Background/aims: Iatrogenic CSF leaks after endoscopic endonasal transsphenoidal surgery remain a challenging entity to manage, typically treated with CSF diversion via lumbar drainage. Objective: To assess the safety and efficacy of high-volume lumbar puncture (LP) and acetazolamide therapy to manage iatrogenic CSF leaks. Methods: We performed a prospective pilot study of four patients who developed iatrogenic postoperative CSF leaks after transsphenoidal surgery and analyzed their response to treatment with concomitant high-volume lumbar puncture followed by acetazolamide therapy for 10 days. Data collected included demographics, intra-operative findings, including methodology of skull base repair and type of CSF leak, time to presentation with CSF leak, complications associated with high-volume LP and acetazolamide treatment, and length of follow-up. Results: Mean patient age was 44.28 years, with an average BMI of 27.4. Mean time from surgery to onset of CSF leak was 7.71 days. All four patients had resolution of their CSF leak at two- and four-week follow-up. Mean overall follow-up time was 179 days, with a 100% CSF leak cure rate at the last clinic visit. No patient suffered perioperative complications or complications secondary to treatment. Conclusion: Although our pilot case series is small, we demonstrate that a high-volume LP, followed by acetazolamide therapy for 10 days, can be considered in the management of post-operative CSF leaks. Iatrogenic cranial cerebrospinal fluid (CSF) leaks after trans-sphenoidal surgery remain a challenging, albeit common, complication to manage for both neurosurgeons and otolaryngologists. Unlike traumatic CSF leaks, which typically resolve with conservative measures , iatrogenic CSF leaks tend to be refractory to these techniques. Consequently, invasive procedures, such as CSF diversion and revision surgery, are often warranted .The role of perioperative lumbar drainage (LD) in preventing this complication continues to be debated ,3,4. LD 2O. The medication was considered well tolerated, though a formal analysis of adverse effects was not performed. Thus, acetazolamide represents a possible modality to manage mild iatrogenic CSF leaks after transsphenoidal surgery as well.Historically, acetazolamide has been used to treat and manage traumatic CSF leaks. A prospective trial of 36 patients with 42 CSF leaks was conducted to evaluate the difference in opening pressure on lumbar puncture 4 h after one 500 mg dose of acetazolamide . AcetazoWe sought to somehow combine these two treatment paradigms to manage our low-grade post-operative CSF leaks after endoscopic transsphenoidal surgery. We initially performed a lumbar puncture to measure the opening pressure in these patients, to rule out hydrocephalus and meningitis, and then started each patient on 10 days of acetazolamide to further reduce CSF production and pressure. This study is focused on the safety and proof of concept of this strategy. We conducted a single-institution retrospective analysis of prospectively collected data to evaluate the safety and efficacy of high-volume lumbar puncture and acetazolamide therapy for the management of iatrogenic cerebrospinal fluid leak following transsphenoidal surgery for tumor resection. Patient were accrued at our institution from June 2018 to June 2019 and their data maintained in an institutional database. The study was performed with the approval of the Institutional Review Board, and the consent process was waived, as it is a retrospective review of patient data stripped of identifying information. ®  dehydrated amniotic membrane (DAM) [After undergoing a full endocrinologic workup and ophthalmologic evaluation, patients underwent a standard endoscopic endonasal transsphenoidal surgical approach for pituitary macroadenoma resection. Each patient had a CSF leak, intra-operatively. According to the grade of leakage as observed during endoscopic evaluation, we divided the patients into two groups: those harboring a low-grade leak, indicated by a small weeping leak confirmed by Valsalva maneuver without an obvious or with only a small diaphragmatic defect; and those with a moderate leak, indicated by a definite diaphragmatic defect and egress of CSF . Each lene (DAM) ,10,11, aEach patient subsequently presented at various time points after surgery with a low-grade CSF leak from the nares on a clinical exam that was found when the patient bent forward, confirmed by both a board-certified neurosurgeon and otolaryngologist. Cerebrospinal fluid was sent for B2-transferrin, which was confirmatory in all cases. Each patient then underwent a CT scan of the brain to rule out pneumocephalus  and hydrPrior to discharge, each patient was evaluated to confirm cessation of their leak and subsequently discharged with strict instructions again to avoid Valsalva maneuvers and strenuous activity, given two weeks of stool softeners, and asked to maintain elevation of the head of their bed for two weeks. These patients were followed up in clinic by otolaryngology and neurosurgery to assess for the presence of CSF leak at two and four weeks, post-operatively. Clinical information was collected retrospectively.3 of pneumocephalus on CT brain non-contrast (Inclusion criteria comprised the following: (1) patients older than 18 years old; (2) patients with less than 1 cmcontrast ; and (3)This study was performed in accordance with PROCESS guidelines .n = 4) were assigned to acetazolamide therapy after they developed iatrogenic cerebrospinal fluid leak from transsphenoidal surgery for tumor resection from June 2018 to June 2019 by the senior neurosurgeon. At our institution, four patients  versus late (n = 29) treatment with acetazolamide in adult and pediatric patients with, or at risk of, a CSF leak due to a skull base fracture. In the early treatment group, patients received acetazolamide with 48 h of admission, and had greater mean of CSF leak resolution rate within 14 days (100% versus 78% in the late treatment group), and a shorter mean CSF leak duration  were subsequently managed with acetazolamide therapy post-operatively. However, 2 of the 24 subjects managed with Diamox became intolerant of the medication, and ultimately required permanent CSF diversion with VP shunt placement. Notably, 22 of the 24 patients (92%) were successfully treated with acetazolamide for their elevated ICP [Chaaban et al., published their results from 46 patients with 56 spontaneous CSF leaks managed over 5 years. Fifty-two CSF leaks (93%) were successfully repaired at first attempt. Of note, opening pressures via lumbar puncture increased significantly following closure of the skull base defect by 8 cm Hated ICP .In our series, we studied the use of acetazolamide with a concomitant high-volume lumbar puncture as salvage therapy to treat iatrogenic CSF leaks after transsphenoidal surgery for tumor resection. Patients were hospitalized for an average of two nights after undergoing lumbar puncture and were subsequently discharged. This stands in strong contrast to the typical prolonged hospital stays associated with lumbar drainage. Further, these patients did not suffer the common side effects of lumbar drainage, such as subdural hematomas or hygromas, meningitis, and low-pressure headache, which can be debilitating in some circumstances ,19. At bNo complications directly attributable to acetazolamide or the high-volume lumbar puncture were encountered. We believe this treatment regimen transiently reduces intracranial pressure, and allows for normal dural scarring to occur, ultimately preventing further CSF leakage. The present study has several limitations. First, our pilot study is inherently limited by a small sample size and the natural biases of a single-surgeon series. Second, there is no control group to compare traditional management with early skull base re-construction to our pilot group managed with acetazolamide and lumbar puncture. Finally, the authors acknowledge that there is no definitive way of determining if lumbar puncture had any bearing on the success of this treatment in this small cohort of patients, or rather, if the acetazolamide was the sine qua non reason for CSF leak resolution. However, these represent a novel approach to managing iatrogenic cerebrospinal fluid leak. To further study and confirm these results, we will use these initial positive data to justify a randomized control trial, looking at the efficacy of this management paradigm.We report one surgeon’s case series of four patients who suffered from iatrogenic postoperative CSF leak following endoscopic endonasal surgery and were all successfully managed with high-volume lumbar puncture and acetazolamide therapy. This approach eliminates the morbidity associated with lumbar drainage, the classical neurosurgical practice to manage post-operative CSF leaks. With close follow-up, these patients can be managed in a relatively conservative fashion, eliminating the need for prolonged hospital stays, lumbar drainage, and additional surgery.


