58.5%; ConclusionThe study demonstrated that the troponin level on presentation to the ED was a viable independent prognostic factor in COVID-19 pneumonia. However, further studies are needed to investigate targeted therapeutic interventions among patients with elevated troponin levels, such as cardioprotective therapies like corticosteroids, immunosuppressants, antivirals, or immunoglobulins. In late 2019, a novel coronavirus was identified as the pathogen responsible for a cluster of pneumonia cases in Wuhan, a city in the Hubei Province of China. This coronavirus disease 2019 (COVID-19) has spread rapidly in China and other countries, resulting in a pandemic declared by the WHO on March 11, 2020 [The spectrum of clinical severity of COVID-19 is very broad. While the clinical manifestations of COVID-19 have been characterized by cough, fever, myalgia, and headache, extrapulmonary involvement has also been frequently observed . CardiacElevated cardiac troponin is a marker of myocardial injury, commonly seen in hospitalized patients with COVID-19 due to unclear reasons . The freThis study aims to determine the frequency of patients with COVID-19 who had elevated troponin levels on presentation to the ED and determine the factors associated with elevated troponin levels. Additionally, the study aims to identify the association of elevated troponin and the outcome of COVID-19.Study designAfter obtaining an ethical review board approval, we conducted a retrospective study wherein the factors associated with elevated troponin levels in COVID-19 pneumonia were evaluated.Study settingThe study was conducted in King Fahad Hospital of the Imam Abdulrahman Bin Faisal University, the single academic hospital in the Eastern Province of Saudi Arabia. It has around 250,000 visits to the ED annually.Study populationThe Hospital Information System was used to identify all visits to the ED from March 2020 to November 2020 for patients who tested positive for SARS-CoV-2 and underwent frontal chest X-ray on their presentation. Patients below the age of 18 years and pregnant patients were excluded.Study objectivesThe study aimed to identify the demographic, clinical, and laboratory parameters associated with elevated troponin levels in COVID-19 pneumonia on presentation. Additionally, it investigated the association of elevated troponin and the outcome of COVID-19, including the need for intensive care, mechanical ventilation, and in-hospital mortality.Data collectionA structured data collection form was used to collect data from the electronic health records. The data collection was conducted by emergency medicine physicians who were given a detailed explanation of the purpose of the study and had training and supervision by the principal investigator. The collected data included age, sex, comorbidities, presenting symptoms, duration of symptoms, and vital signs. In addition, data on the severity of COVID-19 pneumonia were collected, including the disposition decision , the need for mechanical ventilation, the need for ICU admission, thrombotic complication, length of stay, and mortality. The Charlson Comorbidity Index was used to measure comorbidities. This index score ranges from 0 to 37 and includes various medical conditions [Ethical considerationThe study was approved by the Institutional Review Board at the Imam Abdulrahman bin Faisal University (IAU IRB No. 2020-01-384). The data were stored electronically, and only the principal investigator had access to the stored data. Furthermore, the need for informed consent was waived because the patients were de-identified.Statistical analysisAfter checking for completeness and consistency, data were analyzed using IBM SPSS for Windows, version 26 . Categorical variables, presented as percentages and frequency distribution, were compared using the Chi-squared or Fisher’s exact tests. Continuous variables, presented as mean and SD, were compared using the independent sample t-test or ANOVA. Multivariable binary logistic regression analysis was conducted to identify the independent factors associated with elevated troponin. Candidate variables were selected based on medical literature and bivariate analyses. OR with 95% CI were estimated using the full model fit and were reported in comparison with the designated reference group. The presence of multicollinearity was assessed using the bivariate Spearman’s correlation coefficients. The goodness-of-fit of the model was evaluated using the Omnibus and Hosmer-Lemeshow tests. The significance level was defined as α = 0.05.Patient characteristicsThe study involved 214 patients who presented to the ED and had positive results on the SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) test and had troponin-I levels measured. This included 148 (69.2%) men and 66 (30.8%) women. Overall, 92 (43.0%) were aged 51-65 years, and 50 (23.4%) were aged above 