Elevated intracranial pressure (ICP) is observed in association with a range of brain disorders. One of these challenging disorders is idiopathic intracranial hypertension (IIH), characterized by raised ICP of unknown cause with significant morbidity and limited therapeutic options. In this review, special focus is put on the preclinical research performed in order to understand the pathophysiology behind ICP regulation and IIH. This includes cerebrospinal fluid dynamics, molecular mechanisms underlying disturbances in brain fluids leading to elevated ICP, role of obesity in IIH, development of an IIH model and ICP measurements in rodents. The review also discusses existing and new drug targets for IIH that have been evaluated in vivo.ICP monitoring in rodents is challenging and different methods have been applied. Some of these methods are invasive, depend on use of anesthesia and only allow short-term monitoring. Long-term ICP recordings are needed to study IIH but existing methods are hampered by several limitations. As obesity is one of the most common risk factors for IIH, a rodent obese model has been developed that mimics some key aspects of IIH. The most commonly used drugs for IIH have been evaluated in vivo for their efficacy at lowering ICP in the existing animal models. These studies suggest these drugs, including acetazolamide, might have limited or no reducing effect on ICP. Two drug targets that can impact ICP in healthy rodents are topiramate and a glucagon-like peptide-1 receptor (GLP-1R) agonist. However, it remains to evaluate their effect in an IIH model with more precise and valid ICP monitoring system. Therefore, continued evaluation in the preclinical research with refined tools is of great importance to further understand the pathophysiology behind disorders with raised ICP and to explore new drug targets. Elevated intracranial pressure (ICP) is observed in association with a range of brain disorders and their underlying pathophysiology is widely unknown. Actually, there is limited insight into the regulatory mechanisms of this essential balance under normal conditions and consequently also a significant lack of specific treatments. In this review, we aim to focus on the preclinical research behind ICP regulation mainly with Idiopathic Intracranial Hypertension (IIH) as a disease model. Further, we aim to discuss the existing and novel drug targets for IIH that have been evaluated in preclinical models.The regulation of ICP is fundamental for providing a stable environment to enable normal brain function. According to the anatomists Monro and Kellie, ICP is determined by three parameters; the volume of the brain, cerebral blood volume and cerebrospinal fluid (CSF) volume, where cerebral blood volume and CSF are homeostatically controlled and compensate for acute changes in ICP . Several+/K+ ATPase and the Na+-K+-2Cl−-1 cotransporter (NKCC1) are fundamental in CSF secretion at the CP, and are targeted clinically to reduce ICP , organs that reside in the cerebral ventricles and display the characteristic asymmetrical distribution of ion transporters of fluid transporting epithelia  of the superior sagittal sinus are thought to drain CSF directly into the venous system . The preIt has been hypothesised that CSF can be drained via perivascular spaces that penetrate the parenchyma, termed glymphatics, in an Aquaporin-4 (AQP4) dependent manner . This glThere are currently a limited number of studies that assess CSF dynamics in IIH. Several studies utilizing small cohorts of IIH patients identified an increased CSF flow rate through the cerebral aqueduct, indicating increased CSF secretion –23. StudHistological data from cortical biopsies of IIH patients showed increased AQP4 expression at astrocyte end feet, a constituent of the glymphatic drainage. This is suggestive of an altered state of CSF clearance via the glymphatic system . FurtherAlthough it is apparent that ICP and in particular CSF dynamics are altered in IIH, the current literature fails to elucidate conclusively whether IIH patients have CSF hypersecretion, reduced CSF drainage or a combination of these factors. Furthermore the studies do not address the etiology of the alterations they describe, consequently further studies are required.The gold standard for measuring ICP in patients is by using an intracranial probe however, this requires neuro-surgical intervention with risk of infections. Lumbar puncture (LP) opening pressure measured during an LP is less invasive however, the obtained ICP values are less accurate than the gold standard method, and can only provide a short-term pressure reading during the procedure. There are some LP measurement methods that have been shown to be comparable to intraparenchymal ICP recordings –32. The In rodents, ICP values have been reported to vary between 2 and 8 mmHg with a mean value around 6 mmHg , 40. In As mentioned earlier, IIH is a challenging disease with increased ICP of unknown aetiology and was previously called benign intracranial hypertension or pseudotumor cerebri. The most disabling symptoms are chronic headache, impaired vision due to optic nerve compression and cognitive impairment –45. IIH As the molecular regulation of CSF is not yet fully understood, there is a lack of understanding for many pathological conditions associated with abnormal CSF dynamics. Hence, diseases involving elevated ICP are generally poorly understood and managed, due to the lack of specific targeted treatment options. Medical interventions for IIH range from dietary therapy (weight loss) to diuretics and surgical CSF diversion . In IIH IIH has a prominent association with both the female gender and obesity as 90% of patients with IIH are obese , 48, 59.There is also a strong association between abdominal fat deposition and health outcomes consequently adipose distribution in patients with IIH may be of relevance . It has +/K+-ATPase at the CP, suggesting that testosterone may modulate CSF secretion [Currently there are very few animal models with elevated ICP and they are associated with the induction of severe cerebral traumas such as subarachnoid hemorrhage, TBI, brain tumors, kaolin induced hydrocephalus and cerebral ischemia , 70–73. ecretion . It remaAnother fundamental factor in the development of an IIH animal model is the ability to perform long-term ICP measurements in rodents. ICP measurements are widely used for diagnostic purposes and to inform treatment options for conditions with raised ICP such as IIH. However, the invasive nature of performing ICP monitoring in humans limits clinical research. Thereby, there is a great need of consistent and precise ICP recording in animal models, where ICP can be measured across the day, in response to a drug and during disease development. However, it has been very challenging to measure ICP accurately in animal models due to the lack of refined equipment designed specifically to measure ICP in rodents. Various locations and techniques for measuring ICP have been investigated such as epidural, subdural, ventricular cisterna magna placement using a cannula, fluid-filled catheter or fiber-optic pressure transducers –86, summAlthough the implementation of epidural ICP recording using fluid-filled systems has been a major improvement for measuring ICP in rodents, this method has some limitations. In this method the transducer is not permanently implanted, which prevents the possibility of continuous ICP recordings over larger time intervals. Furthermore, this ICP method as the others depends on repeated use of anesthesia/sedation before each recording to immobilize the animals which are additional cofounders affecting ICP. Additionally, the fluid-filled systems can develop air bubbles, resulting in false ICP values. Fiber-optic pressure transducers have been used to avoid these problems, however, it has only been developed for animals under anesthesia . TelemetRecently, telemetric probes with specific ICP transducers have been developed to measure ICP continuously in freely moving rats (Kaha Sciences) . This haIn the management of IIH, the focus is on reducing the elevated ICP by pharmacological therapy. Given that IIH patients often suffer from visual disturbances and headache, management of IIH has been focused on preserving vision and reducing headache morbidity . In thisTopiramate was originally used for epilepsy treatment and as a migraine prophylactic drug , 102. To+/H+ exchanger and/or Na+ channels on the CP epithelial cells [Diuretic agents have long been known to reduce CSF secretion and have therefore been regarded as a treatment option for IIH Fig. . One of al cells , 117. Onal cells . Howeveral cells . ClinicaOctreotide is a synthetic somatostatin analogue and a potent inhibitor of growth hormone. Two uncontrolled clinical studies have shown its efficacy for IIH treatment , 120. In+- and K+-dependent adenosine triphosphatase activity in cell cultures. Subcutaneous injections with exendin-4 showed a reduction in ICP in both healthy and hydrocephalic rats [Glucagon-like peptide-1 receptor (GLP-1R) agonists are used to treat diabetes and promote weight loss. Due to its ability to affect fluid homeostasis in the kidney, we investigated if the GLP-1R agonist, exendin-4, could modulate CSF secretion and reduce ICP in rats . This stlic rats . Importalic rats . Taken tThe role of obesity on ICP regulation has been evaluated in vivo and lead to the first animal model mimicking some aspects of IIH. However, a refined model is needed to further investigate the mechanism behind IIH and also to provide valuable new knowledge regarding both regulation and dysregulation of CSF and ICP dynamics. The most used drugs in the management of IIH have been evaluated in rodents and although with some limitations, some of these are not efficient for lowering ICP. However, their effect on IIH-related symptoms such as visual outcome and headache has not been evaluated in animal models. Preclinical research suggests a GLP-1R agonist as a novel drug target for elevated ICP which needs to be further evaluated. Furthermore, a novel ICP monitoring system has been developed for long-term ICP monitoring in freely moving animals, which is a prerequisite to resolve the molecular origin of IIH-elevated ICP and to evaluate drug targets. In conclusion, preclinical investigations including the development of adequate in vivo models are warranted in order to increase our understanding in IIH pathophysiology and to explore novel drug targets able to reduce ICP.