Copiparvovirus. To our knowledge, this is the first field epidemiologic study on EqPV-CSF and EqCoPV using both serum and fecal samples. Our findings demonstrate the risk factors for infection and could facilitate the development of disease prevention strategies.Equine parvovirus-cerebrospinal fluid (EqPV-CSF) and eqcopivirus (EqCoPV) are new parvovirus species (EqPVs) identified from various tissues  in horses with neurologic and respiratory diseases. In this study, we described the prevalence rate of EqPV-CSF and EqCoPV in 133 and 77 serum and fecal samples, respectively, using polymerase chain reaction. Further, we analyzed the potential risk factors for infection. We calculated the nucleotide and amino acid similarity and constructed phylogenetic trees. There was a moderate-to-high prevalence rate  of each virus in serum; moreover, age, country of foaling, and clinical colic signs were significantly associated with the EqPVs infection. The newly identified EqPV-CSF/EqCoPV genomes had high nucleotide and amino acid identities with previously isolated strains in the USA. In phylogenetic analysis, they clustered and formed a new subgroup in the genus  Copiparvovirus and family Parvoviridae, were identified; namely, equine parvovirus-cerebrospinal fluid , equine parvovirus-hepatitis , and Eqcopivirus  [Since the first isolation of the equine parvovirus in 1985, three equine parvovirus species (EqPVs), which belong to the genus V, 2019) ,4. ExcepV, 2019) ,13,14,15EqPV-CSF was first isolated in 2015 from cerebrospinal fluid (CSF) in a horse with neurological signs and lymphocytic pleocytosis in the USA . In 2019p < 0.05). Our findings could expand the current knowledge regarding EqPV-CSF/EqCoPV and facilitate the control of epidemiologic risk factors. This is the first study to report the prevalence and genetic history of EqPV-CSF and EqCoPV among horses in Korea using serum and fecal samples. Studies have demonstrated that EqPV-CSF circulates in imported horses or offspring of imported dams. Unlike EqPV-CSF, EqCoPV has endemic characteristics and has been detected in local and foreign breed horses with a high prevalence rate. Especially, both viruses were related with clinical colic cases under statical significance , with separation of sera after centrifugation. Fecal sampling was performed per rectum or shortly after defecation for each horse using sterile gloves [g for 30 min. All serum and fecal supernatants were stored at −70 °C until further analysis. The animal protocols were approved by the Institutional Animal Care and Use Committee of Korea Racing Authority (KRA IACUC-2106-AEC-2106).We collected 133 serum and 77 fecal samples from 199 horses  in Jeju Province, which is a major horse-breeding region in Korea. Specifically, 88 horses presented for medical treatment with clinical signs , 111 hore gloves . SubsequViral nucleic acids from serum or feces were extracted with Patho Gene-Spin™ DNA/RNA kit  using 150 μL of serum or fecal supernatants, followed by elution in 30 μL of elution buffer. The specific sequences of EqPV-CSF and EqCoPV were detected through polymerase chain reaction (PCR) using the Maxime™ PCR PreMix kit . We used reported primers targeting nonstructural (NS) genes of EqPV-CSF , as wellWe generated 2 complete viral coding sequences (CDS) of each EqPV-CSF, and EqCoPV isolate using the primer walking method . SupplemWe deposited all sequenced genes of EqPV-CSF  and EqCoPV  into the GenBank database (accession numbers OK422845 to OK422862). To evaluate the evolutionary history of the newly isolated EqPV-CSF and EqCoPV, nucleotide sequences were aligned using the MUSCLE algorithm, and phylogenetic trees were constructed with Molecular Evolutionary Genetics Analysis (MEGA) software version X  . SubstitCopiparvovirus members to determine the similarity of the amino acid and nucleotide sequences. Similarly, sequences were aligned using the MUSCLE algorithm, and p-distances were calculated using MEGA X.We calculated the genetic distances between isolated Korean parvovirus strains and other genus p-value, odds ratio (OR), and confidence interval of the OR. Statistical significance was set at p < 0.05. All statistical analyses were performed in Jamovi software .We performed statistical analyses to determine associated factors for the incidence of EqPV-CSF or EqCoPV. We performed the binomial logistic regression test (age) or chi-square test . We reported the We tested 133 serum and 77 fecal samples for EqPV-CSF and EqCoPV DNA. For the serum samples, 5 (3.8%) and 13 (9.8%) horses were positive for EqPV-CSF and EqCoPV DNA, respectively . One horp < 0.05) (p < 0.05) (p < 0.05) . The logistic regression model revealed a significant impact of age on EqPV-CSF, with older horses showing an increased infection chance ( < 0.05) . Further < 0.05) . Especia < 0.05) .Copiparvovirus and generated a maximum-likelihood tree. Each isolate of EqPV-CSF and EqCoPV was clustered with the previously identified strains  and EqCoPV  in serum . A previp < 0.05)  . These fIn the previous studies, the correlation of clinical disease with EqPV-CSF or EqCoPV infection remains unclear ,16,20. GThe tissue tropism and transmission routes of EqPV-CSF and EqCoPV remain unclear. As demonstrated in an experimental study on another equine parvovirus species, EqPV-H , EqPV-CSCopiparvovirus. The taxonomy of EqPV-CSF and EqCoPV remains to be established [In the phylogenetic tree analyses, the newly isolated Korean EqPV-CSF and EqCoPV strains showed a close relationship with previously reported strains in the USA. Specifically, EqPV-CSF and EqCoPV were divided from a common ancestor and formed a distinct subgroup from other copiparvovirus species. This evolutionary characteristic is consistent with the results of sequence similarity analysis in the NS gene of each virus. EqPV-CSF and EqCoPV showed relatively higher nucleotide and amino acid similarity rates with each other than with other species in genus ablished ,30. MoreTo our knowledge, this is the first field study regarding the prevalence, associated factors, genetic characteristics, and evolutionary history of EqPV-CSF and EqCoPV infections. Our findings expand the current knowledge in the field basis and could inform the transmission and clinical relevance of each virus to some degree. However, there is a need for further studies regarding infectivity, pathogenicity, and transmission as potential risk factors in global horse husbandry.


Point‐of‐care tests may play a valuable role in reducing the risk of donor‐derived SARS‐CoV‐2 transmission in lung transplantation. A major challenge in LTx is the risk of donor‐derived viral transmission. Current guidelines advise screening of deceased lung donors with chest computed tomography (CT) and recommend reverse transcription (RT)‐PCR testing for There are reports about two cases of donor‐derived SARS‐CoV‐2 transmission during LTx in the literature. Based on a negative NP swab, the lungs were accepted but after LTx, viral infection was detected with RT‐PCR on an LRT sample from the donor.Viral RNA can persist in the lung for a long time after the acute phase of infection. We reported a double LTx from a donor who was convalescent from mild COVID‐19 (occurring 3 months earlier) and who tested twice negative on NP swab RT‐PCR. No donor‐derived transmission occurred. RT‐PCR on a biopsy of the donor lung before LTx revealed a low viral load with a Ct value of 35, reflecting persistence of viral RNA. Viral culture on the same sample was negative.Several cases of liver, kidney, and heart transplantation with organs from RT‐PCR positive donors have been reported but no donor‐derived SARS‐CoV‐2 transmission has occurred.Despite the observation of SARS‐CoV‐2 RNAemia, no cases of transmission through blood product or stem cell transfusions have been reported.LTx programs are balancing the risk of donor‐derived transmission with rejecting noninfected, potentially suitable donor lungs. RT‐PCR does not allow to differentiate between persistence of viral RNA, viral shedding, and ongoing viral replication. Access to viral cultures in daily practice is impractical, leaving physicians with the Ct value as an indirect marker for viral load and infectivity. Defining a Ct value as a universal threshold for infectivity is not possible, with laboratories using different protocols and RT‐PCR primer sets . Based on current evidence, lungs from a donor with a positive RT‐PCR result on an LRT sample are not considered acceptable for transplantation. In case of doubt regarding lung donor infectivity, repeated LRT RT‐PCR testing, careful assessment of the recent history, and judicious chest CT evaluation are indispensable.The incubation period of infection with SARS‐CoV‐2 is highly variable.To further reduce the likelihood of donor‐derived viral transmission and narrow the window of uncertainty between the last RT‐PCR and lung procurement, we here propose the use of point‐of‐care tests (POCTs) on a bronchoalveolar lavage (BAL) sample taken by the procuring surgeon during bronchoscopy. Using POCTs may increase the opportunity of detecting a high viral load and, by extension, replication‐competent virions in the lung grafts. Narrowing the window of uncertainty has gained even more importance with the emergence of the B.1.1.529 (Omicron) variant: its incubation time appears to be shorter.POCTs are easy‐to‐use assays that enable quick, on‐site detection of SARS‐CoV‐2. They include rapid antigen tests (RATs) and various types of nucleic acid amplification tests (NAATs). The performance of POCTs in BAL samples has not been reported except for one NAAT , which showed a sensitivity of 96% and specificity of 100%.RATs are easily transportable, provide a rapid answer after a brief set‐up time, and are user‐friendly Figure . For NP Several NAATs consisting of a disposable cassette that is inserted in a portable analyzer have been developed for detection of SARS‐CoV‐2 RNA including the Roche cobas Liat System,The lung procurement team can choose to bring along a RAT or preferably a portable NAAT analyzer to the donor center. After bronchoscopy, the BAL sample is tested and while awaiting the result, macroscopic evaluation of the donor lungs can be performed. When the result of the POCT is positive, the donor lungs are not considered acceptable for LTx.During the COVID‐19 pandemic, the gap between demand and supply of lung donors has increased. Discarding uninfected, potentially suitable donor lungs must therefore be avoided. For NP swabs, the specificity of RATs and NAATs is >95%.We anticipate that the COVID‐19 pandemic will pave the way for a more regular use of POCTs to reduce the risk of donor‐derived transmission of other pathogens that reside in the donor lungs. Timely and on‐site detection of other harmful pathogens such as influenza, aspergillus or mucor could prevent serious morbidity and mortality in LTx recipients.Immunity, Inflammation and Disease. The authors confirm that the work described has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form in English or in any other language, without the written consent of the copyright holder.The authors of this manuscript have no conflicts of interest to disclose as described by Conception and design of the work: Jan Van Slambrouck, Peter Mombaerts, Laurens J. Ceulemans. Acquisition, analysis, or interpretation of data: Jan Van Slambrouck, Joost Wauters, Peter Mombaerts, Laurens J. Ceulemans. Assisted in writing: All authors. Approval final draft: All authors.


