Older patients commonly receive benzodiazepines during anaesthesia despite guidelines recommending avoidance. Interventions to reduce perioperative benzodiazepine use are not well studied. We hypothesized an automated electronic medical record alert targeting anaesthesia providers would reduce administration of benzodiazepines to older adults undergoing general anaesthesia.We conducted a retrospective study of adults who underwent surgery at 5 hospitals within one US academic health system. One of the hospitals received an intervention consisting of provider education and an automated electronic medical record alert discouraging benzodiazepine administration to patients aged 70 years or older. We used difference-in-differences analysis to compare patterns of midazolam use 12-months before and after intervention at the intervention hospital, using the 4 non-intervention hospitals as contemporaneous comparators.The primary analysis sample included 20,347 cases among patients aged 70 and older. At the intervention hospital, midazolam was administered in 454/4,240 (10.7%) cases pre-alert versus 250/3,750 (6.7%) post-alert (p<0.001). At comparator hospitals, respective rates were 3,186/6,366 (50.0%) versus 2,935/5,991 (49.0%) (p=0.24). After adjustment, the intervention was associated with a 3.2 percentage point (p.p.) reduction in the percentage of cases with midazolam administration ; p=0.002). Midazolam dose was unaffected ; p=0.90). In 76,735 cases among patients aged 18–69, the percentage of cases with midazolam administration decreased by 6.9 p. p. ; p<0.001).Provider-facing alerts in the intraoperative electronic medical record, coupled with education, can reduce midazolam administration to older patients presenting for surgery but may affect care of younger patients. One institution recently described a multi-component quality improvement initiative that decreased benzodiazepine administration to patients presenting for surgery.,Nudges are environmental modifications designed to improve decision-makingWe tested the impact of an automated, clinician-directed EMR alert, coupled with educational outreach, on perioperative administration of midazolam to adults aged 70 years or older undergoing elective non-cardiac surgery. We hypothesized that exposure to the alert would be associated with decreased rates of benzodiazepine administration to patients requiring general anaesthesia.This is a retrospective study of data from 5 hospitals in one academic US health system. One of the hospitals received a clinician-directed EMR alert discouraging benzodiazepine administration to surgical patients aged 70 years or older with educational outreach introducing the alert; the 4 remaining hospitals received no interventions and served as a grouped comparator. The effect of the alert on midazolam administration was estimated via difference-in-differences analysis (described below).th June, 2019, an automated EMR alert entitled “Delirium Prevention in Older Patients” was activated within the intraoperative anaesthesia record. The alert displays automatically for every anaesthesia provider who opens the intraoperative component of the anaesthesia record for patients aged 70 years or older. The alert text suggests avoidance of benzodiazepines with midazolam as the prototypical medication  analysis, a widely-used technique in economic research and policy evaluation. Our analytical plan was finalised in November 2020. Analysis began in May 2021. Unlike uncontrolled pre-post analyses, DID uses a contemporaneous comparison group  to account for temporal changes in a particular outcome  that might otherwise confound causal inference .19, 20, 21,21We used descriptive statistics and simple hypothesis tests to compare patient and procedure characteristics and outcomes at the intervention versus comparator hospitals. We fitted a linear probability modelWe evaluated the parallel trends assumption via visual inspection and in a regression model estimating our outcome using data from the 12-month pre-intervention period only. This model predicted the primary study outcome along with an interaction between treatment arm  and whether the case occurred during the first 6 months or the second 6 months of the pre-intervention period, plus all controls listed above. A non-significant estimate on the interaction term indicates parallel pre-intervention trends across the intervention versus control hospitals in the primary outcome.To test the robustness of our findings, we repeated our main model to predict an endpoint of glycopyrrolate administration, a medication that was not specifically targeted by the alert. This served as a falsification testth April, 2019 and 16th June, 2019) and during the period after EMR alert activation  compared to the period before 4th April, 2019.To assess whether the intervention affected younger patients, we fitted the same models in our secondary sample of patients aged 18–69 years who underwent general anaesthesia for elective non-cardiac surgery. We also analysed cases performed under sedation, since clinical decision-making for benzodiazepine use in this context may differ from that used for general anesthesia.Analyses were performed using Stata 17.0 . P<0.05 was our threshold for statistical significance. The strengthening the reporting of observational studies in epidemiology (STROBE) checklist appears in After exclusions , our priIn the period before alert deployment, at the intervention hospital, midazolam was administered in 10.7%  of cases involving patients 70 years or older compared with 6.7%  of cases post-alert (p<0.001). In contrast, at the comparator hospitals, midazolam was administered in 50.0%  of cases versus 49.0%  of cases respectively (p=0.24) . SupplemFor cases at the intervention hospital, the mean midazolam dose per case was 1.69 mg (SD 0.68 mg) pre-alert versus 1.72 mg (SD 0.73 mg) post-alert (p=0.59). At comparator facilities, the mean dose per case was 1.86 mg (SD 1.37 mg) versus 1.93 mg  respectively . After aWhile we did note differences in the incidence of midazolam administration at intervention versus comparator facilities, we confirmed parallel trends over the pre-alert period by visual inspection and by regression analyses using data restricted to the pre-alert period only as above ; p=0.06; 76,735 cases were performed on patients aged 18–69 years . At the 21,718 cases with patients aged 70 years and older were performed under sedation rather than general anaesthesia . The perThe alert did not significantly impact the percentage of cases with glycopyrrolate administration for patients aged 70 years or older , p=0.75; Within one US academic health system, a clinician-facing, automated, EMR alert, preceded by clinician education, reduced the percentage of cases of general anaesthesia in which patients aged 70 years or older received midazolam by an adjusted 3.2 percentage points (p.p.). This finding was robust to adjustments for patient and procedure-level factors. The dose of midazolam, when administered, was not affected. Alert implementation was also associated with a decrease in the rate of midazolam administration for patients between 18 and 69 years undergoing general anaesthesia, despite the alert targeting older patients.Notably, our intervention included both an educational component and an EMR alert, which were rolled out in sequence. We did not observe a change in midazolam administration in the period that immediately following the educational component, but during which the EMR alert had not yet been activated. This argues that impacts of education alone are unlikely to explain our findings. As we did not separately study an EMR alert at a site that did not receive departmental education, we cannot separate the impact of the alert alone versus the alert combined with a preceding educational intervention.,This study adds to prior knowledge of how to reduce perioperative midazolam administration to older adults. Previous studies in perioperative settings have shown EMR-based nudges, such as visual reminders embedded into the intraoperative chart, can improve compliance with guideline-directed patient management such as appropriate surgical antimicrobial prophylaxis.There are multiple possible mechanisms that could explain the impact of our intervention on care patterns. As a visual cue, the alert may have served as a “priming” stimulus to encourage guideline-concordant care.,,Our study has certain limitations. This quality improvement initiative took place at a major academic health system, and specifically at a hospital with relatively low rates of midazolam use. Baseline patterns of midazolam use at each hospital were not known during the design and implementation study phases and were only discovered during retrospective analysis. The generalizability of our findings to hospitals with different patterns of midazolam use remains to be determined. Reassuringly, the alert led to reduced rates of midazolam administration across surgical departments regardless of variable baseline rates of utilization. It is possible that our model failed to account for unobserved factors influencing midazolam usage patterns. For example, the final four months of our study occurred during the first wave of the COVID-19 pandemic, when case and patient mix varied considerably. Likewise, we did not consider variation amongst individual anaesthesia providers. These concerns are somewhat alleviated by the consistent results we obtained with analyses using time and group fixed effects to control for unobserved confounding. While we did not see a major impact of educational outreach in this study, education can powerfully alter provider behaviour, and a stronger outreach effort may have augmented observed effects.,,,Despite these limitations, our work is important for policy and practice. Among older surgical patients, receipt of potentially avoidable medications such as midazolam has been associated with adverse outcomes, including prolonged hospitalization, and guidelines recommend avoidance in this population.In conclusion, an automated EMR alert directed toward anaesthesia providers and preceded by provider education and outreach, was associated with a decrease in perioperative midazolam administration to patients of all ages undergoing surgery. Further research is needed to determine the generalizability of findings and effects on patient outcomes. EMR nudges combined with education may be a useful strategy to reduce administration of benzodiazepines to older surgical patients.All authors  made substantial contribution to conception and design, acquisition of data, or analysis and interpretation of data; drafting the article or revising it critically for important intellectual content; approve the version to be published; and agree to be accountable for all aspects of the work thereby ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.10.13039/100001080Patrick and Catherine Weldon Donaghue Medical Research Foundation, Grant Number 10084485 (M. Neuman). The funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.The The authors declare that they have no conflicts of interest.


