For this purpose, a series of clinically used therapeutic agents were collected and their activation potentials on Nrf2 were assayed by using 293T-Nrf2-luc cell line. Among all tested therapeutic agents, midazolam was found with good Nrf2 activation effect and this agent could significantly ameliorate CCl4-induced damage to HepG2 cells. In vivo animal tests showed that pretreatment with midazolam reduced the liver pathological tissue damage and the serum levels of ALT and AST in CCl4-induced liver injury mice. Further investigations showed that midazolam could strongly up-regulate the expression of both Nrf2 and HO-1 in the mice liver, accompanied by increasing of the levels of antioxidant enzyme SOD and reducing the production of MDA, as well as reducing the pro-inflammatory cytokines  secretion. Collectively, our results clearly demonstrate that midazolam can ameliorate CCl4-induced acute liver injury and oxidative stress via activating the Nrf2 signaling pathway.Oxidative stress contributes greatly to initiation and progression of liver injury. Activation of nuclear-factor erythroid 2-related factor 2 (Nrf2) has been considered as an attractive strategy for preventing and treating the oxidative damage related to liver injury. This study aimed to find an efficacious agent to activate Nrf2/HO-1 signaling pathway from clinically used therapeutic agents and to characterize the usefulness for preventing and treating CCl Oxidative stress is a state of imbalance between oxidation and antioxidant effects in human body, which could result in the generation of a large number of reactive oxygen species (ROS) in cells. Under physiological conditions, excessive ROS will affect a series of cell transduction pathways, influence the production and secretion of oxidative stress molecules, and regulate the balance between proliferation and apoptosis of cells. Meanwhile, it can also affect the function of mitochondria, leading to cell dysfunction, and eventually damage various tissues and organs . Particuin vivo therapeutic effect of antioxidants on liver diseases. Particularly, some sedative and analgesic anesthetics have been widely concerned as treatment methods to reduce oxidative stress and protect important organ functions. Studies have shown that propofol can reduce the release of ROS and malondialdehyde (MDA) from the liver after ischemia-reperfusion, and contribute to the recovery of lung function , which harbors Nrf2 promoter-luciferase reporter gene construct driven by multiple dioxin response elements. After that, 293T-Nrf2-luc cell line was obtained through antibiotic screening.293T-Nrf2-luc cell line was developed 2. The fresh media were replaced every 2 days.Both of 293T-Nrf2-luc cell lines and HepG2 cell lines were grown in DMEM/high glucose medium  containing 10% fetal bovine serum  and 1% penicillin-streptomycin solution . The culture condition was constant temperature of 37°C, a humidified atmosphere of 95% air and 5% CO3 cells per well and grown overnight. Baicalin and iso-dihydromyricetin were prepared in our laboratory  according to the manufacturer’s directions. Luciferase activity was measured on Spectramax M3  with luciferase production as readout. Data was shown as the percentage of fold induction of the test compound against the vehicle control group.Following 24 h incubation, luciferase activity was measured by the Steady-Glo4 cells/well) and grown overnight. To verify the effects of midazolam on HepG2 viability, cells were treated with midazolam at different concentrations  for 24 h. The cells were incubated with CCK-8  solution (20 μl/well) and cultured at 37°C for another 1 h. The absorbance of the dissolved solutions was detected at 450 nm on Spectramax M3.HepG2 cells were seeded in 96-well culture plates  were added to culture for 24 h, and HepG2 cells activity was detected by CCK-8 to determine the IC50 value of CCl4. After 24 h of intervention with different concentrations of midazolam, IC50 concentration of CCl4 was added for another 24 h of intervention, and finally CCK-8 was performed to detect cell activity.In order to verify the protective effect of midazolam on CClThe antioxidant effect of midazolam was tested in adult C57BL/6 mice  provided by Shanghai Slake Experimental Animal Co., Ltd. All mice were fed in the laboratory at a temperature of about 22°C and a relative humidity of 50%, with a light-dark cycle for 12 h. A 1-week acclimatization period was adopted before the experiment, during which the mice also had free access to food and water. All animal experiments were approved by the animal ethics committee of Yueyang Hospital of integrated traditional Chinese and Western Medicine Affiliated to Shanghai University of traditional Chinese medicine (ethics No.: YYLAC-2020-0801).n = 8 in each group): control, CCl4 group , CCl4 plus midazolam 4 mg/kg/day (CCl4 + M4 group); CCl4 plus midazolam 8 mg/kg/day (CCl4 + M8 group). In the CCl4 group, mice were intraperitoneally (i.p.) injected with 0.5% (v/v) CCl4 . In the CCl4 + M4 group or CCl4 + M8 group, midazolam was respectively i. p. injected at 4 or 8 mg/kg for 3 days, and 0.5% CCl4 was i. p. injected at 2 h after the last dose of midazolam. Mice were sacrificed 24 h after CCl4 injection, and blood and liver samples were collected. A part of the liver was immediately fixed in 10% formaldehyde for histopathological observation, while the rest was frozen in liquid nitrogen and stored at −80°C for other experiments.Next, 32 mice were randomly divided into the following groups . Midazolam was i. p. injected at 8 mg/kg for 3 days, and 50% CCl4  were i.p. injected at 2 h after the last dose of midazolam. The mortality of mice in each group was recorded up to 36 h after CCl4 administration.In order to explore the effect of midazolam on CCln = 8) was fixed in 10% neutral buffered formalin for 48 h and embedded in paraffin. Then 4 μm thick sections were cut and stained with hematoxylin and eosin (H&E) according to standard procedure. The samples were observed and images were obtained using an optical microscope .The liver of each mouse (g for 10 min at 4°C and the serum was collected to measure the activity of aspartate aminotransferase (AST) and alanine transaminase (ALT), which were measured by kits . Liver tissue homogenization was performed using tissue homogenizer . The homogenates were centrifuged  at 4°C, and the obtained supernatant was used for subsequent measurement of MDA and SOD by kits . The levels of TNF-α and IL-6 in the liver tissues were measured using ELISA kits according to the manufacturer’s instructions .Blood samples were centrifuged at 3000 × Total proteins were isolated from the liver tissues and cells using protein extraction kit . The cell nucleus proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit . Protein concentrations were measured using BCA protein assay kit . Equal amounts of protein from each sample were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to PVDF membranes. Then, the membranes were blocked with 1 × TBST containing 5% nonfat milk for 2 h at room temperature and continued to incubate with primary antibodies against mice Nrf2 , HO-1 , β-actin  overnight at 4°C. Subsequently, the membranes were washed three times with TBST and incubated with secondary antibody at room temperature for 2 h. Blots were visualized using a hypersensitive chemiluminescence kit . The results were quantified by densitometry using ImageJ software.The 3D structure of Kelch-like ECH-associated protein 1(Keap1) was downloaded from the Protein Data Bank (PDB Code:1ZGK) . AutoDocp-values less than 0.05 were considered statistically significant.The data were analyzed with GraphPad Prism 7.0  and presented as the means ± standard deviation (S.D.). One-way ANOVA analysis was used for differences between groups. The survival rate was analyzed by the log-rank test (p < 0.01)  were evaluated. The results showed that midazolam up-regulated the activity of Nrf2 more than 2-fold compared with the negative control group  < 0.01) .(p < 0.01)  . To deteTo further verify the inductive effect of midazolam on Nrf2, the protein levels of Nrf2 in 293T-Nrf2-luc cells with or without midazolam treatment were determined. As shown in 4-induced HepG2 cells were used as model cells. After pretreatment of HepG2 cells with different concentrations of midazolam for 24 h, CCl4 . By contrast, midazolam could significantly improve HepG2 cells activity compared with the CCl4 group (p < 0.01) even at low concentration (2.50 μM), as depicted in 4-induced hepatocytes.Next, the protective effects of midazolam on hepatocytes were investigated by using CCl4 induced acute liver injury in mice. As shown in 4 group were significantly increased (p < 0.01). In sharp contrast, the activity levels of ALT and AST in serum collected from midazolam pretreatment group were significantly decreased (p < 0.01), when compared with that from the CCl4 group  on CCll4 group . Further reduced . The his4 group were down-regulated evidently when compared to that in the control group. However, following administration of midazolam, the protein levels of both Nrf2 and HO-1 were significantly enhanced. These findings clearly suggested that Nrf2-related signaling pathway in mice liver could be enhanced following administration of midazolam.As shown in (p < 0.05), while SOD activity levels were decreased at 24 h after CCl4 treatment (p < 0.05). Compared with CCl4 group, midazolam pretreatment group reduced MDA levels (p < 0.05) and increased SOD activity levels (p < 0.05) (4 treatment significantly increased TNF-α and IL-6 expression levels compared with the control group (p < 0.01). As expected, midazolam dose-dependently reduced IL-6 expression levels (p < 0.01) when compared with that in the CCl4 group, while the serum levels of TNF-α were also significantly reduced following administration with high dose midazolam (8 mg/kg) (p < 0.05)  . Meanwhi < 0.05) .4. For this purpose, a lethal dose of CCl4 for mice  was used, while midazolam (8 mg/kg) was administrated for three consecutive days (once per day). As shown in 4 group (p < 0.05).To further verify the hepatoprotective effect of midazolam, we investigated the ability of midazolam to affect the survival rate of mice treated with lethal dose of CClvia strong hydrophobic interactions including Alkyl, Pi-Alkyl and Pi-Pi stacked interactions  . Clinicaive role . To concive role .in vitro and in vivo. Cell assays demonstrated that midazolam dose-dependently enhanced Nrf2 expression and showed good protective effects on CCl4-induced HepG2 cells. Animal tests showed that midazolam could significantly reduce the liver pathological tissue damage and the serum levels of both ALT and AST in CCl4-induced liver injury, accompanied by increasing of the levels of antioxidant enzyme SOD and reducing the production of MDA, as well as reducing the pro-inflammatory cytokines  secretion. Molecular docking simulations suggested that midazolam could stably bind to Keap1 and hinder the binding of Keap1 to Nrf2. Collectively, our findings suggested that midazolam may be a good protector for alleviating CCl4-induced acute liver injury via enhancing Nrf2 signaling pathway, which offered a new pharmacotherapy for preventing and treating toxin-induced organ injury.In summary, this study found that midazolam acted as an efficacious agent to activate Nrf2/HO-1 signaling pathway, which could enhance the levels of several key antioxidant proteins both