65 years. In total, 89 (41.6%) patients had a Charlson Comorbidity Index score of 0-1, whereas 38 (17.8%) patients had a score of 4 or more. Diabetes mellitus and hypertension were the most frequent comorbidities, with 96 (44.4%) and 95 (44.4%) patients, respectively. In addition, asthma or chronic obstructive pulmonary disease were seen in 22 (10.3%) patients. Dyspnea (65.9%), fever (54.7%), and cough (51.4%) were the most frequently reported symptoms. Nearly half (48.6%) of patients had an oxygen saturation below 95%, and 147 (68.7%) had a respiratory rate above 20 bpm  patients had elevated troponin levels on presentation. The age and gender of patients were not significantly associated with elevated troponin levels (P > 0.05). However, the Charlson Comorbidity Index score was significantly associated with troponin levels (P = 0.02). For instance, 74 (83.1%) patients with a score of 0-1 had normal troponin levels, whereas 15 (39.5%) patients with a score of 4 and above had elevated troponin levels. Among the comorbidities, the history of chronic kidney disease was the only comorbidity associated with elevated troponin levels (P < 0.01). More patients with chronic kidney disease had elevated troponin levels than those who had not (63.6% vs. 21.2%). There was no significant association between the duration of symptoms and the troponin levels (P = 0.12) . Similarly, 19 (38.0%) patients with elevated troponin levels required mechanical ventilation compared with 31 (18.9%) patients with normal troponin levels (P = 0.01). In total, 36 (76.6%) patients with elevated troponin levels required admission to the ICU compared with 58 45.0%) patients with normal troponin levels (P < 0.01). In addition, the mortality rate during hospitalization among patients with elevated troponin levels was higher than those with normal troponin levels . However, the troponin levels were not found to be significantly associated with the length of stay or the occurrence of thrombotic complications (P > 0.05) . For example, patients who were admitted to the ICU were 3.3-times more likely to have elevated troponin levels compared with those who did not  (Table The study aimed to identify the demographic and clinical factors associated with elevated troponin levels and investigate the association of troponin levels and the outcome of COVID-19 pneumonia. The findings demonstrated that elevated troponin levels were significantly and strongly associated with mortality, the need for ICU, and mechanical ventilation.Our findings were consistent with previous research, which investigated the association of troponin and the outcome in COVID-19 pneumonia. For example, a meta-analysis showed that elevated troponin is strongly associated with death, severe presentation, hospitalization in the ICU, and/or mechanical ventilation. Furthermore, patients with elevated troponin were 10-times more likely to develop these adverse outcomes .In a study involving over 2500 hospitalized patients with COVID-19, it was found that 36% of patients had elevated troponin levels. The elevated troponin levels were more common among patients with previous cardiovascular disease or having cardiac risk factors. Furthermore, it was found that even mildly elevated troponin levels were significantly associated with mortality. Interestingly, greater elevations of troponin levels were associated with a higher risk of mortality [In a retrospective study of 416 patients with COVID-19, it was found that elevated troponin levels were more prevalent among older patients with comorbidities and greater laboratory abnormalities, and extensive radiographic involvement of the disease. Additionally, the risk of death starting from the time of symptom onset was more than four times higher in patients with evidence of myocardial injury on admission [Although the sex of the patient was not significantly associated with elevated troponin levels among patients with COVID-19 in our study, it is worth noting that there is a notable difference in the upper limit of the normal range of troponin among men and women . Hence, This is among a few studies to investigate the demographic and clinical findings concerning the initial troponin levels of patients on their presentation to the ED in Saudi Arabia. However, the study has certain limitations. While it included a relatively large sample size, the study was conducted in a single academic institution in the Eastern Province. The retrospective nature of the study is an important limitation.The study demonstrated that the troponin level on presentation to the ED was a viable independent prognostic factor in COVID-19 pneumonia. However, further studies are needed to investigate targeted therapeutic interventions among patients with elevated troponin levels, such as cardioprotective therapies like corticosteroids, immunosuppressants, antivirals, and/or immunoglobulins.