Classical theories of cerebrospinal fluid (CSF) production and flow are taught throughout medical education. The idea that CSF is produced and/or filtered by the choroid plexus and flows in one direction throughout the ventricular system has been a largely accepted thesis. However, modern studies have called into question the validity of this hypothesis, suggesting that CSF does not move unidirectionally but rather is driven by microvessel contractions in a to-and-fro manner throughout the cerebrospinal system. Moreover, new insights suggest that in addition to CSF production, the exchange of fluids and proteins between the cortical vasculature and the interstitium may function as the brain’s version of a lymphatic system. This comprehensive review provides evidence for a different framework of CSF flow. One that includes perivascular pulsations that push CSF back and forth, allowing exchange between the CSF and interstitium, and with CSF production occurring throughout the cerebrospinal system. These findings could be revolutionary in understanding the pathophysiology of CSF flow and in the treatment of pathologies such as intracranial hypertension, hydrocephalus, Alzheimer's disease, and many others.  Cerebrospinal fluid (CSF) is a clear, colorless fluid usually rich in glucose (60%-80%) . ClassicBulat et al. described the multidirectional flow of CSF based on fluid mechanics in the central nervous system (CNS) microvessels . This hyUnderstanding the process of CSF physiology is essential for the management of CNS disorders. The Bulat-Klarica-Oreskovic model opens the possibility for new approaches to diseases that involve CSF. Hydrocephalus is an illustrative example of the discrepancies between each model. It is defined as an accumulation of CSF that causes the ventricles to swell. It can be communicating, meaning without obstruction, or obstructive, with obstruction. Hydrocephalus is treated based on the classical model, but experiments that have led to the development of the modern hypothesis demonstrate that the success of some procedures may be merely coincidental ,10. SuppAs always, new ideas bring exciting possibilities and the hope that challenging pathologies can finally be conquered, but it is important to evaluate these new theories with a critical mind. This review aims to summarize the proposed mechanisms of CSF dynamics to support the findings by Bulat et al. ,9.ResultsWhile the classical model has been largely accepted since its publication in the early 1900s, there have been several notable studies in the subsequent years that have pushed incrementally toward the modern hypothesis. In this comprehensive review, we have included studies supporting the modern hypothesis based on relevance and estimated influence on the progression toward current theory. Studies were included based on searches for modern CSF pathophysiology, modern CSF theory, CSF dynamics, glymphatics, and the brain’s lymphatic system. Studies that were not available in full text or English were excluded. Some of the first deviations from classical theory were seen in studies performed by Bering et al., Chiro et al., Naidich et al., and Drayer et al. The significance was unknown at the time, but these studies suggested that CSF may not be exclusively, or even primarily, produced within the choroid plexus. Moreover, they opened the possibility that CSF exchange may occur between the perivascular networks within the brain and systemic circulation -17. ConnThe first published description of the modern hypothesis occurred in 1993 when Bulat et al. proposed that CSF flow is not limited to unidirectional flow . In 2008Expanded consideration of resultsIn 1952, Bering published an article demonstrating that large amounts of CSF uptake occur in the periventricular white matter. Deuterium oxide  with 0.8% NaCl was injected into animals (monkeys and dogs) and humans and subsequently monitored. It was discovered that the fluid rapidly accumulated in the cisterna magna and lumbar subarachnoid space before equilibrating within the ventricles and the remainder of the lumbar areas. This suggested that CSF was not produced solely by the choroid plexus, otherwise, the cerebral ventricles would be the first areas to accumulate the D2O tracers. Moreover, patients who underwent choroid plexectomy were found to have negligible alteration in water exchange . Considerable controversy remained about the pathway of CSF exchange. In 1964, Chiro et al. referenced radiolabeled iodine tracer studies demonstrating the possibility of CSF production throughout the cerebrospinal system. Although Chiro admitted at this time, the net of flow is likely forward through the traditional ventricular progression, he added that at least part of the fluid must move in a to-and-fro fashion, with vascular pulsations as a likely mechanism . Naidich et al., in 1976, explored periventricular edema in the setting of hydrocephalus using computerized tomography (CT) imaging. The study suggested that hydrocephalus primarily affects the white matter, while sparing the gray matter. Images from this study revealed minimal disruption of the caudate nucleus and thalamus but significant disruption of the white matter, including fragmentation and swelling. Degeneration of the cortical mantle and edematous changes caused by hydrocephalus led to neuron loss . In 1977Conner et al., in 1984, published research on normal pressure hydrocephalus and its relationship to transmantle pressure. Hydrocephalus was induced in cats using intracisternal injection of kaolin, allowing for measurements comparing the ventricular pressure to the transmantle pressure. Pressure over the convexity of the cerebrum (transmantle) was shown to be elevated while ventricular pressure remained stable over a four-week period, suggesting that transmantle pressure was a determinant of ventricular dilation. Moreover, it was hypothesized that this could be an explanation for pseudotumors, in which the ventricles do not dilate despite increasing pressure . Cats wiBulat et al. officially proposed the modern idea for CSF flow in 1993, in a conference paper entitled, “Dynamics and Statics of the Cerebrospinal Fluid: The Classical and a New Hypothesis”. The author of many papers and extensive research in cerebral biochemistry and pathophysiology, Bulat hypothesized what many of the aforementioned experiments suggested; CSF does not flow unidirectionally, but in fact travels via pericapillary mechanisms in a to-and-fro manner . This waIn 2008, Bulat and his colleagues Lupret, Oreskovic, and Klarica clarified this by hypothesizing that exchange of macromolecules occurs in a bidirectional fashion between the interstitium and perivascular spaces. Moreover, they suggested that extracellular fluid, mainly water, is transferred by cerebral microvessels with systolic-diastolic pulsations. Pathways of macromolecules and water were compared using 3H-Inulin and 3H-water, respectively. 3H-water infusions into the ventricles coincided with significant pressure increases in the cerebral arterial plasma and cisternal CSF, suggestive that 3H-water is absorbed from the ventricles into the periventricular capillaries, which ultimately drain into the confluence of sinuses. Concordantly, the amount of 3H-water that had drained into the venous circulation was greater than what remained in the cisterns and arterial plasma. This provided a roadmap for 3H-water flow from the confluence of sinuses to the systemic circulation, before returning across the cerebral capillary walls into the CSF and interstitial fluid, establishing rapid equilibrium. 3H-Inulin was injected into the lateral ventricles, while once again measuring its presence within the arterial plasma, cisterns, and confluence. In this case, concentrations within the cisterns increased, but 3H-Inulin in the arterial plasma and confluence remained near the baseline. 3H-Inulin was demonstrated to be slowly removed from the CSF into the systemic circulation before ultimately being excreted in the urine . FollowiThe next logical step seemed to be figuring out the mechanism by which fluid is exchanged between the perivascular spaces and interstitium. Igarashi et al. performed a pair of studies using aquaporin-4 knockout mice, demonstrating the dependence of fluid exchange upon these channels. An aquaporin-4 inhibitor, TGN-020  was administered to control mice and knockout mice to compare the effects on regional cerebral blood flow. Following administration of TGN-020, the knockout mice exhibited very little change in regional cerebral blood flow, while the wild type showed a significant increase. Acetazolamide administration, used as a positive control, resulted in an even greater increase in regional cerebral blood flow in the knockout mice, with a similar increase in the wild-type group. Igarashi et al. then performed a similar study, this time including aquaporin-1 and aquaporin-4 knockout mice to measure radiolabeled water influx in the CSF, cortex, and basal ganglia. It was shown that aquaporin-1 inhibition caused minimal change in water influx into the CSF, suggesting that these channels play little role in this process. Aquaporin-1 channels are nearly completely restricted to the choroid plexus, indicating that regulation of water exchange within the CSF is not driven by these channels, but rather more dependent upon aquaporin-4 channels located diffusely throughout the astrocytic endfeet along transcerebral capillaries. CSF carriage of solutes from the brain parenchyma along the perivascular space seemed to be the brain’s equivalent to the lymphatics system and may provide a route for clearance of pathological macromolecules, such as amyloid proteins found in Alzheimer’s disease ,23. ThisModern explorations into the hydrodynamics of the brain include time-slip MRI imaging to more rapidly view CSF changes within the cerebral spaces. Studies by Yamada et al., Takeuchi et al., Shibukawa et al., and several others have used this technology to better understand cerebral pathophysiology. One of the benefits of this technology is that it allows CSF itself to be used as the tracer, rather than relying on radiolabeled analogues, thus preserving natural physiology. Notable from these studies is that CSF is not shown to travel unidirectionally, but rather pulsates back and forth, moving cranially during diastole and caudally during systole, as the Bulat-Klarica-Oreskovic theory hypothesized years earlier. Visualization of these pulsations has been made using time-slip MRI, with a seemingly predictable relationship with stroke volume emerging. Moreover, acceleration of waste removal has been demonstrated during shaking of the head and sleep, suggesting physiological conditions play a role in CSF exchange -26. FurtLimitationsAlthough we have tried to be inclusive of the major chronological discoveries relating to modern CSF pathophysiology, the studies included are not exhaustive. There are studies that may provide evidence supporting and contradicting the modern hypothesis that was not found in the selection process. Moreover, the importance of discoveries was determined based on relevance to the topic and estimated influence on the progression toward the modern theory. These metrics are subjective and limit the use of this study to a summary of discoveries rather than an analysis of the findings of each study. The study of ICP and cerebrospinal fluid dynamics has progressed significantly in the past century. Evidence that CSF flow is multidirectional and that production occurs throughout the cerebrospinal system is accumulating. Techniques like CT imaging, aquaporin-4 knockout mice, radiolabeled tracers, and advanced MRI imaging have provided evidence supporting multidirectional CSF exchange. Modern research points toward vascular pulsations driving the exchange of fluid between the interstitium and CSF, thus providing a new framework for CSF dynamics. If the modern models are accurate, one could imagine the implications in treating conditions such as hydrocephalus, intracranial hypertension, and neurodegenerative diseases like Alzheimer's. Although the reviewed studies provide convincing evidence, the understanding of CSF dynamics is still evolving, and additional research is needed to elucidate the mechanisms involved more completely. It would be helpful to have contemporary studies examining the equilibration mechanisms suggested by earlier research. We now have access to technology that could provide a more instantaneous view and perhaps unlock ways to manipulate CSF exchange when desired. Moreover, research into the clearance of CSF proteins has begun, but experimentation designed to understand the drivers of waste clearance would provide hope toward treating pathologies hallmarked by the accumulation of protein .