Neuro-melioidosis, comprising 4% of all cases of melioidosis carries a risk of high morbidity and mortality. We describe two Sri Lankan patients presenting with long segment myelitis secondary to melioidosis.Burkholderia pseudomallei. Serum melioidosis antibody titre was 1: 320. He was started on IV meropenem with oral cotrimoxazole for 12 weeks followed by oral co trimoxazole. But he had poor clinical recovery.Case 1: 47-year-old male presented with right side hemiparesis which progressed rapidly to quadriparesis. Initial cerebro spinal fluid (CSF) analysis revealed protein 76 mg/dl and glucose 72 mg/dl but without a cellular reaction. MRI spine revealed long segment myelitis with contrast enhancement. The patient was treated with intravenous methyl prednisolone pulses (IV MPP) and plasma exchanges(PLEX) on suspicion of an immune mediated myelitis but without success. A repeat MRI revealed high signal changes in the brain stem and along the entire spinal cord with contrast enhancement. MRI brain after treatment with MPP/PLEX showed enhancing hyper intensities along the corticospinal tracts. The repeat CSF revealed protein 1187 mg/dl, glucose 78 mg/dl, lymphocytes 1600/mm3 and neutrophils 10,200/mm3. CSF culture has become positive for Case 2: 47-year-old female presented with bilateral lower limb weakness for 1-week duration. On examination, she had flaccid paraparesis with a sensory level at T11. Inflammatory markers were elevated. CSF analysis revealed protein 50 mg/dl with lymphocytes 172/mm3. MRI pan spine revealed a long segment myelitis. Serum melioidosis antibody titre was 1: 640. She was treated with IV meropenem for 8 weeks followed by oral co-trimoxazole with an excellent clinical and radiological response.Numerous neurological manifestations have been described with melioidosis, however long segment myelitis with a positive CSF culture is not yet reported. These cases signify the importance of considering melioidosis as a differential in patients with long segment myelitis especially in endemic areas. Melioidosis is an emerging infection in South East Asia and Australia . It has 3 and neutrophils 10,200/ mm3. CSF-Adenosine De Aminase (ADA) was elevated 26 U/L. With the significant CSF glucose drop, cellular reaction and elevated CSF ADA, CNS-TB was considered, and anti-TB treatment was started. However, subsequently, the CSF culture became positive for Burkholderia pseudomallei. The serum melioidosis antibody titre was 1: 320. IV meropenem was initiated with oral cotrimoxazole for 12 weeks followed by maintenance with oral cotrimoxazole. Regardless, the clinical recovery was poor, with persistent quadriparesis despite some imaging improvements.A 47-year-old post man presented with right-sided upper and lower limb weakness, which rapidly progressed to quadriparesis with bladder and bowel involvement. He had no history of fever but had recurrent hiccups. There was no preceding history of tuberculosis. The patient had no history of recent vaccination. There was no significant local or international travel history. An examination revealed flaccid quadriparesis. The upper limb power was MRC grade 3/5 bilaterally and the bilateral lower limb power was MRC grade 0/5. Reflexes were diminished in the upper and lower limbs and he had bilateral up going plantar response. There was no obvious sensory level. Rest of the neurological examination including cranial nerves was normal. The initial haematology screen including inflammatory markers was unremarkable. Repeated blood cultures were sterile. Initial CSF analysis revealed protein 76 mg/dl and 72 mg glucose/dl (Random Blood Glucose 159 mg/dl) but without a cellular reaction. CSF Tuberculosis Polymerase Chain Reaction (TB PCR)/gene Xpert was negative. CSF oligo clonal bands were negative. The initial CSF bacterial culture did not reveal any organisms. A spine MRI showed an enhacing long segment myelitis extending from the lower medulla up to the T10 level  has many differentials, with AQP4-NMOSD being the most common. Other possible aetiologies include Systemic Lupus Erythematous (SLE), Sjogren’s syndrome, vitamin B12 deficiency, intrathecal tumours and such vascular abnormalities as AVM, infective causes like HIV 1 and HTLV and, rarely, intramedullary TB. LETM due to melioidosis has not been reported so far to the best of our knowledge.Burkholderia pseudomallei, is contracted by inoculation of soil and water through wounds or inhalation and is particularly common during and after the wet season. Risk factors for melioidosis are widely documented and include diabetes mellitus, alcoholism, renal disease, immunosuppression and thalassemia [B. pseudomallei infection can be latent for extended periods and then become reactivated, like CNS tuberculosis. Melioidosis is also known as the ‘Vietnamese time bomb’, as it has been reported in US soldiers who served in Vietnam several years before the onset of meningitis due to CNS melioidosis [Melioidosis, which is caused by gram-negative, facultative, soil saprophyte lassemia . This islassemia , as in oioidosis .B. pseudomallei-positive CSF culture report. In the second case, a lymphocytic CSF with elevated protein without a sugar drop was consistent with the commonly found changes in CNS melioidosis. Hence, CSF findings in CNS-Melioidosis can vary. A very high CSF- ADA level (> 10U/L) is of some value in the diagnosis of TB Meningitis (TBM) [In neuro-melioidosis, CSF findings usually show mononuclear cells, high CSF protein and normal glucose levels . In our  level > U/L is ofThe long segment myelitis seen in the spinal cord of both these patients was attributed to neuro- melioidosis, having excluded all other causes. Positive CSF culture and elevated melioidosis antibodies supported the diagnosis. CNS melioidosis shows a propensity to involve and spread along the white matter tracts across the commissural or longitudinal fibres . The sprTherefore, the diagnosis of CNS melioidosis depends on various factors. First, the risk factors must be considered even though they can be absent. CSF findings can vary and can be nonspecific, as described in our patients Table . HoweverDelayed diagnosis and extensive involvement of the spinal cord and brain can make the prognosis poor in this condition, as in our first case Table . On the


For many people, the wait for a life-saving transplant can stretch over many years and never become a reality. Recipients of allografts from living donors have significant advantages over those who receive deceased-donor grafts in terms of graft and patient survival. In this context, one in three potential living kidney donors is ABO-incompatible or crossmatch positive with the intended recipient. Such donors have been traditionally deemed “incompatible,” irrespective of their motivation to donate and their health status, limiting the potential donor pool. Kidney paired donation (KPD) is a potential way to overcome this barrier for this group of donor-recipient pairs. The first paired kidney exchange was performed in 1991 and has since gained popularity all over the world. In its simplest form, KPD involves the simultaneous exchange of organs between two sets of incompatible donor-recipient pairs, and with the growth of registries and collaboration between centers, more complex donor-recipient chains have developed and now even include international donations. All of this is accomplished with excellent donor and recipient outcomes, with patient and graft outcomes after KPD at least comparable to, and probably better than, those after standard living-donor kidney transplantation.Kidney transplantation, whether from a deceased or living donor, improves the quality of life and survival of patients with end-stage kidney disease (ESKD) and advanced chronic kidney disease (CKD) and Bastos et al. debate the merits and potential pitfalls of KPD. The authors of both articles summarize the literature in the field of KPD, with Bastos et al. coming out strongly in favor of KPD, while Medina-Pestana et al. express great caution not only with KPD but also with living donation and perhaps transplantation in general.In spite of the growing popularity of KPD, this practice has not yet caught on in South America. In this issue of the Brazilian Journal of Nephrology, Medina-Pestana et al. cite the positive impact of KPD in facilitating kidney transplantation, especially for patients who are disadvantaged by their blood type or sensitization history. This is supported by published outcomes such as those from the National Kidney Registry (NKR) in the US. KPD has allowed more African American patients, who are historically over-represented on waitlists and who are more often of blood type B, and patients who are highly sensitized, being able to receive a transplant. The results in these higher-risk populations compared favorably with those of non-KPD living-donors, although cold ischemia time and incidence of delayed graft function increase in KPD chains that involve transportation of a living donor organ. They acknowledge that despite its bureaucratic complexity, requiring matching algorithms, organ shipping, and coordination between transplant centers, KPD has been successfully performed in multiple countries, both developed and under developed. Little to no negative political or social consequences have been noted, demonstrating that KPD is a valuable resource for further improving the quality of life and survival of people with end-stage kidney disease.Bastos et al.Despite the growing body of literature, concerns about the practice of KPD remain, and as recently as 2018, the Brazilian Federal Council of Medicine urged caution. It raised concerns that KPD may result in worse outcomes, benefit only a minority in a country with large social-economic disparities, and a negative impact on transplantation rates in the country. raise several concerns, including the fear that only the wealthy will benefit and that devoting resources to promote KPD would divert attention from the deceased donor program. The authors are concerned that living donation poses potential risks to the long-term health of the donor, which may be exacerbated by a lack of medical care, especially in resource-poor countries and therefore advocate for a cautious approach to living kidney donation in general. While donor risk may be ethically justified, they fear that the link between donor and recipient will be diluted in a KPD as all donor-recipient pairs are unrelated. They worry that poor outcomes of KPD might adversely affect organ donation. In conclusion, Medina-Pestana et al. urge caution in blindly assuming that renal transplantation is superior to dialysis, especially as patients are at risk for immunosuppression and for disease transmission with organ transplantation.Medina-Pestana et al.. Intense and lively discourse is crucial, as each society must decide for itself how to prioritize resources for the care of the sick and disadvantaged. Furthermore, with regard to living organ donors, they must decide how best to protect their interests and long-term health. By bringing awareness of KPD to the medical community in Brazil, both Medina-Pestana et al. and Bastos et al. and colleagues are furthering the cause of transplantation in their community. And if one is convinced that kidney transplantation is a life-enhancing and life-extending therapy, then KPD should be considered as an option for all transplant candidates who have a living donor who is medically able but cannot donate a kidney to the intended candidate because of ABO or human leukocyte antigen (HLA) incompatibility.The ethics of KPD are complex