Necroptosis plays an important role in the pathogenesis of acute kidney injury (AKI), and necroptosis-related interventions may therefore be an important measure for the treatment of AKI. Our previous study has shown that augmenter of liver regeneration (ALR) inhibits renal tubular epithelial cell apoptosis and regulates autophagy; however, the influence of ALR on necroptosis remains unclear. In this study, we investigated the effect of ALR on necroptosis caused by ischemia-reperfusion and the underlying mechanism. In vivo experiments indicated that kidney-specific knockout of ALR aggravated the renal dysfunction and pathological damage induced by ischemia-reperfusion. Simultaneously, the expression of renal necroptosis-associated protein receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), and mixed-lineage kinase domain-like protein (MLKL) significantly increased. In vitro experiments indicated that overexpression of ALR decreased the expression of hypoxia-reoxygenation-induced kidney injury molecules, the inflammation-associated factor tumor necrosis factor-alpha (TNF-α), and monocyte chemotactic protein. Additionally, the expression of RIP1, RIP3, and MLKL, which are elevated after hypoxia and reoxygenation, was also inhibited by ALR overexpression. Both in vivo and in vitro results indicated that ALR has a protective effect against acute kidney injury caused by ischemia-reperfusion, and the RIP1/RIP3/MLKL pathway should be further verified as a probable necroptosis regulating mechanism.  CurrentNecroptosis, a new form of programmed cell death discovered in recent years, has characteristics of both necrosis and apoptosis. Like apoptosis, necroptosis is regulated by specific molecules; its morphological characteristics in common with necrosis include cell swelling, plasma membrane permeability, and loss of cell contents . Necroptin vitro model regulates autophagy and ferroptosis [Augmenter of liver regeneration (ALR) was first isolated from the livers of weanling rats. Because it promotes hepatocyte proliferation, ALR is also called a liver stimulating factor . Previouroptosis ,6. HowevWe hypothesized that ALR could alleviate ischemia-reperfusion-induced acute kidney injury by regulating necroptosis. We established an ischemia-reperfusion model through in vivo and in vitro experiments to confirm that ALR can regulate necroptosis through the RIP1/RIP3/MLKL pathway, thereby alleviating acute kidney injury.22.1flox/flox-Ggt1-Cre, referred to as knockout (KO) mice) and control mice  mice) were purchased from Saiye Biotechnology Co. Ltd.,  and raised in the Animal Experiment Center of Chongqing Medical University . The mouse pups were genotyped by polymerase chain reaction (PCR). Briefly, a 0.5 mm tail biopsy was cut, and the deoxyribonucleic acid (DNA) was extracted with a commercial kit . Primers for Neo-del PCR were as follows:  F2: 5′-GCTCAAGCCAATCTCTCAGCTT-3′ and R2: 5′-ATCTAGAGTTTCTAGGTAGGGCCTG-3′. Homozygotes showed one 450 bp band, whereas heterozygotes showed two bands at 450 bp and 327 bp. The wildtype allele showed a 327 bp band. Primers for loxP PCR were as follows:  F1: 5′-GTTTGGAGCACGGGAAGCGACCG-3′ and R1: 5′-GTTGACAGCCTACTAAATAACTTCG-3’; targeted: 305 bp. Primers for Ggt1-Cre PCR were as follows:  F3: 5′-CATCACATCAGGCACCCCAGAA-3′ and R3: 5′-GAACATCTTCAGGTTCTGCGGGA-3’; Cre amplicon: ~420 bp.Specefic pathogen Free (SPF) grade C57 background kidney-specific knockout 23-kDa ALR mice /12 h, and standard water and diet were provided. When the mice reached 8–12 weeks and weighed 22–25 g, the CON mice were randomly divided into CON group and CON+IR group, and the KO mice were randomly divided into KO group and KO+IR group, with 6–8 mice per group. For establishing the ischemia-reperfusion model, mice were anesthetized with sodium pentobarbital intraperitoneally at a dose of 60 mg/kg. Then the back hair was removed, and the mouse body temperature was maintained at 36.5 to 36.7°C on a temperature-regulated heating pad. After iodophor smearing, a small 1 cm incision was made on both sides of the back spine, the skin, and subcutaneous tissues were carefully separated to expose the kidney. A sterile blood vessel clip was used to clamp the renal pedicle. The kidney and incision were kept moist, and the color change in the kidney was observed during the operation . After 22.2Blood samples were collected retro-orbitally from the sacrificed mice and allowed to stand for 6 h at 4°C. After the serum was separated, the blood samples were centrifuged at 900 × g for 10 mins. The upper layer serum was aspirated and transferred into a new eppendorf (EP) tube . Serum c2.3Mouse kidney tissue was fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin sections (4 μm) were used for hematoxylin and eosin (HE) staining and periodic acid-Schiff (PAS) staining ,29. The 2.42 at 37°C [Human kidney proximal tubular (HK-2) cells were purchased from the American Type Culture Collection  and cultured in Roswell Park Memorial Institute 1640 medium  supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin  in a humidified incubator under an atmosphere of 5% CO2.5HK-2 cells were seeded in six‐well plates and grown to 70–80% confluence before transfection. Lentiviral vectors were used for cell transfection. Lentivirus particles containing 23-kDa ALR ; Shanghai Gene Chemistry, China) and lentivirus particle vector  were transfected into HK-2 cells for 8–16 h according to the manufacturer’s instructions. At 72 h after transfection, the cells transfected with the indicated lentivirus vectors were grown in complete medium containing puromycin (3 μg/ml) for selection for 2 weeks. At 72 h after transduction, transduced cells were observed under a fluorescence microscope to verify GFP fluorescence, and cells with an infection efficiency of at least 80% were selected for subsequent experiments . The fol2.62, 5% CO2 and 1% O2 at 37°C). After 6 hours, the cells were transferred to room temperature and cultured in Roswell Park Memorial Institute 1640 complete medium at 37°C and 5% CO2 for the specified time points  [To induce hypoxia and reoxygenation (HR) in vitro, we placed HK-2 cells in D-glucose free and serum-free Dulbecco’s modified Eagle’s medium (DMEM) for 12 h in advance and incubated the cells to synchronize cell growth. Then, the cells were incubated for 6 hours in a three-gas incubator under hypoxic conditions (94% N4 hours) . If nece2.7The postoperative kidney tissues were immersed in glutaraldehyde, fixed with 1% osmium tetroxide, dehydrated with a series of ethanol gradient solutions for 15 mins, embedded in epoxy resin, sliced into 60 nm serial sections, stained with 2% uranyl acetate and 0.04% lead citrate and finally observed with a transmission electron microscope  .4. The samples were then embedded in epoxy resin, sliced into serial sections of 60 nm, stained with 2% uranyl acetate and lead citrate and observed using TEM [HK-2 cells were harvested immediately after the planned treatments. After dehydration with a gradient series of ethanol solutions for 15 mins, HK-2 cells in 10 cm dishes were fixed in 2.5% glutaraldehyde at room temperature for 1 hour, followed by further fixation with 1% OsO2.84 cells per well and observed at 0, 24, 48 and 72 hours [A cell counting kit-8  was used to detect cell viability. According to the manufacturer’s instructions, each group of cells was seeded in 96-well plates at a density of 1 × 10 2.9Protein was extracted from mouse kidney tissue or cells with radioimmunoprecipitation assay (RIPA) lysis buffer, and the protein concentrations were detected with a BCA protein concentration determination kit . An equivalent amount of protein (40 µg) was separated with 10%–15% SDS-PAGE (US Bio-RAD) and transferred to polyvinylidene fluoride membranes with a constant current of 260 mA. The membranes were blocked with skimmed milk powder for 1 hour at room temperature, and specific primary antibodies were added and incubated with shaking overnight at 4°C. After being washed with Tween 20 Tris buffered saline three times, the membrane and peroxidase-conjugated secondary antibody were incubated for 1 h at room temperature. Finally, the membrane was washed three times, and immunoreactive bands were detected with Immobilon Western substrate and a Chemi Doc Imaging System  and quantitatively analyzed in triplicate by normalization of the band intensities to those of the controls in films scanned with ImageJ v1.8.0 software  .The following antibodies were used: DYKDDDDK Tag (D6W5B) rabbit mAb , MLKL (D2I6N) rabbit mAb , RIP (D94C12) rabbit mAb , RIP3 (E1Z1D) rabbit mAb , RIP3 (D4G2A) rabbit mAb , MLKL (ab243142) , MCP1 (ab151538) , TNF alpha (ab215188) , KIM-1  , ALR  and GAPDH (D16 H11) rabbit mAb . Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse were used as secondary antibodies .2.10−ΔΔCt relative quantification method was used [Total ribonucleic acid (RNA) was extracted from HK-2 cells with a total RNA extraction kit . The PrimeScript™ II Reverse Transcriptase Kit (TaKaRa) was used to reverse transcribe total RNA into cDNA for analysis of mRNA levels. Real-time PCR was performed with SYBR Premix (TaKaRa). For quantitative analysis, a CFX Connect Real-Time system was used to amplify the genes with SYBR Premix, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a positive control (Bio-Rad). The CFX Manager primer sequences were as follows: ALR-F 5′-GTG AGG AGT GTG CTG AAG ACC-3′; ALR-R 5′-TGA GCA GTC GAA GTC AGG CTTG-3′; GAPDH-F 5′-GTCTTCCCCTCCATCGTG-3′ and GAPDH-R 5′- AGGGTGAGGATGCCTCTCTT-3′. All experiments were performed in at least triplicate, with three independent replicates for each group. The messenger RNA (mRNA) levels were normalized to GAPDH mRNA. To quantify gene expression, the 2was used .2.11Statistical analysis was performed in GraphPad Prism 7.0 . The data are expressed as mean ± standard deviation of three independent replicates. One-way ANOVA and subsequently Tukey’s post hoc test were used to compare the differences between multiple groups. P < 0.05 was considered to indicate a statistically significant difference.3This study investigated the effect and underlying mechanism of ALR on necroptosis in IR-induced acute kidney injury. We found that kidney-specific knockdown of ALR aggravated IR-induced acute kidney injury with markedly elevated necroptosis-related proteins. In addition, we transfected HK-2 cells with lentivirus in vitro to establish a cell model of HR-induced AKI, and found that overexpression of ALR can inhibit inflammatory burst and regulate necroptosis. The underlying mechanism may be the inhibition of RIP1/RIP3 /MLKL pathway.3.1flox/flox-Ggt1-Cre mice and ALRflox/flox mice after mating are shown in flox/flox was present in the DNA, a 450 bp fragment was amplified; otherwise, two fragments of 450 and 327 were amplified in heterozygotes. The 420 bp fragment was amplified only if the Cre gene was present in the DNA. As shown in flox/flox gene, on the basis of PCR results. In summary, 2, 5, 6, 7, 8, 9 were ALRflox/flox-Ggt1-Cre mice, and 1, 3, 4 were ALRflox/flox mice.The genotype identification results of the progeny of