Non-alcoholic fatty liver disease (NAFLD) occurs in 25% of the global population and manifests as lipid deposition, hepatocyte injury, activation of Kupffer and stellate cells, and steatohepatitis. Predominantly expressed in hepatocytes, the augmenter of liver regeneration (ALR) is a key factor in liver regulation that can alleviate fatty liver disease and protect the liver from abnormal liver lipid metabolism. ALR has three isoforms , amongst which 23-kDa ALR is the most extensively studied. The 23-kDa ALR isoform is a sulfhydryl oxidase that resides primarily in the mitochondrial intermembrane space (IMS), whereby it protects the liver against various types of injury. In this review, we describe the role of ALR in regulating hepatocytes in the context of NAFLD. We also discuss questions about ALR that remain to be explored in the future. In conclusion, ALR appears to be a promising therapeutic target for treating NAFLD. NAFL is characterized as simple steatosis without histological evidence of hepatocyte injury or inflammation, whereas NASH occurs with the presence of hepatic inflammation and ballooning degeneration . When fuWith economic growth and changing lifestyles, the prevalence of NAFLD has increased rapidly to become a global burden. NAFLD occurs in 25% of the general population, especially in developed countries . It is eThe pathogenesis of NAFLD is not fully understood. Currently, the “multiple- or continuous-hit” hypothesis is the most accepted explanation . An unhePNPLA3 gene as the most important genetic factor related to NAFLD to date (PNPLA3(148M) is a major risk factor for fatty liver, as it promotes steatosis through comparative gene identification 58- (CGI-58-) dependent inhibition of adipose triglyceride lipase (ATGL) during the progression of liver disease. CGI-58 is a cofactor of ATGL that significantly enhances the triglyceride (TG) hydrolase activity. Wang et al. speculated that the accumulation of PNPLA3(148M) sequestered the function of CGI-58, thereby limiting its access to ATGL or other lipases  (MBOAT7 gene encodes lysophosphatidylinositol acyltransferase 1 (LPIAT1), which preferentially binds arachidonic acid to PI  , is alsotol (PI) . The MBOid to PI , which ireatment .TM6SF2 gene encodes a protein involved in regulating hepatic TG secretion. A glutamic acid to lysine substitution at amino acid position 167 of the TM6SF2 protein (E167K) disrupts the secretion of very low-density lipoprotein (VLDL). Deletion of TM6SF2 resulted in abnormal VLDL-TG secretion, which progressed to hepatic steatosis  variant. Knockdown of TM6SF2 in Huh7 and HepG2 cell lines reduced the expression of diacylglycerol O-acyltransferase 1 and 2 , lamin A/C (LMNA), and hormone-sensitive lipase genes, are potential therapeutic targets for NAFLD . Simultaneously, the accumulation of FFA promotes intracellular TG accumulation (LMNA(D300N)  . A polymA(D300N) .With economic growth, the global prevalence of metabolic syndromes, such as obesity, diabetes, and dyslipidemia, increases annually . ExcessiMitochondrial dysfunction is frequently related to the development of NAFLD. Indeed, structural and functional alterations of mitochondria significantly contribute to changes in cellular lipid metabolism and oxidant stress responses . DomínguIn addition to hepatocytes, KCs and HSCs are associated with the occurrence of NAFLD and progression to NASH during different stages of the NAFLD spectrum . The KCsChronic inflammation sustains these inflammatory stimuli and induces HSCs to initiate fibrotic processes. Located in the space of Disse in the liver, HSCs are physically quiescent cells that function as a store of vitamin A. Following sustained inflammation, HSCs are activated by cytokines and free radicals released from the surrounding cells, such as hepatocytes, T cells, and KCs. Once activated, HSCs undergo a phenotypic switch from adipocyte-like quiescent cells to myogenic cells with increased protein expression of alpha-smooth muscle cell actin (α-SMA) and extracellular matrix (ECM) proteins . IncreasCollectively, the crosstalk among hepatic cells substantially contributes to the development of NAFLD . Thus, dALR was first identified in 1975 in crude extracts of liver homogenates in weaning rats . InjectiGFER) is located on chromosome 16 and consists of three exons and two introns , forkhead box A2 (FOXA2), early growth response protein 1 (Egr-1), and hepatocyte nuclear factor 4α (HNF4α). Repressors include activator protein 1/activator protein 4 (AP1/AP4), CCAAT/enhancer binding proteins (C/EBPβ), and HNF4α  induces GFER expression (The promoter region of nd HNF4α . Bindingpression . The dowpression .GFER promoter region. An upstream antioxidant response element (ARE) located between −27 and −19 bp induces GFER expression upon binding nuclear factor erythroid 2-related factor 2 (Nrf2) when hepatocytes are exposed to oxidative stress  AMPK-sterol regulatory element binding protein 2- (SREBP2-) low-density lipoprotein receptor pathway. LKB1 is an upstream activator of AMPK. Knockdown of ALR expression inhibits LKB1 phosphorylation, leading to AMPK inactivation and SREBP2 maturation/nuclear translocation. SREBP2 and low-density lipoprotein receptor actions are closely associated with cholesterol accumulation within hepatocytes. Thus, alterations in these events can lead to extensive cholesterol accumulation and the development of lipid metabolism disorders  expression to enhance long-chain fatty acid transport into mitochondria for usage . DynaminMost soluble IMS proteins rely on a mitochondria-targeting sequence for import, and ALR participates in protein import and export by cooperating with Mia40 . Mia40 iGandhi et al. successfully developed mice with liver-specific depletion of ALR (ALR-L-KO), which showed that a lack of ALR accelerated the development of steatohepatitis and hepatocellular carcinoma . Two weeN-acetylcysteine and recombinant ALR rescued anti-oxidative stress responses by suppressing miR-540 expression and lipid accumulation in ALR-deficient hepatocytes. In agreement with these results, the exogenous administration of recombinant ALR to ALR−/−KO mice inhibited miR-540 expression and steatosis , CPT1-α, peroxisomal membrane protein 70 (PMP70), and acyl-CoA oxidase 1 (ACOX1) are a series of antioxidant proteins, which are targeted by miR540 . In ALR-teatosis .in vitro, caspase-3 activity and Bax protein expression were reduced, therefore reducing lipotoxicity. These results indicate that ALR exerted its lipid-lowering and anti-apoptotic actions by elevating the mitochondrial FFA transporter CPT1α, increasing toxic FFA β-oxidation in mitochondria, and decreasing the delivery of toxic FFA metabolites. In vivo, reduced mRNA levels of ALR and FOXA2 (a transcription factor inducing ALR expression) were found in mice fed a high-fat diet, human patients with steatosis, and NASH liver samples. These results demonstrate the role of ALR in reducing lipid deposition and increasing β-oxidation in patients with NASH   .via the microRNA-26a/p-Akt/cyclin D1 pathway  play a protective role in liver regeneration , and a rvia high-affinity receptors. The activation of KCs induces the release of various cytokines that trigger hepatocyte proliferation.Acute response cytokines, such as IL-6 and TNF-α, are mainly released from KCs and are associated with hepatocyte proliferation . As descTherefore, there is no evidence for a direct anti-fibrotic effect of ALR on hepatocytes. Nevertheless, ALR inhibits fibrotic progression in the liver by suppressing hepatic stellate cell (HSC) activation in NAFLD.Among all inflammatory cytokines, TGF-β1 is the most potent stimulator of HSC activation . The binMetalloproteinases, which play a dominant role in ECM degradation, are inhibited by tissue inhibitors of metalloproteinase (TIMP). Of the four TIMPs, only TIMP-1 and TIMP-2 are detected in liver tissue, with TIMP-1 expression being more pronounced than TIMP-2. In a rat hepatic fibrosis model induced by porcine serum injection, the administration of ALR plasmid decreased the expression levels of TIMP-1 mRNA and protein and was accompanied by reduced deposition of collagen I and collagen II in the liver .In vitro, ALR knockdown by shRNA promoted mitochondrial fission and elongation, which led to enhanced ATP production for HSC migration. Moreover, the proportions of F-actin and G-actin were higher in ALR-deficient HSCs following shRNA transfection. Conversely, ALR overexpression slowed HSC migration by reducing energy supply and inhibiting mitochondrial fusion (Cell motility is ATP-consuming and mediated by microfilament assembly. l fusion .The proposed mechanism by which ALR protects hepatocytes in NAFLD is summarized in Nearly 50 years of research on ALR has consistently demonstrated its involvement in the spectrum of NAFLD. Despite this progress, how ALR expression is regulated in the context of NAFLD is not well defined. Moreover, most ALR studies focus on the liver. Thus, whether ALR in other organs, such as the kidney and brain, communicate with the liver to participate in NAFLD remains an open question. Additionally, although 21-kDa ALR is one of the main isoforms, its role is not fully understood. There are still mysteries surrounding ALR worth exploring in the future.In conclusion, ALR promotes mitochondrial homeostasis, protects hepatocyte survival and function, and suppresses macrophage and HSC activation. Collectively, these features make ALR a potential therapeutic target for the treatment of NAFLD.