Arginine-Vasopressin (AVP) is a nonapeptide that exerts multiple functions within the central nervous system and in the blood circulation that might contribute to outcome in critically ill patients. Sex differences have been found for mental and physical effects of AVP. For example, stress response and response due to hemorrhage differ between males and females, at least in animal studies. Data on humans -especially on AVP within the central nervous system (CNS)—are scarce, as cerebrospinal fluid (CSF) which is said to represent central AVP activity, has to be collected by means of invasive procedures. Here we present data on 30 neurocritical care patients where we simultaneously collected blood, CSF and saliva to analyze concentrations in the central and peripheral compartments.30 neurocritical care patients were included  with a median age of 60 years. CSF, plasma and saliva were obtained simultaneously once in each patient and analyzed for AVP concentrations. Correlations between the central compartment represented by CSF, and the peripheral compartment represented by plasma and saliva, were identified. Relations between AVP concentrations and serum sodium and hematocrit were also determined.In the whole patient collective, only very weak to weak correlations could be detected between AVP plasma/CSF, plasma/saliva and CSF/saliva as well as between AVP concentrations in each of the compartments and serum sodium/hematocrit. Regarding the subgroup of postmenopausal females, a significant moderate correlation could be detected for AVP in plasma and CSF and AVP CSF and serum sodium.Absolute concentrations of AVP in central and peripheral compartments did not show sex differences. However, correlations between AVP plasma and CSF and AVP CSF and serum sodium in postmenopausal females indicate differences in AVP secretion and AVP response to triggers that deserve further examination. Arginine vasopressin (AVP) is a nonapeptide which is synthesized in the hypothalamus and travels via axonal pathways to the posterior pituitary lobe. When it is released into the bloodstream, it exerts so-called peripheral effects such as vasoconstriction, reabsorption of water from the kidney and blood-pressure regulation . MoreoveAVP can be detected in various body compartments, including blood, cerebrospinal fluid, and saliva. The blood–brain barrier is assumed to be impermeable for endogenous AVP, and AVP secretion within the central nervous system (CNS) and in the bloodstream are not necessarily coupled. Therefore, it cannot generally be assumed that changes of AVP concentrations in cerebrospinal fluid (CSF) and other body fluids like blood, saliva or urine always move in the same direction , 4. AVP Sex differences in the AVP system have been found in animals as well as humans concerning central and peripheral properties of AVP which also play a role in critical care. For example, a positive correlation of plasma AVP concentration and distress has been found in men, but not in women . In animTaken together, there is strong evidence for sex differences in the secretion and effects of AVP within the CNS and periphery in animal models. However, data on humans are scarce and since CSF can only be obtained invasively, blood or even saliva concentrations are often used as surrogate parameters.The aim of our study was to analyze AVP concentrations in the two peripheral compartments blood and saliva as well as in the central compartment represented by CSF to detect correlations of neuropeptide concentrations that allow to infer from peripheral to central concentrations in neurocritical care patients with ventricular drain. In addition to the whole patient collective, we analyzed men and pre- and postmenopausal women separately to detect differences between groups. Furthermore, we correlated AVP concentrations in the three compartments blood, CSF and saliva with the routinely collected laboratory parameters serum sodium and hematocrit and analyzed for deviations between sexes.The study was approved by the institutional ethics committee of the Medical Faculty of the Technical University of Munich (Project Number: 282/14). We prospectively analyzed simultaneously taken blood, CSF and saliva samples from 30 consecutive neurocritical care patients between October 2018 and February 2019. 13 patients were male, 13 female postmenopausal and 4 female premenopausal. Median age was 60 years (38–86 years). The inclusion criteria were: adult, hemodynamically stable patients (> 18 years) with external ventricular drainage and existing arterial or central venous access for noninvasive collection of blood and CSF. Either the patient himself or his legal representative had to give written informed consent to participate in the study.Patients with acute infections, including acute CNS infections, patients with acute hemorrhages requiring transfusion, and patients showing signs of diabetes insipidus or hypovolemia were excluded.14 patients suffered from aneurysmal SAH, 8 from brain trauma, 3 from brain tumor, 4 had a non-traumatic cause of intracranial hemorrhage and 1 patient suffered from hydrocephalus as a late consequence of a CNS infection. Two patients died during the hospital stay. For each patient, we recorded blood hematocrit and serum sodium from the respective day’s routine lab examination at the timepoint nearest to AVP sample collection and the patients’ Glasgow Outcome Score at discharge from the ICU. Patient data are shown in Table CSF, blood and saliva samples were obtained simultaneously once in each patient. All samples were drawn during regular day duty at the intensive care unit between 07:30 and 16:30.©Salivette) that was placed in the patient’s cheek pocket for 1 min. Samples were put on ice and transported to the laboratory immediately. After centrifugation for 10 min at 1300 g and 4 °C, aliquots were stored at − 80 °C until analysis. Plasma, CSF and saliva were treated identically and analyzed as described before [125I-labelled AVP  was added. The method is described in detail by Landgraf et al. [We took blood from a pre-existing arterial line and CSF from the ventricular drain in pre-chilled EDTA-Tubes. Saliva was obtained by means of a cotton swab . Data were analyzed for normal distribution by Shapiro–Wilk test. Differences in hormone concentrations in the three compartments as well as differences in AVP concentrations, serum sodium and hematocrit between sexes were determined by Friedman test and post-hoc Wilcoxon signed-rank test and Bonferroni correction where appropriate. Correlations of AVP concentrations in the different compartment and correlations of AVP and serum sodium concentrations as well as AVP concentrations and hematocrit were assessed with Spearman’s rank correlation coefficient. In this article, we describe correlation coefficients < 0.1 as “very weak” which means negligible, coefficients between 0.1 and 0.4 as “weak” and coefficients between 0.4 and 0.7 as “moderate” . Only coAll patients showed detectable AVP concentrations in each of the three compartments blood, CSF and saliva which are compatible with those described in the literature , 16, 17.Regarding the whole patient collective, correlation coefficients of AVP plasma/CSF, plasma/saliva and CSF/saliva were weak to very weak. AVP in none of the three compartments correlated significantly with serum sodium or hematocrit either. When analyzing the subgroups, it was noticeable that a significant moderate correlation of AVP in plasma and CSF could be detected in postmenopausal women. A moderate, but not significant correlation was found in this group for AVP in plasma and saliva. Scatterplots of AVP concentrations in plasma and CSF, plasma and saliva and saliva and CSF are shown in Fig. Concentrations of serum sodium and hematocrit showed very weak correlations with AVP concentrations in the three compartments plasma, CSF and saliva. Again, there was an exception in the group of postmenopausal females where AVP CSF showed moderate negative correlations with serum sodium and hematocrit. Correlation coefficients and p-values are depicted in Table AVP concentrations in CSF, plasma and saliva did not differ significantly between the outcome groups defined according to GOS Table . Due to In our patient collective of neurocritical care patients, we found only weak correlations of AVP concentrations in the central and peripheral compartments. A significant correlation between central or peripheral AVP and hematocrit and sodium concentrations in blood could not be demonstrated either. The picture is different in postmenopausal women: Here, a significant positive correlation between AVP in plasma and CSF could be detected. Furthermore, we found moderate inverse correlations between AVP in CSF and sodium and AVP in CSF and hematocrit in this subgroup with the correlation between AVP CSF and sodium being statistically significant. Outcome was not significantly related to either central or peripheral AVP concentrations.The nonapeptide AVP exerts multiple functions. AVP secretion within the central nervous system is considered crucial for maintaining a mental balance, and disturbances in the central neuropeptide system, in which AVP plays an important role, may contribute to the development of psychiatric issues like post-traumatic stress disorder . EspeciaIn the periphery, AVP mediates the maintenance of blood pressure and water and electrolyte homeostasis; additionally, it is involved in the endocrine stress response . StimuliAVP is detectable in various body fluids such as blood, saliva, urine and cerebrospinal fluid. However, the majority of studies do not distinguish between central and peripheral AVP concentrations. As data on CSF are rare, blood concentrations are mostly used as a surrogate and the exact relationship between the concentrations of the nonapeptide within the CNS and in the periphery is still unclear. Although separate central and peripheral secretion mechanisms are assumed, blood or saliva are used for many questions due to their ease of extraction.In a previous work on a comparable—not identical—patient population, our group could already show for oxytocin, which is closely related to AVP, that blood concentrations correlate only weakly with CSF concentrations . In the Another issue is the time of sample collection, which is assumed to influence hypothalamic neuropeptide concentrations especially in the central compartment . HoweverThe limited number of human studies using a wide variety of body fluids complicates the analysis of AVP concentrations. Moreover, AVP is elaborately and costly to analyze, which is the reason that some authors used the equimolar secreted copeptin as a surrogate for blood concentrations in their studies , 26. ThiThe primary objective of our study was to examine AVP concentrations in the blood, CSF, and saliva compartments for any correlations to determine whether CNS concentrations in critically ill patients can be estimated by blood or saliva concentrations.Our primary findings were only very weak to weak correlations between central and peripheral compartments in the entire patient collective. This confirms previous statements in the literature that central and peripheral secretion of neuropeptides are not necessarily coupled and that the blood–brain barrier is largely impermeable to endogenous neuropeptides .The subgroup of postmenopausal women differed from the overall collective in showing a moderate positive correlation of AVP in plasma