ALRAfter establishing the ischemia-reperfusion model in the knockout mice, we detected the blood serum creatinine and urea nitrogen concentrations, as shown in PAS staining and Kidney Injury Score results showed that, compared with the CON group, the CON+IR group exhibited more epithelial cell edema, basement membrane destruction, brush border discontinuity, cell nucleus shedding, renal tubular lumen enlargement, and even tubular and massive infiltration of inflammatory cells (P < 0.001). The damage in the KO+IR group was more evident than the above-mentioned damage in the CON+IR group (P < 0.01), thus suggesting that kidney-specific knockout of ALR significantly aggravated the pathological damage of kidney tissue in AKI mice caused by IR .3.2The death of renal tubular epithelial cells was analyzed by transmission electron microscopy. The results indicated that the renal tubular epithelial cells of mice after acute kidney injury showed signs of ultrastructural necrosis, such as increased volume, intracellular structure disorder, irregular chromatin, swelling of organelles and rupture of the plasma membrane. The ultrastructural characteristics of cell necrosis were more apparent in the KO+IR group . RIP1, R3.3To simulate the in vivo IR model in vitro, we established an HK-2 cell IR model with sugar-free serum-free medium combined with three-gas incubator hypoxia-reoxygenation (HR). Western blotting and RT-PCR were used to detect the expression of ALR at different time points after HR treatment. The protein and nucleic acid levels of ALR were detected at 3 h, 6 h, 12 h and 24 h respectively during the reoxygenation process after 6 h of hypoxia. The expression of ALR protein and nucleic acid gradually increased after HR, peaked at H6R12 and then gradually decreased, thus suggesting that ALR is involved in the HR injury process in renal tubular epithelial cells .Because our previous study indicated that overexpression of ALR had a protective effect on HK-2 cells with AKI, we overexpressed the ALR gene in follow-up experiments. After transfecting HK-2 cells with ALR lentivirus, we used anti-Flag antibody to detect ALR expression levels by real-time PCR and Western blotting. As shown in 3.4We selected different HR time points to obtain total cell protein and used Western blotting to observe the expression of key proteins of necroptosis, including RIP1 RIP3 and MLKL . After HTo further explore the effects of inhibiting necroptosis in AKI, we added the RIP1 inhibitor Nec-1 (50 µm) to the cell culture medium before HR treatment and extracted cellular protein from the H6R6 group. Western blotting was used to detect necroptosis-associated proteins . Compare3.5The effects of overexpressing 23-kDa ALR on cell proliferation were detected by CCK-8 analysis. The proliferation levels of cells in each group were detected at 0, 24, 48 and 72 hours after transfection. After HK-2 cells overexpressed 23-kDa ALR, no significant difference was observed in cell proliferation in the Lv+ALR group compared with the Normal group and the Lv-vector group  after AKI increases, and the long-term mortality rate increases significantly . HoweverTo date there are three ALR isoforms described, 15-kDa, 21-kDa and 23-kDa, in liver and kidney . Furthermore, the 15-kDa ALR is mainly expressed in the cytosol and was found extracellular, whereas the two longer isoforms, 21-kDa and 23-kDa, where found in the ISM and in the cytosol . HoweverThe mechanisms underlying cell death have long been a focus of life science research. Necroptosis is an important form of programmed necrosis discovered in recent years, whose importance has been confirmed in many diseases. Necroptosis has several notable characteristics: (1) It has the morphological characteristics of necrotic cell death, such as loss of cell plasma membrane integrity and loss of mitochondrial membrane potential. (2) Autophagy is a common downstream response to necroptosis, which is often accompanied by autophagosome formation. (3) Inflammatory cell infiltration is more common in tissues that undergo necroptosis, and some necrosis-like cells produce large amounts of reactive oxygen free radicals. (4) Necroptosis is specifically inhibited by the small molecule Nec-1 ,34. Liu Apoptosis, necroptosis and autophagy have been found to be associated. Our previous studies have confirmed that ALR inhibits apoptosis in AKI and regulates autophagy. Consequently, we assumed that ALR might regulate necroptosis in AKI. In this study, we observed typical necrotic morphological changes after IR injury in mice. We conducted Western blotting to detect the changes in necroptotic protein after IR injury in mice. Compared with that in the CON group, the expression of RIP1, RIP3 and MLKL was significantly higher in the CON+IR group, thereby confirming that IR induced necroptosis in AKI. Compared with that in the CON+IR group, the expression of necrosis-related proteins in the KO+IR group was also significantly higher . TherefoWe observed the changes in ALR after hypoxia and reoxygenation. In different periods of hypoxia and reoxygenation, ALR gradually increased and then decreased, and peaked at H6R12; the expression remained high at H6R24 . In agreThrough our in vitro experiments, we found that necroptosis occurred in HK-2 cells after hypoxia and reoxygenation. In different periods of hypoxia and reoxygenation, RIP1, RIP3 and MLKL showed a trend of increasing first and then decreasing, and their levels peaked at H6R6 . ConsequWe transfected 23-kDa ALR lentivirus into HK-2 cells and selected stable 23-kDa ALR overexpressing cells by using purine. Cell viability was detected with a CCK-8 kit. Overexpression of the 23-kD ALR scarcely influenced cell viability. Therefore, we speculate that the function of ALR against IR-induced AKI does not play a role in promoting cell proliferation, thus excluding the effect of proliferation on the experimental results.Nec-1 has been found to specifically inhibit the activity of RIP1 and subsequently block the formation of necrosomes. It is currently the most widely studied inhibitor of necroptosis. To confirm that the inhibition of necroptosis protects against renal damage in AKI caused by HR, we added Nec-1 before hypoxia and reoxygenation treatment in HK-2 cell culture. The results of Western blotting indicated that after addition of Nec-1, the expression of RIP1, RIP3 and MLKL significantly decreased, thus confirming that Nec-1 specifically inhibits RIP1 and necroptosis. The protein expression of the kidney injury molecule KIM-1 and the inflammatory factors TNF-α and MCP-1 revealed that inhibition of necroptosis decreased HR-induced kidney injury, whereas inhibition of necroptosis inhibited the inflammation caused by AKI. Necroptosis is programmed cell death induced by various stimuli both inside and outside cells, including TNF family members, Fas ligand, interferon and oxidative stress. TNF-α-induced necroptosis is the most common form . Recent in vivo experiments, our findings indicated that ALR regulates necroptosis. However, whether ALR directly targets necroptosis-mediated cell death or indirectly regulates the RIP1/RIP3/MLKL pathway is unclear. Therefore, the basic mechanism must be further clarified in future research. The cell death pathway of AKI after renal IR injury is complex and interrelated. The available evidence has shown that multiple cell death pathways including apoptosis, necrosis and ferroptosis are involved in this process. Meaningful protection may require targeting multiple pathways, because inhibiting one pathway might make another pathway sensitive [To further illustrate the regulatory effect of ALR on necroptosis, we observed the changes in the morphology of HK-2 cells after hypoxia and reoxygenation through TEM. After hypoxia and reoxygenation, HK-2 cells experienced plasma membrane rupture, increased permeability, cell swelling and decreased organelle content, findings consistent with the morphological characteristics of necrosis. Simultaneously, we detected the changes in necroptosis-associated proteins. Western blotting indicated that the expression of RIP1, RIP3 and MLKL in the Lv-ALR+HR group was significantly lower than that in the Lv-vector+HR group. In agreement with the results of ensitive . The mosOur research revealed that ALR prevents and ameliorates AKI induced by hypoxia and reoxygenation through the inhibition of RIP1/RIP3/MLKL-mediated necroptosis. Our findings highlight the preventive and therapeutic potential of ALR in the treatment of AKI.5In summary, our results demonstrate for the first time that kidney-specific knockout of ALR aggravates IR-induced acute kidney injury, and that ALR can ameliorate IR-induced acute kidney injury by regulating necroptosis through the RIP1/RIP3/MLKL pathway. This study provides a theoretical basis for the prevention and treatment of AKI caused by IR.


There has been increasing interest in finding non-invasive biomarkers for neurodegenerative diseases such as Alzheimer’s disease (AD). This observational study investigated AD-specific biomarkers in tear fluid. Tear fluid was collected from a total of 65 subjects, including 23 patients with subjective cognitive decline (SCD), 22 patients with mild cognitive impairment (MCI), 11 dementia patients and 9 healthy controls (HC). Levels of amyloid-beta peptides , total-tau (t-tau) and phosphorylated-tau (p-tau) were determined using multiplex immunoassays. Levels of AB40 and t-tau were detectable in the vast majority (> 94%) of tear fluid samples. Cerebrospinal fluid (CSF) was available from a subset of patients. In this group, tear t-tau levels were significantly higher in people with dementia compared to SCD patients. Tear t-tau levels were elevated in patients with neurodegeneration (classified according to the A/T/N system) compared to patients without neurodegeneration. Negative correlations were found between CSF AB42 and CSF t-tau, and between CSF AB42 and tear t-tau. In summary, this study shows the potential of tau proteins in tear fluid to be associated with disease severity and neurodegeneration. AB plaques and NTS can be visualized by cortical amyloid and tau PET ligand binding or postmortem immunohistochemistry3. These key pathological hallmarks are however not restricted to brain tissue. The retina, that is embryologically derived from the neural tube as a protrusion from the brain, shares many similarities with brain tissue. As such, AB plaques and tau accumulation have also been observed in postmortem retina samples of AD patients5. In addition, non-invasive retinal imaging was able to detect amyloid plaques in vivo4. The retina of AD patients is also characterized by widespread retinal ganglion cell losses, retinal nerve fiber layer (RNFL) thinning and optic nerve degeneration10.Aggregation of amyloid-beta (AB) peptide into extracellular plaques together with the presence of neurofibrillary tangles (NTS) of truncated and phosphorylated forms of the microtubule-stabilizing protein tau are central in the molecular pathogenesis of Alzheimer’s disease (AD)13. Low CSF AB42 and elevated CSF phosphorylated tau (p-tau) and total tau (t-tau) are validated diagnostic biomarkers for AB plaques, NTS and neurodegeneration, respectively14. Other, more accessible, body fluids such as blood are known to contain AB and tau16. However, quantification of AD biomarkers in peripheral fluids is challenging, even with very sensitive techniques, because of their low abundance in these biological samples17. Because of its close proximity to the brain, there has been increasing interest in testing ocular fluids such as tear fluid, aqueous humor and vitreous body. Previous studies in cognitively healthy persons found soluble AB42 in aqueous humor18 and AB40, AB42 and neurofilament light (NfL) in vitreous humor20. Another study showed presence of the full-length amyloid-beta precursor protein (APP) in human tear fluid and the lacrimal gland (extraocular gland that produces tear fluid) of healthy persons21. However, it is not clear yet whether other AB species and tau proteins are present in tear fluid, and if they are, whether their concentrations differ between patients and controls.In addition to its presence in AB plaques and NTS, AB and tau are also found as soluble forms in cerebrospinal fluid (CSF)Tear fluid is non-invasively collected via Schirmer’s strips, a paper strip which is gently placed in the lower eyelid where it absorbs tear fluid. This method, which is used as a standard test in clinical practice, is a simple and quick procedure to collect basal tears and is becoming increasingly relevant. Tear fluid  can be used directly in (immuno-)assays.In the present work, we explored whether amyloid-beta peptides , t-tau and p-tau are present in tear fluid and if differences can be found between patients with cognitive impairment and healthy controls.The total study group consisted of 56 patients and nine cognitively healthy controls. Twenty-three patients received a diagnosis of SCD, 22 patients had a diagnosis of MCI and 11 patients had a clinical diagnosis of dementia    compared to patients without neurodegeneration (N negative)  (p = 0.04)  was only detectable in AD samples and undetectable in samples from controls.Previous studies investigating tear fluid samples of AD patients used mass spectrometry and were therefore not able to detect amyloid-beta and tau, since (untargeted) mass spectrometry is less sensitive for low abundant proteins than immunoassays26. “N” refers to a quantitative or topographic biomarker of neurodegeneration or neuronal injury (CSF t-tau). The + /− categorization is based on lab-specific threshold values. Tear t-tau levels were elevated in N positive patients compared to N negative patients. These results suggest that tear fluid may mirror CSF with regards to (t-)tau pathology. Cutoff values for tear fluid are however yet to be established and require further investigation.We found elevated levels of tear AB40 and tear t-tau in patients with cognitive impairment, and those levels increased with increasing disease severity. However, the majority of differences were not statistically significant, most probably due to large sample variability and small sample size. The level of tear t-tau was significantly higher in dementia patients compared to SCD patients in the CSF group, in the same way as CSF t-tau. Interestingly, all these dementia patients displayed CSF t-tau levels above the diagnostic threshold, while this was not the case for all SCD patients, except for one patient. In view of this, we used the A/T/N classification system of AD biomarkers that divides patients into binary classes27 and oligomeric alpha-synuclein28 for Parkinson’s disease and alpha-1 anti-chymotrypsin for multiple sclerosis29. In those studies, the levels of TNF-alpha and alpha-1 antichymotrypsin were also elevated in the CSF of parkinsonian patients and multiple sclerosis patients, respectively. Together, these studies support the hypothesis that tear fluid may predispose to mirror pathophysiological changes in the central nervous system.Our study is in line with others efforts that identified tear biomarkers for different neurological conditions, such as TNF-alpha30 The inter-correlation between the three AB species in tear fluid resembles both CSF22 as well as plasma30, while there was a lack of correlation for t-tau levels between tear fluid and CSF. The latter finding is in agreement with plasma tau31 and together these results suggest that tear fluid seems to resemble better with peripheral fluids, such as plasma, compared to CSF. A larger study comparing those three body fluids for a wide range of biomarkers would clarify this.In our investigation, we were able to correlate biomarkers levels in tear fluid with CSF in a subset of patients from whom CSF biomarker levels were available from clinical diagnostic purposes. We observed that CSF AB42 and tear t-tau were negatively correlated in the same way as CSF AB42 and CSF t-tau. On the other hand, AB42 and t-tau were positively correlated in tear fluid, similarly to what has been found in plasmaLimitations of our study are the small sample size, especially in the dementia group, and the limited availability of information on the etiology of the disease. This is because our study subjects were recruited from a pool of patients who visited the memory clinic because of cognitive complaints. In most cases, their clinical diagnosis is not accompanied with CSF sampling or PET imaging as these are not routinely performed in regular care settings. Although we believe that the current study population is more clinically relevant than comparing severely affected patients with healthy controls (as it are patients with early symptoms that would benefit from future tear-based biomarker tests), larger sample size studies are warranted to confirm and validate our results.A total of 56 patients and nine cognitively healthy controls (HC) were enrolled in this study. Patients were recruited from the BioBank Alzheimer Center Limburg (BB-ACL), Maastricht, the Netherlands. Inclusion criteria were an MMSE score ≥ 20 and a CDR score from 0 to 1, thereby including patients across the whole clinical spectrum (i.e. from subjective cognitive disorder to (mild) dementia). Exclusion criteria at baseline were neurological diseases  or cerebrovascular accident (CVA) (< 2 years), or TIA/CVA with concurrent (within three months) cognitive decline) and a history of psychiatric disorders , or alcohol abuse). Healthy controls were recruited at the University Eye Clinic Maastricht, the Netherlands. The medical ethics committee of the Maastricht University Medical Center, the Netherlands approved the study protocol and this study followed the tenets of the current version of the Declaration of Helsinki. Written informed consent was obtained from all participants.32. A diagnosis of mild cognitive impairment (MCI) was based on the clinical National Institute on Ageing-Alzheimer’s Association (NIA-AA) criteria, which entails that patients have impaired cognitive functioning but sufficiently preserved function performance such that a dementia diagnosis cannot be made33. All other patients were classified as subjective cognitive decline (SCD), indicating the presence of a cognitive complaint initiation the visit to the memory clinic, but having no impairment in cognitive test performances and activities of daily living. The global cognitive status was evaluated using the Mini-Mental State Examination (MMSE)34.During their visit to the memory clinic, all patients underwent standardized clinical assessments including neuropsychological assessments, blood tests, MRI cerebrum and, from a subset of patients, CSF sampling. A diagnosis of dementia was made according to regular DSM-5 criteria for neurocognitive disordersCSF was collected via a lumbar puncture. After collection, CSF was centrifuged at room temperature, aliquoted and stored at −80 °C until analysis. The levels of amyloid-beta-42 (AB42), total tau (t-tau) and phosphorylated at threonine 181 tau (p-tau) were measured using commercially available single-parameter ELISA methods (respectively Innotest® beta-amyloid (1–42) and Innotest® hTAU-Ag; Innogenetics, Ghent, Belgium) at the department of Neurochemistry, Radboud University MC, Nijmegen the Netherlands. Diagnostic cutoff values were 500 pg/mL for AB42, 350 pg/mL for t-tau and 85 pg/mL for p-tau.26. “A” refers to the value of an AB biomarker (CSF AB42); “T,” the value of a tau pathology biomarker (CSF p-tau); and “N,” a quantitative or topographic biomarker of neurodegeneration or neuronal injury (CSF t-tau). The + /− categorization is based on threshold values.We used the A/T/N classification system of AD biomarkers that divides patients into 3 binary classes36. Tear fluid was then eluted by centrifugation and stored at −80 °C until further use. Levels of amyloid-beta peptides , t-tau and p-tau in eluted tear fluid were determined using the Human Aβ triplex Ultra-Sensitive assay and the Phospho(Thr231)/Total Tau duplex assay  at the department of Internal Medicine, School for Cardiovascular Diseases (CARIM), Maastricht University Medical Center, Maastricht, the Netherlands. The absolute concentrations were then normalized to the wetting length (tear fluid volume) and dilution (extraction buffer volume) as described elsewhere37. Values below the lower limit of detection were excluded.Tear fluid was collected from the left and right eye via Schirmer’s tear strips  without topical anesthesia. Tear fluid was extracted from each Schirmer’s strip by agitating small cut pieces of these strips in 60 µL PBS, Tween20 0.5% and cOmplete™ Protease Inhibitor Cocktail  at 4 °C for 1.5 hStatistical analyses were performed using GraphPad Prism version 9.0.0 . Data in tables are presented as medians with interquartile range (IQR). Differences in continuous variables across diagnostic groups were analyzed using Kruskal–Wallis testing with multiple comparison. Differences in continuous variables across A/T/N groups were examined by the Mann–Whitney U test. Differences in categorical variables were analyzed using the Fisher exact test. Correlations were assessed using Spearman Rank correlation analysis. Correlations with less than 10 pairs were omitted.