Neuroimmunology is a rapidly expanding field, and there have been recent discoveries of new antibodies and neurological syndromes. Most of the current clinical studies have focused on disorders involving one specific antibody. We have summarized a class of antibodies that target common neuronal epitopes, and we have proposed the term “anti-neuron antibody syndrome” (ANAS). In this study, we aimed to clarify the clinical range and analyse the clinical features, cytokines/chemokines and predictors in ANAS.This was a retrospective cohort study investigating patients with neurological manifestations that were positive for anti-neuron antibodies.P = 0.0193) and having a chronic onset (P = 0.0306), and predictors of relapses included having lower levels of CSF BAFF (P = 0.0491) and having a larger ratio of serum TGFβ1/serum CXCL13 (P = 0.0182).A total of 110 patients were identified, of which 43 patients were classified as having autoimmune encephalitis (AE) and the other 67 were classified as having paraneoplastic neurological syndrome (PNS). With regards to anti-neuron antibodies, 42 patients tested positive for anti-N-methyl-D-aspartate receptor (NMDAR) antibody, 19 for anti-Hu, 14 for anti-Yo and 12 for anti-PNMA2 (Ma2). There were significant differences between the ANAS and control groups in serum B cell-activating factor (BAFF) levels and in cerebrospinal fluid (CSF) C-X-C motif chemokine10 (CXCL10), CXCL13, interleukin10 (IL10), BAFF and transforming growth factor β1 (TGFβ1) levels. Predictors of poor outcomes included having tumours (Most patients with ANAS had a relatively good prognosis. Having tumours and a chronic onset were both associated with poor outcomes. CSF BAFF and the ratio of serum TGFβ1/serum CXCL13 were associated with relapses. Neuroimmunology is a rapidly expanding field that has led to the discovery of a series of new antibodies and neurological syndromes. Most of the current clinical studies have focused on disorders related to one specific antibody –3 and haANAS is considered to be an immune-mediated disorder and is characterised by the presence of antibodies that target neuronal epitopes. T and B cells might be responsible for the syndrome and participate in key steps . Some cyIn this study, we collected the clinical characteristics and data on the cytokines/chemokines in the sera and CSF and aimed to look for the associations between them and ANAS. Then, we strived to identify predictors for the outcomes and relapses of ANAS. Here we report the data.BR. Our study included 110 patients hospitalized between March 18, 2011, and July 3, 2017. We collected demographic and clinical data, including age, sex, type of onset, magnetic resonance imaging (MRI) fluid-attenuated inversion recovery (FLAIR)/T2, electroencephalogram (EEG)/electromyogram (EMG), CSF leukocyte counts, CSF protein levels and the antibody-specific diagnosis of the patients. The data with regards to associated tumours, outcomes and relapses were obtained from patients in the hospital and during the follow-up. A total of 108 patients had follow-up evaluations between 24 and 99 months (median 54 months), and 2 patients dropped out. All patients were grouped into AE or PNS by clinical syndromes and into good or poor outcomes by the modified Rankin scale (mRS). They were also grouped into tumours or no tumour and into relapses or no relapse. Good outcomes were defined as a grade of 0–2 on the mRS, while poor outcomes were defined as a 3–6.We performed a single-centre, retrospective cohort study at the 960 Hospital of the PLA Joint Logistics Support Force. Our study protocol included all patients with neurological manifestations and had positive anti-neuron antibodies, including amphiphysin, CV2, PNMA2, Ri, Yo, Hu, Recoverin, SOX1, Titin, NMDAR and GABAThe study was approved by the ethics committee of the 960 Hospital (NO.201611). All patients provided informed written consent to allow their medical records and samples of sera and CSF to be used in this study. All patients’ data were processed anonymously.A total of 156 samples were obtained from patients within the first 3 days after admission, which included 105 sera and 51 samples of CSF. The control group included 21 sera that were donated from healthy adults and 20 samples of CSF from apparently healthy adults treated for traumatic fractures of the lower limbs, and the CSF control samples were collected from spinal anaesthesia. All samples were stored at − 80 °C until analysis.BR antibody was performed by recombinant immunofluorescence using an IIFT: Glutamate Receptor Mosaic . Paraneoplastic antibodies , -Ri, -Yo, -Hu, -SOX1, -Titin, -Recoverin) were determined in an immunoblot assay using EUROLINE Neuronal Antigens Profile (IgG) . CXCL10, CXCL13, IL10, and BAFF were analysed using a premixed multiplex system  human magnetic luminex assays. TGF-β1 was tested using ELISA kits  and a human TGF-β1 immunoassay。 