and CSF. This may have several causes: On the one hand, the coupling of central and peripheral AVP secretion mechanisms could differ between sexes. A rodent study for example showed that AVP plasma values increased in male and female rats with heart failure whereas mRNA levels were lower in females than in males . AnotherIn the second part of our study, we attempted to capture, at least roughly, the relationship between AVP and volume and electrolyte status. We chose serum sodium and hematocrit as two parameters from the routine laboratory that seemed suitable for this purpose. This choice based on the fact that sodium concentrations account for a large part of serum osmolality , and an Regarding the whole patient collective, only very weak to weak correlations could be shown between AVP concentrations in CSF, plasma, and saliva and sodium and hematocrit. This somehow reflects the findings of a historical study, where a rise in plasma AVP only after an acute salt load has been shown whereas 90 min after the salt load, no correlation could be found between plasma osmolality and AVP concentration . Our patAgain, the subgroup of postmenopausal females differed from the whole collective. Postmenopausal women showed moderate inverse relations of AVP in CSF and serum sodium and hematocrit, where the correlation with sodium was statistically significant. This finding may be a hint to sex differences in volume and electrolyte regulation. At least in animal studies, plasma AVP response to hemorrhage was higher in female rats whereas pituitary AVP content was lower . A humanIn rats with liver cirrhosis, differences between males and females in AVP release were also detected which provided an explanation for females not developing hyponatremia in that study .Regarding outcome, no significant differences in AVP concentrations of the three compartments blood, CSF and saliva were found according to the Glasgow Outcome Scale (GOS) at discharge from ICU. However, our patient collective included only few patients with good neurological outcome, and the vast majority was GOS 3 which means severe disability and dependency in daily life . TherefoAlthough no sex differences were found in absolute concentrations of AVP in central and peripheral compartments, there is discrete evidence of different secretion and distribution mechanisms in men and women. Our study should be considered a pilot study because of the small number of patients, especially premenopausal women. However, our results encourage further investigation of sex differences in central and peripheral AVP regulation that could account for both physical and psychological outcomes in critically ill patients.


Next generation sequencing (NGS) of human specimen is expected to improve prognosis and diagnosis of human diseases, but its sensitivity urges for well-defined sampling and standardized protocols in order to avoid error-prone conclusions.In this study, large volumes of pooled human cerebrospinal fluid (CSF) were used to prepare RNA from human CSF-derived extracellular vesicles (EV) and from whole CSF, as well as from whole human serum and serum-derived EV. In all four fractions small and long coding and non-coding RNA expression was analyzed with NGS and transcriptome analyses.We show, that the source of sampling has a large impact on the acquired NGS pattern, and differences between small RNA fractions are more distinct than differences between long RNA fractions. The highest percentual discrepancy between small RNA fractions and the second highest difference between long RNA fractions is seen in the comparison of CSF-derived EV and whole CSF. Differences between miR (microRNA) and mRNA fractions of EV and the respective whole body fluid have the potential to affect different cellular and biological processes. I.e. a comparison of miR in both CSF fractions reveals that miR from EV target four transcripts sets involved in neurobiological processes, whereas eight others, also involved in neurobiological processes are targeted by miR found in whole CSF only. Likewise, three mRNAs sets derived from CSF-derived EV are associated with neurobiological and six sets with mitochondrial metabolism, whereas no such mRNA transcript sets are found in the whole CSF fraction. We show that trace amounts of blood-derived contaminations of CSF can bias RNA-based CSF diagnostics.This study shows that the composition of small and long RNA differ significantly between whole body fluid and its respective EV fraction and thus can affect different cellular and molecular functions. Trace amounts of blood-derived contaminations of CSF can bias CSF analysis. This has to be considered for a meaningful RNA-based diagnostics. Our data imply a transport of EV from serum to CSF across the blood–brain barrier.The online version contains supplementary material available at 10.1186/s12967-022-03612-3. In the coming decades aging populations will cause an increased number of people spending more lifetime with disabling neurodegenerative diseases like dementia and Parkinson’s disease . TherefoCerebrospinal fluid (CSF) from lumbar punctures is frequently used for biomolecule-based diagnostics of neurological diseases, and combinations of marker molecules were proven useful for the diagnosis of neurodegenerative, inflammatory and infectious diseases of the central nervous system (CNS). Nevertheless, none of the biomarkers currently in use is exclusively specific for only one disease condition, and the diagnostic value of proteins, DNA and other marker molecules still depend on additional diagnostic findings and the knowledge of the clinical context –6. This In the last decade, the detection and characterization of RNA species in different body fluids reflecting the transcriptome of their sources of origin, has fuelled hope for the development of new, specific prognostic and diagnostic RNA-markers , 8. But In addition to the described hindrances, more general challenges connected to human samples like CSF need to be considered, i.e., the total volume of CSF in adults is approximately 150 ml, and has an average, age-dependent turnover rate of approximately four times a day that depends on the physical activity . This, aHere we show that body fluids and their respective EV have significantly different compositions of long and small RNA, and that miR derived from whole body fluid and respective EV have the potential to affect different cellular and biological processes.CSF samples were collected according to clinical necessities for routine diagnostics in the Department of Neurology, University Medical Center of Göttingen. Only samples from patients who presented in the clinic with a variety of symptoms but finally had no obvious signs of a known disease were included in this study. Before onsets of clinical routine diagnostics, the number of erythrocytes and leucocytes were counted manually in each sample, and within one hour after aspiration the samples were centrifuged for ten minutes at 105 × g. Cell-free supernatants were carefully aspirated for further clinical analysis. Samples with signs of haemolysis before the centrifugation step or samples that had counts above 275 erythrocytes/µl before and/or signs of haemolysis after the centrifugation step were excluded from this part of the study. Furthermore, only samples with leucocyte counts below 8 cells/µl were included in this part of the study. Five samples with leucocyte counts from five to eight leucocytes/µl before centrifugation were also made cell-free by centrifugation and were also included in this part of the study, as their donors had no obvious signs of a known disease. After completion of clinical analysis, the remnants of the CSF samples were stored frozen at − 80 °C until further processing for this study. No CSF samples were specifically collected and no extra CSF samples were drawn from any of the patients for the purpose of our research. No identifying information was acquired for this study, and patients gave prior written consent to the scientific use of their samples. For this part of the study 324 CSF samples were collected, 161 samples  from male and 163  samples from female patients. The average age of the patients was 55,5 ± 21,1 years; for more detailed information about the samples, please refer to Additional file For the purpose of comparison, samples were also collected from CSF with high erythrocyte counts before the 105 × g centrifugation step, or from CSF with obvious signs of haemolysis after the lumbar puncture; in none of samples the number of leucocytes was above 23 before the centrifugation step. These samples were processed completely separate from normal CSF and will be referred to further on as blood-contaminated CSF samples. 36 blood-contaminated CSF samples were collected, 21 samples  from male and 15  samples from female patients. The average age of the patients was 61,45 ± 23,3 years; for more detailed information about the blood-contaminated samples, please refer to Additional file From all CSF samples a 100 µl aliquot was tested for bacterial or fungal contamination for five days at 37 °C in cell culture medium; none of the samples included in the study showed any signs of contamination.CSF samples necessary to make up a total volume of 74 ml were thawed on ice and pooled. The pooled samples were mixed and briefly centrifuged in order to divide the pool in two equal aliquots of 37 ml. One aliquot was used for column centrifugation and the other one for extracellular vesicle (EV) preparation; 20 aliquots for each kind were prepared.Ultraspin columns are molecular size-based filters that amongst others can be used to isolate protein-bound RNA and RNA included in extracellular vesicles , 40. TucThree ml CSF were pipetted into each of four ultrafiltration spin columns with a molecular cut-off of 50 KDa  and centrifuged at 1860 × g at 4 °C until the volume was concentrated to approximately 250 µl. Then to each of the four spin columns another 3 ml of CSF was added, and the columns were again spun until the volumes were concentrated to approximately 250 µl. This step was repeated one more time, but at the last centrifugation the volume was concentrated to approximately 200 µl. The first centrifugation step takes around 10 min, the second about 18 min and the last step approximately 25 min. After centrifugation the four resulting concentrates of the 36 ml CSF were transferred to DNA-low-binding tubes and each emptied concentration chamber of the spin columns was rinsed once with 50 µl ice-cold 10 mM TRIS pH 7, 4. The rinses were then added to the respective concentrates to make up a total volume of 250 µl in each of the four DNA-low-binding tubes; these four CSF-concentrates were finally used for preparation of one RNA sample.11, 2 ml ice-cold PBS was added to 36 ml of pooled CSF samples; the combined volumes were carefully mixed, briefly centrifuged, and divided into four times 11,8 ml, which were distributed to four ultracentrifugation tubes (Beckman coulter). The tubes were balanced with ice-cold PBS and then centrifuged at 4 °C at 180,000 × g for 4 h. The resulting supernatants were aspirated by pipetting and to each pellet 1 ml of Tri-Reagent (Sigma T9424) was added. The tubes were vortexed for 30–60 s, briefly centrifuged and the suspensions were then transferred to 1,5 ml DNA-low-binding tubes. The suspensions were left standing for 5 min at room temperature and then further used for RNA-preparation.To each of the four 250 µl CSF-ultrafiltrates, 0,75 ml Tri Reagent (Sigma T3934) was added; the mixtures were vortexed for 60 s and then left standing for five minutes at room temperature. Then 100 µl of 1-bromo-3-chloropropane was added to each tube, samples were vortexed for 30 s and left standing at room temperature for five more minutes. Samples were then spun at 12,000 × g at 4 °C for 10 to 15 min to separate the watery from the organic phases. 