The cerebrospinal fluid (CSF) is a clear ultrafiltrate of blood that envelopes and protects the central nervous system while regulating neuronal function through the maintenance of interstitial fluid homeostasis in the brain. Due to its anatomic location and physiological functions, the CSF can provide a reliable source of biomarkers for the diagnosis and treatment monitoring of different neurological diseases, including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and primary and secondary brain malignancies. The incorporation of CSF biomarkers into the drug discovery and development can improve the efficiency of drug development and increase the chances of success. This review aims to consolidate the current use of CSF biomarkers in clinical practice and explore future perspectives for the field. Cerebrospinal fluid (CSF) is a clear fluid that protects our brain and spinal cord, and can help diagnose and monitor neurological diseases like Alzheimer's and Parkinson's. Biomarkers in CSF are like clues that help doctors and researchers better understand these diseases. By using CSF biomarkers, doctors can diagnose and monitor patients more accurately, while researchers can develop more effective treatments. This review looks at how we use CSF biomarkers in medicine and how they might help us in the future. Better understanding of CSF biomarkers can improve the lives of people living with neurological diseases. The cerebrospinal fluid (CSF) is a clear, colorless fluid that envelopes the central nervous system. The so-called CSF space is made up of intracerebral ventricles, subarachnoid spaces of the spine and brain, and the central spinal cord canal.Normal healthy adults have approximately 150 ml of CSF that is distributed within the cranial and spinal subarachnoid spaces (∼125 ml), and ventricles (∼25 ml). CSF is secreted at an average rate of 20 ml/h or 400–600 ml/day, most of it by the choroid plexuses of the ventricles. The remainder of the CSF is secreted by the interstitium and the meninges. It maintains a pulsatile flow in a unidirectional rostrocaudal manner in the ventricular cavities, and in a multidirectional manner in the subarachnoid spaces. The CSF circulates through the intracerebral ventricles, cisterns, and subarachnoid space, and is absorbed (resorbed) at the level of arachnoid villi into the internal jugular venous system, and by cranial and spinal nerves, and the ependyma. The entire CSF volume is renewed 4–5-times every 24 h in adults. Interestingly, MRI studies have identified a notable circadian rhythmicity in CSF physiology; there is an approximately fourfold difference in the rate of CSF production between 6 p.m. (12 ml/h) and 2 a.m. (42 ml/h) [+) are regulated stringently to ensure that any K+ lost from the ventricular system is replaced immediately; CSF K+ levels are maintained at a concentration of approximately 2.8 mM. In addition to the different ions, CSF also has cells, proteins, small molecules such as neurotransmitters and their metabolites, glucose, lactate, vitamins B1, B12, C, folate and b2-microglobulin, and nitric oxide secreted from the choroid plexus, as well as circulating DNA, RNA and microRNA.Physiologically, the CSF is an ultrafiltrate of blood secreted via the choroid plexuses. A multitude of studies have examined the various components of CSF, both in health and disease ,3. CSF cThe main function of the CSF is to mechanically protect the brain and spinal cord. CSF passively transports to dural venous sinuses via arachnoid granulations to regulate brain volume and clear antigens . In addiPhysiological CSF pressure varies between 10 and 15 mmHg in adults and 3 and 4 mmHg in infants . Though Apart from the clinical implications of such dysregulation, the CSF may serve as a reliable indicator of the health of the central nervous system. To this end, the CSF finds application as a valuable analyte to probe the brain and spinal cord in a minimally invasive manner. Specifically, the physical and molecular changes in the CSF may be valuable diagnostic and prognostic biomarkers for different neurological diseases . This reDifferent inflammatory or immune molecules including various proteins, other cell-free molecules and nucleic acids are secreted in the CSF. These can serve as quantifiable biomarkers in the CSF and include brain-derived proteins, exosomes, metabolites, small molecules, cell-free RNA, and cell-free DNA. These CSF-based biomarkers may find application in minimally invasive diagnosis and treatment monitoring of primary and secondary neurological diseases .Proteins that exhibit altered expression levels in the CSF during the development and progression of neurological diseases can act as valuable disease-tracking biomarkers. Many synaptic and axonal proteins have been studied and explored as possible biomarkers, both in CSF and blood. Two well studied putative biomarkers are neurofilament light (NFL) polypeptide and neurogranin. NFL is a protein expressed in large, myelinated neurons. Its levels in the blood and CSF have been shown to increase following neurodegeneration . A postsCirculating cells are cytologic biomarkers in the circulatory system and can be detected in blood or in fluids such as the CSF. These cells typically originate from malignancies, either primary tumors or metastases, and are termed as circulating tumor cells (CTCs). Most CTCs are rapidly destroyed in the blood circulation, which in turn limits their metastatic potential – only 2.5% of CTCs form micrometastases, whereas only 0.01% cells induce macrometastases . CTCs inin situ hybridization (FISH) technologies [A promising emerging trend for CSF analysis is liquid biopsy, either using immunofluorescence, or fluorescence nologies . Howevernologies ,18. Addinologies . Other enologies . The mosnologies . AdditioCell-free DNA: Cell-free DNA (cfDNA) in circulation, either in plasma or body fluids such as CSF, pleural fluid, or urine is a rich source of information on the physiological and pathological states of the individual. Though cfDNA in these body fluids is from different tissue origins, a detailed genomic analysis can differentiate and identify the source of origin of these molecules. cfDNA molecules have increasingly become promising biomarkers in the detection and monitoring of many diseases, mainly using next-generation sequencing for genome-wide analyses and single-base resolution. The analyses of cfDNA has facilitated blood-based diagnoses in place of tissue biopsies in the settings of prenatal and cancer care ,22. The cfRNA are gene transcripts that can be detected in the body fluids of cancer patients. In spite of the presence of nucleases in the circulation, cfRNA is surprisingly well protected from degradation, likely due to its packaging into exosomes [cfRNA can be detected in body fluids using microarray technologies or reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) [cfRNA over cfDNA is that the former enables researchers and clinicians to detect fusion genes [Cell-free RNA: Cell-free RNA (cfRNA) is a novel area in the field of circulating biomarkers that is also a challenging avenue in diagnosis and prognosis of diseases. Circulating exosomes . cfRNA cqRT-PCR) ,27. The on genes .miRNA profile is an upcoming area of interest [miR-146a and miR-155 are upregulated in the CSF of patients with Alzheimer's disease [Micro RNA: MiRNAs are small endogenous mediators of RNA interference and key regulatory components of many biological processes. Many miRNAs are deregulated in cancers either as oncogenic (oncomirs) or as tumor suppressors. MiRNAs are found in the blood serum and other body fluids including CSF. MiRNAs have the potential to serve as biomarkers for diseases; cancer diagnostics using interest ,30. The interest ,32. Othe disease .Epigenetics is the study of chemical modifications in the DNA, which regulates the gene expression or cellular phenotype associated with disease. Epigenetic modifications such as methylated DNA are epigenetic aspects that can be exploited to interrogate for specific CSF biomarkers associated with different diseases. The most common method is bisulfite treatment to distinguish between methylated and unmethylated DNA, which converts unmethylated cytosine in DNA to uracil. Further methylation-specific PCR can quantify DNA methylation limited to one genomic locus ,35. ManyThe CSF contains a range of small molecules that can be measured for diagnostic purposes. Neurotransmitters, such as dopamine, norepinephrine and epinephrine, along with their metabolites, can help diagnose certain neurological and psychiatric diseases, such as Parkinson's disease and depression ,37. NeopHaving reviewed the different types of biomarkers that can be detected in the CSF, the second half of this review will focus on CSF biomarkers for neurodegenerative diseases and brain malignancies.Appropriately described by the term “CSF analytic brain” over 25 years ago, it has been clear for a long time that pathologies of the central nervous system are in most cases associated with changes in the CSF that may be detected through examination . With inFor CNS pathologies such as neurodegenerative disorders and brain malignancies, it is very difficult to obtain tissue-based biopsies for diagnosis; it is even more difficult to obtain multiple biopsies for longitudinal studies. It is in such cases where CSF-based diagnostics will play an important role. To this end, CSF and blood are being studied as potential analytes for many CNS disorders. Biomarkers have been identified, tested, and