Toxic oligomeric α-synuclein  has been suggested to play a central role in the pathogenesis of Lewy body diseases such as Parkinson’s disease (PD). Cerebrospinal fluid (CSF) levels of αS, O-αS, total and phosphorylated tau, and amyloid β 1–42 (Aβ1–42) are thought to reflect the pathophysiology or clinical symptoms in PD. In this study, we examined correlations of the CSF levels of these proteins with the clinical symptoms, and with each other in drug-naïve patients with PD.Twenty-seven drug-naïve patients with PD were included. Motor and cognitive functions were assessed using the Unified Parkinson’s Disease Rating Scale (UPDRS), Montreal Cognitive Assessment (MoCA), and Neurobehavioral Cognitive Status Examination (COGNISTAT). CSF levels of total αS, O-αS, Aβ1–42, total tau and tau phosphorylated at threonine 181 (P-tau181p) were measured. CSF levels of these proteins were compared with clinical assessments from the UPDRS, MoCA and COGNISTAT using Spearman correlation analysis. Spearman correlation coefficients among CSF protein levels were also evaluated.p < 0.05) and bradykinesia (p < 0.01), and positively correlated with COGNISTAT subtest of judgement (p < 0.01) and CSF levels of Aβ1–42 (p < 0.001), total tau (p < 0.001) and P-tau181p (p < 0.01). Lower CSF levels of Aβ1–42, total tau and P-tau181p were significantly related to worsening of some motor and/or cognitive functions. The CSF level of O-αS showed no correlation with any motor and cognitive assessments or with CSF levels of the other proteins.CSF levels of αS were negatively correlated with UPDRS part III (motor score)  contains supplementary material, which is available to authorized users. Parkinson’s disease (PD) is a common neurodegenerative disorder, but diagnosis based on assessment of clinical symptoms and radiological findings can be difficult, especially in the early phase. Therefore, biomarkers reflecting the pathophysiology of PD are required, and pathogenic proteins related to neurodegeneration in cerebrospinal fluid (CSF) may be candidates as such biomarkers. The pathological hallmark of PD is the presence of Lewy body, that is, abnormal aggregates of α-synuclein (αS) in the brain. Recently, oligomeric α-synuclein (O-αS) has been shown to have neurotoxic effects, and such oligomers may play a central role in the pathogenesis of PD –3. Most In Alzheimer’s disease (AD), the CSF level of amyloid β (Aβ)1–42 (Aβ1–42) is decreased and that of tau is increased . These pMany studies in PD patients at various clinical stages and under different medication have shown correlations between CSF levels of the candidate biomarker proteins and clinical symptoms, but the results have not necessarily agreed among studies. Correlations of CSF levels of these proteins with motor and cognitive functions are rarely compared in drug-naïve PD patients. Therefore, in this study, we examined correlations of motor and cognitive functions, and CSF protein levels only in drug-naïve PD patients.Twenty-seven patients with de novo PD were enrolled in the study. Diagnosis of PD was made according to MDS clinical diagnostic criteria for PD . PatientLumbar puncture was performed at the vertebral interspaces of L3-L4 or L4-L5 in a fasting state before treatment. The first 5 ml of CSF was used for routine examination, and the next 10 ml was used for this study. The CSF sample was collected in a sterile polypropylene tube and centrifuged for 10 min at 400 g within 10 min after completion of lumbar puncture. The supernatant was packed in a polypropylene microtube and stored at − 80 °C until assays were performed.CSF total αS was analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit  , 14. Bri3, pH 9.6 (100 μL/well). The plate was washed with phosphate buffered saline containing 0.05% Tween20 (PBS-T) and incubated with 200 μL/well of blocking buffer, which is PBS-T containing 2.5% gelatin, for 2 h at 37 °C. After washing 200 μL/well of PBS-T, 100 μL CSF containing 1% cocktail of protease inhibitors  and 5% heterophilic antibody inhibitor  were added to each well, and then incubated at 37 °C for an additional 3 h. Biotinylated 211 monoclonal antibody diluted to 1 μg/mL in blocking buffer was added and incubated at 37 °C for 2 h. The plate was washed and then incubated for 1 h at 37 °C with 100 μL/well of ExtrAvidin-Peroxidase  diluted 1:1000 in blocking buffer. The plate was washed and incubated with 200 μL/well of an enhanced chemiluminescent substrate , after which chemiluminescence in relative light units was immediately measured with a microplate luminometer . All the samples were measured in 1 plate to avoid plate-to-plate variations.The levels O-αS in CSF samples were measured using a single antibody sandwich ELISA, as described previously with some modification , 15. An For both immunoassays, the samples were screened in blind fashion and were randomly tested.We used the multiplex xMAP Luminex platform with Innogenetics immunoassay kit-based reagents for the CSF biomarker measurements of P-tau181p, total tau, and Aβ1–42  , 17. BriThe Unified Parkinson’s Disease Rating Scale (UPDRS)  was usedp < 0.05 (two-tailed probability).CSF levels of proteins were compared with clinical assessments from the UPDRS, MoCA and COGNISTAT. Correlations among CSF protein levels were also evaluated. Correlation coefficients were calculated using Spearman correlation analysis. The level of significance was The participants included 14 males and 13 females. Three patients did not undergo the UPDRS and MoCA, and 8 did not take the COGNISTAT. CSF samples of all participants were watery clear and not bloody. Red blood cells were not observed microscopically in sediments of the CSF. CSF levels of P-tau181p in 2 patients were below the detection sensitivity and were excluded in analyses using P-tau181p. The number of participants in calculation of each correlation coefficient is shown in Tables p < 0.05). For clinical symptoms, CSF levels of total αS were negatively correlated with UPDRS scores for part III ; total tau was negatively correlated with the UPDRS subscore for bradykinesia (p < 0.05) and positively correlated with COGNISTAT subscores for language-naming and judgment (both p < 0.05); and P-tau181p was negatively correlated with UPDRS scores for part III, rigidity, and bradykinesia .Spearman correlation coefficients of each CSF protein level with patient background and motor symptoms are shown in Table p < 0.001, Fig. p < 0.001, Fig. p < 0.01, Fig. Spearman correlation coefficients among CSF protein levels are shown in Table The results of this study showed that CSF levels of total αS are negatively correlated with UPDRS part III and bradykinesia scores, which indicates that these motor functions worsen as the CSF level of total αS decreases. This result agrees with those from the Parkinson’s Progression Marker’s Initiative (PPMI) study, which showed that the CSF αS level is negatively correlated with MDS-UPDRS part III (motor score) and the Hoehn and Yahr scale in drug-naive PD patients . TherefoAlthough O-αS is toxic and mediates neural damage –3, no clThe CSF levels of total tau and P-tau181p as well as total αS were negatively correlated with bradykinesia scores. Bradykinesia is the earliest and most fundamental motor symptom in PD . These cIn the present study, the CSF level of Aβ1–42, an AD biomarker, was positively correlated with MoCA score, which suggests that cognitive function deteriorates as the CSF Aβ1–42 level decreases. In AD, a decrease in CSF Aβ1–42 is thought to be due to its aggregation in the brain . Therefon = 7, r = 0.929, p < 0.01), but not in patients with a disease durations of ≥1 year . On the other hand, CSF level of total αS correlated with total tau  and P-tau181p  in patients with a disease durations of ≥1 year and these results agree the PPMI study [In the PPMI study, a correlation between CSF levels of αS and Aβ1–42 was not observed . The PPMMI study . DiffereThis study includes only drug-naïve PD patients and the effect of medication is eliminated. Our results support a central role for interaction and aggregation of αS with Aβ1–42, tau, and phosphorylated tau in the pathogenesis of PD. However, the study has limitations. The number of patients is relatively small and we did not include a normal control group. A larger number of participants and control group would have made our conclusions more comprehensive and meaningful. However, recruitment of patients who agree to lumbar puncture is difficult, since most patients with parkinsonism want to be examined without lumbar puncture, and we could not recruit healthy volunteers who agreed to undergo lumbar puncture. Consequently, only 27 patients agreed to participate in the study. However, therapies targeting pathogenic proteins are under development, and knowledge of the relationship between pathogenic proteins and clinical symptoms is important. Therefore, we believe our report is significant and contributes to understanding of the pathophysiology of PD.CSF levels of αS correlate with some clinical symptoms and with CSF levels of Aβ1–42, total tau and P-tau181p in drug-naïve PD patients. These correlations suggest a central role for interaction and aggregation of αS with Aβ1–42, tau, and phosphorylated tau in the pathogenesis of PD. Although O-αS has been shown to have neurotoxic effects, CSF levels do not reflect clinical symptoms or levels of other proteins in cross-sectional assessment.Additional file 1:All data used during this study. (XLSX 13 kb)


Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare pulmonary disease caused by functional deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF therapy in aPAP has been reported effective in some studies. This meta-analyses aimed to evaluate whether GM-CSF therapy, including inhaled and subcutaneous GM-CSF have therapeutic effect in aPAP patients.We analyzed 10 studies searched from PubMed, EmBase, Web of Science, Wiley Online Library and Cochrane Collaboration databases to evaluate the pooled effects of GM-CSF treatment in aPAP patients.p < 0.001), relapse rate , PaO2  and P(A-a)O2  showed that GM-CSF treatment was effective on aPAP patients. Further analyses showed that inhaled GM-CSF treatment was more effective than subcutaneous GM-CSF therapy, including a higher response rate , more improvements in PaO2  and P(A-a)O2 .Ten observational studies involving 115 aPAP patients were included. The pooled analyses of response rate O2 than subcutaneous GM-CSF treatment in aPAP patients, suggesting inhaled GM-CSF therapy could have more benefits on aPAP patients. Therefore, GM-CSF therapy, especially inhaled GM-CSF, might be a promising therapeutic option in treating aPAP.As two routes of exogenous GM-CSF treatment, inhaled and subcutaneous were both proven to have effect on aPAP patients. Furthermore, inhaled GM-CSF therapy showed a higher response rate, more improvements on PaOThe online version of this article (10.1186/s12931-018-0862-4) contains supplementary material, which is available to authorized users. Pulmonary alveolar proteinosis (PAP), first described by Rosen et al. in 1958 , 3. The Approximate 90% PAP cases were autoimmune pulmonary alveolar proteinosis (aPAP) characterized by elevated levels of antibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF) in blood and alveoli, and the antibodies could not be detected in health controls –9. In reWhole lung lavage (WLL) has been used as the standard treatment for aPAP for decades. However, the performance of WLL therapy was limited due to the requirement of anesthesia and invasive procedure . In receThe purpose of this meta-analyses was to evaluate the effect of GM-CSF therapy including inhaled and subcutaneous routes in aPAP patients.The search flow diagram of included studies for this meta-analyses is shown in Fig. We included both controlled and uncontrolled studies in English using GM-CSF therapy in aPAP patients aged 18 years or older. The aPAP patients were diagnosed by open lung biopsy or transbronchial lung biopsy, and anti GM-CSF antibody level was examined. The minimum time for GM-CSF therapy should be more than 4 weeks, because there were delays to observe the effect of GM-CSF therapy –19.Patients diagnosed with secondary or congenital PAP were excluded. In addition, studies not reporting the outcomes of interest, studies with < 3 participants or follow-up time < 3 months were excluded.n < 3). In addition, six studies reporting the same population as other studies were also excluded. And one study was excluded as participants were less than 18-year old. Furthermore, five studies were excluded because their full texts were not available. At last, 10 observational studies satisfying the criteria were included in this meta-analyses.A total of 817 studies met the above selection criteria. In the process of screening, 45 case reports were excluded due to low number of participants (Two researchers (Gaohong Sheng and Peng Chen) independently performed literature search and extracted the data from the published studies according to prespecified inclusion/exclusion criteria.The studies were included or excluded when both researchers made the same judgement for inclusion or exclusion. Disagreements between them were resolved by arbitration of principal investigator (Hui-Lan Zhang). Data on study characteristics, baseline characteristics of patients, and clinical endpoints were extracted and analyzed.2), alveolar-arterial oxygen gradient (P(A-a)O2), pulmonary function, and disease severity score (DSS) before and after the treatment of GM-CSF. Through synthesizing these parameters, we evaluated therapeutic effects and calculated the response/relapse rate based on the number of response/relapse patients provided in each study. The response criteria were considered as the improvement in PaO2, P(A-a)O2, pulmonary function, radiology, or symptoms. During the follow-up period, we considered the responders as relapse when these parameters were pejorative.The endpoints of this study were alveolar oxygen partial pressure  criteria . Each st2 and P(A-a)O2 were analyzed using weighted mean difference (WMD) with 95% CI. Random effects model was performed to compute the pooled analyses because of the large variety in the GM-CSF therapy. The heterogeneity of the studies was assessed using Cochran’Q statistic, and the magnitude of heterogeneity was estimated using the I2 statistic [Data on studies and patients were collected by the two investigators independently. The response and relapse rates were assessed by calculating proportion with 95% confidence intervals (CI) for each study. PaOtatistic . The impWe also realized that different GM-CSF therapy routes might achieve different effects. Therefore, subgroup analyses was performed according to the routes of GM-CSF therapy. Random effects model was performed in each subgroup to compute the pooled analyses, and differences between the subgroups were analyzed. In addition, we also conducted subgroup analyses according to the proportions of WLL therapy, age of patients, proportions of men and proportions of smokers.p < 0.05 were recognized as statistical significance. STATA software 14.0 was used to perform statistical analyses.All comparisons were two-tailed, and Ultimately, 10 observational studies were included in this meta-analyses. In these studies, participants in 5 studies received subcutaneous GM-CSF therapy , 22–25, 2, P(A-a)O2, pulmonary function and the severity of symptoms of the included studies were listed in Additional file A total of 115 patients were included in this meta-analyses, the baseline characteristics of the patients in the included studies were listed in Table p < 0.001)    Fig. . The degp = 0.009)    Fig. . The deg2 pre- and post-GM-CSF therapy in total 55 patients. Compared to baseline PaO2, the PaO2 after GM-CSF therapy was improved by 13.76 mmHg     Fig. . The deg2 pre- and post-GM-CSF therapy were available in 8 studies with a total of 103 patients. Through synthetic analyses, one study [2. And the P(A-a)O2 was decreased by 19.44 mmHg    One study  was obvi01) Fig.  between We also performed analyses on diffusing capacity of the lung for carbon monoxide (DLCO), total lung capacity (TLC), vital capacity (VC), forced expiratory volume in one second FEV1), forced vital capacity (FVC), DSS, and 6 min’ walk distance (6MWD) , while the inhaled subgroup was 89%  . These data suggested that inhaled GM-CSF was more effective in aPAP patients.The subcutaneous subgroup showed a response rate of 71%  Fig. , suggestp = 0.14)  .There were 5 studies assigned to subcutaneous subgroup and the pooled relapse rate was 24%  Fig. . The oth05) Fig. . The dif2 pre- and post-therapy. Significant increase in PaO2 was observed in both subcutaneous group  and inhaled group  .In total, five studies showed data on PaO01) Fig. . The dif2) were significant in both subcutaneous group  and inhaled group  .In the studies of subcutaneous subgroup, sensitivity analyses showed one study  had a si05) Fig. . The meap > 0.05), respectively.Results of meta regression analyses suggested effects of GM-CSF therapy were independent of the age of patients, proportions of men, history of smoking, and proportions of combined WLL therapy (2 and P(A-a)O2 were found, and the differences between these subgroups were significant. And response rate was significantly higher in the high initial dose subgroup. Relapse rate was found significantly higher in the subgroup with younger age and higher anti GM-CSF antibody titer. The P(A-a)O2 improvement was more significant in the subgroup which more patients using WLL therapy. The details were shown in Table We performed subgroup analyses based on the age of patients, and the proportions of men, smokers, anti GM-CSF antibody titer, initial dose and patients combined with WLL therapy. In the studies with more smokers, higher response rate, more improvements in PaO2 and P(A-a)O2 indicated that GM-CSF therapy was effective on aPAP patients. Moreover, inhaled GM-CSF therapy had a higher response rate , more improvements in PaO2  and P(A-a)O2  than subcutaneous GM-CSF therapy, suggesting that inhaled route was more effective than subcutaneous route in patients with aPAP.This meta-analyses including 10 studies enrolling 115 patients aimed to evaluate whether GM-CSF therapy including inhaled and subcutaneous routes were effective on aPAP patients. The pooled outcomes of response rate, relapse rate, PaOThe WLL therapy, whose response rate ranged from 70 to 84%, has been used as standard treatment for aPAP patients for decades , 13, 31.p = 0.023). Furthermore, we also compared relapse rate, the values of PaO2 and P(A-a)O2 pre- and post-therapy in inhaled and subcutaneous subgroups, the results confirmed that aPAP patients would benefit more from inhaled GM-CSF therapy.In 2012, one meta-analyses reported that inhaled GM-CSF therapy showed a trend toward higher response rate in aPAP patients comparing with subcutaneous route, suggesting inhaled route might be a better route for GM-CSF therapy . In thisp = 0.262). We found a significant higher relapse rate in the patients younger than 45 years old than that of older than 45 years old (p = 0.006) by subgroup analyses (Table p = 0.045)   were found, however, full texts of these RCTs were not available.2 and P(A-a)O2 than subcutaneous route. These results provided a convincing evidence that GM-CSF therapy might be a promising therapeutic option in treating aPAP, furthermore, inhaled GM-CSF would have more benefits on aPAP patients than subcutaneous route.Pooled analyses of available studies suggests that treatment of GM-CSF were effective on aPAP patients with response rate (81%). However, further analyses demonstrated that inhaled GM-CSF therapy showed a higher response rate (89% vs. 71%), more improvements of PaOAdditional file 1:Table S1. Basic characteristics of all included studies. Table S2. Modified AHRQ Quality Assessment Criteria for Observational Studies. Table S3. The endpoints of included studies before and after GM-CSF therapy. Table S4. Subgroup analysis of side effects with GM-CSF therapy on patients with aPAP. Figure S1. (a). Funnel plot of response rate. (b). Begg’s funnel plot of response rate. (c). Egger’s publication bias plot of response rate. (d). Sensitive analysis of response rate. Figure S2. The similarity between random and fixed effect models shows no significant influence on the pooled effect of response rate. Figure S3. (a). Funnel plot of relapse rate. (b). Begg’s funnel plot of relapse rate. (c). Egger’s publication bias plot of relapse rate. (d). Sensitive analysis of response rate. Figure S4. The similarity between random and fixed effect models shows no significant influence on the pooled effect of relapse rate. Figure S5. (a). Funnel plot of PaO2. (b). Begg’s funnel plot of PaO2. (c). Egger’s publication bias plot of PaO2. (d). Sensitive analysis of PaO2. Figure S6. (a). Funnel plot of P(A-a)O2. (b). Begg’s funnel plot of P(A-a)O2. (c). Egger’s publication bias plot of P(A-a)O2. (d). Sensitive analysis of P(A-a)O2. Figure S7. Sensitive analyses of P(A-a)O2 in subcutaneous group shows one study was significantly different with the others. Figure S8. (a). DLCO improved by 8.49% after GM-CSF therapy. (b). TLC improved by 13.04% after GM-CSF therapy. (c). VC improved by 4.83% after GM-CSF therapy. (d). FEV1 improved by 12.58% after GM-CSF therapy. (e). FVC improved by 8.45% after GM-CSF therapy. (f). DSS improved by 2.1 after GM-CSF therapy. (g). 6MWD improved by 51.96 after GM-CSF therapy.