350 µl from each upper watery phase were transferred to a 2 ml DNA-low-binding tube. The remaining watery phases of the first extractions, were reextracted with 400 µl of 10 mM TRIS pH 7,4 , and 450 µl of the reextracted watery phases were then combined with the 350 µl volumes of the first extraction step. To each sample 4 µl of glycoblue (15 mg/ml) and 27,5 µl 3 M sodium acetate pH 5,2 were added. Samples were mixed carefully and then 800 µl of -20 °C cold isopropanol  were added to each sample; samples were again vortexed and then stored for RNA-precipitation overnight at − 20 °C. The next day one of the four samples was centrifuged for 45 min at 4 °C at 13,000 × g, the supernatant was decanted and the content of another tube from the precipitation step was pipetted onto the pellet of the first tube. The tube was again centrifuged for 45 min at 4 °C at 13,000 × g and the supernatant was decanted; this was repeated until the content of all tubes from the precipitation step were concentrated in one tube, resulting in a pellet that combines the RNA of 36 ml whole CSF. After washing the pellet once with 1000 µl 75% ethanol, it was resuspended in 75% ethanol and kept at -80 °C until to the last precipitation step. In the last step the RNA was pelleted by centrifugation for 45 min at 4 °C at 13,000 × g and each pellet was dissolved in 8 µl 10 mM TRIS pH 7,4 for NGS analysis.Extracellular vesicle preparation of 36 ml aliquots of human CSF with ultracentrifugation. Then 100 µl 1-Bromo-3-Chlor-Propane were added to each tube and the suspensions were vortex for 15 s. Tubes were again left standing for five minutes before they were then treated as described for RNA-preparation from ultrafiltrated CSF (see above).The four tubes containing the 1 ml Tri-Reagent and the extracellular vesicle RNA were then treated as described in Sixteen samples of 15 ml of blood were collected from healthy volunteers of our research group and volunteers who donated blood to the blood bank of the University Medical Center of Göttingen. Serum was separated with serum separator tubes at 2000 × g for ten minutes at 4 °C. After centrifugation the serum was aliquoted and stored frozen at -80 °C. A 100 µl aliquot of each serum sample was used to determine the hemoglobin content, and only samples with hemoglobin concentrations below the limit of detection of the routine analysis (< 5 mg/dl) were used for further processing. For more detailed information about the age and sex distribution of the sample donors, please refer to Additional file RNA-preparation of ultrafiltrated CSF, resulting in four independent RNA preparations that finally were pooled to one RNA sample.The preparation of RNA from serum concentrates of ultraspin columns is hampered by an approximately 200fold higher protein concentration in serum as compared to CSF; this results in long centrifugation times and extremely viscous concentrates that are difficult to pipette. Therefore, total RNA of serum was only prepared by ultraspin columns to proof the principle of the method, whereas total RNA of serum for NGS was exclusively extracted with Tri-Reagent (Tri-Reagent BD (T3809) for blood). For this purpose, 1 ml human serum samples from six donors were thawed on ice-water. From each single 1 ml sample four aliquots of 250 µl were added to four 1,5 ml DNA-low-binging tubes containing 750 µl Tri-Reagent. The tubes were vortex for 30 to 60 s and left at room temperature for five minutes, then 100 µl 1-bromo-3-chloropropan were added to each tube and the mixtures were again briefly vortexed and incubated for five minutes at room temperature. Then the RNA was prepared from each sample exactly as described in One millilitre of serum was added to 9 ml of ice-cold PBS in ultracentrifugation tubes, the tubes were carefully mixed and briefly centrifuged to collect all liquid; tubes were then balanced with ice-cold PBS and centrifuged for 4 h at 180,000 × g at 4 °C. After centrifugation the supernatants were carefully pipetted from the pellets and 1 ml Tri-Reagent was added to each tube. In contrast to the extracellular vesicle pellets of CSF, the extracellular vesicles of serum formed visible pellets, and the resuspension of these pellets was achieved by vortexing and holding the tubes briefly in an ice-cold ultrasonic bath. After resuspension and five minutes incubation at room temperature, 100 µl 1-bromo-3-chloropropan were added to each tube, and then samples were exactly treated as described for RNA-preparation from ultrafiltrated CSF.The non-coding RNA sequencing (ncRNA-seq) and its primary analysis were performed by the NGS Integrative Genomics Core Unit . For RNA-sequencing RNA samples were subjected to non-stranded mRNA library preparation using the TruSeq RNA Sample Prep Kit v2 with minor modifications (ligation and PCR amplification cycles). Fragment sizing of final libraries were analyzed via Fragment Analyzer (average of 300 bp). Libraries were sequenced ) on the HiSeq 4000 platform. For miR library preparation we used the QIAseq miR Library Kit, a gel-free miR sample according to manufacture recommendations. Fragment sizing of final libraries were analyzed via Fragment Analyzer (average of 70 bp). Libraries were sequenced ) on the HiSeq 4000 platform.https://www.ensembl.org/Homo_sapiens/Info/Index) using bowtie2 version 2.3.4 with default parameters [The whole RNA from each sample was used for both, the small and long RNA NGS approach. Sequenced reads were initially trimmed for Qiagen Small RNA 3’ Adapter using cutadapt version 2.10 . The trirameters . High-qurameters  using trrameters , where thttps://www.ensembl.org/Homo_sapiens/Info/Index) using STAR version 2.5.2 with default parameters [Sequenced reads of long RNA were aligned to the Homo sapiens sapiens genome assembly hg38 from ENSEMBL (rameters . The resrameters . Similarhttp://ftp.ensembl.org/pub/release-97/gtf/homo_sapiens/Homo_sapiens.GRCh38.97.gtf.gz). The ENSEMBL gene IDs were mapped to their comparable miRBase IDs using the R package biomaRt. Utilizing the miRBase IDs of miR of interest as input, the R package multiMiR was used to extract target genes of those miR, where validated targets relied on the databases miRecords [While miR could be tested for deregulation between particular conditions, determining their biological context was more challenging, since direct association of miR and functional terms (gene ontology categories or pathways) were not available, thus making a direct enrichment analysis of biological terms impossible. Therefore, the analysis involved initially annotating the miR to their target coding genes, and then using those target genes for the enrichment analysis. In brief, all transcripts tested for a particular comparison (e.g. whole CSF vs. CSF-derived EV) were overlayed with their ENSEMBL gene IDs from the human gene set version 97 ; from small RNA-sequencing all libraries were included in the analysis. Statistical differences between groups were analyzed with the Mann Whitney test with Prism 7 for Mac.The time necessary for routine analysis of CSF depends on clinical requirements; i.e., some CSF samples can be analysed within hours, whereas several days might be necessary for more detailed analyses. Therefore, CSF samples have to be kept at 4 °C for varying periods until the remnants can be stored at − 80 °C. We thus analyzed the effect of long-time storage of CSF at 4 °C on the RNA content. CSF samples were divided in 2 equal aliquots, one was directly stored frozen at − 80 °C after clinical analysis and the second was left for 14 days at 4 °C; RNA from these parallel aliquots was then isolated with spin columns as described. Additional file Next, we determined the total RNA concentrations in the different body fluids. Additional file RNA gel analysis of all CSF and serum samples used in this study  RNA and miR comprise approximately two thirds of all detected species Fig. A. WhereaMiR act concerted, i.e. a single miR can target different transcripts and a single transcript can be targeted by various miR. As miR are known to be regulators of metabolism and are linked to human diseases , 55, we Similar to the small RNA fractions, Fig. The clustering- and the two-dimensional PCA plot , there are eight different sets of differentially expressed miR targets in whole CSF  (Additional file The differences in the long RNA-profiling seem less profound than the small RNA-profiling, and most long RNA transcripts found in whole CSF are also found in the three other fractions. Nevertheless, the generally higher number of significantly differentially expressed long RNAs leads to comparable total numbers of up- and down-regulated transcripts in both preparations Table , and thuThis study shows that the composition of small and long RNA differs significantly between whole body fluid and its respective EV fraction. Differentially expressed long RNAs belong to different transcript sets involved in distinctive biological functions, and differentially expressed miR can target specific transcripts specific to different cellular and molecular functions. We show that trace amounts of blood-derived contaminations of CSF can bias RNA-based CSF diagnostics and the presented data imply a transport of EV from serum to CSF across the blood–brain barrier. These aspects are important for the search of RNA-based diagnostic markers from CSF and serum. A future collaboration of hospitals with access to CSF analysis could allow the establishment of age- and sex-dependent standard RNA patterns for CSF diagnostics, similar and in addition to the already established protein patterns.Additional file 1: Age distribution of patients, volume size, and erythrocyte and leucocyte numbers of CSF and serum samples. Fig. S2 RNA content of 100 KDa column concentrates and filtrates. Fig. S3 Age- and sex-distribution of serum samples. Fig. S4 RNA content of CSF samples in dependence of the storage temperature and RNAse treatment. Fig. S5 RNA content of CSF and serum samples. Fig. S6 Gel analysis of all RNA samples. Fig. S7 Electropherogramms of the RNA samples from serum. Fig. S8 Electropherogramms of the RNA samples from CSF. Fig. S9 Comparison of RNA content and electropherogramms of the RNA samples from CSF and blood-contaminated CSF. Fig. S10 Venn diagrams and UpSet plots form small and long RNA. Fig. S11 Heat maps of small RNA read counts not shown in figure 4. Fig. S12 GO Slim summaries of miR targets from whole CSF, CSF-EV, whole serum, and serum EV. Fig. S13 Heat maps of read counts encompassing all possible comparisons of down- and up-regulated long RNA transcripts. Fig. S14 Venn diagrams of significantly expressed small RNA transcripts, expressed miR, and target transcripts of expressed miR. Fig. S15 Distribution of small RNAs in body fluids and their respective EV.Additional file 2: Read counts of differentially expressed small RNA transcripts listed in table1.Additional file 3: Target sets of miR expressed in serum and CSF fractionsAdditional file 4: Read counts of differentially expressed long RNA transcripts listed in table 2 .Additional file 5: Transcript sets of mRNA expressed in serum and CSF fractions.