validated for cancer and for neurodegenerative diseases. For many CNS disorders such as neurodegenerative diseases, CSF provides a distinct advantage over blood owing in large part to its proximity to the brain parenchyma. Due to this physical proximity, proteins can be secreted from the brain extracellular space into the CSF, which can then be collected through spinal tap. Similarly, in the case of malignancies, nucleotides can be released into the CSF that can be detected by various novel techniques. CSF is routinely collected in the clinical setting, primarily for diagnoses of many brain disorders including CNS infections and degenerative diseases. Once collected, CSF analyses can help detect and characterize these secreted biomarkers biochemically. Please see Alzheimer's disease (AD) represents an unmet medical need owing to a severe lack of effective therapies. Many of the so-called disease-modifying drugs that looked promising have failed to make an impact on the lives of patients. To develop better therapeutic options for patients with AD, it is important to identify and characterize well-validated biomarkers for early detection and accurate diagnosis. This is because failure to successfully develop effective treatment options in part reflects on the inefficiency of current clinical trials that may not be targeting or enrolling the “appropriate” patient populations. There are numerous scientists currently looking at biomarkers in multiple biological fluids (such as CSF and blood) in combination with imaging and neuropsychological testing.Genetically, AD is classified into two subtypes depending on the age of onset – early age of onset (before the age of 65 years and often in the late 40 s or early 50 s) labeled as early-onset Alzheimer's disease (EOAD) and comprising 1–5% cases; and disease developing after the age of 65 years classified as late-onset Alzheimer's disease (LOAD).Beta-amyloid (Aβ) protein was identified to be present in plaques, while phosphorylated Tau aggregates were detected in tangles, in the brains of patients with AD ,44. WhilExtensive research in this area has so far identified a few CSF-based biomarkers. These include Aβ isoform Aβ1–42 (Aβ42), total Tau, phospho-Tau (p-Tau), and neuro filament light chain (NFL), measured by ELISA . The maiIn some Alzheimer's cases, autoantibodies have been detected by ELISA against certain brain-derived proteins. One such autoantibody that was validated included the Aβ autoantibody in cognitively normal older population. Anti-Aβ42 autoantibodies were significantly higher in cognitively normal individuals when compared with age-matched Alzheimer's patients ,49. BaseParkinson's disease (PD) is a progressive neurodegenerative disease that affects ∼1% of the adults over 60 years of age . The priα-Synuclein has been shown to aggregate and is found in Lewy bodies in patients with PD. This has made α-Synuclein a prime target for biomarker studies in both CSF and blood. In a few previous studies using large patient cohorts, it was observed that there were lower levels of α-synuclein in the CSF of patients with PD when compared with healthy controls . The levIn addition to α-Synuclein, studies have demonstrated that familial PD is associated with mutations in DJ-1 (PARK7) . ImportaAn interesting finding from a recent study highlights biomarkers that are originally being studied in the context of AD. This study from a cohort of untreated PD patients collected sequential samples till patients received levodopa therapy. They found that following levodopa treatment, those patients who had a higher p-Tau and a p-Tau/Aβ42 ratio in the CSF developed cognitive decline subsequently . This fiAmyotrophic lateral sclerosis is a paralytic disease characterized by progressive motor neuron degeneration in the brain and spinal cord. The pathologic hallmark of this disease is motor neuron death in the motor cortex and spinal cord. The clinical presentation of ALS is heterogeneous, which makes clinical diagnosis difficult. Moreover, there is no diagnostic test that can definitively confirm or rule out ALS. Though the exact pathophysiological mechanisms underlying ALS are not clear, multiple genes have been implicated by gene mapping and DNA analyses . CurrentNumerous proteins in the CSF and blood are being evaluated as potential biomarkers for ALS ,62. NFL TARDBP (TAR DNA-binding protein) gene is mutated in some cases of familial ALS, TDP-43 (TAR DNA-binding protein-43) is a core component of cytoplasmic inclusions in ALS and FTLD. Hence CSF levels of TDP-43 are also being studied to evaluate its utility as a biomarker for ALS. Using antibody-based approaches, TDP-43 was found to have increased levels in ALS patients; interestingly, lower levels of TDP-43 were associated with worse prognosis and reduced survival [Apart from neurofilament proteins, survival .Based on the inflammatory responses seen in ALS, different mediators of inflammation are also being studies in both CSF and blood, such as IL-8, wide-range C-reactive protein, etc . Based oCurrently, brain malignancies are challenging to both diagnose and treat, and are associated with high rates of mortality and morbidity . EspeciaIn contrast, the physical proximity makes CSF a suitable analyte for biomarkers in brain malignancies. Moreover, CSF can be accessed readily and relatively non-invasively (via lumbar punctures) for longitudinal disease monitoring during and after therapy. While cytological analysis of CSF has low sensitivity, it is also non-quantitative . Hence wCSF-based biomarkers can be used for early diagnosis of brain malignancies, early identification of recurrence, monitoring therapeutic responses, and personalized medicine strategies for targeted therapies. There are two main modalities for CSF biomarkers: ctDNA and miRNA.Circulating tumor DNA that is shed from the tumor can be detected in the CSF. Recent studies have demonstrated that the detection rate of ctDNA in CSF is higher than that in tumor cells by next generation sequencing  and dropctDNA detection in the CSF has been demonstrated in both primary brain malignancies and in metastatic cancers; these analyses can be either restricted to a single gene or can involve a panel of multiple genes depending on the specific mutations in the tumor under consideration . A myriaWhile this is an extremely powerful tool in detection and monitoring of brain malignancies, there are some significant limitations as well, to ctDNA-based CSF biomarkers, primarily the lack of sensitivity in case of some malignancies. The location of the tumor, mainly its proximity to the CSF may be an important factor that drives the levels of ctDNA in the CSF, particularly in the case of primary CNS tumors ,87.Many studies that have demonstrated that miRNAs can function as oncogenes (oncomirs) and/or tumor suppressors; significantly, dysfunctional expression of miRNAs is a common feature of many types of cancer ,99. ImpoEGFR, IDH1/2 and TP53 genes have also been associated with brain cancers and can be detected in the CSF. A recent review has highlighted the significance of these biomarkers [Other biomarkers such as tumor markers CEA, CA-125, and AFP can be produced by the brain tumors and detected in the CSF. Additionally, genetic mutations in omarkers .The presence of these biomarkers in the CSF can aid in the diagnosis of brain cancer, facilitate monitoring of disease progression, and inform treatment decisions. Nevertheless, the interpretation of CSF biomarkers should be performed in conjunction with other clinical and radiographic findings, as well as histopathological analysis of tissue samples.Rigorous scientific approaches are crucial to identifying, characterizing, and validating CSF-based biomarkers for clinical use. It is essential to demonstrate their sensitivity and specificity before they can be applied in the clinical neurological setting. The confluence of various -omics technologies will undoubtedly facilitate and drive the development of CSF-based biomarkers and their clinical translation to address the various unmet needs in this challenging clinical space.CSF biomarkers should not be limited to the clinic but should also be incorporated into the drug discovery and development process early on to make drug development more efficient. Using appropriate biomarkers in clinical trial settings will improve chances of success through patient population stratification, identification of patient subpopulations most likely to respond, appropriate dose selection, demonstration of drug efficacy, and shortening the length of clinical trials. CSF biomarker-driven clinical strategies will make clinical trials time and cost efficient in both early and late stages.CSF serves as a protective and regulatory fluid for the central nervous system, aiding in diagnosis and prognosis of neurological diseases.CSF is a reliable source of biomarkers for neurodegenerative diseases and brain malignancies.CSF-based biomarkers can reflect pathophysiological changes related to a disease and aid in diagnosis and monitoring.nucleic acids, or metabolites and can diagnose diseases such as Alzheimer's, Parkinson's and brain malignancies.Biomarkers in CSF can be proteins, Demonstrating sensitivity and specificity of CSF biomarkers is crucial for clinical translation, and innovative technologies can facilitate their development and validation.Incorporating CSF-based biomarkers into drug development can enhance target identification and drug efficacy optimization, leading to improved success rates and efficiency.