Monoclonal antibodies play an important role in the treatment of various diseases. However, the development of these drugs against neurological disorders where the drug target is located in the brain is challenging and requires a good understanding of the local drug concentration in the brain. In this original research, we investigated the systemic and local pharmacokinetics in the brain of healthy rats after either intravenous (IV) or intracerebroventricular (ICV) administration of EGFRvIII-T-Cell bispecific (TCB), a bispecific monoclonal antibody. We established an experimental protocol that allows serial sampling in serum, cerebrospinal fluid (CSF) and interstitial fluid (ISF) of the prefrontal cortex in freely moving rats. For detection of drug concentration in ISF, a push-pull microdialysis technique with large pore membranes was applied. Brain uptake into CSF and ISF was characterized and quantified with a reduced brain physiologically-based pharmacokinetic model. The model allowed us to interpret the pharmacokinetic processes of brain uptake after different routes of administration. The proposed model capturing the pharmacokinetics in serum, CSF and ISF of the prefrontal cortex suggests a barrier function between the CSF and ISF that impedes free antibody transfer. This finding suggests that ICV administration may not be better suited to reach higher local drug exposure as compared to IV administration. The model enabled us to quantify the relative contribution of the blood-brain barrier (BBB) and Blood-CSF-Barrier to the uptake into the interstitial fluid of the brain. In addition, we compared the brain uptake of three monoclonal antibodies after IV dosing. In summary, the presented approach can be applied to profile compounds based on their relative uptake in the brain and provides quantitative insights into which pathways are contributing to the net exposure in the brain. The applied method allowed for serial sampling of drug concentration over 48 h in serum, CSF and ISF of brain parenchyma. In addition, we compared the brain uptake of EGFRvIII-TCB with two additional tool compounds (DP47-TCB and DP47-IgG) after IV administration. All three tested mAbs show no cross-reactivity with targets in the rat and therefore no target-binding is expected, so the disposition of the molecules can be compared based on their molecular properties. We selected the molecules since both, EGFRvIII-TCB and DP47-TCB have the same molecular format and molecular weight (MW = 195 kDa) but different antigen binding fragment (Fab) regions exhibiting different biophysical properties i.e. charge patches. DP47-IgG has a lower molecular weight (MW = 143 kDa) and contains the same two Fabs as DP47-TCB but lacks the CD3-binding Fab. For quantitative interpretation of the PK data and to provide insights into physiologically relevant processes in the brain uptake, we developed a reduced Physiologically-Based PK model (rPBPK) which was built and refined based on previous published models  or intravenous (IV) administration in rats using a push-pull microdialysis technique with large pore membranes. In this study, we tested EGFRvIII-TCB , a T-celd models . In concThree different monoclonal antibodies were used as tool compounds in this study: EGFRvIII-TCB , DP47-TCB (MW = 195 kDa) and DP47-IgG (MW = 143 kDa). EGFRvIII-TCB was designed in the 2 + 1 heterodimeric format that consists of two antigen binding fragments (Fab) specific for the EGFRvIII and one Fab recognizing the CD3Ɛ chain of the T-cell receptor as described  and an Fin vitro recovery experiments. Probes were positioned in a beaker containing 1,000 ng/ml of antibody diluted in artificial CSF  + 0.2% BSA. The beaker contents were continuously stirred and kept at a constant temperature of 37°C. The probes were positioned in the beakers and perfused with aCSF +0.2% BSA at a flow rate of 0.5 µl/min. Following pre-stabilisation, six microdialysis samples were collected in 30 min intervals. In addition, beaker content reference samples were collected at the start and the end of the experiment. Recovery was calculated as the average concentration in the microdialysate samples divided by the average concentration in the beaker samples. For EGFRvIII-TCB, an in vitro recovery rate of 14 % and 10% for DP47-IgG and for DP47-TCB were determined. The measured drug concentration in microdialysate was converted into the respective drug concentration in ISF of PFC by accounting for the respective in vitro recovery.The relative recovery of antibodies using the push pull PP-PE 6/4 probes  was determined by in vivo experiments. Experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals  and were in accordance with European Union directive 2010/63 and the Dutch law. The experiments were carried out under a license, issued by the national committee for licensing of animal experiments  and were approved by the Animal Care and Use Committee (Instantie voor Dierenwelzijn) of CRL Groningen.A total of 25 adult male Wistar rats  were used for the ad libitum.Following arrival, animals were housed in groups of up to five in polycarbonate cages (65 × 33 × 20 cm) with wire mesh top in a temperature (22 ± 2°C) and humidity (55% ± 15%) controlled environment on a 12 h light cycle (07.00–19.00 h). After surgery, animals were housed individually in polypropylene cages (30 × 30 × 40 cm). Standard diet  and domestic quality mains water were available Animals were anesthetized using isoflurane (2.5%–3% and 800 ml/min O2). Before surgery, flunixin  was administered once for analgesia during surgery and the post-surgical recovery period. A mixture of bupivacaine and epinephrine was applied to the incision site and to the periost of the skull for local analgesia.Each animal was placed in a stereotaxic frame  and PP-PE 6/4 microdialysis probes  were positioned in the left prefrontal cortex . In addition, an injection guide suited for compound administration , was positioned in the right lateral ventricle . All coordinates were based on .In the same surgical procedure an indwelling cannula was placed in the jugular vein  to allow for blood sample collection. A stainless steel cannula was placed in the cisterna magna  to allow for CSF sample collection. The probe, injector guide, and cannula were fixed in position and attached to the skull with stainless steel screws and dental cement.via the tail vein). Microdialysates were collected in 30-min intervals during the first 8 h post-dose. From 8 h post-dose until 48 h post-dose samples were collected in 4-h intervals. Blood samples and CSF samples were collected at t = −0.5, 0.5, 1, 3, 4.5, 6, 12, 24, and 48 h post-dose via the respective cannulae positioned in the jugular vein and cisterna magna. Blood samples were allowed to clot and centrifuged to obtain serum.After a day of recovery, the push pull probes were connected to a microperfusion pump  with PEEK tubing . The probes were perfused with a CSF +0.2% BSA at a flow rate of 0.5 µl/min. Following 2 h of pre-stabilization, microdialysate sample collection was initiated. At t = 0 min, antibody in physiological buffer formulation  was administered intracerebroventricular via the injection guide  or intravenously  and inflammation markers  were at baseline after 1 day of recovery. Push pull microdialysis procedures are performed using a gravity pull and concomitant validation of the flow by weighting each sample, rather than the active pull using a perfusion pump. Both in house historic and published data  support The experiments were performed in freely moving awake animals which were continuously monitored. No behavioral change was observed during or immediately following the administrations by infusion suggesting there was no marked change in pressure on the ventricle. In addition, prior to dose administration a basal CSF sample was collected which will have temporarily caused a minor decrease in total CSF volume, and thus reducing potential overall relative volumetric concerns. Similar administrations in the lateral ventricle have shown no effects on pressure or change to ventricular size as followed by MRI .Bioanalytical assays were developed for measurement of the three tested drugs  in rat serum samples using an enzyme-linked immunosorbent assay (ELISA). Capture of EGFRvIII-TCB was done with biotinylated EGFRvIII antigen, while DP47-TCB or DP47-IgG was captured by an anti-DP47 antibody. The bound drug was detected using digoxigenin-labeled anti-PG antibody  followedNoncompartmental analysis (NCA) was performed in MATLAB Simbiology (version R2021b) using the linear trapezoidal rule for interpolation. Each rat was treated as an individual subject and NCA parameters have been reported as mean ± standard deviation. Areas under the concentration versus time curves (AUC0→t-last) in serum, ISFbrain, and CSF were calculated for the tested antibodies. The maximal drug concentration in serum (Cmax) and time to maximal concentration (Tmax) were derived.A reduced brain PBPK (rPBPK) model was built to describe the uptake of mAbs into CSF and ISFbrain after ICV and IV dosing. The final model structure is shown in The following components of the structural model were included: 1) the model accounted for the physiological compartments such as brain vascular and interstitial space of the brain parenchyma, the CSF compartment consisting of lateral ventricles (LV), third and fourth ventricles (TFV), cisterna magna (CM), and subarachnoid space (SAS), serum, lymph, and a lumped rest-of-body compartment. 2) The brain compartment was described with volumes of CSF and ISFbrain compartments. 3) Brain uptake was described by convection through large pores and transcytosis. These processes are governed by the physiological flows in the brain. A reflection coefficient (1- σ) with σ ∈  is also added to account for permeability limitations for molecules across the brain barriers. These reflection coefficients are drug-related parameters and vary between different mAbs. 4) The model further assumes a barrier function between the CSF and ISFbrain, which is captured with another reflection coefficient limiting the flow of antibody from CSF to the ISFbrain. The final structural model is presented in 1) The brain is described by three main components: brain vasculature, CSF compartment, and the ISFbrain in the brain parenchyma. The CSF compartment is subdivided into the lateral ventricles (LV), third and fourth ventricles (TFV), cisterna magna (CM), and subarachnoid space (SAS). This was done to account for ICV dosing that is administered into the LV, while CSF sampling took place in CM. The respective physiological parameters such as CSF flows and compartment volumes were taken from . Except 2) Since there is only negligible elimination through lysosomal degradation expected in the brain , there w3) The uptake into the CSF and ISFbrain of the brain parenchyma were described as net uptake, and no distinction between transcellular and paracellular transport was made. The net brain uptake was described as a lumped or hybrid reflection coefficient (σuptake) across either the BBB or BCSFB.4) A reflection coefficient was included to restrict the flow of antibodies between the CSF and ISFbrain (σCSF_ISF).The model structure has the following assumptions:In line with existing knowledge on CSF flow physiology  and as cThe newly developed reduced PBPK model consists of 13 ODEs and 32 parameters. Of those parameters, 28 were fixed to reported physiological values  and fourn = 15). First, the model was fit to serum PK data after IV dosing in order to estimate the endosomal degradation rate (kdeg). A proportional error model was used. The obtained parameter value was then fixed and three parameters related to brain uptake  were estimated by fitting the model simultaneously to CSF, and ISFbrain data sampled from the three dosing groups of EGFRvIII-TCB . A constant error model was assumed.The rat PK data from EGFRvIII-TCB, DP47-IgG, and DP47-TCB were modeled separately and using a two-staged approach. For EGFRvIII-TCB, a naive-pooled approach was used to fit the model to the rat data (n = 5) and DP47-TCB (rats n = 5). First, the model was fit to serum PK data in order to estimate the systemic elimination kdeg. A proportional error model was used. In the second