Tourist destinations with cultural heritage have arisen as a prominent issue in tourism literature. Creating a positive image of the destination can influence tourists’ satisfaction and willingness to return. The goal of this research is to investigate the relationship between destination image formation (DIF), tourist satisfaction (TS), and tourist trust (TT). As a result, the structural relationships between local community participation (LCP), authenticity (A), access to local products (ALP), TS, and TT were investigated in this study. This study used a quantitative approach based on a survey of 644 domestic and foreign tourists visiting the Guangdong cities of Guangzhou, Foshan, and Shenzhen. The statistical software SmartPLS 3.3.3 was used to determine the relationship between variables in the research model using structural equation modeling. The outcomes show a positive correlation between LCP, A, and ALP, which led to tourist satisfaction and, eventually, tourist trust. It is concluded that the DIF and TS may result in increased tourist trust. There is also a discussion of additional theoretical contributions, practical implications, and limitations. The outcomes of this study will help to shed light on the variables that encourage and promote tourism in developing countries. Today, tourism is among the most vital industries on the planet . It is aThe tourism sector has become a key contributor to the Chinese economy since starting the reforms and opening in the early 1800s . The booThe term “destination image” refers to the process through which people try to form an impression of a particular vacation destination. The destination image encompasses beliefs, feelings, perceptions, and knowledge about a destination, in addition to direct and indirect information gained while traveling to the destination, such as through associations, tourism-related channels, social platforms, and the Internet facility. The significance of developing destination images for managers stems from the fact that they have a considerable impact on tourists’ purchasing decisions and actions . FurtherThe effect of DIF and TS on TT has been a popular subject of tourist studies. When making strategic marketing decisions for tourism locations, it is crucial to determine the destination’s image due to the assumption that a positive DIF creates trust toward tourist destinations and increases tourists’ satisfaction . Trust iThree conceptual tourism destination research gaps will be addressed in the present study. (a) It will first look into the individual link between DIF and TT. (b) It will investigate the connection between DIF and TS. (c) It will look at the relationship between TS and TT. (d) It will examine the role of TS as a mediator between DIF and TT. Previously, very few studies have explored such kinds of relationships. This is the first study investigating how destination image formation, directly and indirectly, affects tourist trust in the Chinese heritage tourism context.An integrated model is presented in this study, which adds to the existing body of tourist literature by providing a fresh perspective about the role of DIF in affecting the TS that leads to TT. The findings can help those in charge of marketing tourist destinations in designing and implementing market-driven approaches that promote consumer satisfaction and trust through strategies that focus on destination image formation represented in Authenticity, LCP, and ALP.The absence of actual studies involving Asian tourists is the second conceptual research gap. It is anticipated that Asia will become the globe’s most popular vacation destination and largest tourist-producing region in the near future. Notably, Tourism in PRC was almost non-existent prior to 1978, when it unlocked its gates to the outside world. Since then, China has become a significant tourism marketplace. China’s tourism officials have been concentrating their efforts on evolving national tourism , and thiIn addition to addressing the first two gaps, this study addresses the third gap, which is the need for extended research into visitors’ perceptions and behavior regarding their experiences in Heritage Sites , ParticuRQ1: How does DIF and tourist satisfaction influence tourist trust?RQ2: How do DIF influences tourist satisfaction impact on tourist trust?RQ3: How does tourist satisfaction mediate the relationship between DIF and tourist trust?This paper’s structure is as follows: section “Review of literature” includes a literature review. Second, section “Conceptual Framework” covers how the hypotheses were generated, while section “Research Methodology” explains the research methodology and the statistical analysis of this study, respectively. Section “Discussion” contains the discussion and closing remarks. The study comes to a close with a review of the study’s limitations as well as future research prospects.Destination image formation is defined as the collection of an individual’s beliefs and thoughts about a specific environment or setting. In the same vein, the environmental and social aspects of a destination’s image quality are two of the most important factors in determining its perceived quality . PreviouIn the tourism industry, a great deal of research has been conducted on customer satisfaction . The pleTrust is defined in tourism literature as the dependability and credibility of important factors associated with tourist destinations . It is cPrevious research has confirmed that TT has an effect on tourists’ risk perception and emotional bonding to a site , and insTourists are more inclined to pay a visit to places they think to be reliable and reputable. In the long run, tourists may form a personal connection with trustworthy places . TraveleThe current study highlights how destination image formation positively and significantly influences the tourist trust using tourist satisfaction as mediating variables. Previously Destination Image Formation , TS, and Tourist Trust have significant research attention . The theSeveral studies indicated that DIF  positively influence TT preferences . SimilarIn addition, it is widely acknowledged that the tourists can attain a better image through receiving destination image better and getting more satisfaction, especially in emerging nations like China. While TT is defined broadly as the confidence, a visitor develops in a product or service provider during a visit to a tourist destination due to a relationship exchange between two parties . They coSimilarly, Most studies explored that destination image formation in services firms found that image influences consumer trust and loyalty . Then DaFurthermore, another study explored the significant role of authenticity in formulating TT. The empirical results indicated the importance of online reviews, perceived authenticity, and trust in the tourism framework . SeveralH1.1: LCP has a beneficial and significant impact on TT.H1.2: A has a positive and significant impact on TT.H1.3: ALP has a positive and significant impact on TT.Tourist satisfaction refers to a tourist’s emotional or affective assessment of a tourist destination’s product or service utility. Alternatively, it establishes a sense of comfort, delight, and acceptance for the products or services being used or consumed . PreviouSeveral studies explored that DIF  influences tourists’ perceived value and tourist satisfaction . SimilarH2.1: LCP has a positive and significant impact on TS.H2.2: A has a positive and significant impact on TS.H2.3: ALP has a positive and significant impact on TS.Previous research has highlighted the importance of satisfaction due to its remarkable accuracy in predicting a traveler’s or tourist’s future aspiration . AccordiH3: TS has a positive and significant impact on TT.According to H4.1: TS mediates the relationship between LCP and TT.H4.2: TS mediates the relationship between A and TT.H4.3: TS mediates the relationship between ALP and TT.This research has been conducted in the tourism sector of China as an emerging nation. Currently, several studies have been concentrated on destination image formation, tourists’ satisfaction, and tourist trust in advanced countries such as America, Austria, Spain, South Korea, and France, etc. , with coFor the empirical testing of hypotheses, a deductive technique was used in conjunction with a positivist philosophical paradigm. We found the fundamental patterns and correlations between variables, and we generalized our findings. The results of the testing of hypotheses contribute to the relevant realm of knowledge. To minimize bias, positivism was chosen, as it examined the sole external reality objectively. To get a representative sample from a broad population, a survey was undertaken . The resThis study used destination image formation dimensions  as independent variables, tourist trust as the dependent variable, and TS as a mediator. The study used three characteristics of DIF as independent variables , TT as a dependent variable, and TS as a mediator. The questionnaire’s initial section described the study’s purpose and included instructions. Additionally, this section included anonymity and privacy disclaimers and inquired about respondents’ personal information, such as gender, age, income level, and field of education. The second part described the items associated with DIF (15 items), TS (7 items), and TT variables (6 items).All assertions were evaluated using a seven-point Likert scale (from 1 to 7: strongly disagree to strongly agree). Before collecting the final data, the questionnaire items were translated into Chinese using the back-translation method. A pilot study of 30 participants was conducted to determine the questionnaire’s reliability and validity. All participants in the pilot study shared similar demographic characteristics with the primary study sample, allowing for the trial of final data analysis. The pilot study participants were aware of the study’s purpose, and they gave some suggestions for minor changes to the questionnaire. We made modifications in response to their feedback to ensure that all participants comprehended all items and could effectively complete the questionnaires. The amended questionnaire was used to obtain the final data.The LCP-related Items were adapted from a variety of sources. The authenticity-related items were altered from The six ALP-related items were altered from The TS-related items were adapted from The TT-related items were altered from Guangzhou (Canton), a rich metropolis bursting with vitality, it is the provincial capital and largest city of Guangdong Province, which borders the South China Sea on its eastern shore. Located on the Pearl River, which is accessible to the South China Sea, the city is Guangdong’s political, economic, scientific, educational, and cultural capital. A multifunctional metropolis since the Qin Dynasty (221–207 BC), Guangzhou