Streptococcus pneumoniae is isolated as a causative microorganism, the ceftriaxone dose may be continued or reduced to a single dose of 2 g every 24 h, per institutional preference. There is no clear guidance that indicates the superiority of one regimen over the other. The objective of this study was to evaluate the susceptibility of S. pneumoniae in the cerebral spinal fluid (CSF) of patients with meningitis and the relationship between ceftriaxone dose and clinical outcomes. We identified 52 patients with S. pneumoniae meningitis with positive CSF cultures who were treated at the University Hospital, Bern, Switzerland, over a 19-year period. We collected clinical and microbiological data for evaluation. Broth microdilution and Etest methods were performed to test penicillin and ceftriaxone susceptibility. All isolates were susceptible to ceftriaxone. Ceftriaxone was empirically used in 50 patients, with a starting dosing regimen of 2 g every 24 h in 15 patients and 2 g every 12 h in 35 patients. In 32 patients started on a twice-daily regimen (91%), doses were reduced to once daily after a median of 1.5 (95% CI 1–2) days. The overall in-hospital mortality was 15.4% (n = 8), and 45.7% of patients reported at least one sequela of meningitis at the last follow-up . We found no statistical difference in outcome between the 2 g every 24 h and the 2 g every 12 h ceftriaxone dosing regimens. A ceftriaxone total daily dose of 2 g may be associated with similar outcomes to a 4 g total daily dose, provided that the causative organism is highly susceptible to ceftriaxone. The persistence of neurological and infection sequelae at the last follow-up underscores the need for optimal treatment of these complex infections.The recommended empiric ceftriaxone dosing regimen for acute bacterial meningitis in adults is 2 g every 12 h. After penicillin-susceptible  Streptococcus pneumoniae and Neisseria meningitidis. S. pneumoniae is responsible for two-thirds of meningitis cases in Europe and the United States ) after 2 g q24h at day 1 is 239 (SD ± 26) mg/L and at day 4 is 260 (SD ± 24) mg/L, and the minimum concentration  at day 1 is 13 (SD ± 5.5) mg/L and at day 4 is 15 (SD ± 6.2) mg/L [In healthy adults, the maximum plasma ceftriaxone concentration (C.2) mg/L . The eli.2) mg/L ,19. The .2) mg/L . However.2) mg/L . Both el>MIC) [min/MIC of >1) over the first 24 h of therapy based on plasma albumin concentrations and renal clearance. Of note, the elimination of the unbound fraction may also be enhanced with augmented renal clearance [90 for ceftriaxone among the S. pneumoniae isolates investigated in this study was 0.0625 mg/L (BMD) and 0.023 (Etest).Schleibinger et al.  investig>MIC) . In Schl>MIC) , in all >MIC)  investiglearance ,22. For learance . For comCSF/AUCserum of 0.7% [High (unbound) plasma concentrations of ceftriaxone led to higher absolute concentrations beyond the blood–brain barrier. For the treatment of meningitis, however, more than the aforementioned pharmacokinetic and dynamic parameters have to be taken into consideration. The penetration of ceftriaxone into the CSF in the absence of meningeal inflammation is relatively poor. Studies reported a CSF/plasma ratio of 1.5% for ceftriaxone  or an AU of 0.7% . Within  of 0.7% . Moreove of 0.7% . Thus, t of 0.7% . The est of 0.7% .Conversely, the penetration of ceftriaxone into the CSF is increased in bacterial meningitis because the inflammatory cascade leads to strong alterations of the blood–brain barrier ,20. In aTaken together, these results show that in acute bacterial meningitis, the unbound proportion of ceftriaxone increases with increasing total plasma concentration and in critically ill patients with hypoalbuminemia. Furthermore, ceftriaxone penetrates 10 times as much or more into the CSF in the presence of meningeal inflammation as it does in a condition without meningeal inflammation. The absolute ceftriaxone concentration achieved in the CSF is from 100 to 1000 times or more than that of the MICs of penicillin-susceptible pneumococci. Elimination from the CSF is relatively slow; hence, the half-time in this compartment is considerably prolonged.t > MIC for ceftriaxone 2 g q12h [t > MIC in the CSF approached ≥90% only when the ceftriaxone MIC was ≤0.03 mg/L. The implications of pharmacodynamic studies need to be reexamined in the clinical setting, as certain assumptions made in such studies could be questioned.To our knowledge, no study has systematically investigated the outcome of pneumococcal meningitis with a ceftriaxone dose reduction strategy when the causative strain is susceptible. A Monte Carlo simulation estimated the probability of target attainment for total drug CSF concentrations at 50% and 100% 2 g q12h . The prot > MIC estimates were calculated from theoretical extrapolations of ceftriaxone pharmacokinetic and dynamic studies, as well as from our MIC susceptibility testing results. Because of the retrospective nature of the study, we were unable to reconstruct the rationale for why the initial empiric dose was ‘only’ 2 g q24h in 15 cases or why 2 g q12h was continued in 3 cases despite the fact that the causative microorganism was highly susceptible. Similarly, we were unable to evaluate other factors that may have influenced mortality . The uneven distribution of the dosing groups and relatively low sample size did not allow for firm statistical conclusions. However, the clinical outcome results of our study are comparable to those of others, indicating that the reducing the ceftriaxone dose after susceptibility test results are available does not necessarily lead to a poorer outcome. The early recognition of signs of meningitis and the rapid administration of antibiotics are still pivotal to the prognosis.Our study has limitations. No semiquantitative or quantitative method was applied to evaluate the growth or concentrations of microorganisms in CSF, and no ceftriaxone drug concentrations were measured in plasma or CSF in these cases. Hence, we were unable to perform pharmacometric studies. The S. pneumoniae strains with proven culture growth in CSF obtained from adult patients who were referred or treated for bacterial meningitis at the University Hospital of Bern , Bern, Switzerland. The rationale for this selection is that we could evaluate the clinical outcome of pneumococcal meningitis in association with ceftriaxone dosing and include the embedded discussion on MICs and antibiotic penetration into the CSF. We included isolates obtained from 2000 to 2018. The rationale for this time span is that it included the publication year of the ESCMID guidelines for bacterial meningitis [Clinical pneumococcal isolates from normally sterile body sites  have been collected in our institution since 2000. For this study, we included only ningitis  and a clS. pneumoniae and meningitis [All isolates were identified according to the defined protocol of the Swiss National Center for Pneumococci . Isolateningitis : penicilS. pneumoniae isolates. Only adult patients (≥18 years of age) were included. Access to charts was available only for patients treated at the University Hospital Bern , Bern, Switzerland. The following variables were obtained: age, gender, comorbidities, diabetes mellitus, impaired renal function, chronic heart disease, immunosuppression, chronic alcohol abuse, evidence of a recent respiratory infection, and travel history. In addition, symptoms and findings were obtained, as well as laboratory parameters at clinical presentation.To evaluate the association between microbiological and clinical data, we reviewed patient charts corresponding to the Treatment analysis included antibiotic therapy  in comparison to the outcome. The primary outcome was defined as in-hospital death. Secondary outcomes included in-hospital complications , neurological impairment at discharge, and sequelae at the latest follow-up. The maximum follow-up was defined as the latest available contact at the University Hospital Bern , irrespective of the cause of contact.p-values of <0.05 were interpreted as being statistically significant. GraphPad Prism was used for statistical analysis and the graphical depiction of the obtained data.For all variables, the number and proportion of available results were obtained. Patients with more than 10% missing variables were excluded from the clinical analysis. Continuous variables were registered as mean and SD. Categorical variables were registered as counts, and groups were compared via the chi-square test and Fisher’s exact test. S. pneumoniae isolates obtained from CSF, susceptibility to penicillin and ceftriaxone was high, and the ceftriaxone MIC values were low. The vast majority of patients from whom these isolates were obtained were eventually treated with ceftriaxone at a dose of 2 g q24h. Mortality was 15.4%, and 45.7% of patients reported at least one sequela in the follow-up examination, indicating that acute pneumococcal meningitis is a severe disease. However, these results are consistent with those reported by others. Our study indicated that the ceftriaxone dosing regimens of 2 g q24h or 2 g q12h may have a similar outcome, provided that the causative microorganism is highly susceptible to ceftriaxone. Thus, reducing the ceftriaxone dose from 4 g daily (empiric therapy) to 2 g daily (targeted therapy) after susceptibility results are available may not influence the outcome.In our cohort of