step, only two drug-related parameters  were estimated while the third parameter (σCSF_ISF) was fixed to the value obtained from EGFRvIII-TCB. The model was fitted simultaneously to CSF, and ISFbrain data sampled after 15 mg/kg IV dosing. A constant error model was assumed. In order to provide a quantitative assessment of model performance, we calculated the prediction error (PE%) using the equationA similar modeling strategy was applied, independently, for DP47-IgG  were similar for EGFRvIII-TCB , DP47-IgG , and DP47-TCB . However, total drug exposure reported as AUC0-48h of EGFRvIII-TCB was 40%–46% lower than those of DP47-TCB and DP47-IgG, respectively. The exposure in ISFbrain versus serum was overall higher for EGFRvIII-TCB (0.41–1.71%) compared to DP47-IgG (0.19%–0.87%) and DP47-TCB (0.16%–0.64%). The NCA results are summarized in Next, we compared how well the recently proposed minimal PBPK (mPBPK) model  predictsBased on these insights, we developed and evaluated our proposed reduced brain PBPK model  and asseFurthermore, the model described the PK of EGFRvIII-TCB in serum, CSF, and ISFbrain after IV administration of 15 mg/kg , and aftvia the CSF-ISF barrier  amino acid sequence and therefore biophysical properties like surface charge. While EGFRvIII-TCB and DP47-TCB have the same molecular format (2 + 1) and molecular weight (MW = 195 kDa), EGFRvIII-TCB has a positive charge patch in the Fab regions specific for the EGFRvIII  which isvia BBB or BCSFB as well as their respective relative contributions to the uptake into the ISFbrain. The proposed model has a reduced complexity and may facilitate the quantitative interpretation of drug uptake through the BBB and BCSF barriers. This will in turn allow for easier cross-compound comparison, as we illustrated with the three compounds investigated.In the present work, we conducted a mechanistic PK study to assess and quantify the brain uptake of EGFRvIII-TCB after IV and ICV dosing in rats. In addition, we compared the brain uptake after IV dosing of EGFRvIII-TCB with two other tool compounds. In order to quantify the local drug exposure in the brain, we collected CSF by sampling from the cisterna magna and applied a push-and-pull microdialysis system using microdialysis probes with high molecular weight cutoff membranes that allow recovery of antibodies and proteins from the interstitial fluid  of the pin vivo measurement of free antibody concentration in selected regions of the brain in freely moving animals  to better reflect the dosing and sampling sites, which were spatially separated as suggested by . Since tgligible  and sincgligible  that incgligible  confirmegligible . The par and Lee  propose lleagues  have desDP47 Fab .It has been discussed that the CSF may contribute to the drug uptake into ISFbrain . To our It is generally accepted that free fluid transfer is possible between the CSF and ISFbrain across the ependymal cell layers in the ventricles, which distinctively lack tight junctions . The comWhile we were able to estimate the reflection coefficient σCSF-ISF from simultaneous fitting of ICV and IV data, we could not conclude if this is a drug-dependent parameter that may also vary between the compounds. It should also be considered that the parameter estimate may be confounded by experimental conditions. CSF was sampled from the cisterna magna, which is proximal to the site of entry into the ISFbrain. ISFbrain was sampled from the prefrontal cortex. When we consider the distal location of the prefrontal cortex relative to the cisterna magna, and the multidirectional CSF flow within the subarachnoid space as well as CSF outflow at the arachnoid villi and towards the lymphatics, it is possible that part of the antibodies in SAS got diluted and cleared. Additionally, there is a high tortuosity within the brain’s extracellular space, which prevents bulk flow . This imA limitation of our experimental design was the large injection volume required for ICV dosing, which exceeded the volume of a lateral ventricle. Although no changes in animal behavior were observed during as well as immediately after the administration and basal CSF sample collection decreased the relative volume prior to start of the infusion, it is unclear which impact the additional volume has on CSF hydrostasis and how this may influence the PK of EGFRvIII-TCB.Lastly, a key limitation of the proposed reduced PBPK model is that is relies on physiological parameters with uncertainty in their respective values such the flow and production rates of CSF and ISF . In addivia BBB. For further investigation, additional studies are proposed to analyze more compounds that differ in size and with varying biophysical properties.A recent study by Chang and colleagues  suggestsIn this work, we conducted a mechanistic PK study to investigate the disposition of antibody based therapeutics to the brain appling microdialysis. The PK data in CSF, ISFbrain and serum have been analyzed with a reduced brain PBPK model to assess the relative contributions of net uptake via BBB and BCSFB barriers. The modeling results suggest that there is a limited transfer of antibody directly from CSF to ISFbrain. This apparent barrier between CSF and ISFbrain could be explained by preferential size exclusion in the perivascular spaces, but is likely also the result of antibody dilution and removal from the CSF, leading to lower than expected levels of subarachnoid-parenchymal transport. This may explain why ICV dosing did not result in higher ISFbrain exposure as compared to the IV route of administration and may have implications on the choice of the preferred administration route. The proposed model provides mechanistic insights into physiological processes related to brain uptake such as the relative contributions of the blood-brain and blood-CSF barriers to the total brain uptake.


Background: Anterior pituitary hormones in blood follow a circadian rhythm, whichmay be influenced by various factors such as intracranial pathologies. Incerebrospinal fluid (CSF), pituitary hormones have been collected onlyselectively and circadian rhythm has not yet been investigated. This pilot studyanalysed diurnal variations of anterior pituitary hormones in patients inneurocritical care to determine whether circadian rhythmicity exists in thesepatients. Possible influences of intracranial pathologies were alsoinvestigated. Blood and CSF concentrations were assessed simultaneously toexplore the value of blood concentrations as a surrogate parameter for CSFlevels. Methods: Blood and CSF samples of 20 non-sedated patients were collectedat 06:00, noon, 18:00 and midnight, and analysed for adrenocorticotropic hormone(ACTH), cortisol, thyroid-stimulating hormone (TSH) and insulin-like growthfactor-1 (IGF-1) concentrations at each of the four time points. ACTH and IGF-1were measured by sandwich chemiluminescence immunoassay. Cortisol and TSH weremeasured by electrochemiluminescence immunoassay. Results: Results showedinconsistent circadian rhythms. Less than 50% of the patients showed a circadianrhythmicity of ACTH, cortisol, TSH or IGF-1. Significance of diurnal variationswas only present for blood concentrations of TSH. Correlations between blood andCSF concentrations were strong for cortisol and TSH. Conclusions: CSFconcentrations were only in the measurable range in some of the patients. Noclear circadian rhythmicity could be identified, except for TSH in blood.Absence of significant diurnal variations could be explained by the underlyingpathologies or disturbing influences of the intensive care unit. Bloodconcentrations of cortisol and TSH may be suitable surrogate parameters forCSF. A more complex rhythm with several maxima and minima, is expected for thesomatotroph axis, especially for growth hormone (GH), whereas IGF-1 levels in bloodshowed maxima in the morning and minima in the early evening.2 Disturbance of pituitary hormone homeostasis can havefar-reaching consequences such as influencing heart rate, blood pressure and glucosehomeostasis, and account for muscle wasting and memory impairment.1In physiological conditions, the pituitary gland contributes to proper body functions andhomeostasis. The assumed circadian rhythm of anterior pituitary hormones seems to be animportant part of the hormonal balance. Cortisol release triggered byadrenocorticotropic hormone (ACTH) forms the main part of the body's ‘stressresponse’, which is crucial for a patient's reaction to critical events such astrauma or operations. The circadian rhythm of serum cortisol and ACTH is described inthe established literature with minima at midnight and maxima in the earlymorning.4 As many body functions are modulated bydiurnal variations of plasma glucocorticoid levels, it might be possible thatdisturbances of circadian rhythmicity lead to hormonal imbalances that essentiallyinfluence clinical outcome.Circadian rhythmicity can be disrupted by elements such as brain injury, medication orunnatural environments including those with noise and artificial light, which are commonin a modern intensive care unit (ICU), and these disruptors have been discussedextensively in the literature.6Central nervous system (CNS) studies on pituitary hormones and peptides mostly concernsex steroids and the hypothalamic neuropeptides oxytocin and arginine-vasopressin. Thesesubstances seem to exert cognitive and behavioural as well as neuroprotectiveeffects.8 and they are mostly confined to daily samples; studiesperforming serial measurements are scarce.9As the brain is not directly accessible and cerebrospinal fluid (CSF) collection involvesinvasive procedures, little is known about the occurrence and metabolism of anteriorpituitary hormones in the CNS of humans. Only a few predominantly older studies on thistopic existThe aim of this study was to investigate circadian rhythmicity of pituitary hormones ofawake patients with intracranial pathologies in the absence of sedatives. We wished todetermine: 1) whether anterior pituitary hormones are detectable in CSF using commonmethods; 2) whether these CSF concentrations reflect blood concentrations; and 3)whether there is also a circadian rhythmicity in the CNS, as described for bloodconcentrations of certain hormones such as cortisol.The study cohort included non-sedated, awake patients with external ventriculardrainage. Only patients who were orientated and able to give written informedconsent were included in the study. Exclusion criteria were underage patients,application of catecholamines or sedatives, CSF infections , cortisolsubstitution and refusal to participate in the study. Patients underwent follow-up 3months after disease onset. All but three patients had good neurological outcome andwere largely independent in everyday life. Thus, we were able to recruit arelatively homogeneous cohort for the study. The study was approved by the ethicscommittee of the Technical University of Munich, School of Medicine (referencenumber 5459/12).Blood and CSF were collected from a pre-existing arterial line and externalventricular drainage. Collection was scheduled at 06:00, noon, 18:00 and midnight.Each sample was analysed for cortisol, ACTH, TSH and IGF-1 levels, amounting to atotal of eight data points per patient per point in time.The samples were analysed by the institutional Department of Clinical Chemistry. Forblood concentrations, the laboratory reference range was taken as the benchmark fornormal values. For CSF concentrations, no reference ranges exist in the routinediagnostics of our hospital.® XL analyser.Both CSF and blood levels of ACTH and IGF-1 were measured by sandwichchemiluminescence immunoassay using a Liaison® analyser  for cortisol and a cobas8000® analyser  for TSH.Cortisol and TSH levels in blood and CSF were determined by electrochemiluminescenceimmunoassay using a cobas e411Hormone concentrations in blood and CSF were first analysed at an individual level.The analysis determined the detectability of the hormones with common analyticalmethods and then, for blood concentrations only, the position within or outside thereference range. The temporal course in the individual patients was graphicallydisplayed and visually checked for the presence of a possible circadian rhythm. In asecond step, cumulative analysis of the entire patient cohort was carried out.1 For IGF-1, maxima inthe early morning and minima in the early evening are described.3Circadian rhythm was defined as minima and maxima at the time points described in theestablished literature. This means that cortisol and ACTH show maxima in the earlymorning and minima at midnight; TSH would have its maximum at midnight and minimumaround noon.