has seen numerous transformations during the last two thousand years. Furthermore, it is one of China’s most famous tourist destinations. Foshan is a progressive city. Following China’s open-door policy, it was one of the first ports in China to engage in foreign trade. It has also grown to become the third-largest city in Guangdong Province due to manufacturing and tourism. In recent years, it has invested resources to further develop its thriving tourism business. It is located on the northern bank of the Pearl River, approximately 20 kilometers (12 miles) north of Guangzhou in Guangdong Province. The city, which dates all the way back to nearly 5,000 years ago, was named after three Buddha statues discovered in this location during the Tang Dynasty (618–907). Shenzhen is a young and modern metropolis in southern China adjacent to Hong Kong. It is best known for its fast development over the last decades; it attracts flowing business people to seek opportunities, especially cheap but good-qualified electronic products. It is also a great holiday destination with fabulous tourist attractions and markets for traditional local products.Guangdong province in China was the location of the data collection, with three cities chosen for study: Guangzhou, Foshan, and Shenzhen; A non-probability sampling method was used for the present study since it is difficult to obtain a representative sampling frame for social science studies or to find potential respondents from target populations to address research questions . The resThe researchers used robust analysis to ensure the validity, reliability, and credibility of the findings using two statistically efficient software such as SPSS and SmartPLS 3.3.3. The descriptive analysis was done through SPSS. SmartPLS 3.3.3 was used during measurement modeling and structural modeling analyses . For thiDemographic data for the present study can be found in The statistical software SmartPLS 3.3.3 was applied to determine the association among variables used in the research model via structural equation modeling. SmartPLS 3.3.3 was selected because of its statistical efficiency and low sensitivity to sample size than other software for covariance-based SEM . CovariaThis study aimed to ascertain the relationship between DIF, TS, and TT. Thus, we investigated the model’s constructs for validity and reliability prior to evaluating the expected influences. The constructs’ validity and reliability are summarized in Henseler and Ringle questioned Fornell and Larcker’s method of determining discriminant validity, claiming that it cannot be relied upon to determine discriminant validity . They adWhen analyzing structural equation modeling, ensure that the issue of collinearity has been resolved. When the Variance Inflation Factor (VIF) exceeds 5, it indicates that there may be a collinearity issue between the dimensions . In thisThe R2 value is considered when evaluating a model’s explanatory ability. The R2 value ranges from 0 to 1—the greater the explanatory power, the greater the value. When the R2 value is close to 0.50, the model has modest explanatory power. When the R2 value is close to 0.75, the model has a high degree of explanatory power. According to f2 is used to detect the effect of exogenous variables on endogenous variables. When 0.02 < f2 ≤ 0.15, it is a small effect. When 0.15 < f2 ≤ 0.35, it is a medium effect. Furthermore, when f2 > 0.35, it is a large effect. It can be seen from f2 of Authenticity to TS is 0.490. It displays a large-effect explanatory ability. Access to local products to TS is 0.191 indicating medium effects. Local community participation to TS is 0.041 indicating a smaller effect. f2 of Tourist Satisfaction to TT is 0.019, indicating a smaller effect. Authenticity to TT is 0.076. It displays a smaller-effect explanatory ability. Access to local products to TT is 0.034, indicating smaller effects. Local Community participation to TT is 0.055, which also showed a smaller effect.The explanatory impact value Q2 value (Q2 (=1–SSE/SSO), greater than zero. Redundancy analysis was run through blindfolding in PLS-SEM to measure the predictive criterion accuracy based on Stone– Geisser’s Q2 value , which aSmartPLS-SEM 3.3.3 was utilized in this investigation in order to ascertain the associations between variables in the research model . Partialp < 0.05), indicating that hypothesis H1.1 is supported. Likewise, authenticity has a substantial positive influence on TT , supporting hypothesis H1.2. ALP has a beneficial effect on the TT , confirming hypothesis H1.3. LCP displays a substantial positive connection with TS , indicating that hypothesis H2.1 is supported. Furthermore, there is a substantial positive association between authenticity and TS , which supports hypothesis H2.2. ALP exhibits a substantial positive connection with TT , supporting hypothesis H2.3. Similarly, TS displays a substantial positive connection with TT , implying that hypothesis H3 is accepted.The three DIF constructs directly influence TT. Furthermore, LCP exhibits a significant positive connection with TT , A , and ALP  on TT. As a result, TS was demonstrated to have substantially and favorably mediated between LCP, A, and ALP, validating our hypotheses H4.1, H4.2, and H4.3  and TT. As illustrated in H4.3 see .The results derived from this study are meaningful because they were generated using a synthesized model framework. Only a few studies on rising countries like China have been published prior to this one. In addition, the few studies that have been done in emerging countries demonstrate a lack of focus on the tourism sector, despite its critical role in a country’s socioeconomic development. To the best of the author’s knowledge, this is one of the first studies investigating the impact of destination image formation on tourist trust in the Chinese heritage tourism context. The present study explores the direct relationship between DIF and TT. Additionally, we measured the direct relationship between DIF and TS. Likewise, we also measured the direct connection between tourist satisfaction and tourist trust. Moreover, the current research also measured the indirect relationship of destination image formation on tourist trust through TS in the Chinese tourism context.We first focused on the direct relationship between DIF practices  and TT. The results revealed that all three dimensions of DIF pillars  significantly influence TT, which approved our hypotheses H1.1 to H1.3. Prior studies confirmed that DIF positively influenced on TT . SimilarSecond, the study looked at the direct link between DIF  and TS. The results indicated that DIF  positively and directly affect TS, which approved hypothesis H2.1–H2.3. Prior studies confirmed the relationship between DIF  and TT . SimilarThird, the current study investigated the direct effect of TS on TT. The findings support hypothesis H3 by demonstrating that TS has a significant and positive effect on TT. Prior studies have also shown that TS positively influences TT. Finally, the study measured the indirect effect of DIF . The results confirmed that TS significantly and positively mediates between DIF  and TT, significantly supporting our hypotheses H4.1 to H4.3. Previous research has also shown that TS mediates between DIF and TT . StavriaThe results of this empirical study have valuable implications for managers and researchers. The present study’s findings enhance the discussion on jointly measuring the relationship among TS between DIF , TS, and TT. The results confirmed the relationships between DIF , TS, and TT, and TS also have mediating effects. We concluded the results of this study as follows: First, LCP plays an effective and positive role in building TT at destinations. Similarly, it was also considered that increased authenticity has a plosive and influential role in enhancing TT.Additionally, ALP is also considered the predictor of TT. Second, LCP has an influential role in enhancing TS. Similarly, authenticity has an influential role in increasing the level of TS. Furthermore, ALP is also considered the predictor of TS. Third, TS has a positive role in enhancing the role of TT. Fourth, TS has an intervening role in strengthening the relationship between LCP and TT. Similarly, TS has an intervening role in strengthening the relationship between A, TT, and TS has an intervening role in strengthening the relationship between ALP and TT.The image formation concept has received substantial focus in recent years as a key problem in the field of tourism research. However, many research gaps, especially the impact of tourist destination image formation , on tourist trust, and tourist satisfaction was used as a mediated variable were yet to be explored. Understanding these processes will have the benefit of providing destination marketers and managers with the knowledge they need to differentiate their destination offerings and foster a lifelong emotional connection with tourists. For this purpose, this study proposed a synthesized research model and evaluated it empirically. The results confirmed the strengthened proposed model, which is the first to include these factors and their relationships.First and foremost, the findings of this study indicate that trustful visitors bring major advantages in terms of both competitiveness and economics. Key elements of the tourist connection, for instance, satisfaction and attachment, should be considered in addition to physical features by destination marketers or managers in order to better suit travelers’ actual and symbolic requirements. Destination managers’ concern for guests’ well-being and interests is shown in these characteristics. Tourist sites with a good reputation have a competitive advantage over others because of the trust that tourists have in it, such as whether or not their travel expectations are met or surpassed and how many related uncertainties are minimized, for example. An emotional connection can be formed between a place and its visitors if they have confidence in it, which can lead to increased return visits and recommendations from those who have been there. Therefore, destination marketers must rely on trust, developing a destination’s image and customer satisfaction to build long-term relationships with travelers that will lead to a loyal customer base.Second, the current research demonstrates empirical evidence of relationships between DIF, TS, and TT. Previous scholars have asserted that destination image and TS are inextricably linked to trust . Others,Third, the current research contributes to tourist research by investigating the impact of DIF on TT while taking Tourist Satisfaction into account as a mediator. Prior research has consistently argued that in broad tourism settings, TS mediates the link between destination image and loyalty , and ourIn this respect, we contribute to the literature on tourism through