To this end, electrophysiological effects of CSF from AE patients or hCSF were measured by in vitro neuronal network activity (ivNNA) recorded with microelectrode arrays (MEA). CSF from patients with either N-methyl-D-aspartate-receptor-antibody  or Leucine-rich-glioma-inactivated-1-Ab  associated AE suppressed global spiking activity of neuronal networks by a factor of 2.17 (p < 0.05) or 2.42 (p < 0.05) compared to hCSF. The former also suppressed synchronous network bursting by a factor of 1.93 (p < 0.05) in comparison to hCSF (n = 13). As a functional diagnostic test, this parameter reached a sensitivity of 86% for NMDAR-Ab- and 100% for LGI1-Ab-associated AE vs. hCSF at a specificity of 85%. To explore if modulation at the NMDAR influences effects of hCSF or pathological CSF, we applied the NMDAR-antagonist 2-Amino-5-phosphono-pentanoic acid (AP5). In CSF from NMDAR-Ab-associated AE patients, spike rate reduction by AP5 was more than 2-fold larger than in hCSF (p < 0.05), and network burst rate reduction more than 18-fold (p < 0.01). Recording ivNNA might help discriminating between functional effects of CSF from AE patients and hCSF, and thus could be used as a functional diagnostic test in AE. The pronounced suppression of ivNNA by CSF from NMDAR-Ab-associated AE patients and simultaneous antagonism at the NMDAR by AP5, particularly in burst activity, compared to hCSF plus AP5, confirms that the former contains additional ivNNA-suppressing factors.The intent of this study was to investigate if cerebrospinal fluid (CSF) from autoimmune encephalitis (AE) patients regulates  Limbic encephalitis, the most important clinical form of AE, is characterized by subacute (<3 month) alterations in mental status or psychiatric symptoms, seizures and typically magnetic-resonance imaging (MRI) findings with frequent but not inevitable inflammatory signs in CSF. Positive antibody testing can confirm the diagnosis. Therapy of AE consists of immunosuppression/-modulation with steroids, intravenous immunoglobulins, plasmapheresis or long-term regimes with rituximab or cyclophosphamide2.Onconeuronal antibodies (Abs) against intracellular proteins like Hu, Ma or CV2 and those against cell-surface or soluble proteins like the NMDAR, LGI1 or contactin-associated-protein-like-2 (CASPR2) can be associated with AE3. Moreover, clinical symptoms may be non-specific so that only the subacute course favors the diagnosis. Pathological results from MRI, electroencephalography and CSF are not AE specific, and autoimmune-Abs are sometimes clinically irrelevant, for instance in patients with small lung cancer and Hu-Abs without neurological disease4. Monitoring AE is important in order to schedule adequate duration and strength of immunosuppressive therapy, but accurate Ab-titers in serum and CSF are not always available and rarely correlate to clinical conditions5 to inform clinical decisions. Healthy human control CSF (hCSF) has been recently associated with increased neuronal activity and survival in several in vitro systems7.However, the diagnosis of AE can be challenging, and a substantial portion (up to 44%) of patients are negative for known antibodies9, including one case with NMDAR-Ab-associated encephalitis10. In the present study, we investigated, if ivNNA is able to discriminate between CSF from patients with AE associated with LGI1-Abs (n = 6) and hCSF (n = 13) and consolidate the data concerning AE associated with NMDAR-Abs (n = 7). Additionally, we investigated effects on ivNNA by a CASPR2-Ab CSF sample.A functional read out system could be helpful in order to measure disease activity. In recent years, we could show that ivNNA measured by the MEA system  significantly increased absolute values of global spike activity, the number of network bursts, also illustrated in the spike raster plots (SRP), and the peak firing rate (PFR), compared to baseline activity under aCSF Fig. . This phNMDAR and pCSFLGI1, but not in hCSF, bind to β-tubulin+ mouse neurons.Using immunohistochemistry, we verified the presence of neuronal-antibodies in CSF samples from NMDAR- and LGI1-encephalitis patients. As illustrated in Fig. NMDAR, the ratio of the spike rate was only 1.39 (±0.28) and in pCSFLGI1 only 1.25 . These results represent a significantly reduced increase of activity in the presence of CSF from the patients compared to samples from healthy controls . In the hCSF situation, ratios of spike rate, burst rate and the PFR were 3.02 (±0.42), 1.45 (±0.13) and 3.00 (±0.70), respectively. By contrast, in pCSFLGI1 and pCSFNMDAR .PFR ratios were not different between all groups. Representative SRPs illustrate network firing patterns from hCSF, pCSFDAR Fig. . InteresNMDAR on ivNNA could be NMDAR-mediated, we applied 50 µM of AP5 to hCSF and pCSFNMDAR.To examine whether the differential effects of hCSF and pCSFNMDAR even further, to 0.29 of initial level , while in hCSF AP5 only reduced activity to 0.66 (±0.08). Network bursting was even more prominently suppressed in pCSFNMDAR to 0.07 of initial level , compared to hCSF at 1.2  of NMDAR antibodies found reduced NMDAR activity (reduced NMDAR currents)15. As long-term effects might be related to NMDAR internalization, it remains unclear if NMDAR antibodies exert direct electrophysiological effects. Our study supports the possibility of direct and rapid effects. Direct receptor-mediated effects are further supported by our experiments with AP5, that indicate a stronger dependence of ivNNA on NMDAR function under the influence of pCSFNMDAR in comparison to hCSF. Since NMDAR antibodies have been reported to lead to prolonged open states of NMDAR16 this effect would then be specifically mediated by the NR1 subunit of the NMDAR, which is known to be the target of antibodies in NMDAR encephalitis patients14. However, it could also be postulated that the NMDAR antibodies lead to a reconfiguration of the network function that is consequently more drastically modified by blocking NMDAR which is described in other networks17.Antibodies to neuronal surface proteins now play an important role in the diagnosis of subacute forms of encephalitis, but not all patients with clinically-defined autoimmune encephalitis have positive antibodies and the acute functional effects have not been examined. Here we show that ivNNA of primary neuronal mouse cultures measured by MEA is suppressed by pCSFLGI1 also suppressed ivNNA. Again, the rapid effects reported here and the role of LGI1-Ab on voltage-gated potassium channel complexes suggest direct pCSFLGI1-Ab-related mechanisms. Interestingly, under pCSFLGI1, only global spiking activity was suppressed, while network bursting was preserved, indicating a different mechanism in comparison to pCSFNMDAR.pCSFNMDAR and pCSFLGI1, we do not assume that additional factors in pCSF, such as drugs administered to the patients, play a major role for the observed effects. We would then also expect a higher variability between patients, since they presumably received different drugs at distinct dosages. The clinical data at the time of CSF sampling were not systematically available and drug levels were not measured, so that we could not correlate drugs that were applied to patients to measured effects on ivNNA. Nevertheless, we cannot exclude such drug effects contaminating our results.Because of different effects in pCSF18, direct effects of CASPR2-Ab on ivNNA are conceivable. Thus, functional CSF diagnostics with ivNNA appears to be sensitive to AE CSF samples from a spectrum of individual patients.From receiver-operating characteristic (ROC), we derived a tentative spike ratio threshold (rel. to aCSF) below 1.9 for discriminating pCSF against hCSF. With this setting, we obtained a specificity of 85% for both at a sensitivity of 86% in NMDAR-Ab-associated encephalitis patients  or 100% in LGI1-receptor-encephalitis patients. If this threshold was applied, an additional CASPR2-Ab sample (spike ratio 0.56) that was also measured on MEAs for the actual study lay in the pathological range. As CASPR2 is essential for correct localization of voltage-gated potassium channels in the central nervous system9, also in AE, application of ivNNA can image functional effects of pathological CSF. In those diseases, pathological CSF led to diversely regulated ivNNA. For instance, in AE, network bursting was reduced but preserved while it was nearly abolished in TBI. In DLB, spiking activity was reduced, while it remained unchanged in CSF samples from Parkinson´s disease patients. In NMDAR-Ab-associated encephalitis, many previous reports have shown that NMDAR-Ab cause loss of NMDARs and result in reduced NMDAR function that appears within days15. Suppression of ivNNA, however, is rapid within minutes, and thus, possibly related to additional mechanisms that are directly receptor-mediated. In TBI, NMDAR-modulating substances like small amino acids are elevated in CSF and may cause rapid ivNNA suppression. Our LGI1-Ab data also provide electrophysiological evidence, demonstrating a direct effect of pCSFLGI1 on functional neuronal networks.In summary, we could demonstrate an overall correlation between ivNNA and AE diagnosis. Like in traumatic-brain-injury (TBI) and Dementia-with-Lewy-Bodies (DLB)9. To show similar correlations of clinical conditions and ivNNA for NMDAR-Ab-associated encephalitis patients, further investigations of cognitive states at the time of taking CSF and its effects on ivNNA are necessary. Such effects could be shown during the course of the disease for a single patient with NMDAR-Ab-associated AE10. As a perspective, functional CSF diagnostics could be developed further to help monitor AE response to treatments, and to identify patients with novel antibodies.In TBI patients, we were able to demonstrate quantitative correlations of ivNNA to a clinical score, the Glasgow-Coma-ScaleAll experimental methods were carried out in accordance with relevant guidelines and regulations and were approved by an institutional committee. Experiments were approved by the local Animal Care and Use Committee .19. Briefly, pregnant females were killed using CO2, and embryos were quickly taken out and decapitated. By microsurgical dissection, hippocampi were isolated and washed three times with 4 °C magnesium- and calcium-free HBSS (PAA Laboratories GmbH) before treatment for 15 minutes with 2.5% trypsin. Subsequently, tissues were rinsed in DMEM with fetal bovine serum (Biochrom AG), L-glutamine (Invitrogen) and penicillin/streptomycin (Invitrogen) to block the trypsin reaction. 