®version 25  and R® version 3.5.2 and3.1.0 . Thesignificance of diurnal variations of hormone levels was determined by Friedman testand, if appropriate, verified by Wilcoxon and Bonferroni test as posthoc analyses. Correlations between blood and CSF levels were determinedusing Spearman's rank correlation coefficients. A p value of <0.05 wasconsidered statistically significant.Statistical analysis was performed using IBM SPSS StatisticsFor statistical calculations, concentrations below the assays' detection limit werehandled as being equal to the threshold.A total of 20 patients were included in the study . Regarding diagnoses, 15 patients suffered aneurysmaticsubarachnoid haemorrhage (Hunt and Hess II–IV), three patients showedhydrocephalus malresorptivus after resection of intracranial tumours, and twopatients suffered traumatic subarachnoid haemorrhage.Table 1). CSFcortisol and TSH concentrations corresponded to values described in theliterature, whereas ACTH and IGF-1 concentrations were lower in ourstudy.13In most cases, hormone concentrations in the blood could be determined. In theCSF, ACTH and IGF-1 were below the detection limit in the vast majority of cases were found in 12 patients. Onesample at 18:00 was missing due to an intervention outside the ward.Figure1).1Four patients showed physiological rhythm in blood as described inestablished literature, which is comparable to the assumed rhythm of ACTH.Only one patient showed ACTH levels in CSF above the threshold ofdetermination at all sampling time points. CSF concentrations ranged from4.4 pg/mL to 5.6 pg/mL. A total of 13 patients consistently had nomeasurable ACTH levels in CSF. No circadian rhythm could be shown.Figure 1).1Six patients experienced the assumed circadian rhythm in blood, with maximain the early morning and minima around midnight .CSF cortisol levels were measurable for all patients. Concentrations rangedfrom 0.1 μg/dL to 8.0 μg/dL, with a median of 1.58μg/dL. Seven patients experienced a circadian rhythm of cortisol inCSF similar to that assumed in blood  at each timepoint.Figure 2).Five patients experienced the assumed circadian rhythm in blood, with maximaat midnight and minima around noon .1TSH CSF concentrations were entirely above the threshold of determination.Concentrations ranged from 0.01 μlU/mL to 0.52 μlU/ mL in CSF,with median of 0.03 μlU/mL. One patient showed a circadian rhythmsimilar to that assumed in blood . No patient showed reducedIGF-1 concentrations compared with the normal range. IGF-1 in blood rangedfrom 80 ng/mL to 413 ng/mL, with a median of 171.5 ng/mL.1 .Two patients showed a circadian rhythm of IGF-1 in blood, with maxima in theearly morning and minima in the early eveningFigure3).Three patients showed measurable IGF-1 levels in CSF consistently. For 11/20patients, IGF-1 levels could not be determined in any CSF sample.Concentrations ranged from 3 to 8 ng/mL, with a median of 4 ng/mL. Onepatient showed a value of 153 ng/mL in CSF. Considering the other IGF-1concentrations in CSF, we viewed this value as an outlier value. Nocircadian rhythm was apparent . CSF ACTH and IGF-1 levels were below the detection limit in more than 50% ofthe collected samples, and therefore we did not perform an analysis fordiurnal fluctuations in the whole patient cohort in these cases.Figure 4 shows asummarized representation of the blood and CSF concentrations of allavailable measurements at the individual sampling times in the form ofboxplots. Medians and interquartile ranges are depicted in Table 2.Figure5). The strongest correlation between blood and CSFvalues was found in midnight samples . All correlationcoefficients are shown in Table3.Correlations between blood and CSF concentrations varied depending on hormone andtime point. Four independent data sets, each consisting of 20 data points, couldbe constructed from the sample data. No significant correlations were foundbetween blood and CSF values of ACTH or IGF-1 at the different time points.Cortisol and TSH exhibited a significant positive correlation for all points intime (0.61<r<0.89)  patients had their maxima at midnight or 06:00, with minima at noon or18:00.Furthermore, only 20% of the patients in our study showed a circadian rhythm of ACTHin blood. For IGF-1 blood levels, there were even fewer patients (10%) showingcircadian rhythmicity in the blood.17Therefore, our results are similar to the findings of Schneider et al. who showedthat impairment of pituitary hormones after traumatic brain injury and aneurysmalsubarachnoid haemorrhage has a sequence: ACTH is mainly affected, TSH was influencedleast.18 Disturbances of circadian rhythm in patients inthe ICU have been described previously but, in contrast to our study, focused onanalgosedated patients.19In general, brain injury due to trauma, surgery or ischaemia may causehypothalamic–pituitary–adrenal axis disturbances, with subsequentimpaired hormone secretion and disturbed circadian rhythm.14 The authors investigated 20patients, sampling blood for cortisol and ACTH at 06:00, noon, 18:00 and midnight.Results were categorized into normal, reversed and unspecific diurnal pattern.However, the authors' definition of a physiological circadian rhythm differs fromthe description in the current article. Zetterling et al. defined‘normal’ as maximum cortisol levels at 06:00 or noon, and‘inversed’ as minimum at 06:00 or noon.14 If this definition is applied to the currentstudy, 13 out of 20 patients (65%) would exhibit a ‘normal diurnalpattern’. Thus, a change of definition would lead to markedly changedfindings regarding the prevalence of a circadian rhythm for these data.Above all, the definition of circadian rhythmicity is questionable. As conclusions onthe physiological circadian rhythm cannot directly be drawn from our patients, acomparison with similarly designed studies is reasonable. Of particular interest isthe study by Zetterling et al., because of the analogous study design.20 Theirdefinition of circadian rhythm of TSH was less strict than for the corticotroph axisand is still the current reference for a circadian rhythm of TSH. The authors'findings within a group of six subjects showed maxima at between 02:00 and 04:00,and minima between 18:00 and 20:00, results that were partially confirmed by theresults of the current study.Fundamental research of rhythmicity of TSH and the somatotroph axis was performed byPatel et al. in 1972.21 however, diurnal variations of IGF-1concentrations have, to the best of our knowledge, not been investigated todate.IGF-1 was preferred over GH in our study because the rhythmicity of GH has previouslybeen described as very complex. The role of IGF-1 in intracranial pathologies isrelatively well studied, and endogenous production in the CNS has beendescribed;2 The authorsincluded 12 patients and described a pulsatile secretion pattern. In contrast, somelaboratory datasheets point out that IGF-1 measurement is possible throughout thewhole day because of lack of circadian pattern, proving diverging views of circadianbehaviour of IGF-1.22 In thecurrent study, as far as a circadian rhythmicity of IGF-1 is concerned, 90% of thepatients showed no significant diurnal variations.Our understanding of the circadian rhythm of the somatotroph axis is based on work byWiner and Shaw 1990.23 Data onACTH in CSF are scarce and have included patients with various diseases; however,findings suggest that CSF ACTH levels are slightly higher than plasma levels andthat the blood–brain barrier is impermeable to ACTH.24 Circadian rhythmicity with a peak in the evening could be shownin primates.25 The obtainablemeasurements in our study suggest no rhythm in CSF for ACTH. Cortisol showed theexpected rhythm in CSF in only seven out of 20 patients. Our findings did do notsuggest any kind of rhythm of TSH in CSF. For IGF-1, a full set of measurements wasonly obtained for three patients. The other measurements were again below thedetection limit. Therefore, circadian rhythm of IGF-1 in CSF can be neitherconfirmed nor refuted based on these data.As CSF sampling always requires invasive procedures, little is known about pituitaryhormones in the CSF and their possible circadian fluctuations. Blood and CSFconcentrations differ: pituitary hormones in CSF are only present at a fractionalamount of serum levels.26 Therefore, thepermeability of the blood– brain barrier, as well as the blood–CSFbarrier, could also influence CSF concentrations of pituitary hormones. Thispermeability may be also affected by stress and, therefore, stressful conditions mayalter CSF concentrations. However, this fact is discussed controversially in theliterature.27 In patientswith traumatic brain injury, strong correlations of cortisol and progesterone myindicate impaired blood–brain barrier or disturbed hormone metabolism withinthe brain.28Hormone concentrations in CSF may depend not only on the molecular size but also onblood levels.29 or refuting24 a correlation. Inthe current study, the statistical evaluations showed different results for eachhormone. For cortisol and TSH, the moderate-to-strong positive correlation allows usto draw inferences about CSF levels from blood sampling. This would allow theinvestigation of larger patient cohorts in different contexts without the need forexternal CSF drainage. ACTH and IGF-1 showed only weak correlations between bloodand CSF. However, for these hormones, analysis was hindered by the fact that manyvalues were below the detection limit. This suggests that electrochemiluminescenceimmunoassay cannot be recommended for determination of ACTH and IGF-1 CSFconcentrations as this method does not seem to be sensitive enough. However, wechose these analytical methods because they can be easily applied within clinicalroutine in a hospital's laboratory. For detection of CSF ACTH and IGF-1 levels, moresophisticated methods are needed, but cortisol and TSH can be detected in the CSF bythe common routine assays used in our study.Correlation of serum and CSF levels of pituitary hormones is discussedcontroversially, with several authors either confirming5Upregulated expression of IGF-1 within the brain is described as a response topost-traumatic brain damage.21Impaired cerebral hormone metabolism could be a cause of poor outcome in acutecerebral disease. Strong positive correlations of blood and CSF concentrations mayindicate impaired integrity of the blood–brain barrier. Alternatively, a lackof correlation may hint to endogenous hormone synthesis and independent metabolismwithin the brain, as discussed for sex steroids, oxytocin andarginine-vasopressin.6 Differing diurnal secretion patternsin blood and CSF may also suggest independent peripheral and central hormonemetabolism. As circadian rhythmicity was absent in a large percentage of patients inour study, this question cannot be answered satisfactorily.Little is known about pituitary hormones and their metabolism in the CNS. Forexample, sex hormones are supposed to be synthesized within the CNS and to exertneuroprotective functions.Our study was designed to contribute to a better understanding of pathophysiologicalprocesses in neurocritical care patients, but the number of included patients wastoo small to evaluate practical implications. However, it is conceivable that theintegrity of the blood– brain barrier and the circadian rhythm may be worthmonitoring to improve patients' internal rhythm with targeted application ofhormones. This could lead to re-establishment of a patient's physiologicalcondition. Furthermore, improved monitoring may provide more information aboutprognosis and recovery processes. Until now, routine hormone measurement in blood,and especially in CSF, has been too expensive and time consuming. The results ofthis pilot study indicate that it is worth investigating these conditions in moredetail in further studies.Several limitations to our study are