An involvement of the central-nervous and peripheral, innate and adaptive immune system in the pathogenesis of Parkinson's disease (PD) is nowadays well established.We face several open questions in preparation of clinical trials aiming at disease-modification by targeting the immune system: Do peripheral (blood) inflammatory profiles reflect central (CSF) inflammatory processes? Are blood/CSF inflammatory markers associated with CSF levels of neurodegenerative/PD-specific biomarkers?1−42, t-Tau, p181-Tau and NFL. All analyses were stratified by sex as the immune system shows relevant sex-specific differences.Using a multiplex assay we assessed 41 inflammatory markers in CSF/serum pairs in 453 sporadic PD patients. We analyzed CSF/serum correlation as well as associations of inflammatory markers with clinical outcome measures  and with CSF levels of α-synuclein, AβCorrelations between CSF and serum were sparse and detected in only 25% (9 out of 36) of the analysable inflammatory markers in male PD patients and in only 38% (12 out of 32) of female PD patients. The most important pro-inflammatory mediators associated with motor and cognitive decline as well as with neurodegenerative/PD-specific biomarkers were FABP, ICAM-1, IL-8, MCP-1, MIP-1-beta, and SCF. Results were more robust for CSF than for serum.Levels of central-nervous and peripheral inflammatory markers might be regulated independently of each other with CSF inflammatory markers reflecting CNS pathology more accurately than peripheral markers. These findings along with sex-specific characteristics have to be considered when designing clinical trials aiming at disease-modification by targeting the immune system. Moreover, a growing number of epidemiological and genetic studies as well as post-mortem and fluid-biomarker analyses provide evidence for a relevant influence of inflammation on incidence and progression in PD –4. An ins in CSF , 6. Micrs in CSF . Moreoves in CSF .Several post-mortem and biofluid  studies reported increased inflammatory profiles to be associated with clinical subtypes of PD, promoting an accelerated motor and non-motor phenotype , 10–13. 1−42, Tau and NFL? Knowledge on these questions will help to stratify patients and enrich cohorts for clinical trials.Despite this clear role for inflammation in the pathogenesis of PD, we face several open questions in preparation of clinical trials aiming at disease-modification by targeting the immune system: 1. Do peripheral (blood) inflammatory profiles reflect central (CSF) inflammatory processes indicative of a cross-talk between periphery and brain? 2. Are blood/CSF inflammatory markers associated with CSF levels of neurodegenerative/PD-specific biomarkers such as α-synuclein, AβBetween 2005 and 2018, CSF/serum pairs of 453 sporadic PD patients recruited from the outpatient clinic and/or ward for PD at the University Hospital of Tübingen were collected. Fourty-eight neurodegenerative healthy elderly  served as control individuals.1−42 676, t-Tau 230, p-Tau 41, NFL 1025, total α-synuclein 569. Female PD patients presented with mean age of 66 years, mean age at onset of 59 years, mean disease duration of 7 years, mean HY of 2.1, mean UPDRS-III of 26, mean MoCA of 25, mean LEDD of 533. Mean CSF levels in pg/ml were as follows: Aβ1−42 684, t-Tau 257, p-Tau 43, NFL 1143, total α-synuclein 689. For more demographic and clinical details  and from 2013 on, we used low protein-binding polypropylene cryovial 2D barcode cryovials FluidX (Article Nr. 68-0703-01) for storage. Samples with abnormal routine CSF diagnostics  were excluded.http://rbm.myriad.com). Mean storage-time until measurement was 6 years. Storage-time showed a negative correlation in CSF for ICAM-1 (−0.155 p = 0.004), Interleukin-4 (−0.146 p = 0.007), Interleukin-7 (−0.255 p < 0.001), PSA-f (−0.155 p = 0.029) and TF (−0.108 p = 0.045) and a negative correlation in serum for CKMB (−0.115 p = 0.032) and BDNF (−0.214 p < 0.001). For measurements, samples were thawed at room temperature, vortexed, spun at 18.000 x g for 1 min and pipetted into a master microtiter plate. After dilution with assay diluents in a manner of 1:5, an aliquot of 10 μl diluted sample was introduced into one of the capture microsphere multiplexes followed by incubation at room temperature for 1 h. Reporter antibodies were added followed by incubation for an additional hour at room temperature. Streptavidin-phycoerythrin solution was added followed by incubation for another hour at room temperature. For control purposes, calibrators and controls were included on each microtiter plate. Standard curve, control, and sample QC were performed to ensure proper assay performance. Samples were tested in singles. Analysis was performed using the Luminex 100/200 instrument. Data were interpreted using the software developed and provided by Myriad RBM.Levels of 41 inflammation-associated markers were measured in CSF/serum pairs using the multiplexed immunoassay by Myriad RBM, Austin, TX, USA (The Lower Limit of Quantitation (LLOQ) means the lowest concentration of an analyte in a sample that can be reliably detected and at which the total error meets the laboratory's requirements for accuracy. NMI's requirement for accuracy is the concentration of an analyte at which the coefficient of variation of replicate standard samples is 30%.CSF LLOQ: CA 125 U/ml: 2; IgE U/ml: 2,04; TSH ulU/ml: 0,0195; Alpha Fetoprotein pg/ml: 110; CEA pg/ml: 95,8; CKMB pg/ml: 157; FABP pg/ml: 618; Factor VII pg/ml: 562; GH pg/ml: 36,2; ICAM-1 pg/ml: 644; Leptin pg/ml: 60,4; MMP-3 pg/ml: 42,4; MMP-9 pg/ml: 7,840; PSA-f pg/ml: 4,32; TF pg/ml: 89,6; TNF-alpha pg/ml: 3,58; Brain-Der Neu Fac pg/ml: 23,2; ENA 78 pg/ml: 19,2; GM-CSF pg/ml: 19,1; IL-1alpha pg/ml: 1; IL-1beta pg/ml: 0,358; IL-2 pg/ml: 6; IL-3 pg/ml: 4,08; IL-4 pg/ml: 5,22; IL-5 pg/ml: 2,3; IL-6 pg/ml: 0,862; IL-7 pg/ml: 8,44; IL-8 pg/ml: 2,74; IL-10 pg/ml: 2,28; IL-12 p40 pg/ml: 73,8; IL-12 p70 pg/ml: 18,1; IL-13 pg/ml: 2,04; IL-15 pg/ml: 187; IL-16 pg/ml: 12,3; IL-18 pg/ml: 12,4; Lymphotactin pg/ml: 85,4; MCP-1 pg/ml: 8,66; MDC pg/ml: 5,82; MIP-1 beta pg/ml: 13,6; SCF pg/ml: 26,2; TNF-beta pg/ml: 2,62; TPO pg/ml: 420.Serum LLOQ: CA 125 U/ml: 5; IgE U/ml: 5,1; TSH ulU/ml: 0,0489; Alpha Fetoprotein pg/ml: 276; CEA pg/ml: 240; CKMB pg/ml: 394; FABP pg/ml: 1,550; Factor VII pg/ml: 1,410; GH pg/ml: 90,5; ICAM-1 pg/ml: 1,610; Leptin pg/ml: 151; MMP-3 pg/ml: 106; MMP-9 pg/ml: 19,600; PSA-f pg/ml: 10,8; TF pg/ml: 224; TNF-alpha pg/ml: 8,95; Brain-Der Neu Fac pg/ml: 58; ENA 78 pg/ml: 47,9; GM-CSF pg/ml: 47,7; IL-1alpha pg/ml: 2,5; IL-1beta pg/ml: 0,895; IL-2 pg/ml: 15; IL-3 pg/ml: 10,2; IL-4 pg/ml: 13,1; IL-5 pg/ml: 5,75; IL-6 pg/ml: 2,16; IL-7 pg/ml: 21,1; IL-8 pg/ml: 6,85; IL-10 pg/ml: 5,7; IL-12 p40 pg/ml: 185; IL-12 p70 pg/ml: 45,2; IL-13 pg/ml: 5,1; IL-15 pg/ml: 468; IL-16 pg/ml: 30,7; IL-18 pg/ml: 31; Lymphotactin pg/ml: 214; MCP-1 pg/ml: 21,7; MDC pg/ml: 14,6; MIP-1 beta pg/ml: 34,1; SCF pg/ml: 65,5; TNF-beta pg/ml: 6,55; TPO pg/ml: 1,050. For a list of all assessed markers see .1−42, t-Tau and p181-Tau were measured using ELISA kits from INNOTEST, Fujirebio GmbH, Germany. CSF levels of NFL were measured using the UmanDiagnostics NF-light®assay. Intra-assay coefficients of variation for each CSF parameter were below 15%. CSF levels of total α-synuclein were assessed using an ELISA kit for human α-synuclein . Intra-assay imprecision of 4.4% was calculated from duplicate analyses and expressed as median of the range to average of the duplicates. Inter-assay imprecision of <10% was determined using two quality control CSF pool samples.CSF levels of AβThe study was approved by the Ethics Committee of the University of Tuebingen . All participants gave written informed consent.kathrin.brockmann@uni-tuebingen.de.Anonymized data are available upon request to: 1−42, t-Tau, p181-Tau, and NFL. As this study was exploratory, we did not correct for multiple testing. However, only correlations with at least 10 valid sample pairs and a correlation coefficient ρ > 0.20 were considered meaningful .Statistical analysis was performed using SPSS 26.0 software for Windows (IBM). All analyses were stratified by sex as the immune system shows relevant sex-specific differences of inflammatory profiles . Group cOf the 41 analyzed inflammatory markers some were not detectable in CSF and/or Serum. Specifically, Interleukin-5 and Interleukin-12p70 weren't detectable in CSF and Serum in healthy controls while Interleukin-12p70 wasn't detectable in CSF in PD patients. BDNF wasn't detectable in CSF in any of the cohorts. Therefore, these 3 metabolites were excluded from the respective group analyses.Moreover, some inflammatory markers were measurable only in a small number (<10) of CSF/Serum pairs and/or of inflammatory/PD-biomarker pairs. These are listed below and were also excluded from the respected analyses due to lack of meaningful outcome.For CSF/serum correlations of inflammatory markers, the following metabolites were excluded as the number of measurable CSF/serum pairs were <10:n = 7), TNF-alpha (n = 5), Interleukin-1 alpha (n = 0), Interleukin-1 beta (n = 0), Interleukin-2 (n = 0), Interleukin-3 (n = 0), Interleukin-7 (n = 1), Interleukin-10 (n = 9), Interleukin-12p40 (n = 4), Interleukin-13 (n = 7), Interleukin-15 (n = 8), Lymphotactin (n = 2), MDC (n = 2), TPO (n = 9), Alpha-fetoprotein (n = 2), CEA (n = 3), CKMB (n = 3), CA-125 (n = 5) and IgE (n = 0).Males: FactorVII (n = 6), Interleukin-1 alpha (n = 8), Interleukin-1 beta (n = 4), Interleukin-2 (n = 7), Interleukin-3 (n = 0), Interleukin-7 (n = 5), Lymphotactin (n = 4), MDC (n = 1), Alpha-fetoprotein (n = 1), CEA (n = 4), CKMB (n = 2), PSA-f (n = 8), CA-125 (n = 3) and IgE (n = 3).Females: FactorVII (For correlations between CSF inflammatory markers and CSF PD-biomarkers the following metabolites were excluded as the number of measurable pairs were <10:n = 7), MMP9 (n = 8), TNF-alpha (n = 4), Interleukin-1 alpha (n = 4), Interleukin-1 beta (n = 0), Interleukin-2 (n = 1), Interleukin-3 (n = 1), Interleukin-7 (n = 4), Interleukin-12p40 (n = 8), Lymphotactin (n = 2), MDC (n = 2), TPO (n = 9), Alpha-fetoprotein (n = 2), CEA (n = 3), CKMB (n = 3), CA-125 (n = 5), IgE (n = 0).Males: FactorVII (n = 6), Interleukin-1 beta (n = 4), Interleukin-3 (n = 0), Lymphotactin (n = 6), MDC (n = 1), Alpha-fetoprotein (n = 1), CEA (n = 2), CKMB (n = 2), CA-125 (n = 3), IgE (n = 4).Females: FactorVII (For correlations between serum inflammatory markers and CSF PD-biomarkers the following metabolites were excluded as the number of measurable pairs were <10:n = 4), Interleukin-2 (n = 2), Interleukin-3 (n = 1), Interleukin-7 (n = 2), Interleukin-13 (n = 7).Male: Interleukin-1-alpha (n = 7), Interleukin-3 (n = 0), Interleukin-7 (n = 7).Female: Interleukin-2 , Interleukin-3 (n = 0), Interleukin-5 (n = 0).Males: Interleukin-2 (n = 9), Interleukin-2 (n = 3), Interleukin-3 (n = 0), Interleukin-5 (n = 0), MDC (n = 6), PSA-f (n = 7) and IgE (n = 6).Females: Interleukin-1-beta , Interleukin-5 (n = 2), MDC (n = 9).Males: Interleukin-3 (n = 9), Interleukin-3 (n = 1), Interleukin-5 (n = 3), MDC (n = 6), IgE (n = 6).Females: Interleukin-1-beta , Interleukin-3 (n = 7).Males: Interleukin-2 (n = 4), Interleukin-3 (n = 4), Interleukin-5 (n = 7), Interleukin-12p70 (n = 9).Females: Interleukin-2 (n ≥ 10) for meaningful correlation analysis. A significant correlation between CSF and Serum was found only in a small proportion of the analyzed markers. Interleukin-4 and Leptin were positively correlated between CSF and serum in males and in females . In males, ICAM-1 (rho = 0.553 p = 0.032), MIP-1 beta (rho = 0.521 p = 0.047), PSA-f (rho = 0.738 p = 0.004) and TSH (rho = 0.567 p = 0.043) were positively correlated between CSF and serum. In females, Interleukin-6 (rho = 0.423 p = 0.039), MCP-1 (rho = 0.424 p = 0.019), FABP (rho = 0.389 p = 0.050) and Interleukin-12p40 (rho = 0.832 p ≤ 0.001) were positively correlated between CSF and serum.In males, 18 out of 41 and in females 24 out of 41 inflammatory markers reached a sufficient number of