The testing was performed according to the manufacturer’s instructions. As reported  of 1. We performed all the statistical analyses with SAS, version 9.4 .Demographic information and clinical features are expressed as numbers (N) and percentages (%). Levels of cytokines/chemokines are presented as the median . The Fisher’s exact or Wilcoxon rank sum tests were used to test for significant differences among the different patient groups as appropriate. Variables associated with outcomes or relapses (6/L) and 30 (27.3%) had elevated CSF protein levels (> 450 mg/L) , and 54 (49.1%) of the patients were women. Fifty-two patients (47.3%) had an acute onset, 45 (40.9%) had a chronic onset and 13 (11.8%) had a subacute onset. Thirty-three patients (30%) were identified as having MRI (FLAIR)/T2 abnormalities (excluding ischaemic lesions), 52 (47.3%) had EEG/EMG abnormalities, 41 (37.3%) had a leucocytosis in the CSF (white blood cells > 5× 10BR) were classified as AE, and the other 67 patients (60.9%) were classified as PNS. Factors significantly associated with PNS patients included having a chronic onset (P < 0.0001), an elevated CSF protein level (P = 0.0116), tumours (P = 0.0005), no relapse (P = 0.0113) and poor outcomes (P = 0.0004) when compared to AE patients. In this study, 40(36.4%) patients had a tumour. Nine tumours (8.2%) were non-small cell lung cancers, 6 (5.5%) were small cell lung cancers, 5 (4.5%) were breast cancers, 3 (2.7%) were ovarian teratomas and 17 (15.5%) were other tumours.Regarding the anti-neuron antibodies in the patients, 42 patients (38.2%) tested positive for anti-NMDAR antibodies, 19(17.3%)for anti-Hu, 14 (12.7%)for anti-Yo, 12 (10.9%) for anti-PNMA2, 8(7.3%) for anti-Amphiphysin, 8 (7.3%) for anti-CV2 and 7 for others (6.4%). As reported , 43 (39.P = 0.0002), an elevated CSF protein level (P = 0.0075), tumours (P < 0.0001) and no relapse (P = 0.0081). During the follow-up, 33 patients (30%) had clinical relapses, including 23 patients  that relapsed once and 1 patient that relapsed 8 times. We also recorded significant positive correlations between having a relapse and an acute onset (P = 0.0163), MRI (FLAIR)/T2 abnormalities (P = 0.0345) and a normal CSF protein level (P = 0.0012).At the final follow-up, 37 patients (33.6%) had poor outcomes. In patients with the poor outcomes, we recorded significant relevant factors, including having a chronic onset (P = 0.0431) and in CSF CXCL10, CXCL13, IL10, BAFF and TGFβ1 levels (P < 0.05) between the ANAS and control group , IL10 (P = 0.0091) and TGFβ1 (P < 0.0001) between the AE and PNS patients. We detected significant inverse relationships between IL10 levels and patients with tumours (P = 0.0124). In patients with poor outcomes, the significant relevant factors included higher levels of CXCL10 (P = 0.0258) and BAFF (P = 0.0409) than those with good outcomes. In patients with relapses, we recorded significant positive correlations for TGFβ1 (P = 0.0032), whereas we detected significant inverse relationships for CXCL10 (P = 0.0279) compared with no relapse .In the univariate analysis of the serum cytokines/chemokines levels, there were significant differences in CXCL10 ( P = 0.014. In patP = 0.0002), an elevated CSF protein level (P = 0.0075), tumours (P < 0.0001), no relapse (P = 0.0081), PNS (P = 0.0004), higher levels of serum CXCL10 (P = 0.0258) and serum BAFF (P = 0.0409) (Table P = 0.0193) and a chronic onset  (Table P = 0.0163), MRI (FLAIR)/T2 abnormalities (P = 0.0345), a normal CSF protein level (P = 0.0012), AE (P = 0.0113), a good outcome (P = 0.0081), lower levels of serum CXCL10 (P = 0.0279) and higher levels of serum TGFβ1 (P = 0.0032) (Table P > 0.05).In all patients, the univariate analysis revealed factors associated with poor outcomes, including having a chronic onset P = 0.000, an elevP = 0.000, higher P = 0.0606), a chronic onset (P = 0.0010), an elevated CSF PRO level (P = 0.0123), tumours (P < 0.0001), and PNS (P = 0.0372) (Table P = 0.0047) (Table P = 0.0048), serum TGFβ1/serum CXCL13 (P = 0.0455), serum TGFβ1/serum BAFF (P = 0.0279), CSF TGFβ1/serum TGFβ1 (P = 0.0205), serum TGFβ1/CSF BAFF (P = 0.0076), higher levels of serum TGFβ1 (P = 0.0202) and other factors were identified that might be associated with relapses, including having a normal CSF protein level (P = 0.0632), AE (P = 0.0737), lower levels of CSF IL10 (P = 0.0913) and CSF BAFF (P = 0.0990) (Table P = 0.0491) and a larger ratio of serum TGFβ1/serum CXCL13   classified as PNS, and the common antibodies found were anti-NMDAR, anti-Hu and anti-Yo. A total of 36.4% of patients had tumours, and the majority of the tumours were lung cancers and breast cancers. As