150,000 cells in 110 µl of solution were plated on MEAs coated with poly-D-lysine solution  and 500 µl of DMEM with fetal bovine serum (Biochrom AG), L-glutamine (Invitrogen) and penicillin/streptomycin (Invitrogen). After cells had settled down for 4 h, MEAs were flooded with Neurobasal culture medium (Invitrogen) supplemented with B27 (Invitrogen), glutamine, and penicillin/streptomycin.We plated dissociated hippocampal neural cultures prepared from E17 mice on microelectrode arrays . Number of spikes per minute was calculated as global measure of action potential activity in the entire network. Coordinated firing across the entire network was quantified by the number of network bursts per minute.MEAs (Multichannel Systems) had a square grid of 60 planar Ti/TiN electrodes of 30 µm diameter and 200 µm spacing. Electrodes had an input impedance of 30–50 kΩ according to the specifications of the manufacturer. Signals from all 60 electrodes were simultaneously sampled at 25 kHz, visualized, and stored using the standard software MC_Rack provided by Multi Channel Systems. Spike detection was performed off-line by the SPANNER software suite . The networks show sparse or coordinated spike activity; the latter is defined as rhythmic spiking across multiple spatially distributed extracellular electrodes leading to synchronous network bursts  was smoothed using a normalized Gaussian kernel with standard deviation 100 ms. Network burst onset and termination were determined when this 100 ms smoothed network firing rate exceeded the slowly varying 1-second moving average, using the actual spike time stamps of the first and last spike in the time interval considered. Consecutive network bursts were merged into a single burst, if firing rate valleys between bursts were too shallow, or if bursts were less than 200 ms apart. Finally, the peak firing rate (PFR) of a given network burst was defined as the maximum of the 100 ms smoothed network firing rate within this burst. Very low-firing bursts were discarded, if their PFR did not exceed 10% of the average of the top five peaks, or if less than four electrodes contributed. Network burst detection was eventually checked by visual inspection. In this way, synchronous network activity on time scales of tens to several hundreds of milliseconds could reliably be capturedglial fibrillary acidic protein . Appropriate secondary antibodies coupled to Cy2 or Cy3  were applied. Cell cultures were counterstained for one minute with 4′,6-Diaminodino-2-Phenylindol  to visualize cell nuclei. To verify the presence of neuronal antibodies in pCSFNMDAR and pCSFLGI1, we applied pure pCSF on β-tubulin III-positive (Cy3) mouse hippocampal neurons  and detected human antibodies by anti-human secondary antibodies .For immunocytochemical investigations of cell composition from E17 mice hippocampi on MEAs, cultured cells were washed in phosphate-buffered saline (PBS) and fixed for 15 min in 4% paraformaldehyde. Primary antibodies were monoclonal mouse antibodies to β-tubulin III  and In vitro NNA develops by multiple interconnected neuronal sub-types and astrocytes. After a 3-week maturation period cultures were used for experiments.Images were collected with a confocal-laser scanning microscope . Cultures are illustrated in Fig. 1 and including antibody positivity of NMDAR-Ab or LGI1-Ab in their CSF and serum (7 NMDAR samples: 6F (86%), 1M; average age: 49.3 years; 6 LGI1 samples: 2F (33%), 4M; average age: 53.2 years). Some of those patient CSF samples showed a slight inflammatory syndrome with slight pleocytosis and slightly elevated total protein. Specific antibody testing was performed by indirect immunofluorescence tests with primary antibodies from patients biofluids and a commercial second IgG-Fc-region binding fluorescence antibody. These routine procedures were performed with commercially available specific transfected HEK cells  or live cell-based assays (Oxford) for each antibody, respectively.The collection and use of CSF for this study was approved by the Ethics committee of the university Tübingen . Samples were collected by lumbar puncture during diagnostic work up. The use of external CSF samples for research purposes was approved by local internal review boards and informed consent was obtained from all subjects. In particular, we withdrew at least 12 ml, centrifuged the CSF at 4000 g for 10 minutes at 4 °C. 100 µl aliquots were frozen at −80 °C within 2 hours. For each experiment we used one 100 µl aliquot and never reused, refroze or rethawed CSF samples after application to MEAs. Healthy human control CSF (hCSF) was taken from healthy individuals that underwent lumbar puncture to exclude acute neurological diseases and had normal CSF results concerning cell-count, total protein, glucose and lactate concentrations (13 control samples: 9F (69%), 4M; average age: 50.5 years). All patients with limbic encephalitis were diagnosed according to typical clinical signs and symptoms8. Briefly, MEAs with cultures aged 21–28 DIV were transferred from the incubator to the amplifier headstage, networks were covered with a Potter cap to prevent evaporation and keep osmolarity constant and were left to equilibrate for 10–20 minutes, controlled by visual inspection. Each experiment then lasted 25 minutes, starting with a 5-minute recording in culture medium, followed by two 5-minute recordings of baseline activity in artificial CSF , and immediately followed by two 5-minute recordings in either human CSF  or patient CSF  after complete liquid exchange. We always performed a full medium exchange by gentle pipetting to avoid shear strain on the networks. MEA recordings were interrupted before medium exchange and restarted shortly afterwards, so that no pipetting artifacts entered into the analysis. After exchanging either culture medium to aCSF, or aCSF to human control/patient CSF, we observed network equilibration during the first 5 minutes and checked stable activity levels during minutes 6 to 10. Only stable activity from minutes 6 to 10 was used for further analysis, and ratios “(activity under pCSF)/(activity under aCSF)” were calculated for each individual MEA  are presented in text and figures.For a binary CSF classification as either “control” or “NMDAR/LGI-1 patient” CSF, sensitivity was defined as the proportion of true control CSF samples that had been correctly classified as controls: Sens = TP/(TP + FN), and classification specificity as the proportion of true patient CSF samples that had been correctly classified as patient CSF: Spec = TN/(TN + FP). Here, true positive (TP) denotes true control CSF correctly classified as control CSF, true negative (TN) denotes true patient CSF correctly classified as patient CSF, while false positive (FP) denotes true patient CSF falsely classified as control CSF and false negative (FN) denotes true control CSF falsely classified as patient CSF. Varying the threshold between “predicted control CSF” and “predicted patient CSF” in the change of spike activity level, i.e. the ratio (# spikes/min under h- or pCSF)/(# spikes/min under aCSF), the receiver-operating characteristic (ROC) curve was obtained by plotting sensitivity vs. 1-specificity. From this curve, an optimal classification threshold was obtained by maximizing Youden’s index (=sensitivity + specificity − 1), yielding a spike activity ratio threshold of 1.9 for discriminating between control and patient CSF.


Background: Neurofilament light chain protein (NFL) and chitinase3-like1 (CHI3L1) have gained importance recently as prognostic biomarkers in multiple sclerosis (MS).Objectives: We aimed to investigate NFL and CHI3L1 cerebrospinal fluid (CSF) profiles in multiple sclerosis and the informative and prognostic potential of the individual and combined measures.Methods: CSF NFL and CHI3L1 levels were measured in a cross-sectional cohort of 157 MS patients . Clinical relapse and/or gadolinium-enhanced lesions (GEL) in MRI within 90 days from CSF collection by lumbar puncture (LP) were registered and considered as indicators of disease activity. Longitudinal treatment and disability data were evaluated during medical visits with a median follow-up of 50 months.Results: CSF levels of NFL and CHI3L1 were higher in MS patients compared to non-MS controls. In RRMS and SPMS patients, increased NFL levels were associated with clinical relapse, and gadolinium-enhanced lesions in MRI (p < 0.001), while high CHI3L1 levels were characteristic of progressive disease (p = 0.01). In RRMS patients, CSF NFL, and CHI3L1 levels correlated with each other (r = 0.58), and with IgM-oligoclonal bands . In addition, CSF CHI3L1 concentration was a predictor for 1-point EDSS worsening {HR = 2.99 [95% CI ]} and progression during follow-up {HR = 18 [95% CI ]}. The pattern of combined measure of biomarkers was useful to discriminate MS phenotypes and to anticipate clinical progression: RRMS more frequently presented high NFL combined with low CHI3L1 levels, compared to SPMS (HR 0.41 [0.18–0.82]), and PPMS (HR 0.46 [0.19–0.87]), while elevation of both biomarkers preceded diagnosis of clinical progression in RRMS patients (log rank = 0.02).Conclusions: Individual measures of CSF NFL and CHI3L1 are biomarkers of disease activity and progression, respectively. The pattern of combined measure discriminates MS phenotypes. It also predicts the subset of RRMS patients that will progress clinically allowing early intervention. Neurofilament light protein (NFL), a cytoskeletal polypeptide of the axon , and chiNFL is not a specific biomarker but a reflection of axonal destruction in several neurological diseases –6. In MSCHI3L1 expression is not restricted to the CNS, but its presence in the CSF has been related to endogenous secretion by astrocytes, and microglia/monocytes . Its incIn relapsing-remitting MS (RRMS), the presence of CSF oligoclonal IgM bands (OCMB) has been associated with more substantial T2 lesion load, increased gadolinium-enhancing lesions (GEL), higher relapse rate, more neurological disability, and brain volume loss over time. OCMB have also been related to earlier conversion to secondary progressive MS (SPMS) and can help to identify a subset of primary progressive MS (PPMS) patients with a more inflammatory phenotype –31.Although literature regarding the role of NFL and CHI3L1 in MS is profuse, there is limited information about their relationship with OCMB and little is known about the diagnostic and predictive role of the combined assessment of CSF levels of both NFL and CHI3L1. In this work, with a cohort of 157 patients including RRMS and progressive phenotypes  and with prospectively collected disability data, we aimed to demonstrate that the combined measure of both biomarkers in the CSF might have value, not only in identifying distinct MS phenotypes but also in predicting accrual of disability and further diagnosis of progressive disease in RRMS patients.The study included all MS patients with available samples of CSF, magnetic resonance imaging (MRI), and longitudinal disability data, seen at two university hospitals in Valencia, Spain  between 2008 and 2017 . DemograDiagnosis of clinically definite MS was made according to 2017 McDonald criteria . Active Patients with clinically definite MS were treated with first-line DMT, chosen at physician discretion, unless one of the following circumstances occurred: (i) two clinical attacks in 1 year, (ii) a clinical attack and/or a new GEL within 3 months after the bout, (iii) a disabling clinical attack with residual EDSS of at least 2 points. In these cases, and those with treatment failure, second line DMT was administered. Non-responders to first- and second line DMT proceeded to autologous stem cell transplantation (ASCT).CSF samples were stored at −80°C in the Biobank La Fe with the approval of the Ethics and Scientific Committees (PT17/0015/0043). NFL and CHI3L1 levels in CSF were assessed by enzyme-linked immunosorbent assays using commercially available kits according to manufacturer's instructions . The mean intra-assay coefficients of variation for NFL and CHI3L1 were 4.5 and 6.5%, and inter-assay 3.3 and 5.2%, respectively.Brain and spinal cord 1.5/3.0 Tesla MRI were performed at diagnosis and during the follow-up. CSF oligoclonal IgG bands and oligoclonal IgM bands (OCMB) against lipids were studied by isoelectric focusing and immunoblotting, as previously described . Serum at-test, and a one-way analysis of covariance (ANCOVA) using age, sex, and disease duration as covariates, with no significant changes in the estimated effects found for NFL, and CHI3L1. Bonferroni post-hoc correction method was performed for multiple comparisons. Correlations were analyzed with the Pearson's product-moment correlation test. Spearman's rank correlation method was additionally performed as a sensitivity analysis. To analyze the contribution of paired values of biomarkers, patients were categorized into four groups with a median split method : group 1 included patients with CSF NFL and CHI3L1 levels below median values, group 2 and 3 included patients with only NFL or CHI3L1 values above median, respectively, and group 4 comprised patients with CSF NFL, and CHI3L1 concentrations above median levels. Bivariate analysis with Kaplan Meier survival curves was performed for the probability of 1-point EDSS worsening, diagnosis of progressive disease, escalation of treatment, and occurrence of first relapse after LP in RRMS patients. Predictors were age, sex, disease activity, EDSS, logNFL, and logCHI3L1 , presence of OCMB in the CSF, and treatment at the time of LP. Elastic net penalized Cox proportional hazard regression models were performed in the RRMS subgroup for the same outcomes and predictors. After the selection of the predictors with the elastic net algorithm, the model was refitted with a standard Cox regression to get approximate p-values and estimates of the effects. Additionally, a