our findings by proving the critical role of Destination Image formation  and perceived value generated by it on TS and tourist Trust. We suggest that these variables should be considered when determining a tourism destination’s competitive advantage.The present study also has a few limitations that can affect the interpretation of its results. First, participants have come from a single country (China), which can create cultural biases and limit the generalizability of the results. More empirical pieces of evidence in other cultural contexts are required. We conducted this study to explore the impact of tourist DIF  on tourist trust, and tourist satisfaction was used as a mediated variable. Future studies should explore the mediating role of service quality, destination facilities, and employees behavior.It cannot be denied that the current paper has several shortcomings that need to be addressed in future research. First and foremost, we only focus on tourist destinations in Guangzhou , Foshan , and Shenzhen , which are inside Guangdong Province, China. As a result, the context is limited to these areas and may differ in other locations. Moreover, surveys were conducted during COVID-19’s proactive measures, like quarantine and travel restrictions. Thus, longitudinal studies are necessary to achieve a better generalization of results.Second, our study’s findings might not be relevant to other Asian tourist locations due to the fact that tourism features vary per country. Similar research in other tourist areas within China or in other countries with different cultures is necessary to generalize our findings. Additionally, this study is one of the few to inspect the effect of DIF on the TS and TT from the standpoint of a tourist’s decision-making process. The model should be replicated and validated in more areas to approve its use and validity.Third, only three respondents over 61 were included in the study, which did not represent the overall Foreign and Chinese population, so it may not be possible to generalize the findings. This study had a relatively narrow scope and concentrated on a few key cultural heritage sites in Guangdong, China. As a result, the context is exclusive to this location and may differ in other locations. A sample size of greater than 30 but less than 500 is considered adequate for the majority of research , and thiFinally, more research is required to examine the effects of additional variables so as to have a better comprehension of the dynamics that drive TS and TT. Finally, tourist satisfaction was investigated as a possible mediator of the relationships between DIF and tourist trust, as well as between TS and trust. Nonetheless, the impacts of additional possible mediators  ought to be researched in order to offer a more complete framework.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Methods: Sixteen TN patients treated with microvascular decompression and 16 control patients undergoing spinal anesthesia for urological conditions were included. Lumbar CSF was collected preoperatively for the TN patients and before spinal anesthesia for the controls. A multiplexed proximity extension analysis of 91 CSF proteins was conducted using Proseek Multiplex Development 96, including biomarkers of cell communication, cell death, neurogenesis, and inflammation Results: The TN patients and the controls were of similar age, sex, and burden of co-morbidities. The TN patients exhibited higher concentrations of Clec11a, LGMN, MFG-E8, and ANGPTL-4 in CSF than the controls (q < 0.05). Conclusions: TN patients exhibited increased CSF biomarkers indicative of peripheral demyelinating injury (Clec11a), immune tolerance and destruction of myelin (LGMN), neuronal cell death (MFG-E8), and disturbances in myelin clearance (ANGPTL-8). Our findings are hypothesis-generating for candidate biomarkers and pathophysiological processes in classical TN.Trigeminal neuralgia (TN) is a severe type of facial pain. A neurovascular conflict between cranial nerve V and a nearby vessel is the main pathophysiological mechanism, but additional factors are likely necessary to elicit TN. In this study, the primary aim was to explore differences in protein expression in the cerebrospinal fluid (CSF) of TN patients in relation to controls.  Trigeminal neuralgia (TN) is characterized by brief and intense episodes of sharp facial pain, usually located in the V2/V3 dermatome of the trigeminal nerve (CN V) . The estIn the current study, the primary aim was to further explore potential pathophysiological mechanisms in classical TN by CSF analyses of protein biomarkers using the Proseek Multiplex Development 96 panel . The main findings were that TN patients exhibited elevated CSF biomarkers of neuroinflammation and neuronal injury.In this prospective observational study, patients referred to the Department of Neurosurgery at Uppsala University Hospital, who fulfilled the criteria for classical TN, according to the beta version of the International Classification of Headache disorders, third edition , and werIn the TN group, CSF was collected from the day before surgery by lumbar puncture without anesthesia. In the control group of urological patients, the lumbar CSF sample was collected by free flow into a sterile tube before the spinal anesthesia was administered. The CSF samples from both groups were handled according to the same protocol and were stored in a −70 °C freezer in both groups. The CSF samples from Cluj, Romania, were shipped to Uppsala, Sweden, by professional cargo, and kept frozen at −70 °C during delivery.Multiplexed proximity extension analysis (PEA) was conducted using Proseek Multiplex Development 96 including 92 protein biomarkers. The Development panel analyzed a different subset of biomarkers as compared to the Inflammation panel that was used in a previous study by our group . The curThe procedure has been described in detail in a previous study by our group . One µL Five µL of the primer pairs were loaded into the left side of the 96.96 Dynamic Array IFC, and the protein expression program was run in a Fluidigm Biomark reader.The primary aim of this study was to explore potential CSF biomarkers in TN in relation to a control group. The categorical and ordinal/continuous variables were presented as numbers (proportions) and medians (interquartile range (IQR)), respectively. Differences in demographic variables between the TN patients and the controls were assessed with the Chi-square or Mann–Whitney U-test, depending on the type of data. A principal component analysis (PCA) was performed to investigate if there were global differences between TN and controls. Only biomarkers with less than 20% missing values were included.Differences in CSF biomarker concentrations between TN and controls were assessed with linear regression analyses for each biomarker, adjusting for age and sex. To account for multiple comparisons, the false-discovery rate  procedure was used. Only biomarkers with a q-value < 0.05 were considered statistically significant. Furthermore, a multivariate partial least squares discriminant analysis (PLS-DA) classification model was trained to investigate if biomarker concentrations can be used to distinguish between TN and controls. Only biomarkers with less than 20% missing values were included in the model. The predictive ability of the PLS-DA models was evaluated by 10 5-fold cross-validations. The variable importance (VIP) was reported for the analyzed biomarkers.The study was conducted in accordance with the Helsinki declaration and its later amendments. The study was approved by the Regional Research Ethics Committee in Uppsala  for the TN patients and by the Institutional ethics committee of University of Medicine and Pharmacy, Cluj-Napoca, Romania  for the controls. Written informed consent was obtained by all study participants.Demographic details of the TN patients and the controls are described in Of the 91 biomarkers measured using Proseek Multiplex Development 96, 20 were excluded from the analyses since more than 20% of the patients had missing values (under the limit of detection).The PCA analysis of the CSF biomarker concentrations showed no clear differences between TN patients and controls .The PCA analysis A and theTo investigate if biomarker concentrations differed between TN patients and controls, a linear regression analysis was performed, adjusted for age and sex . Out of A PLS-DA model was trained to separate between TN and controls . The modThe four most important proteins in the model all had a mean VIP score >1.5 , and werIn this exploratory study of CSF biomarkers, we found that TN patients exhibited different protein concentration profiles as compared to controls. Particularly, Clec11a, LGMN, MFG-E8, and ANGPTL-4 were significantly higher in CSF in TN patients. These biomarkers are involved in neuroinflammation and myelin turnover and may reflect the demyelination and nerve atrophy that is commonly seen in TN.Clec11a is generally known as a glycoprotein involved in hematopoiesis and osteoblast maturation . There iLGMN, also known as osteolectin, is a protease located in the endoplasmatic reticulum, golgi apparatus, and the lysosome. In pathological conditions, it may be translocated to the cytosol and extracellular compartment . One majMFG-E8 is a multifunctional glycoprotein that is mainly present in lactating mammary glands, but also in many other organs such as the heart, lungs, vessels, and the brain . One funANGPTL-4 is a glycoprotein ,26 that 1 Although TN is usually explained by demyelinating lesions of the trigeminal pathways in the pons in MS, an NVC at the REZ is occasionally present and has even been suggested to drive demyelination in some of these MS patients [Altogether, our findings indicate increased neuroinflammation and disturbances in myelin turnover in TN. Particularly, many of these proteins have been studied in MS. This is interesting considering the relatively high burden of TN in MS patients.patients ,32. HencLastly, our findings should also be viewed in light of important contributions regarding CSF analyses in TN patients. Others have found increases in substance P, calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP) as well as reductions in β-endorphin, serotonin, and dopamine in CSF ,34, reflIt is evident that NVC is not sufficient to elicit classical TN, and there is a need to elucidate further pathophysiological mechanisms. There is so far a paucity of studies assessing CSF biomarkers that may be involved in these processes . This obTN patients exhibited increased CSF biomarkers indicative of peripheral demyelinating injury (Clec11a), immune tolerance and destruction of myelin (LGMN), neuronal cell death (MFG-E8), and disturbances in myelin clearance (ANGPTL-8). Our findings are hypothesis-generating for candidate biomarkers and pathophysiological processes in classical TN.