analysable CSF/serum pairs (n ≥ 10) for meaningful correlation analysis. A positive correlation between CSF and Serum in male and in female PD patients was seen for ICAM-1 , MMP3 , Interleukin-6 , MCP-1 , MIP-1 beta , TSH , Leptin , Interleukin-4  and Interleukin-12p40 . In females also Interleukin-8 (rho = 0.288 p = 0.003), Interleukin-13 (rho = 0.450 p = 0.004) and CKMB (rho = 0.669 p = 0.003) were positively correlated  of the analysable inflammatory markers in male PD patients and in only 38% (12 out of 32) of female PD patients. Of these markers, ICAM-1, Interleukin-4, Interleukin-6, Interleukin-12p40, MCP-1, MIP-1 beta, MMP3, Leptin, and TSH were associated in CSF and serum in both sexes whereas Interleukin-8, Interleukin-13 and CKMB were additionally correlated only in females.Higher CSF levels of FABP, Interleukin-8, MCP-1, and SCF were associated with older age and with cognitive impairment as measured by MoCA scores in males and/or females. Higher CSF levels of Interleukin-8 were also associated with motor impairment in UPDRS-III and H&Y staging.t-Tau, p181-Tau, and α-synuclein in males and in females.Higher CSF levels of FABP, ICAM-1, MMP3, SCF, and TF were associated with higher CSF levels of neurodegenerative/PD-specific biomarkers, namely Higher serum levels of CA-125, FABP, and TNF-alpha as well as lower serum levels of BDNF were associated with cognitive impairment as measured by MoCA scores and with motor impairment assessed by H&Y staging in males and/or females.t-Tau and p181-Tau  others were correlated with NFL . Notably, all of these were only found in female PD patients and were not the same as those associated with the PD-specific biomarker CSF α-synuclein .Whereas some inflammatory serum markers were positively associated with CSF levels of Overall, associations between inflammatory markers with clinical outcomes and with CSF levels of neurodegenerative/PD-biomarkers were stronger and more robust for CSF than for serum.GBA) gene (PDGBA)  . Here, w (PDGBA) . Now, weIn-vivo and in-vitro experiments show an up-regulation of the Stem Cell Factor SCF in neurons of injured brain tissue paralleled by neural stem/progenitor cell migration highlighting that SCF is involved in self-renewal and cell survival  showed a huge overlap with the markers assessed in our study so that these 2 studies are nicely comparable. Importantly, results in our large PD cohort support and expand findings from that study: (I) Only a fraction of markers is robustly detectable in both, CSF and serum. (II) Correlations of cytokines between CSF and blood are sparse indicating that levels of central-nervous and peripheral inflammatory markers might be regulated independently of each other and that changes may not simply reflect passive diffusion of circulating cytokines into or out of the CNS. (III) Associations between inflammatory markers with CSF levels of neurodegenerative/PD-biomarkers are primarily seen with t-Tau, p-Tau and α-synuclein but not with Aβ1−42 and are more robust for CSF than for serum. These findings suggest that CSF levels of inflammatory markers better reflect central-nervous-system pathology as compared to peripheral inflammatory markers.So far, there is only one small study published assessing a variety of inflammatory markers by multiplex assay in CSF/serum pairs with further analyses in relation to clinical outcomes and CSF levels of neurodegenerative/PD-biomarkers . This spBased on results from the present study, further analyses are needed in order to design clinical trials aiming at disease-modification by targeting the immune system. Such studies should be longitudinal and address the following questions: Do blood/CSF inflammatory profiles change over the course of the disease and do they mirror the degree of neuroinflammation, respectively? Which blood/CSF inflammatory profiles are best associated with longitudinal phenotypical trajectories and what is their prognostic value? Which inflammatory markers in blood/CSF are most meaningful with regard to patient stratification and outcome measures? These might not necessarily be the same and it might not be a single marker but rather a combination. Promising candidates identified in the present study are FABP, ICAM-1, IL-8, MCP-1, MIP-1-beta, and SCF.The strength of the present study is the large monocentric collection of high-quality CSF/serum pair samples according to standardized procedures which minimizes variance in sample collection and processing.Limitations of our study are: (I) The single measurement of inflammatory markers so that variations in measurement cannot be accounted. (II) The mean storage-time until measurement of inflammatory markers was 6 years which might impact detectability of markers that are present at low concentrations.kathrin.brockmann@uni-tuebingen.de.Anonymized data are available upon request to: The studies involving human participants were reviewed and approved by the Ethics Committee of the University of Tuebingen . The patients/participants provided their written informed consent to participate in this study.SL: conception, statistical analysis, manuscript preparation, and critical review of the manuscript. MZ, IW, BR, FF, and TG: execution of the study  and critical review of the manuscript. CD: execution of the study and critical review of the manuscript. KW and IL: execution of the study (hSYN ELISA) and critical review of the manuscript. MJ, TJ, and NS-M: analysis of inflammatory markers and critical review of the manuscript. KB: conception, design and execution of the study , statistical analysis, and manuscript preparation. All authors contributed to the article and approved the submitted version.Influence of Inflammatory profiles on PD Phenotype and Progression. Moreover, it was partially funded by the PD-Strat project (FKZ 031L0137B) which was supported by the German Federal Ministry of Education and Research (BMBF) in the frame of ERACoSysMed2.This work was funded by the Michael J. Fox Foundation for Parkinson's Research (MJFF) within the grant KW and IL are employed by Roboscreen GmbH which manufactures the ELISA kit for measurements of total human α-synuclein used in the present study. The remaining 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.


The relationship between cerebrospinal fluid (CSF) biomarkers and the clinical features of idiopathic normal pressure hydrocephalus (iNPH) has been inconclusive. We aimed to evaluate CSF biomarkers reflecting Alzheimer’s disease (AD)-related amyloid β (Aβ) aggregation, tau pathology, neuroinflammation and axonal degeneration in relation to the clinical features of pre- and post-shunt surgery in iNPH patients.42, Aβ40, Aβ38); total tau (T-tau); phosphorylated tau (P-tau181); neurofilament light (NfL) and monocyte chemoattractant protein 1 (MCP1).Mini Mental State Examination (MMSE) scores and gait velocity were evaluated pre- and postoperatively in cohorts of 65 Finnish (FIN) and 82 Swedish (SWE) iNPH patients. Lumbar CSF samples were obtained prior to shunt surgery and analysed for soluble amyloid precursor protein alpha (sAPPα) and beta (sAPPβ); amyloid-β isoforms of 42, 40 and 38 (Aβp < 0.0001). Low CSF T-tau and absence of apolipoprotein E ε4 predicted over 20% gait improvement postoperatively (p = 0.043 and p = 0.008). Preoperative CSF T-tau, P-tau181 and NfL correlated negatively with MMSE scores both pre- (p < 0.01) and post-surgery (p < 0.01). Furthermore, T-tau, NfL and Aβ42 correlated with MMSE outcomes (p < 0.05). Low preoperative CSF P-tau181 (p = 0.001) and T-tau with NfL (p < 0.001 and p = 0.049) best predicted pre- and postoperative MMSE scores greater than or equal to 26.Preoperative patient characteristics showed no significant differences between patients in the FIN and SWE cohorts. Patients in both cohorts had significantly improved gait velocity after shunt surgery (CSF biomarkers of neurodegeneration appeared to correlate with pre- and postoperative cognition, providing a window into neuropathological processes. In addition, preoperative CSF neurodegeneration biomarkers may have potential in the prediction of gait and cognitive outcomes after shunt surgery. Idiopathic normal pressure hydrocephalus (iNPH) is characterized by a symptom triad of gait dysfunction, dementia and incontinence, accompanied by enlarged ventricles , 2. SympThere are several CSF biomarkers assessed for diagnostic and predictive purposes in the field of neurodegenerative diseases. For iNPH patients, it has been shown that the lumbar CSF composition of low tau and APP-derived proteins together with high MCP1 can distinguish iNPH patients from cognitively intact individuals and patients with other neurodegenerative diseases . However181 collected preoperatively [181, the Aβ42/P-tau181 ratio and the Aβ38/Aβ42 ratio [181 correlating with a poorer cognitive outcome. In another study [42 was shown to correlate with postoperative MMSE values.In the current literature, postoperative gait velocity has been found to be associated with lumbar CSF T-tau and P-tauratively , 13. Reg42 ratio , 13, witer study , CSF Aβ4The role of the axonal degeneration biomarker neurofilament-light (NfL) as a symptom predictor in iNPH patients has not been studied widely either. In AD patients, higher plasma NfL levels have been shown to be associated with poorer Mini Mental State Examination (MMSE) scores , and plaSince there are tendencies for biomarkers to enhance the accuracy of diagnosis and prediction of outcome in iNPH, establishing a more precise, specific preoperative biomarker combination able to predict a favourable shunt surgery outcome would be highly beneficial.In this study, we aimed to evaluate preoperatively-obtained CSF biomarkers reflecting AD-related Aβ aggregation, tau pathology, neuroinflammation and axonal degeneration and their associations with pre- and postoperative clinical features in Finnish and Swedish iNPH cohorts.APOE genotyping, and DNA was isolated using a commercial kit according to the manufacturer’s protocol . The extracted samples were analysed by the standard PCR method [In all, 65 and 82 consecutive shunted patients with probable iNPH diagnosed by the Relkin criteria  at KuopiR method .Fig. 1FlPreoperative CSF samples were obtained by lumbar puncture in both cohorts and retained in 10 ml polypropylene tubes. Samples were centrifuged, aliquoted and frozen in a temperature-controlled − 80 °C freezer. There was no blood contamination seen in the samples obtained.181 were measured by INNOTEST ELISAs using the kit manufacturer’s protocol . For Aβ38, Aβ40, Aβ42, sAPPα, sAPPβ and MCP1 analysis, electrochemiluminescence (ECL) assays were used, as described by the manufacturer [The CSF biomarker measurements were performed at the Clinical Neurochemistry Laboratory at Sahlgrenska University Hospital, Mölndal, Sweden. The laboratory technicians were board-certified and blinded to the clinical data. All experiments were executed on the same plate in one round of experiments and using the same batch of reagents. Commercial kits were used to perform the biomarker concentration analysis. CSF T-tau and P-taufacturer  (Meso