We provide evidence that ablation of GSTK1 does not change the basal intraperoxisomal redox state but significantly extends the recovery period of the peroxisomal glutathione redox sensor po-roGFP2 upon treatment of the cells with thiol-specific oxidants. Given that this delay (i) can be rescued by reintroduction of GSTK1, but not its S16A active site mutant, and (ii) is not observed with a glutaredoxin-tagged version of po-roGFP2, our findings demonstrate that GSTK1 contains GSH-dependent disulfide bond oxidoreductase activity.Peroxisomes serve as important centers for cellular redox metabolism and communication. However, fundamental gaps remain in our understanding of how the peroxisomal redox equilibrium is maintained. In particular, very little is known about the function of the nonenzymatic antioxidant glutathione in the peroxisome interior and how the glutathione antioxidant system balances with peroxisomal protein thiols. So far, only one human peroxisomal glutathione-consuming enzyme has been identified: glutathione S-transferase 1 kappa (GSTK1). To study the role of this enzyme in peroxisomal glutathione regulation and function, a GSTK1-deficient HEK-293 cell line was generated and fluorescent redox sensors were used to monitor the intraperoxisomal GSSG/GSH and NAD Complex living systems can only survive within a very narrow range of physicochemical conditions . However2O2) and lipid peroxides (LOOH), thereby being converted to a disulfide-bonded glutathione dimer (GSSG) [An important molecule for redox homeostasis is the tripeptide γ-glutamyl-cysteinyl-glycine, commonly known as glutathione, whose thiol group supplies reducing power for numerous cellular redox reactions . This mer (GSSG) , (ii) mor (GSSG) , (iii) dr (GSSG) ,9, (iv) r (GSSG) , and (v)r (GSSG) .2O2-producing oxidases as well as catalase (CAT), a major H2O2-decomposing enzyme, their activity also contributes to cellular redox balancing and regulation [Peroxisomes are organelles that play a pivotal role in cellular lipid metabolism . Given tgulation . Howevergulation , it is qgulation ,14, and gulation . Furthergulation , but no gulation ,13. Currgulation ,18.GSTK1, also sometimes referred to as disulfide-bond A oxidoreductase-like protein (DSBA-L), is a dimeric member of the glutathione S-transferase protein family that diverges from almost all other human GSTs in terms of sequence similarity and protein structure ,20,21. O2O2 detoxification [2O2 [This study focuses on the role of GSTK1 in peroxisomal glutathione homeostasis. Given that (i) GSTK1 makes use of GSH as an obligate cofactor or cosubstrate ,42, (ii)fication , and . The pSpCas9(BB)-2A-GFP [The plasmids encoding peroxisomal (po-) roGFP2 , cytosolA-roGFP2 , and po-A-roGFP2  have bee)-2A-GFP  plasmid )-2A-GFP , c-cpYFP)-2A-GFP , c-iNAP1)-2A-GFP , and c-i)-2A-GFP  were proggatccaccatgggcaaccggaagtggggcctgtgc-3′ and 5′-ggggggcggccgctcacagtttggacttgcccatcatctcctcccgccac-3′; and for po-iNAP1 and po-iNAPc, 5′-cgagggatccaccatggatccgatgaaccgg-3′ and 5′-cgaggggcggccgctcacagtttggacttgcccatcatctcctccc-3′ , and ligating the BamHI/NotI-digested PCR products into similarly-digested pEGFP-N1 . The plasmids encoding po-HsGSTK1-mCherry or po-HsGSTK1S16A-mCherry were generated by amplifying the po-mCherry coding sequence via PCR (oligonucleotides: 5′-cgagagaagcttaccatggtctctaagggcga-3′ and 5′- ggcggggcggccgcttacagtttagatttgtacagttcatcc-3′) and ligating the HindIII/NotI-digested PCR product into the backbones of the similarly digested po-HsGSTK1-roGFP2- and po-HsGSTK1S16A-roGFP2-encoding plasmids, respectively. To construct the GSTK1 guide RNA-encoding pSpCas9(BB)-2A-GFP plasmids, two oligonucleotides  were annealed and subsequently cloned into the BpiI-digested pSpCas9(BB)-2A-GFP plasmid.The plasmids encoding po-SoNar, po-iNAP1, or po-iNAPc were generated by amplifying the corresponding coding sequences via polymerase chain reaction (PCR): oligonucleotides for po-SoNar, 5′-cgagTOP10F’ E. coli strain  was used as a cloning and plasmid amplification host. Oligonucleotides , restriction enzymes , and Platinum Pfx DNA polymerase  were commercially obtained, and the correctness of the plasmid inserts was verified by Sanger sequencing .The v/v) fetal bovine serum , 2 mM Ultraglutamine-1 , and 0.2% (v/v) Mycozap ) at 37 °C in a humidified 5% CO2 incubator. HEK-293 cells were electroporated  with the Neon Transfection System (Thermo Fischer Scientific) in combination with a homemade sucrose-based RF1 buffer [The DD-DAO Flp-In T-REx 293 cell line routinely used in this research (here referred to as HEK-293) has been described elsewhere . Cells w1 buffer .2O2 inside peroxisomes through expression and activation of a destabilization domain (DD) tagged version of D-amino acid oxidase (DAO), the HEK-293 cells were processed as described elsewhere [2O2 , Triton X-100 , or Dulbecco’s phosphate-buffered saline (DPBS) containing 3-amino-1,2,4-triazole , D-alanine , or L-alanine  were added at the concentrations and time intervals shown in the figure legends.To selectively generate Hlsewhere . For othSubcellular fractionations of HEK-293 cells were performed as described . Based oGSTK1 gene in DD-DAO Flp-In T-REx 293 cells [The CRISPR-Cas9 genome editing technology (as described by ) was use93 cells . To thisThe following primary antibodies were used for immunoblotting: rabbit anti-3-ketoacyl-CoA thiolase (ACAA1) , anti-CAT , anti-cytochrome c oxidase subunit 4 (COX IV) , anti-enhanced green fluorescent protein (EGFP) (1:1000) , anti-pew/v) trichloroacetic acid and 0.0125% (w/v) deoxycholate, followed by acetone-washing. Thereafter, the protein samples were processed for reducing or nonreducing SDS-PAGE and immunoblotting as described elsewhere [Proteins samples were precipitated by treating DPBS-resuspended cell suspensions with 6%  . For thep-value lower than 0.05 was considered statistically significant. Considering this is an exploratory study, no Bonferroni corrections were applied.Statistical analysis was performed using GraphPad Prism . The statistical tests used are specified in the figure legends. A Given the contradictory findings on the subcellular location of GSTK1 ,24,25,26To study the potential role of GSTK1 in peroxisomal glutathione redox metabolism, we selectively disrupted the corresponding gene in HEK-293 cells (genetic background: DD-DAO Flp-In-T-REx 293 ) by usinA heterozygous  and a homozygous  knockout clone were selected and their correctness was validated at the protein level by immunoblot analysis . If cons+/NADH, NADPH, and H2O2 levels under basal conditions. To monitor possible changes in these metabolites, we used compartment-specific forms of the ratiometric fluorescent redox sensors roGFP2 [+ or NADH [+/NADH or NADPH levels lead to higher F480/F400 ratios for SoNar or iNAP1, respectively. Since iNAP1 and SoNar are pH sensitive, it is critical to conduct parallel measurements with sensors that react similarly to pH but not to NAD(H) or NADPH . In the case of roGFP2 and roGFP2-Orp1, oxidation results in the formation of an intramolecular disulfide bridge, which shifts the excitation maximum from 488 nm to 405 nm [2O2 levels, respectively. No significant alterations in any of the examined redox metabolites could be found between control and ΔGSTK1 cells, neither in peroxisomes nor in the cytosol (Given that (i) the overarching aim of this study was to examine the role of GSTK1 in peroxisomal glutathione redox homeostasis, (ii) organellar glutathione pools rely entirely on cytosolic glutathione import , and FP2-Orp1 . Second,o-roGFP2 , a findiGiven the apparent difficulty in producing insults that po-roGFP2 can detect, we transiently exposed the cells to AT-4 and diamide, two oxidants whose mode of action involves a direct reaction with thiols ,68 and tw/v) Triton X-100, a nonionic detergent that can permeabilize cell membranes. Under those conditions, po-roGFP2 responded similarly as observed in AT-4-treated control and ΔGSTK1 cells  GRXs, which catalyze the thiol-disulfide exchange between the GSH-GSSG and roGFP2 redox pairs . Given tWT or po-GSTK1WT-mCherry as compared to GSTK1S16A or po-GSTK1S16A-mCherry (Previous studies have identified serine-16 (S16) as a critical residue for GSTK1 activity ,31,33. T-mCherry A,C, the -mCherry B and per-mCherry D of the 2O2 or AT-4. However, neither internal nor external oxidative insults caused noticeable disulfide bond-induced conformational changes, as evaluated by a redox electrophoretic mobility shift assay (Given that (i) human GSTK1 has two cysteine residues (C27 and C176), and (ii) the latter cysteine has been reported to be a glutathionylation target in the mouse orthologue , we alsoft assay . Note th2O2 [Proteins inside the peroxisome lumen must be constantly safeguarded against the potentially damaging effects of H2O2 , and one2O2 . However2O2 ,74, virt2O2 ,18.https://www.proteinatlas.org/ENSG00000197448-GSTK1/subcellular; accessed on 26 April 2023).We first confirmed that peroxisomes in HEK-293 cells do indeed contain GSTK1 , an obse−/− mice had normal amounts of reduced and total glutathione in their livers and kidneys [Next, we examined how GSTK1 inactivation affected the expression levels of a specific set of peroxisomal and mitochondrial proteins , mitocho kidneys . As othe kidneys , we also16XXS19; Ser16) is lacking a cysteine [As the po-roGFP2 recovery phenotype cannot be observed in ΔGSTK1 cells expressing po-GRX1-roGFP2 , our fincysteine , this macysteine  and Ure2cysteine . In addicysteine .In summary, this study provides evidence that the peroxisomal pool of GSTK1 possesses GRX-like activity. This implies that, after the GST-Omega class , the GSTIn this study, we investigated the role of GSTK1 in peroxisomal redox metabolism in HEK-293 cells. We provide evidence that, while the protein appears to be unnecessary for peroxisomal redox homeostasis under basal conditions, the peroxisomal pool of GSTK1 possesses a glutaredoxin-like activity towards the glutathione redox sensor roGFP2. The discovery of this new biochemical function of GSTK1 opens up new avenues to advance our understanding of how changes in its expression contribute to aging and disease.