shown in other research on PNS, the common antibodies and tumours were anti-Yo, anti-Hu, and anti-titin antibodies and thymoma and lung cancers, respectivel y, which w+ cells, such as activated T cells, in CNS inflammation,[In general, ANAS is considered to be an immune-mediated disorder and has certain antibodies targeting neuronal epitopes. T and B cells might be responsible for the syndrome and participate in key steps . Some cyammation, and theyammation,. BAFF isammation,. The accThe measurement of cytokines/chemokines in the sera and CSF were the key points of the study. Our study aimed to screen several cytokines/chemokines as predictors of the outcomes and relapses of ANAS. As reported in previous studies , 23, PNSIn this study, some cytokines/chemokines related to the activation or inhibition of T and B cells were observed. We tested the levels of cytokines/chemokines in the CSF and sera and then further investigated the relationship between cytokines/chemokines and ANAS. In the univariate analysis, the elevation of CXCL10 and BAFF in the sera indicated a poor outcome, and a decrease of CXCL10 and an elevation of TGFβ1 in the sera was associated with relapses. In the multivariate analysis, the factors associated with relapses included lower levels of CSF BAFF and a larger ratio of serum TGFβ1/serum CXCL13. These cytokines/chemokines might be predictors for outcomes and relapses of ANAS. In this research, we aimed to identify some cytokines/chemokines that were able to predict the prognosis. We also explored more associated factors and calculations that might be related to the prognosis. The ratios of two cytokine/chemokine levels were counted as factors. Then, we observed whether they were associated with outcomes and relapses in the univariate and multivariate analyses. In our study, the ratio of serum TGFβ1/serum CXCL13 was associated with relapses. Multicentre clinical trials and verifications with larger samples might be helpful in order to clarify the relationship between these ratios and prognosis of ANAS. These ratios in this study may indicate a novel way of determining prognosis. In this study, we mainly observed the phenomenon including the difference in factor level in different ANAS groups and between ANAS and normal population. In the future experiment, we will analyse the function and quantity of T and B cell subsets by flow cytometry. At the same time, the interaction in different inflammatory factors would be studied to explore the mechanism in depth.There were many factors associated with the outcomes and relapses of many diseases, and some clinical features have been reported in previous studies. Conscious disturbances and CSF antibody titres have been shown to be predictors of poor outcomes in anti-NMDAR encephaliti s, and aggMuch evidence has been collected in previous studies of AE and PNS , 17, andOur study had the limitations of not being randomized, having fewer cases and having a lack of complete data regarding the cytokines/chemokines in the CSF, which reduces the level of evidence in this ANAS study. Some other factors might influence the levels of cytokines/chemokines found in the sera and CSF, including sampling time, storage conditions, test method, etc. The interactions with different cytokines/chemokines are complex pathological processes. In particular, there is a lack of data on the function and quantity of T and B cells. It is difficult to reflect the complex changes in the process of disease solely using the levels or ratios of cytokines/chemokines in the sera or CSF. However, we proposed the clinical range of ANAS, summarized the features and cytokines/chemokines in the sera and CSF, and then screened possible predictors associated with outcomes and relapses. This might be helpful for the in-depth study of ANAS.Our study focused on studying the association of disorders with a class of antibodies targeting common neuronal epitopes, and this study highlights the fact that ANAS is a clinical neurological syndrome, not a clinical diagnosis. The term ANAS was proposed for the first time and the clinical range was clarified. The study of ANAS might be a beginning for the understanding of these conditions and should be further promoted. This might provide a novel idea for clinical study in neuroimmunology.Our study proposed the term ANAS and clarified the clinical range of this condition. We analysed the association between these diseases, the clinical features and levels of cytokines/chemokines. Then, we screened possible factors associated with poor outcomes, including having tumours, a chronic onset and factors associated with relapses, which included having lower levels of CSF BAFF and a larger ratio of serum TGFβ1/serum CXCL13. These factors might be related to the prognoses of the diseases.