Scfacturer .2 or Wilcoxon’s signed ranks test for discrete variables. Pearson correlation coefficients (r) were assessed for linear dependency analyses. Since CSF concentrations of T-tau, P-tau181, NfL, Aβ38, Aβ40, Aβ42, sAPPα, sAPPβ and MCP1 were concordant in both cohorts, we pooled the cohorts for correlation analysis. For pre- and postoperative comparisons, we used analysis of covariance (ANCOVA) with the confounding covariates of age, sex and APOE genotype. Receiver operating characteristics (ROC curves) and binary logistic regression were used to model MMSE performance and gait outcome. Pre- and postoperative MMSE with gait outcome were dichotomized by cut-offs of 26 by the mild dementia value commonly used and 20% improvement in gait. ROC analysis of biomarkers, dichotomized MMSE and gait was performed. We aimed to identify optimal biomarker cut-offs by estimating Youden’s indices and favouring balanced sensitivity and specificity. Univariate modelling was performed, and significant variables with age, sex and APOE genotype were used in the multiple regression model. Multicollinearity diagnostics were used for variables included in the multiple regression model. All tests calculated were two-sided and considered significant with a p value less than 0.05. The statistical analyses were performed in IBM SPSS Statistics 27.00 for IOS.The group comparisons performed for mean values and frequencies were performed by t tests for continuous variables and xAPOE genotype and mean CSF biomarker values are presented in Table APOE genotype between the Finnish and Swedish cohorts. In addition, CSF concentrations of T-tau, P-tau181, NfL, Aβ38, Aβ40, Aβ42, sAPPα, sAPPβ and MCP1 were concordant in both cohorts. Correlation analysis was assessed for pooled cohorts  was seen in low P-tau181 group during the follow-up whereas the high P-tau181 group remained similar (mean score 21.8) . In addition, the P-tau181 groups showed divergent Aβ42 concentrations of 321 ng/l in the low P-tau181 group and 415 ng/l in the high P-tau181 group (p < 0.001). The APOE genotype correlated only with Aβ42  p < 0.0001)  and T-tau  patients were more likely to have MMSE above 26 preoperatively (p < 0.05). Comparable results were seen for postoperative MMSE results: patients in the low CSF P-tau181 , T-tau , NfL , Aβ38  and Aβ40  groups were more likely to have MMSE scores above 26 (p < 0.05), respectively compared to the scores of patients in the high concentration groups. Favourable outcome of 20% increase in gait velocity was 2.8-, 2.7-, 0.4- and 0.4-times more likely for low CSF T-tau (cut-off 206 ng/l), NfL (1050 ng/l), Aβ42 (301 ng/l) and sAPPβ (239 ng/l) concentration patients compared to high (p < 0.05). The most significant multivariate model for evaluating preoperative MMSE included P-tau181, NfL, APOE, sex and age . In this model, low CSF P-tau181 patients were 3.4 times more likely to have MMSE above 26 (CI (1.3–8.7), p = 0.01). For postoperative MMSE, the combination of T-tau, NfL, Aβ38, age and sex best predicted the performance . The low CSF T-tau and NfL patients were 13.6 (CI 3.4–55.0) and 2.4 (1.0–5.8) times more likely to have MMSE above 26 (p < 0.05). The model with a more specific CSF T-tau cut-off (306 ng/l) predicted MMSE to be below 26 postoperatively by 96.7% specificity, 37.0% sensitivity and 63.9% overall accuracy. In this model, high T-tau patients had a 17.0-fold  greater risk of having MMSE below 26 postsurgery. Gait improvement over 20% postoperatively was best predicted by T-tau, sAPPβ, APOE, age and sex . The patients with low CSF T-tau and absence of APOE ε4-allele were 3.1 (1.0–9.4) and 3.9 (1.4–10.4) times more likely to improve their gait by 20% postsurgery (p < 0.05).Regression modelling was utilized for dichotomized biomarker concentration variables with MMSE performance and gait velocity outcomes as dependent variables Table . Low CSF181 was the only biomarker to correlate with the NPHS outcome . In the Finnish cohort, MCP1  correlated with postoperative outcome. Interestingly, MCP1 was found to negatively correlate with the incontinence outcome of NPHGS in the Finnish cohort .In the follow-up from pre- to postsurgery, both cohorts improved in NPHGS (p = 0.025) and NPHS (p < 0.0001). In the subdomains of both NPHGS and NPHS, a significant change was seen in gait , along with cognition (p < 0.0001). In the Swedish cohort, P-tau181 and NfL are associated with cognitive and gait performance in iNPH patients. Furthermore, T-tau and NfL were able to predict postoperative MMSE results, and T-tau was able to predict an improvement of 20% or more in postoperative gait velocity.To our knowledge, this is the first study to report this broad preoperatively-collected CSF biomarker profile of iNPH patients in comparison with their MMSE scores and gait velocity. Our data showed that T-tau, P-tau181 to predict postoperative MMSE values and thus support the ability of T-tau and P-tau181 to predict postoperative cognition [181 correlated only to the postoperative MMSE, we found P-tau181 to correlate with both pre- and postoperative MMSE values. This difference may be due to the larger number of patients within our study. Additionally, we performed CSF P-tau181-derived group comparison (27.5 ng/l cut-off) against the pre- and postoperative MMSE values. The results were similar to those previously found [181 group resulting in significantly worse postoperative MMSE score , where the low CSF P-tau181 and T-tau groups had a significantly increased chance of MMSE above 26 postoperatively (Table 181 could predict gait outcome measured with NPHGS. Since the gait domain of NPHGS is derived mostly from gait velocity, we consider our results to partly support these earlier findings.The role of biomarkers in defining clinical symptoms has not been established previously with iNPH patients. Nonetheless, our findings verify previous results of the ability of preoperatively obtained lumbar CSF P-tauognition –14. Contognition , who reply found , as the 42, as previously reported [42 (Fig. APOE genotype, we found a weak association with CSF Aβ42, as expected. In regression modelling, the absence of the APOE ε4 allele predicted gait improvement of 20% postsurgery. The correlation seen for CSF MCP1 to outcome and incontinence in the Finnish cohort was interesting as well, evoking the question of whether underlying inflammatory mechanisms are frequently seen amongst Finnish iNPH patients. This finding was not seen in the Swedish cohort contesting this result to occur by coincidence.For amyloid precursor protein-derived biomarkers, we found no direct association with shunt response, corroborating the results of a previous study . Howeverreported . Interes[42 Fig. C. Regard181. In logistic regression modelling, patients with low CSF NfL were significantly more likely to have postoperative MMSE scores above 26 (Table 181 (Table 181. We consider this difference to derive from the different origins of NfL being a large calibre myelinated axon protein [181. In previous studies, NfL has been stated to be elevated in several neurodegenerative diseases, including iNPH, when compared to healthy individuals, mirroring both acute and subacute changes in brain metabolism [The NfL concentrations showed similar associations with MMSE and gait velocity values as T-tau and P-tau26 Table . Furthertabolism . Consequ181 and NfL reported is their ability to reflect acute and subacute neuropathologic processes [181 and NfL CSF concentrations and more severe symptomatology probably represent ongoing damage to the brain parenchyma. More precisely, the progressive neurodegeneration seen in iNPH might derive from reported impaired CSF circulation [A possible explanation for the prognostic features of T-tau, P-taurocesses , 29. Therocesses , 30. Theculation . With sh181, with the MMSE and gait velocity observed postoperatively is potentially caused by other underlying neuropathological processes, such as AD. Low Aβ42 and elevated P-tau181 concentrations of CSF are characteristics of comorbid AD in iNPH [181-derived group comparison (Fig. 181 concentrations obtained presurgery. When comparing the Aβ42 compositions between the P-tau181 groups, a consistently lower concentration of Aβ42 was seen in the high-P-tau181 group. On the other hand, the improvement of MMSE in the low-P-tau181 group might present the established hypothesis of pure iNPH pathophysiology with reversible cognitive impairment since the low CSF tau proteins are stated to be a characteristic domain for iNPH [The other, more frequently discussed hypothesis for the negative correlation of CSF tau proteins, especially P-tau in iNPH . This hy in iNPH . In addifor iNPH , 33. RegOur second intent was to determine plausible differences between two separate cohorts of iNPH patients from different populations of Sweden and Finland. Our data showed that both cohorts were comparable in baseline characteristics and had equivalent shunt surgery outcomes despite the diverse diagnostic protocols Table , Fig. 6.Our challenge in this study was the various measures for symptoms since the Kuopio University Hospital patients were graded using iNPHGS and the Sahlgrenska University Hospital patients were graded using the iNPH scale although the diagnostic tools of MMSE and gait velocity used in both centres were comparable. However, using the MMSE only most likely fails to consider all the cognitive dimensions seen with iNPH patients . Further181 was the best predictor of preoperative MMSE scores. NfL and T-tau combined with Aβ38 predicted postoperative MMSE results with the highest accuracy. The notable finding was also that a higher T-tau cut-off of 306 ng/l could predict at least mildly demented patients postoperatively by a specificity of 96.7%. In gait improvement, T-tau and APOE genotype were found to be the only significant variables in the multivariate model to have the ability to predict 20% postoperative gait improvement. These results highlight the usefulness of NfL- and tau-derived CSF biomarkers. Moreover, a better prognostic value is achieved by using the combination of CSF biomarkers that are reflecting different components of neurodegenerative processes. However, we cannot rule out shunt operations based on this study and further research is still needed for the clinical applications of these biomarkers.We performed univariate and multivariate logistic regression modelling with ROC curve-derived biomarker cut-offs to validate the best predictive CSF biomarker combination for pre- and postoperative MMSE among patients with over 20% improvement in postoperative gait velocity Table . To our 181 appear to be the best predictors both for pre- and postoperative MMSE scores. In addition, preoperative T-tau may have potential for the prediction of gait velocity outcomes after shunt surgery.The patients in the study cohorts were comparable in baseline characteristics and showed equivalent shunt surgery outcomes. CSF biomarkers of neurodegeneration appeared to correlate with pre- and postoperative cognition, providing a window into neuropathological processes. CSF T-tau and P-tau