This study investigates the feasibility of the indocyanine green–guided near-infrared fluorescence (ICG-NIRF) imaging technique in evaluating testicular blood flow during testicular torsion (TT) surgery in pediatric cases.We retrospectively analyzed the eight pediatric patients with TT who underwent surgery in our hospital between February and July 2023. The intraoperative two-step method of ICG-NIRF imaging and testicular incision was used to evaluate the testicular blood flow, followed by a selection of different surgical methods. The removed testes were pathologically examined after surgery, and all patients were followed up 1 month after surgery to evaluate testicular blood flow using gray-scale ultrasound and color Doppler flow imaging (CDFI).P > 0.05). The postoperative recovery was uneventful, with no complications during the follow-up period of 1 month. The postoperative histopathological results of three patients who underwent orchiectomy showed extensive hemorrhage, degeneration, and necrosis of the testicular tissue. Among the five patients who underwent orchiopexy, a gray-scale ultrasound and CDFI examinations showed uniform internal echo of the testes and normal blood flow signals in four patients. One patient with no testicular blood vessel imaging on the torsional side showed uneven internal echo of the testis and no blood flow signals.Eight pediatric TT patients aged 1–16 years, with a median age of 11.5 years, were enrolled. Time from the onset ranged from 4 to 72 h (mean 26.13 ± 25.09 h). A total of eight testes were twisted, including four on the left side and four on the right side. The twisting direction of the testes was clockwise in four cases and counterclockwise in four cases. The rotation of torsion was 180°–1,080° (mean 472.5° ± 396°). There was no statistically significant difference in the imaging time between the four patients with testicular blood vessel imaging on both the torsional and normal sides (ICG-NIRF imaging is a feasible method to evaluate testicular blood flow during TT surgery. Testicular blood vessel imaging within 5 minutes after ICG injection might be the basis for testicular retention during TT surgery. Testicular torsion (TT) is a urological emergency. Most scholars agree that the “golden time” for testes survival is within 6 hours from the onset of TT. The earlier the time of treatment, the greater the chance of saving the testes. With the prolongation of the onset time, the possibility of retaining the testes gradually decreases , 2. It hThe selection of surgical methods in TT surgery is mainly based on observing the testicular blood flow after detorsion. At present, testicular blood flow is assessed by intraoperative observation of testicular color , 4. It cIndocyanine green (ICG) is a three-carbon cyanine dye that can emit fluorescence when irradiated by near-infrared (NIR) at a specific wavelength and can be received and converted to imaging by a specific device , 4. An IThis study aims to report the application of the ICG-NIRF imaging technique in assessing testicular blood flow during TT surgery in Children's Hospital of Capital Institute of Pediatrics and to explore the feasibility of the ICG-NIRF imaging technique in evaluating testicular blood flow during TT surgery in pediatric cases.This study retrospectively analyzed pediatric patients with evaluated testicular blood flow using the ICG-NIRF imaging technique during TT surgery in our center from February to July 2023. Eight patients with TT (two with cryptorchidism) aged 1–16 years, with a median age of 11.5 years, were enrolled. Time from the onset ranged from 4 to 72 h (mean 26.13 ± 25.09 h). A total of eight testes, including four on the left side and four on the right side, were torsional. The twisting direction of the testes was clockwise in four cases and counterclockwise in four cases. The rotation of torsion was 180°–1,080° (mean 472.5° ± 396°) .The patients were in the supine position after general anesthesia. A transverse incision was performed on the torsional side of the central scrotum to expose the torsional testis. Then, a warm saline-soaked gauze was applied to the testis for 10 min after detorsion. After intravenous injection of ICG (dosage 0.3 mg/kg) , the bilt-test was performed for further analysis. A P-value of <0.05 was considered statistically significant.The data were presented as the mean ± standard deviations, calculated using GraphPad Prism version 9.0. The Shapiro–Wilk test was performed to evaluate the data distribution. Due to the normal distribution of the data, a paired P > 0.05)  . All pat > 0.05) . Among t > 0.05) .As an organ with both endocrine and reproductive functions, the testis is of great importance to men. Jacobsen et al. reviewed articles examining changes in testicular function after TT and stated that there is no evidence that it affects sex hormone levels including luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone. However, it can affect semen quality. These patients experience decreased sperm motility and reduced total sperm count, possibly leading to reduced fertility. Studies in animals and humans have also confirmed the presence of damage to the contralateral testis after TT, which may result from multiple factors . Other sInappropriately selecting surgical methods has a great impact on the future life of patients. It is particularly important to choose the right intraoperative surgical methods. In terms of endocrine functions, Arap et al. compared the effects of different surgical methods on the endocrine function of patients with TT, and they found that the average FSH level in blood samples in the orchiectomy group is obviously higher than that in the orchiopexy group. There is no statistical difference in serum LH and testosterone levels between the two groups . SimilarThere is still some controversy about whether to perform contralateral orchiopexy during TT surgery. The guidelines for pediatric urology of the European Association of Urology have pointed out that the contralateral orchiopexy should be performed in children with TT. However, they did not mention the reason . StudiesIn 1959, the US Food and Drug Administration approved ICG for clinical use , 12, 24.At present, the feasibility of applying the ICG-NIRF imaging technique to evaluate testicular blood flow during TT surgery remains unclear, and only three case reports have been published , 15, 27.In the present study, four cases undergoing orchiopexy showed bilateral testicular blood vessel imaging within 1 min after ICG injection, similar to those reported above. In the other four cases, no blood vessel imaging was observed in the torsional testes within 5 min after ICG injection. Therefore, the testes were incised to observe whether the testicular parenchyma hemorrhaged. Orchiectomy was performed in three cases without hemorrhage, and orchiopexy was performed in one patient with testicular parenchymal hemorrhage. Four cases with testicular blood vessel imaging on the torsional sides showed uniform internal echo and normal blood flow signals during the 1-month follow-up. The histopathological results of three cases who underwent orchiectomy showed testicular necrosis, consistent with the intraoperative observation. An ultrasound follow-up of the other patient at 1 month after surgery showed uneven internal echo and no blood flow signals in the torsional testis. We consider that this result might be because hemorrhage after testicular incision originates from venous congestion or hemorrhage in the tunica albuginea blood vessels. Arda et al. reported that venous and arterial hemorrhage should be distinguished when performing a testicular incision to observe hemorrhage and that tunica albuginea hemorrhage could not be used as a true indicator of testicular activity . HoweverThere was no statistical difference in the time of testicular blood vessel imaging between the torsional sides and the normal sides, suggesting that there was no significant difference in the testicular blood flow between the torsional testis after detorsion and warm compress and the normal testis, which may indicate that the good blood flow of the torsional testis and the testis could be retained. The postoperative follow-up ultrasound in four cases also confirmed this finding.In summary, after testicular detorsion, we recommend that a warm saline-soaked gauze could be applied to the testis for 10 min, followed by intravenous ICG injection. The testes showing blood vessel imaging within 5 min after ICG injection should be retained, and those that are not imaged within 5 min should be removed .This is the first study to evaluate testicular blood flow during TT surgery using the ICG imaging time. This study suggests that the ICG-NIRF imaging technique could provide an objective basis for evaluating testicular blood flow during TT surgery. However, it has several limitations. First, the small sample size of this study and experience from a single center may lead to biases in interpreting the results. Moreover, the postoperative follow-up time is relatively short, and the long-term evaluation of the torsional testes function is lacking. Therefore, we will further increase the number of study cases and carry out a long-term follow-up.The ICG-NIRF imaging technique is safe and easy to operate. We point out that the ICG-NIRF imaging technique is feasible for evaluating testicular blood flow during TT surgery. The application of this technique may help avoid the removal of functional testes and the retention of necrotic testes during TT surgery. The torsional testis with blood vessel imaging within 5 min after ICG injection might be the basis for testicular retention during TT surgery. Those that are not imaged within 5 min might be the basis for testicular removal.


Bacterial meningitis is a serious complication after neurosurgery and has a high mortality rate. Early diagnosis and timely treatment are crucial or will have disastrous consequences. The classic triad of bacterial meningitis lacks sensitivity and specificity. Therefore, the diagnosis of post-neurosurgical bacterial meningitis relies on cerebrospinal fluid. But traditional cerebrospinal fluid parameters are not completely reliable in diagnosing post-neurosurgical bacterial meningitis. Previous studies have found that CSF lactate concentration is related to bacterial meningitis. But, after the craniocerebral operation, the cerebrospinal fluid of most patients is bloody. Whether red blood cells interfere with diagnosing PNBM based on lactate concentration is limited. In the current study, we further analysis on whether red blood cells interfere with diagnosing PNBM based on lactate concentration. This study aimed to investigate the value of cerebrospinal fluid lactate concentrations in post-neurosurgical bacterial meningitis. A prospective observational study was performed on 62 patients at Kunshan First People’s Hospital’s intensive care unit affiliated with Jiangsu University. We found that erythrocytes do not affect cerebrospinal fluid lactate, and elevated lactate concentrations can be used as a marker for postoperative bacterial meningitis.The online version contains supplementary material available at 10.1186/s12883-023-03428-8. Approximately 120 cases of bacterial meningitis occur worldwide every year . The incThe classic triad of bacterial meningitis includes fever, neck stiffness, and mental state changes. This triad lacks sensitivity and specificity, and many patients do not have these three characteristics , 7. TherAn ideal marker should be highly sensitive. When CSF infection exists, leukocytes increase anaerobic glucose metabolism, producing lactic acid and reducing pH. Bacterial metabolism, neutrophil glycolysis and anaerobic metabolism in brain tissues during bacterial meningitis lead to lactate accumulation in CSF –20. LactProspective observational diagnostic study.To evaluate the diagnosis value of CSF lactate as a marker in postsurgical patients.①To evaluate whether red blood cells affect cerebrospinal fluid lactate.②To evaluate the correlation of CSF lactate with other CSF markers in PNBM.