The classical cerebrospinal fluid (CSF) circulation theory has been accepted as an established theory of CSF physiology. It describes bulk CSF flow from production site to absorption site. However, much controversy remains regarding the basic CSF physiology and the mechanisms behind the development of hydrocephalus. In the recent observations made using advanced magnetic resonance imaging (MRI) technique, namely, the time spatial inversion pulse (Time-SLIP) method, CSF was used as internal CSF tracer to trace true CSF movement. Observation of the CSF dynamics using this method reveals aspects of CSF dynamics that are different from those of classical CSF circulation theory. Cerebrospinal fluid shows pulsation but does not show bulk flow from production site to absorption site, a theory that was built upon externally injected tracer studies. Observation of the exogeneous tracer studies were true but misinterpreted. Causes of misinterpretations are the differences between results obtained using the true CSF tracer and exogenous tracers. A better understanding of the real CSF physiology can be significant for the advancement of medical sciences in the future. Revisiting CSF flow physiology is a necessary step toward this goal. The dynamics of cerebrospinal fluid (CSF) can be regarded as one of the basic physiological processes of the body. An accurate account of the CSF physiology can provide new insights for understanding human body homeostasis and excretion of waste products from the brain, for developing new concepts of pharmacokinetics effects, and for devising treatments for CSF-related lesions. Unfortunately, the dynamics of CSF can also be the most misunderstood area of human physiology. After Cushing and Weed proposed the classical CSF circulation theory in 1914 , many qu2O17 study  cisternography. Radio-labeled albumin was injected into CSF intrathecally and intraventricularly in a human model . The lab17 study , and O17ed water  using MRIt has been intensively studied that brain capillaries were the site of CSF exchange with the blood -25. In tWith the advent of the Time-SLIP method, it is now possible for the first time to repeatedly trace the real human CSF pulsations without disturbing the normal physiological condition. In particular, the real-time Time-SLIP method provides continuous high-speed imaging of CSF (48 msec/frame), enabling detailed observation of CSF dynamics in its physiological state.Cerebrospinal fluid shows the characteristic movements particularly at locations where the passage is narrow (bottleneck), such as the foramen of Monro and the cerebral aqueduct of Sylvius. Cerebrospinal fluid moves cranially during diastole phase and caudally during systole phase of a cardiac cycle. It is called “to and fro” motion of the CSF. In the foramen of Monro, the CSF from the third ventricle flows along the lateral wall of the lateral ventricles with pulsation to replace the CSF in the lateral ventricle . It can Previous experiments have clearly shown that tracers injected into the CSF in laboratory animals do not follow imagined CSF flow . In the. This puIn the third ventricle (and the fourth ventricle), turbulent flow of CSF is observed . In the In the fourth ventricle, CSF is basically stirred in the same way as in the third ventricle. In this case, the bottlenecks are the cerebral aqueduct of Sylvius and the foramen of Magendie and Luschka. A depiction of the detailed flow movement of CSF in real time has become possible using the MRI Time-SLIP method because the CSF itself is used as a tracer and the achievement of high-speed continuous imaging. Other methods of MRI CSF observation, such as standard phase contrast (PC) MRI, require synchronization with the cardiac pulse. It is then not possible to observe the pulsations-induced mixing, which does not necessarily correlate in phase with that from the regular cardiac cycle.Pulsation of CSF associated with the cardiac beat and the respiration can be observed during surgery or during spinal tap. The net flow has been calculated from the stroke volume by adding and averaging the CSF pulsation in PC MRI. In PC MRI, the movement of CSF due to respiration is assumed negligible. However, it is well known that even for nominal measurement there are large variations of the deduced quantitative values in the individual measurement -35. ObviAfter the MRI Time-SLIP was developed to visualize the movement of CSF due to respiration , furtherIn addition, biphasic movements are sometimes observed in the cerebral aqueduct of Sylvius. 