multinomial logistic regression model was performed including log-transformed CSF NFL, and CHI3L1 levels as predictors. MS clinical phenotypes  and diagnosis of SPMS were used as response variables with RRMS as the reference group. A plot of marginal effects was drawn to ease the interpretation of multinomial logistic regression model. All analyses were conducted using SPSS 21.0 v and R version 3.4.3 (The CRAN project).Statistical analysis was performed by the Biostatistics Department of the Research Health Institute La Fe. Categorical variables were described by counts (n) and percentages (%), continuous, and ordinal variables by median (first and third quartiles). CSF NFL/CHI3L1 levels were log-transformed (logNFL and logCHI3L1) to meet the normality assumption. Comparisons between groups were performed with U-Mann Whitney, Student We studied 157 MS patients, 99 RRMS (63%), and 58 progressive MS (37%), of which 35 were SPMS (22%), and 23 PPMS (15%) .Clinical and demographic features are shown in p = 0.03).Ninety-three patients (60%) had documented clinical attack and/or GEL in the MRI within 90 days from LP. Median time from a clinical attack to LP and from MRI to CSF collection was 38 days  and 22 days , respectively. At the time of analysis, 14 patients in the RRMS cohort had sustained an increase in disability without clinical attacks and, therefore, were reassigned to SPMS phenotype. Disease duration in these patients was significantly higher compared to that in patients who did not progress during follow-up (p < 0.001). The distribution was highly dispersed in the three clinical MS forms with no significant differences between them (p = 0.6) (p < 0.001), and differed between MS phenotypes being 119 ng/ml  in RRMS, 139 ng/ml  in SPMS, and 180 ng/ml  in PPMS (p = 0.01) (post-hoc correction showed a significant difference between RRMS and PPMS (p = 0.009).Overall, the median CSF levels of NFL were 536 pg/ml  in MS patients and 158 pg/ml  in non-MS controls (p = 0.6) . The med = 0.01) . Bonferrp < 0.001; p = 0.016) and SPMS (p = 0.007) but not in PPMS (p = 0.265). Considering only the presence of GEL, NFL levels differed depending on whether GEL was present (740 pg/ml)  or absent (385 pg/ml)  (p < 0.001), but there was no evidence of a correlation with the number of this type of lesions (p = 0.395).At the time of LP, 93 patients were considered active  according to the given definition of active disease . CSF NFL levels in patients with and without active disease were 710 pg/ml , and 329 pg/ml , respectively , SPMS (p = 0.390), or PPMS (p = 0.267). CSF CHI3L1 levels in patients with or without GEL were not significantly different, being 133 ng/ml  and 135 ng/ml  (p = 0.705), and the number of GEL did not correlate with CHI3L1 levels (p = 0.395). Although, overall, median levels of CSF NFL, and CHI3L1 were high in MS, extreme values of both biomarkers were characteristic of RRMS patients with active disease , which was not significantly different from patients without inflammatory activity [140 ng/ml ]  compared to SPMS , and PPMS . There was a trend between decreased CSF NFL levels and disease duration at the time of LP  or time from clinical relapse . NFL was higher in the presence of OCMB  only in the RRMS subgroup of patients  and higher EDSS at the time of LP , although correlations were weak. The presence of OCMB was more frequent with higher levels of CHI3L1 only in the RRMS subgroup , as it occurred with NFL  or presence of GEL  separately, but patients with both conditions had OCMB in the CSF more frequently . The mean number of GEL in the presence of OCMB was 2.53, while in its absence it was ere 1.36, but this observation did not reach statistical significance (p = 0.121).Higher concentrations of CSF CHI3L1 were associated with increased age .At the time of LP, 109 patients were not treated, and 49 patients were treated . Twenty-six patients were under first-line DMT , 20 were on second-line DMT , and the remaining 3 had undergone ASCT in the previous 5 years after treatment failure. Median CSF NFL and CHI3L1 levels did not differ between patients being treated at time of LP (576 pg/ml and 135 ng/ml) and untreated patients  as potential predictor with a tendency for NFL (log rank p = 0.0523) and disease activity (log rank p = 0.109) to differ in 1-point EDSS worsening. After multivariable analysis, only CHI3L1 persisted as independent predictor {HR = 2.99 [95% CI ]}. NFL had no impact on this outcome  experienced a 1-point increase in disability assessed with EDSS after LP. Bivariate Kaplan Meier analysis showed CHI3L1 (log rank  outcome .p = 0.0001), NFL (log rank p = 0.015), and disease activity (log rank p = 0.001) as potential predictors for diagnosis of SPMS. With Cox analysis, only higher CHI3L1 levels, {HR = 18 [95% CI ]}, and active disease {HR = 0.186 [95% CI ]} were shown to be associated with disease progression after LP (Fourteen RRMS patients (14%) progressed to SPMS. Bivariate Kaplan Meier analysis detected CHI3L1 (log rank after LP . NFL wasp = 0.043), and EDSS (log rank p = 0.047) to be potential predictors for the need of treatment escalation. None had predictive value for this outcome after multivariate analysis.Overall, escalation to second line DMT was needed in 57 patients during follow-up . Bivariate Kaplan Meier analysis found gender (log rank p = 0.043) and EDSS (log rank p = 0.046) as potential predictors for clinical relapse. None had predictive value for this outcome after multivariate analysis.Forty-two RRMS patients (42%) had a clinical relapse after LP. Bivariate Kaplan Meier analysis detected gender (log rank p = 0.02) compared to being included in any of the other groups of patients (p = 0.094), need for escalation therapy (log rank p = 0.173) or relapse after LP (log rank p = 0.783).With the purpose of investigating the value of the combined measure in MS prognosis, we stratified patients in four groups according to both NFL and CHI3L1 values with respect to their median in the RRMS cohort in the absence of disease activity . Bivariate analysis using biomarker profiles as predictors showed that being a patient in Group 4 was predictive of diagnosis of progressive disease during follow-up , in density maps with a raster diagram, showed that peak density of patients was distinctly distributed between non-MS controls and MS patients, and within MS, between distinct MS clinical forms . RRMS paWe additionally tested the interaction between both biomarkers to discriminate MS phenotypes and predict progression using a multinomial logistic regression model . The anaThis study shows that MS clinical phenotypes and disease activity are associated with CSF NFL-CHI3L1 levels when assessed both individually and combined. NFL levels alone seemed to be more indicative of disease activity, whereas CHI3L1 levels were related to progression of disability as previously shown. As a novelty, here we state that both biomarkers in combination could be helpful to classify MS patients into distinct clinical forms, and to predict diagnosis of SPMS in the RRMS cohort.We confirmed previous reports in which NFL levels were higher in MS patients compared to non-MS-controls, and further increased when clinical attack and/or GEL occurred . As timeCSF CHI3L1 levels were also high in MS compared to non-MS controls as previously reported , 40. RecCSF CHI3L1 is also very dependent on age . PatientOCMB is a known CSF biomarker of inflammatory disease and implies a worse prognosis in MS . In our Regarding the value of both CSF biomarkers in prognosis, recent reports in a CIS/RRMS cohort did not find a prognostic influence for these biomarkers in progression . AlthougFurther, we analyzed the usefulness of the combined measure of CSF NFL and CHI3L1 levels. Both values provided supplementary information of the MS phenotype with higher values of NFL that seemed to correspond more frequently to active patients (most in the RRMS subgroup) susceptible to respond to current DMT, while higher values of CHI3L1 were associated with progressive MS (more evident for PPMS). Eleven over a total of 14 RRMS patients diagnosed of SPMS during follow-up had high levels of both NFL and CHI3L1. This led us to investigate the combination of biomarkers and the ability to predict the diagnosis of progressive MS in patients from the RRMS cohort. We found that having high levels of CSF NFL and CHI3L1 was predictive of switching to the SPMS group. However, the number of patients that progressed during the study was low and CSF was collected at distinct time points from disease onset, hence we cannot infer from our data whether biomarkers could inform likeliness of conversion or were, indeed, detecting subclinical progression. Supporting the latter was the assessment of longer disease duration in these patients.A disadvantage of measuring biomarkers in the CSF is the invasiveness nature of LP. This prevents for the use of CSF NFL and CHI3L1 levels in longitudinal studies. Currently, new detection methods allow precise detection of NFL in serum, and therefore allow for multiple measures during follow-up . NeverthTo conclude, individual measures of CSF NFL and CHI3L1 are biomarkers of disease activity and progression, respectively. The pattern of combined measure discriminates MS phenotypes and predicts the subset of RRMS patients that will progress clinically allowing early intervention. Whether early intervention guided by biomarkers can change the natural history of the disease is unknown, and should be the focus of future studies.The datasets generated for this study are available on request to the corresponding author.All patients provided written informed consent. The study was approved by the Institutional Ethics Committee in Hospital Universitari I Politècnic La Fe (reference number PI17/01544).SG-P and BC have designed the study, analyzed data, and wrote the manuscript. SG-P additionally carried out the data collection and statistical analysis. LC-N and JC-V have processed CSF and performed ELISA. BC has been attending physician and designed the database, the treatment protocol, and had been responsible with FP-M, FG, CA, and JD for the clinical follow-up. JG-M helped in collecting data. RG helped in designing figures and revising the manuscript. DH supervised the statistical analysis and helped in figure design.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although Seoul orthohantavirus is the only globally spread hantavirus pathogen, few confirmed human infections with this virus have been reported in Western countries, suggesting lower medical awareness of the milder, transient, and often chameleon-like symptoms of this zoonosis. We describe lesser known clinical and laboratory characteristics to help improve underreporting of this virus. Recent reports from several Western countries, including the United States, have described an ill-known hantavirus disease, commonly called hemorrhagic fever with renal syndrome (HFRS), induced by Seoul orthohantavirus (SEOV) and spread by infected wild, laboratory, and pet rats. These reports might bring to an end the long-maintained concept that human hantavirus infections were to be distinguished between HFRS in the Old World and hantavirus cardiopulmonary syndrome (HCPS) in the New World (Rattus norvegicus) and black (R. rattus) rats. SEOV likely arose in northern China, then spread to Europe and subsequently to the Americas in the 18th century . Because NSAIDs are often prescribed for the influenza-like myalgiae preceding HFRS, these drugs can obscure the real ensuing cause of AKI  Table.In contrast to severe HCPS caused by SNV or Andes orthohantavirus (ANDV), HFRS cases can be a diagnostic puzzle involving several swollen organs and including the lungs (Finally, laboratory confirmation of diagnosis, even by an expert clinician, can be confounded by use of current commercial serologic assays that use antigens having weak or no cross-reactivity with murine SEOVs, such as European arvicoline Puumala orthohantavirus or American sigmodontine SNV/Andes orthohantavirus. Close attention should be paid to the multifaceted diagnosis of SEOV infection  Table inAdditional information on clinical characteristics of ratborne Seoul hantavirus disease.