③To describe the optimal CSF lactate cut-off value.62 patients undergoing craniocerebral operation from February 2018 to February 2020 in the intensive care unit of Kunshan First People’s Hospital affiliated with Jiangsu University were selected. The study complied with the Helsinki Declaration and local laws. These postoperative patients all were suspected of having bacterial meningitis. According to the 2008 diagnostic criteria of PNBM of the United States Centers for Disease Control and Prevention/National Care Safety Network (CDC/NHSN)  These diPatients are classified according to predefined definitions. According to qualitative erythrocyte results in CSF, the patients were divided into hemorrhagic CSF and non-hemorrhagic CSF groups. Patients were divided into the PNBM and non-PNBM groups according to the PNBM standard formulated by CDC/NHS in 2008.In this study, all patients underwent emergency surgery and received routine prophylactic antibiotics before and after craniotomy, using first-generation cephalosporin.The measurement variables are expressed by the median . Comparisons between the two groups are performed using Student’s t-test for variables with a normal distribution and Wilcoxon two-sample test for variables with a non-normal distribution. Classification variables are expressed by frequency (%) and comparisons between groups are made by chi-square test or Fisher’s exact method. Mann-Whitney tests were used to estimate the area of the ROC curve where infection occurred, and the cutoff value was used when the index reached the maximum. Both used bilateral tests and statistical analysis software SAS were used. The difference was considered statistically significant when < 0.05.During the 2-year study period, there were 62 suspected cases of PNBMTable . There wTo analysis on whether red blood cells interfere with diagnosing PNBM based on lactate concentration. We compared cerebrospinal fluid parameters between hemorrhagic and non-hemorrhagic cerebrospinal fluid groups. We found that lactate concentration  and protein level  are increased in the hemorrhagic cerebrospinal fluid group compared with the non-hemorrhagic cerebrospinal fluid group; In contrast, the glucose concentration  is lower in the hemorrhagic cerebrospinal fluid group compared with the non-hemorrhagic cerebrospinal fluid group. Still, the difference is not statistically significant. While the white blood cell count in the two groups is different, the difference is statistically significant. The difference in blood glucose concentration (8.632 mmol/L in Hemorrhagic CSF group vs. 8.554 mmol/L in Non-hemorrhagic CSF group) and blood lactate concentration (2.387 mmol/L in Hemorrhagic CSF group vs. 2.238 mmol/L in Non-hemorrhagic CSF group) between the two groups was not statistically significant . and protein concentrations in the PNBM group were increased compared with the non-PNBM group, whereas the glucose concentration  in the PNBM group was lower than that in a non-PNBM group. The blood glucose concentration  did not show a statistically significant difference between the PNBM group and the Non-PNBM group, while the blood lactate concentration was significantly higher in the PNBM group (2.458 ± 0.590 mmol/L) compared to the Non-PNBM group (2.037 ± 0.671 mmol/L) (P = 0.016). The difference in blood glucose concentration was not significant in the PNBM group (P = 0.087) or in the Non-PNBM group (P = 0.851). However, there was a significant difference in blood lactate concentration within the PNBM group (P = 0.004), while no significant difference was observed in the Non-PNBM group (P = 0.271) (Supplementary Table 1).We also evaluated the correlation between CSF lactate and other parametersFig. . AccordiROC curve analysis of CSF parameters shows that CSF lactate levels exhibit good diagnostic efficiency Fig. . When thCerebrospinal fluid acidosis caused by bacterial infection is well described. When CSF infection exists, leukocytes increase anaerobic glucose metabolism, producing lactic acid and reducing pH. Bacterial metabolism, neutrophil glycolysis, and anaerobic metabolism of brain tissues during bacterial meningitis lead to CSF lactate accumulation –20. LactAfter the craniocerebral operation, the cerebrospinal fluid of most patients is bloody due to primary disease, operation, or lumbar puncture injury. The presence of red blood cells in CSF will lead to an increase in lactate levels . This rePrevious studies have also discussed the relationship between cerebrospinal fluid lactate levels and mortality in patients with meningitis. Abassi et al . found tLeib et al.  first reIn our study, we observed that the average blood lactate and blood glucose concentrations are higher in patients of the PNBM group. Zheng et al ., in a mOur research shows that CSF lactate mainly reflects infection and is not related to the number of red blood cells in cerebrospinal fluid. CSF lactate concentration is a reliable indicator for the diagnosis of PNBM. When the diagnostic threshold of CSF lactate is 4.95 mmol/L, the specificity is 100%, which is useful for excluding bacterial meningitis after neurosurgery under appropriate clinical conditions.Below is the link to the electronic supplementary material.Supplementary Material 1


Neurofilament light chain (NfL) reflects axonal damage in neurological disorders. It has recently been evaluated in idiopathic intracranial hypertension (IIH). A biomarker indicating the severity of optic nerve damage in IIH could support diagnostic accuracy and therapeutic decisions.We retrospectively reviewed NfL concentrations in the cerebrospinal fluid (CSF) of 35 IIH patients and 12 healthy controls, who had received diagnostic workup for IIH in our clinic. The diagnosis of IIH was made according to the modified Friedman criteria for IIH and for IIH without papilledema Friedman DI et al Neurol 81:1159–1165 (2013) [1]. NfL in the CSF (CSF-NfL) was correlated with the severity of papilledema and with CSF opening pressure.CSF-NfL correlated with CSF opening pressure at the time of collection. In patients with IIH and moderate or severe papilledema, CSF-NfL was significantly increased compared to patients with mild or no papilledema. Healthy controls with raised intracranial pressure showed no relevant elevation of CSF-NfL.CSF-NfL appears to correlate with the severity of papilledema in IIH and with CSF opening pressure and may therefore be a predictor of optic nerve damage in IIH patients. Idiopathic intracranial hypertension (IIH) is a rare headache syndrome mainly observed in overweight women of childbearing age –4. The dNeurofilament light chain (NfL) is a polypeptide that gives structural support to the axon and regulates its diameter. NfL marks axonal loss in the central nervous system in various neurodegenerative disorders , 14 and For this retrospective study, we reviewed medical charts of adult patients who had received diagnostic evaluation for suspected or already diagnosed IIH at our neurological department  between January 2020 and June 2021. Only patients who had received ophthalmological and neurological examinations, including a lumbar puncture with CSF opening pressure measurement and measurement of CSF-NfL, were included. Diagnosis of IIH was made according to the modified Friedman criteria for IIH and for IIH without papilledema (IIHWOP) . PatientThis research study was conducted retrospectively from data obtained for clinical purposes. Retrospective data extraction and analysis was approved by the ethics committee of Charité – Universitätsmedizin Berlin (Application No. EA4/004/21).t-test was performed for group comparisons of continuous variables and a chi-square test or Fisher’s exact test for group comparisons with regard to sex. Correlation between CSF-NfL ratios and the CSF opening pressure was tested for linear correlation using the Pearson correlation coefficient. A two-tailed p-value of < 0.05 was considered statistically significant.All statistical analyses were performed in SPSS 27 . Continuous variables were described by the mean and standard deviation. Variables were checked for normal distribution using the Shapiro–Wilk test and Q-Q-plots. The CSF-NfL showed a right-skewed distribution and was log-transformed for subsequent analyses (log 10 = lg). CSF-NfL ratios were calculated from the quotient of the CSF-NfL and the age-adjusted normal value . For the statistical analysis, an unpaired n = 33, IIHWOP: n = 2) and 12 patients, for which the suspected diagnosis of IIH was rejected and no other disease could be found, who are subsequently referred to as healthy controls. Five of the IIH/IIHWOP patients had unilateral abducens nerve palsy at the time of examination, three in the IIH-group and the two IIHWOP patients. Patients and healthy controls did not significantly differ with respect to age and sex. Detailed patient characteristics are given in Table We identified 59 patients with suspected IIH, of which 53 had received measurements of CSF-NfL and ophthalmological and neurological examinations. Six patients were excluded because alternative neurological diseases  were found. The remaining 47 patients consisted of 35 with newly made or pre-existing diagnoses of IIH/IIHWOP  in all patients. CSF-NfL had a moderate correlation with age . CSF-NfL did not significantly differ with respect to sex.CSF-NfL ratios had a moderate correlation with CSF opening pressure . CSF-NfL ratios rose with the degree of papilledema . The two groups (group 1: no and mild papilledema vs. group 2: moderate and severe papilledema) did not significantly differ with respect to age and sex. In 10 IIH patients, papilledema had already resolved at the time of examination. In this group, CSF-NfL ratios were lower when compared to IIH patients with papilledema at the time of examination .CSF-NfL ratios in IIH/IIHWOP-patients were significantly higher than in healthy controls . In one patient, CSF-NfL was normal; in the other patient, it showed a mild, 1.3-fold, increase. Three other patients had sixth nerve palsy and moderate (n = 2) or severe (n = 1) papilledema, all with increased CSF-NfL ratios .This study included two patients with IIHWOP (no papilledema, but with abducens nerve palsy and with ICP of 25 and 31 cmHIn line with a prior study , we founWe can only hypothesize concerning the pathophysiological mechanism underlying CSF-NfL release in IIH, but the association of CSF-NfL with both papilledema and CSF opening pressure seems to imply that it results from pressure-induced axonal damage. It still remains unknown whether CSF-NfL in IIH is due to optic nerve damage alone or whether it reflects ubiquitous axonal damage caused by raised intracranial pressure. However, in this study, IIH patients with resolved papilledema had normal CSF-NfL. Moreover, none of our healthy controls showed raised CSF-NfL, despite some of them having slightly increased CSF opening pressures, a finding also reported by Beier et al. . Our stuAs mentioned above, CSF-NfL has been explored in other neurological disorders, with comparatively high CSF-NfL in inflammatory disorders of the central nervous system. CSF-NfL in inflammatory optic neuritis shows much higher levels than our group of IIH patients . Unlike A major limitation of our study is the small cohort and its retrospective nature. More detailed ophthalmological findings, such as perimetric testing and the dependence of CSF-NfL on retinal nerve fibre layer thickness, would be desirable. Moreover, our study lacks correlation with functional visual outcomes and can, therefore, only prepare the ground for future prospective studies. A selection bias may have caused more severely affected IIH patients to be part of this study because only patients who had received a lumbar puncture with CSF-NfL measurement and ophthalmological examination were included. Seemingly less affected patients would not have received such extensive workup.Summarizing, our study confirms the relationship between CSF-NfL concentrations and severe papilledema in IIH patients. Furthermore, we report that CSF-NfL rises with the grade of papilledema. In addition, a correlation of CSF-NfL with CSF opening pressure was observed. We conclude that CSF-NfL may represent pressure-induced axonal damage to the optic nerve in IIH, making it a biomarker for optic nerve damage.Further studies will need to assess the temporal connection of CSF-NfL release with optic nerve damage, papilledema and CSF opening pressure. Depending on the blood assay being used, the correlation between CSF-NfL and serum NfL has been reported to be strong . In the