evident. First, due to the invasive natureof CSF sampling, only a small sample size could be generated.Second, blood and CSF samples taken at the four time points were put on ice andimmediately transported to the laboratory for further processing. A controlgroup of healthy individuals would therefore have had to be hospitalized for theobservation period of 24 hours. Unfortunately, this was not possible fororganizational and logistical reasons, so that a control group had to be omittedin this study design, even if only blood samples had been taken from it.Third, patients included in the study showed intracranial pathologies that mightcontribute to hypothalamic–pituitary disturbances. To mitigate thisfactor, only non-sedated patients undergoing normal (in the context of ICU)sleep–wake cycles were included, as we assumed their circadian rhythmswere more likely to be intact. Thus, we achieved a relatively homogeneous studycohort. Most of the patients had a good neurological outcome, so that acorrelation analysis between hormone concentrations and neurological outcomewould not have yielded a satisfactory result. Further studies with more severelyill patients are necessary to investigate associations between hormoneconcentrations and neurological status.31Furthermore, electrochemiluminescence immunoassay/sandwich chemiluminescenceimmunoassay methods, as commonly used in routine diagnostics, are often notsensitive enough for CSF hormone levels. Studies employ heterogeneous methods todetermine CSF hormone levels and a standard method is yet to evolve.A circadian rhythm for hormones in blood was only present in a minority of patients.Either the rhythm is disturbed by brain injury or by other factors originating fromthe ICU.Significant diurnal variations of hormones in blood at the cohort level were onlyshown for TSH. For other hormones, no definitive statement can be made as CSFconcentrations in particular were often below the detection limit. Disturbed hormonemetabolism and disruption of the blood–brain and blood–CSF barriersmay be a possible influencing factor.CSF and blood levels for TSH and cortisol were positively correlated. Therefore,blood concentrations of these hormones could act as an indicator for CSFconcentrations, which would facilitate further studies.


Complete separation of the deferent duct from the epididymis in cryptorchid testes residing in the abdomen is an extremely rare variant of developmental disorders of the testis and epididymis. Available sources mention only three clinical cases similar to our observations. The unique anatomic aspects of this disorder hamper the correct diagnosis of an intra-abdominal cryptorchid testis. Two boys with nonpalpable left-sided cryptorchidism underwent diagnostic laparoscopy, revealing an intra-abdominally located testis. The epididymis was completely separated from the deferent duct, and the epididymis and testis were supplied by testicular vessels. Exploration of the inguinal canal revealed blind-ending deferent ducts. The testis was brought down through the inguinal canal and fixed in the scrotum in both boys. The follow-up examination at 6 months revealed no signs of testicular atrophy or malposition of the testis in either patient. With our observations in mind, the exclusive use of a transscrotal or transinguinal approach as the initial surgical exploration in the treatment of patients with nonpalpable forms of cryptorchidism may be inappropriate. Careful laparoscopic examination of the abdominal cavity is indispensable in children with suspected testicular regression syndrome or nonpalpable forms of cryptorchidism. The incidence of cryptorchidism among full-term newborns ranges from 1.0 to 4.6% at the time of birth ,4,5,6,7.Virilization of the male genital system starts around the 7th week of gestation. The spermatogenic cord develops in the fetal testis, and the Mullerian duct disappears. At 12 weeks of gestation, cords from the rete testis give rise to the efferent ducts, which fuse with the mesonephric tubules close to the testis to form the epididymal duct. The vas deferens develops from the proximal vas precursor, which represents the central portion of the mesonephric duct .Congenital absence of the vas deferens (CAVD) occurs in 0.1% of men. In the majority of men suffering from CAVD, at least one cystic fibrosis-causing gene mutation is found . HoweverUndescended testes are frequently associated with fusional anomalies of the testis and epididymis, and these malformations are nowadays made responsible for infertility in men suffering from undescended testes .Complete separation of the deferent duct from the epididymis is an extremely rare variation of developmental disorders of the testis, epididymis, and deferent duct. Failure of fusion of the epididymis and testis with complete separation of the epididymis has rarely been described in the literature ,17,18,19These unique anatomic aspects may make it difficult to locate the intra-abdominal testis. Thus, we feel that the usual surgical approach based on the assumption that the connection between the epididymis and its deferent duct is inseparable requires some correction.We aimed to make pediatric surgeons and pediatric urologists aware of this very rare disorder characterized by separation of the epididymitis and deferent duct in cryptorchid testes located abdominally. Unidentified testes may remain in the abdominal cavity rather than being transferred to the scrotum.Patient 1, aged 21 months, suffered from nonpalpable cryptorchidism. During diagnostic laparoscopy, we noted that the internal ring of the left inguinal canal was obliterated and the deferent duct with hypoplastic “testicular” vessels entered the internal inguinal ring .We first established the diagnosis of testicular regression syndrome and planned to perform transinguinal exploration to search for the so-called “testicular lump”. During further diagnostic laparoscopy, we noted an ipsilaterally located abdominal testis and epididymis, which were located in the small pelvis on a vascular pedicle but were detached from the deferent duct .The distance between the internal inguinal ring and the crossing of testicular vessels from the retroperitoneal to the intraperitoneal positions was approximately 4 cm. We opted for orchiopexy because the testicular vessels appeared to be sufficiently long. An incision was made in the left inguinal area to open the inguinal canal. Exploration of the inguinal canal revealed a blind-ending deferent duct A,B.The left testis was brought down through the external inguinal ring into the upper third of the scrotum and fixed using the Shoemaker’s technique .Patient 2, aged 48 months, underwent diagnostic laparoscopy for a nonpalpable left-sided testis. Laparoscopy revealed an intra-abdominal testis and epididymis attached to the testicular vessels. The testis and epididymis were completely separated from the deferent duct. The distance between the internal inguinal ring and the crossing of testicular vessels from the retroperitoneal space to the intraperitoneal position was 4 mm to 5 mm, and the processus vaginalis was patent .Exploration of the inguinal canal revealed a blind-ending deferent duct arrow; .The testis was brought down through the external inguinal ring into the middle third of the scrotum and fixed using Shoemaker’s technique. No intra-operative complications were encountered, and the postoperative course was uneventful. Both patients were discharged on day 2 after surgery, and the follow-up examination at 6 months revealed no signs of testicular atrophy or malposition in either patient.None of our patients showed any signs of cystic fibrosis or congenital renal agenesis.Anomalies of connection between the testis, epididymis, and deferent duct are associated with cryptorchidism in most cases . In the The embryogenesis of the urogenital system must be well understood to appreciate the mechanism of development of this anomaly. The testis and deferent duct originate from different embryologic structures. The head of the epididymis and testis develop from the urogenital ridge, consisting of the genital and mesonephric ridges. The efferent ducts and rete testis develop from the mesonephric tubules. The epididymal duct and the deferent duct develop from the Wolffian (mesonephric) duct ,19. CompTo locate the testis in nonpalpable forms of cryptorchidism, two surgical approaches are used. In the absence of vicarious hypertrophy of the contralateral testis, diagnostic laparoscopy is the method of choice. During laparoscopy, surgeons may mistakenly interpret the deferent duct entering the “closed” deep inguinal ring as testicular regression syndrome. As a result, surgeons may opt for the transinguinal approach to search for the testis. The blind end of the vas deferens detected in the inguinal canal may be erroneously interpreted as a testicular remnant. In turn, misleading intraoperative findings may result in the testicle being left in the abdomen.In accordance with the findings of Barthold and Redman, the partial agenesis of the vas deferens was associated with cryptorchidism in our cases . Male duAbdelmohsen et al. described a patient whose distal part of the right vas deferens was missing . The autBilateral congenital absence of the vas deferens was considered a virtually untreatable cause of male infertility before microsurgical aspiration of sperm from the epididymis and vasa efferentia for in vitro fertilization and embryo transfer, as described by Silber et al. , became Although our clinical observations represent a very rare anomaly in the development of the testis, epididymis, and vas deferens based on the limited literature evidence, the stereotypes of laparoscopic images should be challenged to avoid diagnostic errors. Thus, a thorough laparoscopic search for the nonpalpable gonad is indispensable.An alternative surgical technique to locate a nonpalpable gonad involves the transscrotal or transinguinal approach as the starting option. Since 1969, several authors have published articles on the role of vicarious testicular hypertrophy in unilateral, nonpalpable cryptorchidism as an indicator of the state of the cryptorchid gonad. This procedure assumed that hypotrophy or atrophy of an organ that exists as a pair, such as the ovaries, kidneys, and adrenal glands, would lead to compensatory hypertrophy of the contralateral healthy organ. Studies revealed that the hypertrophy of a healthy testis completely compensates for the absence, hypotrophy, or atrophy of the contralateral testis. Hurwitz et al. found that in 90.3% of patients with unilateral nonpalpable testis, an increase in the testicular size of the contralateral testis to 1.8 cm or more was indicative of the missing testis . SnodgraWhen using the transscrotal or transinguinal approach in children with vicarious hypertrophy of the contralateral gonad, surgeons should be aware of the rare developmental anomaly described in this article. Inspection of the inguinal canal may reveal a vas deferens with a small lump at the end (hypotrophic body and tail of the epididymis), which may be misinterpreted as a testicular remnant.Thus, the question arises whether we may have treated certain patients for suspected testicular regression syndrome in the past and overlooked the intra-abdominally located gonad in certain boys who suffered from dissociation of the deferent duct from the epididymis associated with cryptorchidism. This diagnostic error may have been the consequence of our former conviction that the testis and epididymis cannot exist without being connected to the deferent duct. Although complete dissociation of the epididymis and vas deferens as described here is a very rare anomaly in the development of the epididymis and vas deferens, the small likelihood of its occurrence should be borne in mind when treating patients with nonpalpable forms of cryptorchidism.With our observations in mind, the exclusive use of a transscrotal or transinguinal approach as the initial surgical exploration in the treatment of patients with nonpalpable forms of cryptorchidism may be inappropriate. Careful laparoscopic examination of the abdominal cavity is indispensable in children with suspected testicular regression syndrome or nonpalpable forms of cryptorchidism.