1-40 may diminish due to aging. This experimental method is generally adopted in the experimental model of the glymphatic system (The ISF of the brain and the CSF have the same composition and can freely exchange. Recently, as the glymphatic system , it has It is possible that shaking the head greatly agitates the ISF and CSF, and accelerates the drainage of the waste products from the brain. The resting state indicates a sleeping state, and the state of shaking the head is related to the states of daily living. Both diagnostic imaging and animal experiments were generally observed in a stationary state or under anesthesia. The Time-SLIP images presented here suggest that there can be completely different CSF dynamics in actual daily living. In the sleeping state, only the CSF adjacent to the CVO shows active movement. The waste product in the brain that cannot be absorbed by the local blood-brain barrier carries more into the CSF by stirring during the day. Some of these waste products drain into the lymphatic system from CSF. Indeed, CSF diversion surgery has been attempted to promote drainage of waste products to treat Alzheimer disease ,49. AlthAcute obstruction of the CSF pathway is a clinically very critical situation. The classical CSF bulk flow theory seems to fit well in explaining the cause of acute obstructive hydrocephalus, in which the so-called upstream ventricles expand with obstruction of the ventricular CSF pathway. For example, in the cases where intra-cerebral or intra-ventricular hemorrhage causes acute obstruction of the cerebral aqueduct of Sylvius, emergency drainage is required. In fact, if you are a neurosurgeon chances are that you would have witnessed such a situation and have performed the emergency drainage many times. There is no doubt that this procedure is effective, but the question is whether the cause of ventricular enlargement is the continuous production of CSF from the choroid plexus in the ventricles, which can cause abnormal accumulation upstream of the site of obstruction. If the cause of ventricular enlargement is the continuously generated CSF from the choroid plexus with no outlet, then ventricular enlargement should have been observed in all cases without exception. However, many neurosurgeons have experienced cases where acute hydrocephalus does not occur even with an obstructed cerebral aqueduct of Sylvius . There wIn cat experiments, a cannula was inserted into the cerebral aqueduct of Sylvius from the fourth ventricle. Cannula was kept in physiological level and to collect CSF from the third ventricle. They found not even a single CSF droplet from the cannula ,51,52. IA better understanding of the real CSF physiology can be significant for the advancement of medical sciences in the future. Revisiting CSF flow physiology is a necessary step toward that goal.


The use of oral (PO) antibiotics and lipoglycopeptides are challenging the previous standard of osteomyelitis (OM) treatment, but there is currently a paucity of comparative data between these approaches.Staphylococcus aureus on culture, utilization of outpatient parenteral antibiotic therapy (OPAT) services (IV group only), and concomitant PO therapy (lipoglycopeptide group only) were included in a bivariate analysis and variables with a p-value < 0.2 were included in a multivariate regression model.This retrospective study included patients diagnosed with OM treated with intravenous (IV) antibiotics, PO antibiotics, or lipoglycopeptides between January 1, 2010 and June 1, 2020. Patients in the PO group could receive no more than 14 days of IV antibiotics prior to the PO course, and inclusion into the lipoglycopeptide group required at least 2 doses of drug to be administered. The primary outcome was occurrence of clinical failure within six months of completion of therapy, which was defined as new antibiotics or unplanned surgical intervention for an infection at the same site. Secondary outcomes included in-hospital length of stay (LOS), amputation within 6 months of therapy completion, and incidence of drug and line-related adverse effects. Previous osteomyelitis at index site, surgical intervention as a part of initial management, presence of The IV group included 257 patients, while the PO and lipoglycopeptide groups included 20 and 15 patients respectively. In the IV group, 89 (35%) of the patients experienced clinical treatment failure compared to 5 (25%) in the PO group and 5 (33%) in the lipoglycopeptide group (p=0.71). Median LOS was significantly shorter in the PO group compared to the IV and LGP groups . No difference between groups was observed for amputation within 6 months or incidence of adverse effects. The only variable included in the multivariate regression model was previous osteomyelitis at index site .PO and lipoglycopeptide therapy resulted in similar outcomes compared to IV antibiotics. Only previous OM at the same site was identified as an independent risk factor for failure.Ryan P. Moenster, Pharm.D., FIDSA, AbbVie (Speaker’s Bureau)Melinta