Therefore, this study highlights that the CSF-1 receptor is fully functional in a prostate cancer cell and may be a potential therapeutic target for the treatment of prostate cancer.Prostate cancer is a major public health concern and one of the most prevalent forms of cancer worldwide. The definition of altered signaling pathways implicated in this complex disease is thus essential. In this context, abnormal expression of the receptor of Macrophage Colony-Stimulating Factor-1 (M-CSF or CSF-1) has been described in prostate cancer cells. Yet, outcomes of this expression remain unknown. Using mouse and human prostate cancer cell lines, this study has investigated the functionality of the wild-type CSF-1 receptor in prostate tumor cells and identified molecular mechanisms underlying its ligand-induced activation. Here, we showed that upon CSF-1 binding, the receptor autophosphorylates and activates multiple signaling pathways in prostate tumor cells. Biological experiments demonstrated that the CSF-1R/CSF-1 axis conferred significant advantages in cell growth and cell invasion in vitro. Mouse xenograft experiments showed that CSF-1R expression promoted the aggressiveness of prostate tumor cells. In particular, we demonstrated that the ligand-activated CSF-1R increased the expression of  Prostate cancer is the most frequently diagnosed cancer in men worldwide. It represents a major public health concern in Western countries, and its incidence is increasing rapidly in Asia . DespiteReceptor tyrosine kinases (RTKs) are key regulators of normal cellular processes since they lie at the head of a signal transduction cascade that modulates cell survival, proliferation, differentiation, adherence, and migration. Consequently, uncontrolled/biased RTK signaling plays a critical role in the majority of cancers, which made many RTKs attractive targets for cancer therapy ,5, inclufms proto-oncogene [The macrophage colony-stimulating factor (M-CSF or CSF-1) receptor (CSF-1R), encoded by the c-oncogene ,8, belononcogene . This reoncogene ,11. CSF-oncogene ,13, whiconcogene ,15. The oncogene ,17.In addition to their role as an important regulator of hematopoiesis and bone resorption, CSF-1 and CSF-1R play a role in numerous cell types of non-hematopoietic origin. CSF-1R is expressed in normal placental trophoblast epithelium, and its activation by the locally high levels of CSF-1 produced by the endometrial epithelium is essential for normal embryonic implantation and placental development . CSF-1R spp1 gene that encodes for osteopontin, a major actor in prostate carcinogenesis.In normal mouse prostate, CSF-1R expression is restricted to early development. Though, CSF-1R expression has been reported in mouse and human prostate cancer tissues and cell lines ,24. ThisTo investigate the effects of CSF-1 receptor (CSF-1R or Fms) expression and activation in prostate cancer cells, the murine C2H prostate tumor cell line was used as a model. This clonal epithelial cell line was established from a prostate tumor of the transgenic adenocarcinoma mouse prostate (TRAMP) model . The C2HWe then evaluated the effect of CSF-1R expression on cell growth in the presence or absence of exogeneous CSF-1. As shown in We next tested both parental and CSF-1R-expressing C2H subclones for their growth in vivo after subcutaneous injection in male immunodeficient SCID mice. The results obtained with the cells inoculated in both flanks, either as parental cells or those expressing the CSF-1R, are shown in We next compared the transcriptome profiling of prostate cancer cells stimulated or not by CSF-1 for 24 h or 48 h. For this purpose, the N18-Fms cells were chosen. The analysis of the 60K Agilent microarrays revealed 1258 and 1117 mRNA were significantly differently expressed between unstimulated cells and 24 h or 48 h CSF-1-stimulated cells, respectively A,B. A toGenes upregulated in both 48 h and 24 h upon CSF-1 treatment when compared to untreated control  is a secreted protein produced by numerous cell types, including macrophages . Since CTo determine if CSF-1R is also functional in human prostate cancer cells, we selected two malignant prostate cancer cell lines. M12 is a subline derived from the P69 prostate cell line and displays a mesenchymal-like, highly tumorigenic, and metastatic phenotype ,33. InteAbnormalities in RTK expression and activation are associated with a wide range of diseases. In cancer, RTK plays a key role in the development, progression, and resistance to therapy . Of partFew studies have investigated the expression of CSF-1R and its ligand in prostate cancer. Ide et al.  describeHere we have shown that in response to CSF-1, CSF-1R autophosphorylated and activated two major signaling pathways, ERK1/2 and AKT. Thus, signaling pathways downstream of CSF-1R in prostate cancer cells were similar to those in myelomonocytic cells . Interesspp1 gene was a CSF-1R-responsive gene in a macrophage cell line, a mammary cell line, but not in a fibroblast cell line. This suggests that spp1/osteopontin is one example of CSF-1-responsive genes shared between myelomonocytic lineage and carcinoma cells expressing CSF-1R. Considering the complex relationship between CSF1/CSF-1R-expressing carcinoma cells and macrophages, synthesis of OPN is of particular interest since OPN is a chemoattractant for macrophage and regulates its adhesion, migration, differentiation, and production of cytokine [While conducting cDNA microarray assays for differentially expressed genes downstream of CSF-1R signaling, we identified spp1/osteopontin as a gene of interest. Osteopontin is a multifunctional phospho-glycoprotein secreted in the microenvironment and a key regulator of tumor progression and immunomodulation ,66. We scytokine ,67,68. Tcytokine . In proscytokine . In proscytokine . OPN hascytokine . Messex cytokine . Thus, OIn summary, this study has demonstrated the functionality of CSF-1R in prostate cancer cells and has unveiled osteopontin as a CSF-1R-target gene. Given that CSF-1R also plays a critical role in the tumor immune landscape, more research is warranted in this area for a better understanding of how CSF-1R regulates prostate cancer development and how this knowledge could be used in terms of therapeutic strategies.2. The murine hematopoietic interleukin-3 (IL3)-dependent cell line FDCP1-Fms was maintained in DMEM-10% FBS supplemented with IL3 and antibiotics [TRAMP-C2 cell lines were established from the spontaneous TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) model . TRAMP Cibiotics . NIH 3T3ibiotics . Brieflyibiotics . The humibiotics  was a giMurine wild-type Fms receptor was introduced in different murine cells by retroviral infection using 0.45 μm-filtered supernatants of packaging Psi-2 Fms cells in the presence of polybrene (4 μg/mL) as previously described . Infecte4 cells per plate in a 6-well plate in 2 mL of culture medium with or without 50 ng/mL of recombinant murine CSF-1 (rmCSF-1) . After 72 h, cells were isolated after trypsinization using trypsin–versene (EDTA) solution, and cell counting was performed under the microscope using a Malassez counting chamber.Cell proliferation was evaluated by plating 2 × 10® matrix diluted to half in serum-free DMEM, then incubated for 1 h at 37 °C. For the experiment, each condition was performed in triplicate; 500 μL of DMEM-1% FBS containing 104 cells were deposited on each insert. Inserts are introduced into insert companion plate containing 750 μL of DMEM-1% FBS with or without rmCSF-1 (50 ng/mL). After five days at 37 °C in 5% CO2 atmosphere, the invasive potential of cells was measured by fluorescence using FLUOstar OPTIMA plate reader  after cell staining with calcein AM  for 1 h at 37 °C.Invasion assay was performed using inserts BD Falcon FluoroBlok  with 8 μm pore size PET membrane on 24-well insert companion plate . Inserts were coated with 50 μL of Matrigel4 cells per well were plated in 1 mL of culture medium in 6-well plates for 48 h. Conditioned medium was collected, filtered 0.45 μm, and subjected to ELISA using commercially available mouse M-CSF ELISA Kit  according to the manufacturer’s instructions. The CSF-1 secretion was measured with a standard curve.For CSF-1 quantification, 5 × 10−ΔΔCt) method. Experiments were performed in duplicate. Primer sequences are presented in Total RNA was isolated using the RNeasy mini kit . The QuantiTect reverse transcription kit (Qiagen) was used to convert RNA to cDNA as per manufacturer’s instructions. Standard PCR was performed in 0.2 mL thin-walled tubes (Thermo Fisher Scientific) using KAPA2G hot start kit . Thermal cycling was carried out in an MJ Research PTC-100 instrument  in a 25 μL reaction volume. PCR products were size-fractionated on a 1.5% ethidium bromide-containing agarose gel. For quantitative PCR (qPCR), diluted cDNAs were transferred to 96-well PCR optical plates (Axygen). KAPA SYBR FAST qPCR kit  was used. qPCR was performed using the Agilent Mx3000P detection system . Relative mRNA levels were determined following normalization to mouse RPLP38 or human RPLP0 housekeeping genes and analysis of the comparative threshold cycle (2® software version GX12.0 (Agilent Technologies). All these experiments were performed by Imaxio Company . Further investigations were carried out using the GO biological process and Reactome pathways databases.N18-Fms cells were stimulated by rmCSF-1 (100 ng/mL) for 12 h or 24 h or left unstimulated. Total RNA from two independent experiments was extracted with the RNeasy Plus Kit (Qiagen) and was quantified with the NanoDrop ND-1000 (Thermo Fisher Scientific). Quality of the extracted RNA was evaluated with the Agilent 2100 Bioanalyzer system (Agilent Technologies). cRNA labeling, hybridization, and detection were carried out according to supplier’s instructions (Agilent Technologies). For each microarray, Cyanine 3-labeled cRNA was synthesized with the low input Quick Amp WT labeling kit from 100 ng of total RNA. RNA Spike-In was added to all tubes and used as positive controls of labeling and amplification steps. Labeled cDNA samples (600 ng) were used for 17- hour hybridization at 65 °C to the SurePrint G3 mouse GE 8 × 60K Microarrays (Agilent Technologies). All procedures were carried out according to the manufacturer’s instructions. The hybridized microarrays were scanned with the Agilent DNA Microarray Scanner (G2505C). Signal intensity was quantified from the scanned image by using Feature Extraction software version 11.0.1 (Agilent Technologies). Statistical comparisons and filtering were achieved with the Genespring® #9106), anti-Erk2 (C14) , and anti-actin (C4) . After incubation with the appropriate species-specific horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit (#711-035-152), ant-mouse (#115-035-146), or anti-goat (#705-035-003) , the antigen-antibody complexes were detected using Super Signal West Dura Extended Duration Substrate . Luminescence was captured by digital imaging with a cooled-charge-coupled device camera . For all immunoblot images presented throughout this manuscript, the membranes were cut into horizon pieces according to the estimated molecular weight of proteins of interest and probed with the indicated antibodies. All cropped blots were run under the same experimental condition.Prostate cell lines were starved overnight at 37 °C in DMEM-0.5% FBS medium. FDCP1-Fms cells were starved in DMEM-1% FBS for 3 h at 37 °C. Cells were washed in phosphate-buffered-saline  and stimulated by 2500 U/mL of recombinant murine CSF-1  or 500 n5 C2H-derived clones were resuspended in 150 μL of PBS before subcutaneous injection into flanks, with Fms-expressing cells on the left flank and control cells on the right flank of the same mice (n = 8). Tumor volume was calculated as follows: tumor volume = width × length × (width + length)/2. In order to determine the tumor volume using a bioluminescence imager , each mouse was monitored at the indicated time after intraperitoneal injection of luciferase substrate luciferin  at 150 mg/kg body weight.Eight-week-old male severe combined immunodeficient (SCID) mice were purchased from Pasteur Institute of Lille  and were maintained in accordance with the Institutional Animal Care and Use Committee procedures and guidelines. Further, 1.5 × 10t-test; p values < 0.05 were considered to be significant.Data are presented as mean values ± standard deviation (s.d.) of at least 3 independent experiments. The statistical analysis was performed by using a 2-sided Student


The results are discussed in relation to the beneficial effects of VP-shunt treatment. Furthermore, the possibility that CSF drainage may reduce an inhibitory effect of transiently increased pressure on the metabolic capacity of neuronal cells in the brain is discussed. This capacity includes the elimination of cholesterol by the 24S-hydroxylase mechanisms.Idiopathic normal pressure hydrocephalus (iNPH) is the most common form of hydrocephalus in the adult population, and is often treated with cerebrospinal fluid (CSF) drainage using a ventriculoperitoneal (VP) shunt. Symptoms of iNPH include gait impairment, cognitive decline, and urinary incontinence. The pathophysiology behind the symptoms of iNPH is still unknown, and no reliable biomarkers have been established to date. The aim of this study was to investigate the possible use of the oxysterols as biomarkers in this disease. CSF levels of the oxysterols 24S- and 27-hydroxycholesterol, as well as the major metabolite of 27-hydroxycholesterol, 7 alpha hydroxy-3-oxo-4-cholestenoic acid (7HOCA), were measured in iNPH-patients before and after treatment with a VP-shunt. Corresponding measurements were also performed in healthy controls. VP-shunt treatment significantly increased the levels of 7HOCA and 24S-hydroxycholesterol in CSF ( Idiopathic normal pressure hydrocephalus (iNPH) is a treatable neurological disorder affecting the elderly. The condition is characterized by gait and balance impairments, urinary incontinence and cognitive decline. Diagnostic imaging shows signs of disturbed cerebrospinal fluid (CSF) dynamics, including ventricular enlargement and widened sulci. These findings and the clinical symptoms are thought to reflect impaired CSF outflow and increased intracerebral pressure pulsatility ,2. The mSimilarly, the pathophysiology resulting in the triad of iNPH symptoms is also not clearly understood. However, clinical features of the disease include psychomotor slowing, executive dysfunction, impaired attention, decreased verbal fluency and worsened working memory, all of which correlate with the typical patterns of frontal lobe dysfunction ,4,5. RadThe symptoms of this disease are potentially reversible if recognized early and treated with a ventriculoperitoneal (VP)-shunt. However, it is notable that the positive effects of the drainage of CSF are not accompanied by a reduction in ventricular size .To evaluate the effect of drainage before VP-shunt surgery, CSF drainage through a lumbar puncture is routinely performed as a diagnostic tool. The patients are evaluated for relevant symptoms including gait, balance, cognition and incontinence before and after the lumbar puncture. The purpose is to identify those that are improved by CSF drainage and consequently have a greater chance of benefitting from VP-shunt treatment. A VP-shunt is a silicone tube with one end placed through the brain into the enlarged ventricles and the other end in the abdominal cavity. The rest of the tube is placed subcutaneously, and a valve defines the flow and pressure gradients needed for drainage.iNPH is regarded as one of the most misdiagnosed diseases worldwide and may occur with varying combinations or degrees of other neurological conditions, including Alzheimer’s disease, Parkinson’s disease and vascular dementia . DiagnosThe most common oxysterols in CSF include 24S-hydroxycholesterol (24-OH), 27-hydroxycholesterol (27-OH) and the major oxysterol metabolite 7 alpha hydroxy-3-oxo-4-cholestenoic acid (7HOCA) ,16. The In humans, 24-OH is almost exclusively formed in the brain and there is a continuous flux of 24-OH from the brain into the circulation. There is also a modest flux of this oxysterol from the brain into the CSF. The 24-OH present in CSF has a mixed origin: part of it originates directly from the brain and part of it originates from the circulation. This means that the integrity of the blood–brain barrier is important for the levels of 24-OH in CSF .There is very little formation of 27-OH in the brain, and most of this oxysterol in the brain and CSF originates from the circulation. Despite the relatively high uptake of this oxysterol by the brain the levels in both brain and CSF are low, reflecting a very efficient metabolism. Given the negative effects of this oxysterol on various brain functions ,26,27,28Previous studies have shown normal levels of 7HOCA in patients with Alzheimer’s disease or vascular dementia . In contBased on the above findings, this study aims to investigate the possible use of the oxysterols 24-OH, 27-OH and 7HOCA as biomarkers in iNPH. In addition, the findings may further our mechanistic understanding of the effects of CSF drainage in iNPH.All adult patients (≥18 years) who were offered surgical treatment for iNPH between 2020 and 2022 were eligible for inclusion. The study hospital is a publicly funded and owned tertiary care center, serving a region of roughly 2.3 million inhabitants, and the only neurosurgical center in the region. As part of routine management, referrals to the study center for iNPH were evaluated at a multidisciplinary conference. Cases matching the clinical and radiological criteria for a diagnosis of iNPH were further evaluated in accordance with international guidelines , which iSeventeen patients with iNPH  accepted to participate in the study and signed informed consent after having received both written and oral information . CSF samAlbumin was measured using Tina-quant Albumin Gen.2 kit . Analytical standards (pure > 98%) of each oxysterol were used for calibration and quantification. 7HOCA was measured by isotope dilution mass spectrometry as described previously ,30. The p value < 0.05), it is presented as a median (range) and categorical data as numbers (proportion). The Mann–Whitney U test was used to compare pre- and postoperative metabolites in the treatment group to non-treated controls (non-matched continuous data), and the Wilcoxon signed-ranks test was used to compare metabolites in each treated patient before and after surgery (matched continuous data). All analyses were conducted using the statistical software program R. Statistical significance was set at p < 0.05.The Shapiro–Wilk test was used to evaluate the normality of the data. As all continuous data significantly deviated from a normal distribution pattern . The CSF from anonymous control subjects was obtained from the clinical chemical routine lab. Only information about age and sex was given to the lab analyzing oxysterols.The oxysterols 7HOCA, 24-OH, and 27-OH as well as cholesterol, and albumin were measured in 17 iNPH patients and 28 controls. In 10 patients, a repeated analysis was performed at median 4.5 months after VP-shunt surgery.The pre-operative concentrations of cholesterol, 24-OH and 27-OH were significantly lower in iNPH patients compared to controls, while no difference was seen for albumin or 7HOCA . For theIn this study, we demonstrate that the CSF levels of 24-OH and 27-OH are lower in iNPH patients compared to controls, and that the levels of the metabolite 7-HOCA significantly increases in iNPH patients after VP-shunt treatment.It should be emphasized that the major fluxes in oxysterols to and from the brain do not directly include the cerebrospinal fluid, but rather occur between the brain and the circulation. It is believed, however, that changes in these major fluxes as well as metabolic changes in the brain are reflected in the levels of oxysterols in the cerebrospinal fluid ,35. We hypothesized that measurements of oxysterols in CSF may give useful diagnostic information in iNPH, and that the beneficial effects of CSF drainage may be reflected in changes in the levels of specific oxysterols. Before treatment, the CSF levels of 24-OH and 27-OH were significantly higher in controls compared to iNPH patients . VP-shunAn increase in the levels of 7HOCA has previously been suggested as a marker for blood–brain barrier dysfunction . In thisThe levels of 27-OH in CSF are dependent upon the flux in this oxysterol from the circulation into the CSF. Since 27-OH is a precursor to 7HOCA, this is also the case for 7HOCA. The flux of 27-OH across the blood–brain barrier is, however, also combined with a corresponding flux in albumin across this barrier. Albumin is a strong binder of 7HOCA and thus a disrupted blood–brain barrier would be expected to result in increased levels of 7HOCA in the CSF by these two mechanisms .Zhang et al studied the metabolism of oxysterols in cultured rat astrocytes, Schwann cells and neurons. They found that 27-OH, but not 24-OH, was metabolized into 7HOCA in all cell types. Adding 27-OH to the cell cultures resulted in elevated levels of 7-HOCA in astrocytes compered to neurons. Adding 24-OH to the cultures showed an increased metabolization to other cholesterols in astrocytes. This may be of relevance in the context of iNPH, since its pathophysiology may include disturbances in the glymphatic system that particularly affect astrocytes ,38,39. ILiu et al demonstrated that hypercholesterolemia in the CNS is involved in inducing hydrocephalus . FurtherThe increased level of 7HOCA in CSF after VP- shunt treatment is noteworthy, and considering the neurotoxic effects of 27-OH on both neurons and astrocytes ,28, it sThe main limitation of this study is the limited sample size. Another limitation is the lack of simultaneous analysis of oxysterols in the blood, which could have provided valuable comparative data. This issue needs to be addressed in future studies. A strength of this study is the prospective patient inclusion, as well as the robust collection and analysis of the oxysterol concentrations.Neither the etiology of iNPH nor the mechanisms explaining the beneficial effects of VP-shunt surgery are fully understood. This study analyzed CSF-levels of oxysterols before and after VP-shunt surgery, and showed that CSF from iNPH patients compared to controls had a significantly lower level of oxysterols before surgery and that there were significant increases in 24OH and 7HOCA after VP-shunt treatment. CSF drainage may reduce an inhibitory effect of transiently increased pressure on the metabolic capacity of neuronal cells in the brain. This capacity includes the elimination of cholesterol by the 24S-hydroxylase mechanisms. Thus, oxysterols may potentially be used as a biomarker in the diagnosis and management of iNPH.


Lumbar puncture is among the investigations used to identify various neurological conditions, including some that can cause cardiac arrest (CA). However, CA per se may alter cerebrospinal fluid (CSF) characteristics. Few studies have investigated CSF findings after CA. In this descriptive work, we assessed the frequency and risk factors of abnormal CSF findings after CA and the contribution of CSF analysis to the etiological diagnosis.We retrospectively studied data from prospectively established databases of consecutive patients who were admitted to two French ICUs in 2007–2016 with sustained return of spontaneous circulation (ROSC) after CA and who underwent lumbar puncture as an etiological investigation.P = 0.013). A no-flow time above 6 minutes  and a respiratory cause of CA  were not statistically associated with CSF abnormalities. Nonspecific CSF abnormalities were not significantly associated with poor outcomes .Of 1984 patients with sustained ROSC, 55 (2.7%) underwent lumbar puncture and were included. Lumbar puncture identified a neurological cause of CA in 2/55 (3.6%) patients. Nonspecific CSF abnormalities were noted in 37/53 (69.8%) patients. By multivariate analysis, postresuscitation shock was positively associated with CSF abnormalities (OR, 6.92; 95% confidence interval [95%CI], 1.62–37.26; Lumbar puncture, although infrequently performed, may contribute to the etiological diagnosis of CA, albeit rarely. Nonspecific CSF abnormalities seem common after CA, notably with postresuscitation shock, and may be related to blood-brain barrier disruption. These findings may help to interpret CSF findings after CA. Further studies are warranted to assess our results. Cardiac arrest (CA) is among the most common causes of death in Europe and the United States despite advances in resuscitation and intensive care . During According to recent data, 7% of CAs are due to neurological causes . CerebraWe therefore designed a descriptive, retrospective study of prospectively established databases to evaluate the diagnostic contribution of CSF findings and to describe the frequency and risk factors of CSF abnormalities in patients admitted to the ICU with sustained ROSC after CA.We used two prospectively collected databases established at the Cochin hospital and Versailles hospital #NCT03594318), respectively. Both are CA referral centers that serve the southern and southwest areas of the Paris conurbation (France). Data collection was approved by the ethics committee of the French Intensive Care Society (#CESRLF_12–384 and 20–41), and the data were collected anonymously in compliance with French data protection legislation (French Data Protection Authority #MR004_2209691) ) or mean (range) and qualitative parameters as number (percentage). We compared categorical variables using Fisher’s exact test and continuous variables using the Wilcoxon rank-sum test.P values that were lower than 0.05 or were clinically relevant. Continuous variables were transformed into dummy variables. We applied the Hosmer-Lemeshow goodness-of-fit test and used the C-statistic to estimate the area under the receiver operating characteristics (ROC) curve. Finally, we looked for associations linking CSF abnormalities to survival and functional outcome at ICU discharge.We first described CA characteristics in the patient groups defined by whether CSF analysis contributed to identify the cause of CA. We then described the CSF characteristics in the group whose CSF analysis did not contribute to identify the cause. To assess risk factors for CSF abnormalities after CA, we compared patients with vs. without CSF abnormalities, first by univariate analysis then by building a multivariate model using variables associated with p values <0.05 were considered significant. Analyses were performed using R statistical software version 3.1.2 .All tests were two-sided and LP was assessed in cases of suspicion of a neurological cause for cardiac arrest in 74.5% of patients. CSF analysis helped to identify a neurological cause of CA in 2/55 (3.6%) patients; of these 2 patients, 1 had encephalitis (direct contribution) and the other subarachnoid hemorrhage with possible confusion on the cerebral CT-scan between blood and contrast from previous coronary angiography.3. CSF lactate was above >2.4mmol/L in 10 (91%) of 11 patients.Among the 53 patients whose CSF analysis did not help to identify the cause of CA, 37 (69.8%) had CSF abnormalities . CSF proP = 0.013). A no-flow time longer than 6 minutes  and a respiratory cause of CA . S2 Table in p = 0.017), with higher values in patients who experienced worse outcomes.Overall ICU mortality was 65% (36/55). CSF analysis was performed in only 2.7% of patients admitted to the ICU with stable ROSC after CA and contributed to the etiological diagnosis in only 2 (3.6%) patients. Among patients whose CSF analysis did not contribute to the etiological diagnosis, over two-thirds had nonspecific CSF abnormalities, among which the most common was protein elevation. Postresuscitation shock was an independent and significant risk factor for abnormal CSF. The CSF/serum protein quotient was significantly associated with a poor outcome.LP was performed only very rarely in our population. Few previously published data are available with which to compare our results. Most studies of CSF analysis after CA focused on the neuroprognostication accuracy of CSF biomarkers reflecting neuronal damage , 6, 18. 3. Knowledge of the range of CSF abnormalities seen after CA may help to interpret CSF results. Indeed, CSF cell count above the range reported here should prompt further investigations for a neurological cause of CA. Although several studies assessed the usefulness of various CSF biomarkers for neuroprognostication [Over two-thirds of our patients without identified neurological causes of CA had nonspecific CSF abnormalities, of which the most common was protein elevation (94%), followed by white-cell count elevation (27%), with the highest recorded value being white-cells 144/mmtication , 6, nonetication .All the hypotheses put forward to explain these findings focus on the blood-brain barrier (BBB), which is composed of cerebral endothelial cells, a capillary basement membrane, pericytes, and astrocytes. The BBB regulates the penetration of plasma constituents into the cerebral parenchyma , 24. In First, CSF changes may be due to systemic inflammation. Elevated levels of proinflammatory cytokines have been found in the CSF after CA , 28. In P = 0.4 by univariate analysis). Further studies in a larger population are needed to clarify the links between cerebral anoxia and CSF characteristics. Finally, we found no associations linking CSF abnormalities to sepsis before LP, spinal-cord compression, diabetes, hematological malignancy, or epilepsy before LP.Second, CSF changes may occur in response to primary brain injury with anoxic neuronal damage. Cerebral anoxia may lead not only to BBB disruption but also to the production of specific cerebral proteins, resulting in CSF abnormalities. High-mobility group box 1 (HMGB1) released by necrotic brain cells may act as an early inflammation trigger inducing the local recruitment of proinflammatory cytokines, independently of BBB alterations . IncreasICU mortality was 65% in the overall population of patients with LP after CA. Both patients whose CSF analysis provided etiological information died. Identifying a neurological cause of CA has been reported to carry a very poor prognosis , 9. In oOur study has several limitations. First, given the retrospective design and small sample size, the extent to which our findings apply to the full spectrum of patients with CA is unclear. We included consecutive patients with LP after ICU admission with stable ROSC after CA. However, LP was not performed routinely in patients meeting predefined criteria and was contraindicated in many patients due to anticoagulant exposure, notably due to previous angiography. Moreover, both participating ICUs were high-volume centers whose recruitment may not reflect that of ICUs overall. Second, we considered only LPs done at the early phase after CA, as part of the emergent etiological workup. Delayed CSF analysis may provide important information. One study found that the CSF/serum albumin quotient increased between 24 h and 72 h after ROCS, and others evidenced an increase in CSF protein levels after 2–3 weeks , 33. MorIn conclusion, although rarely performed after CA, LP may contribute to the identification of a neurological cause of CA. In our study, CSF analysis as a second-line investigation identified a neurological cause in 3.7% of patients. Nonspecific CSF abnormalities were common after CA with postresuscitation shock, perhaps due to BBB disruption. These findings may help clinicians to interpret CSF findings after CA. Further studies are warranted to clarify the links between CA and CSF abnormalities.S1 File(DOCX)Click here for additional data file.