This study defines the transit of cefepime between plasma and cerebral spinal fluid (CSF) in a rat model. Male Sprague-Dawley rats received cefepime intravenously. Plasma samples were obtained via a second dedicated intravenous catheter. CSF sampling occurred via an intracisternal catheter. Drug exposures and transfer from the plasma to the CSF during the first 24 h were calculated. The median CSF/blood percentage of penetration was 19%. Cefepime transit to the CSF is rapid and predictable in the rat model. This model will be highly useful for understanding the therapeutic window for cefepime and neurotoxicity. 0–24] and maximum concentration of drug in serum from 0 to 24 h [Cmax 0–24]) were calculated from Bayesian posteriors. CSF penetration was estimated by comparing the exposure profiles between plasma and the CSF. Eleven rats contributed PK data. A four-compartmental model with a lag compartment for CSF fit the data well for both plasma (Bayesian [R2 = 0.956]) and CSF (Bayesian [R2 = 0.565]). Median parameter values (with the coefficient of variation percentage [CV%] in parentheses) for the rate constants to CSF from the lag compartment (K34), to the central compartment from the CSF compartment (K41), and to the lag compartment from the central compartment (K13) were 2.96 h−1 (116.27%), 0.47 h−1 (54.86%), and 0.13 h−1 (23.42%), respectively. The elimination rate constant (kel) was 3.15 h−1 (7.5%). Exposure estimation revealed a plasma median (with interquartile range [IQR] in parentheses) half-life, AUC0–24, and Cmax 0–24, of 1.7 (1.5 to 1.9) h, 111.3 (95.7 to 136.5) mg · 24 h/liter, and 177.8 (169.7 to 236.4) μg/ml, from the first dose, respectively. Exposure estimation of CSF demonstrated a median (with IQR in parentheses) AUC0–24 and Cmax 0–24 of 26.3 (16.6 to 43.1) mg · 24 h/liter and 6.8 (5.2 to 9.4) μg/ml, respectively. The median CSF/blood percentage of penetration was 19%. Cefepime transit to the CSF is rapid and predictable in the rat model. This model will be highly useful for understanding the therapeutic window for cefepime and neurotoxicity.This study sought to define the transit of cefepime between plasma and cerebral spinal fluid (CSF) in a rat model. Male Sprague-Dawley rats received cefepime intravenously. A total daily dose of 150 mg/kg of body weight/day was administered as a single injection every 24 h for 4 days. Plasma samples were obtained via a second dedicated intravenous catheter. CSF sampling occurred via an intracisternal catheter. Cefepime levels in plasma and CSF were quantified via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic (PK) analyses were conducted using Pmetrics for R. PK parameters and exposures during the first 24 h  in a rat model. Male Sprague-Dawley rats received cefepime intravenously. Plasma samples were obtained via a second dedicated intravenous catheter. CSF sampling occurred via an intracisternal catheter. Drug exposures and transfer from the plasma to the CSF during the first 24 h were calculated. The median CSF/blood percentage of penetration was 19%. Cefepime transit to the CSF is rapid and predictable in the rat model. This model will be highly useful for understanding the therapeutic window for cefepime and neurotoxicity. Antibiotic-resistant Gram-negative bacillus (GNB) infections are characterized by high morbidity and mortality. These infections have reached epidemic proportions—infecting at least 2 million Americans annually and resulting in 23,000 attributable deaths . These i4–T>MIC [∼60 to 70% ∫T>MIC])  index most predictive of clinical outcomes for cefepime is the time fraction of unbound drug above the MIC of the pathogen (∫C ∼60 to 0% ∫T>MIC∫T>MIC]) . Faced w∫T>MIC]) –11, as aT>MIC]) –. DespiteT>MIC]) –.Several animal and human studies have evaluated and described the transit of cefepime from the central distribution  to the target areas  –15. Huma–A total of 11 rats received cefepime and had plasma and CSF concentrations sampled. All rats had nine plasma concentrations (complete sampling) and a median of six CSF concentrations sampled over the 4-day protocol. Four animals had intracisternal catheter failures before the collection of a single CSF sample. All plasma samples collected were utilized for model building, with the exception that one rat had a single plasma concentration 100-fold higher than anticipated ; this concentration was excluded from analysis.kel), volume of central compartment (cV), volume of CSF compartment (VCSF), the rate constant to the peripheral from the central compartment (K12), the rate constant to the central from the peripheral compartment (K21), the rate constant to the CSF compartment from the lag compartment (K34), the rate constant to the central compartment from the CSF compartment (K41), and the rate constant to the lag compartment from the central compartment (K13) were as follows (with the coefficient of variation percentage [CV%] in parentheses): kel, 3.15 h−1 (7.52%); cV, 0.11 liter (22.9%); VCSF, 0.14 liter (64.4%); K12, 18.20 h−1 (40.17%); K21, 41.98 h−1 (10%); K34, 2.96 h−1 (116.27%); K41, 0.47 h−1 (54.86%); and K13, 0.13 h−1 (23.42%) (R2) were, respectively, −0.239 μg/ml, 0.425 (μg/ml)2, and 0.956 for plasma and −0.409 μg/ml, 1.94 (μg/ml)2, and 0.565 for CSF  . In the  for CSF .0–24 of 111.3 (95.7 to 136.5) mg · h/liter, and Cmax 0–24 of 177.8 (169.7 to 236.4) μg/ml, from the first dose. PK exposures revealed a CSF median (IQR) AUC0–24 of 26.3 (16.6 to 43.1) mg · h/liter and Cmax 0–24 of 6.8 (5.2 to 9.4) μg/ml. The median percentages of cefepime penetration into the CSF were 19% as calculated from AUC0–24 and 3% as calculated from Cmax 0–24. The complete list of percentages of cefepime penetration into CSF for each animal can be found in The overall pharmacokinetic exposures for all rats are summarized in 0–24 in CSF to plasma was 19%, and collectively the results from animal studies agree with results from human studies (range of 8 to 23%)  PK in neonatal rats  or menin to 23%) –15, 17. to 23%) –.Rodent models have been utilized for neurodegenerative diseases for many years given they possess similar brain structure connectivity and release similar neurotransmitters. Furthermore, the rat has been used in many phenotypic seizure models . AdditioFor translational purposes, it is important to note that our study utilized total drug concentrations of cefepime. As free concentrations were not measured, we did not employ a population value to correct for protein binding as this could be potentially misleading. Notably, human plasma protein binding for cefepime is low (∼10 to 20% –24), and–K13 + K34 = 3.09 h−1), indicating that steady state is approached within an hour (0.693/3.09 × 3 = 0.67 h). This finding is similar to results from a previous study by Lodise and colleagues that described the mass transit of cefepime in 7 patients with external ventricular drains (K13) in their study was nearly identical . Thus, the rat model highly replicates the human condition. It should be noted that the predicted median t1/2 of 1.7 h was calculated from the terminal elimination phase as described in Materials and Methods. When t1/2 is calculated from kel in our model, a substantially different result is obtained . The compartmental based t1/2 calculated is more relevant to acute elimination phase, and those wishing to utilize calculations from our study should understand the differences in calculations for their own application.We identified that peak concentrations in the CSF are blunted and produce different estimates of penetration compared to AUC-based evaluations. The latter is more relevant in our opinion as β-lactams are time-dependent drugs pharmacodynamically, and this has been the standard for defining cefepime penetration in the literature . Our anaWe identified two potential CSF concentration outliers in one animal during the evaluation of each animal’s pharmacokinetic profile  and b. TCmax), demonstrating moderate interanimal variability. These results substantiate the need to study PK at the individual animal level and that variability is similar to that of human studies. Third, four animals had occluded intracisternal catheters before the experimental portion of the study began, and CSF could not be sampled; however, seven animals had more complete data, with ≥4 CSF samples each. Fourth, our model did not utilize infected animals where inflamed meninges can increase the amount of cefepime that reaches the CSF  penetration. This would require further study to explore; however, this has not been the case for the majority of β-lactams studied clinically. Second, animals displayed a CV% of <50%  across exposure measurements  study was conducted at Midwestern University in Downers Grove, IL. All study methods were approved by the Institutional Animal Care and Use Committee  and conducted in an AAALAC-accredited animal facility.n = 11, mean weight, 306 g) were obtained from Charles River . All catheters  for the animals were surgically implanted  for injection. Artificial CSF  and normal saline (Abbott [lot no. C950212]) were used as in the sampling methods described below. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) standard curves were generated using commercially obtained cefepime hydrochloride (USP)  with a purity of greater than 99.5%. Ceftazidime pentahydrate  was used as an internal standard for the cefepime quantification. Formic acid was obtained from Thermo Fisher Scientific . Acetonitrile (BDH [lot no. 16I011254]) and methanol (BDH [lot no. 16L204024]) were obtained from VWR Analytical . Milli-Q water was obtained from Midwestern University via an Aqua Solutions water purification dispensatory. Frozen, nonmedicated, nonimmunized, pooled Sprague-Dawley rat plasma (anticoagulated with disodium EDTA) and CSF were used for calibration of standard curves .n = 5 blood and n = 2 CSF samples per animal. The full sampling strategy over the 4-day study can be found in Blood samples were drawn from a single right-sided internal jugular vein catheter in a sedation-free manner when possible. CSF was collected via an intracisternal catheter. Isoflurane gas was used for temporary sedation when needed . Within the first 24 h, the target samplings were Each sample  was replaced with an equivalent volume of either normal saline or artificial CSF (as appropriate) to maintain euvolemia. Blood and CSF samples from cefepime-treated animals were processed similarly to previous reports , 30.g , 75 μl of the supernatant was collected and reserved for analysis. A Poroshell  120 EC-C18 column  was utilized, with the column temperature held at 25°C for both analyses. Two microliters was injected into an Agilent 1260 LC attached to Agilent 6420 triple quadrupole mass spectrometer. Mass spectrometry analysis was conducted with electrospray ionization in positive mode (ESI+). The MS source conditions were as follows: gas temperature set to 350°C, gas flow set to 13 liters/min, nebulizer set at 40 lb/in2, fragmentor set at 68 and 78 V , and cell accelerator voltage set at 4 V. Collision energies for the cefepime quantifier and qualifier were set at 30 and 10 eV. Ceftazidime collision energy was set at 10 eV. The following transitions (m/z) for cefepime and ceftazidime were identified and utilized: 241.1 → 84.1 for cefepime quantifier, 241.1→86.1 for cefepime qualifier, and 274.1 → 80.2 for cefepime quantifier. A weight of 1/x was applied to the regression analysis (R2 = 0.998) and CSF concentrations of 0.125 and 40 μg/ml (R2 = 0.99). Precision was <7.6% for all measurements, including intra- and interassay measurements. Greater than 92% of the analyte was recovered in all samples tested, with overall mean assay accuracies of 100% for plasma and 98% for CSF. Any samples measuring above the upper limit were diluted with corresponding matrix so that the analyzed concentration was within range of the standard curve.Plasma and CSF concentrations of cefepime were quantified by LC-MS/MS using individual standard curves for each matrix. Milli-Q water containing 0.1% formic acid and acetonitrile (flow rate of 0.5 ml/min) were used as aqueous (A) and organic (B) solvents, respectively, at the following ramping transitions: 0.00 min A (90%) → B (10%), 1.50 min A (90%) → B (10%), 2.50 min A (10%) → B (90%), 5.40 min, A (10%) → B (90%), 5.50 min A (90%) → B (10%), and 10 min A (90%) → B (10%). Plasma and CSF sample volumes of 40 μl were combined with 4 μl of internal standard (10 μg/ml ceftazidime) and subjected to protein precipitation using 455 μl methanol plus 1% formic acid. Following centrifugation for 10 min at 16,000 × analysis , 32. TheC0 + C1Y (with inputs for plasma at C0 = 0.25 and C1 = 0.15 and CSF at C0 = 0.0625 and C1 = 0.15 ng/ml and where Y = observed concentration). Model performance was quantitatively described using observed versus predicted concentrations to calculate bias, imprecision, and coefficients of determination (The simplest base model considered was a 2-compartment model with a plasma compartment and a CSF compartment. Three- and four-compartment models with/without a lag constant were similarly fit (as described below) using the nonparametric adaptive grid (NPAG) algorithm within the Pmetrics package version 1.5.0  for R version 3.2.1  , 34. Themination . The fina posteriori probability (MAP) Bayesian cefepime plasma and CSF concentration estimates at 12-min intervals over the 24-h study period using each animal’s measured cefepime concentrations, exact dose, and dosing schedule. Bayesian posteriors for each animal were used to determine plasma and CSF exposures over the first 24-h period  using the “makeAUC” function within Pmetrics   for plasma and CSF were used to calculate the percentage of penetration into the CSF for each animal /k for the last 3 predicted concentrations. Summary statistics were calculated using GraphPad Prism version 7.02 . PK exposure measure variability was calculated as the coefficient of variation percentage (CV%).The best-fit model was utilized to obtain median maximum les, CA) , 36. Theh animal –15. Tmax0–24 and Cmax 0–24) for plasma and CSF were used to calculate the percentage of penetration into the CSF for each animal. This method was used similar to previous studies (–CSF/AUCplasma and Cmax CSF/Cmax plasma were used for percentage of CSF penetration. Only animals with CSF concentrations sampled were used for estimation of CSF penetration.The estimated cefepime PK exposures (AUC studies –15, 25.


Cerebrospinal fluid (CSF) is a clear fluid circulating in the intracranial and spinal compartments. Under normal conditions, the composition of CSF remains constant. However, in various neurological disease especially in acute conditions, the composition, quantity and its pressure can be altered. By measuring the levels of various CSF components using relevant techniques, diagnosis, severity and prognostication of neurological conditions like infections, subarachnoid hemorrhage, demyelinating conditions, tumor like conditions, etc. can be done. In this review, alterations in CSF components and its relevance to the emergency care physician to help in the management of patients are enumerated.Hrishi AP, Sethuraman M. Cerebrospinal Fluid (CSF) Analysis and Interpretation in Neurocritical Care for Acute Neurological Conditions. Indian J Crit Care Med 2019;23(Suppl 2):S115–S119. It also supplies nutrients as well as helps in removal of various substances like amino acids, neurotransmitters, metabolic byproducts and cells.Cerebrospinal fluid is a clear fluid which is formed as a ultra filtrate of plasma. CSF is present in both the intracranial and spinal compartments. It is continuously being secreted by the choroid plexus at a constant rate inside the ventricles of the brain and circulates in the subarachnoid space of the brain and spinal cord through CSF pathways. The total volume of CSF in the adult is approximately 140 mL. CSF is produced at a rate of 0.2–0.7 mL per minute or 500–700 mL per day.2 In some clinical conditions, lumbar puncture and drainage of can be a therapeutic measure also.3The composition of the CSF and its pressure is maintained relatively constant by various mechanisms. However in disease conditions the composition and pressure of CSF can be altered. Hence analysis of CSF by various methods will help in diagnosis as well as prognostication and response to therapy. CSF analysis is particularly useful in various acute neurological conditions and helps in rapid diagnosis of the conditions and initiate therapeutic measures. CSF analysis usually consists of opening pressure measurement, biochemical analysis, cytology, biomarkers assay, and microbiological evaluation.1  of CSF consists of the following1 ;4 In patients with seizures suspected to be due to certain metabolic illness, a fasting LP is taken for CSF analysis. In patients whose sensorium obtunded, CT scan of brain must be done to rule of signs of raised ICP, mass lesions due to risk of brain herniation due to LP. Sedation may be required in uncooperative patients and children, and fasting guidelines must be ensured to prevent aspiration of gastric contents.It is important to time the lumbar puncture (LP) to get maximum diagnostic yield  and 3. MLP can be performed either in lateral or sitting posture. Usually under sterile precautions, 22–24 G spinal needle is inserted after identifying the lumbar L2–3 or L3–4 space and local infiltration. Once the spinal subarachnoid space is identified using loss of resistance, controlled removal of stylet is done to prevent excessive drainage of CSF. A manometer can be connected if CSF opening pressure measurement is planned. Color of CSF is noted and if blood stained due to traumatic puncture, it may be needed to wait for the blood to be cleared before samples are collected. Samples are usually collected in three to four test tubes each of 3–5 mL CSF for analysis. Samples are transported to lab in biohazard bag without refrigeration.5Subarachnoid Hemorrhage (SAH) is one of the acute neurological condition needing intensive care admission of the patient. The causes for SAH can be due to rupture of intracranial aneurysms, arteriovenous malformations (AVM), vasculitis, traumatic or idiopathic. Most commonly patients present with acute onset severe headache increasing intensity within one hour of onset and often describing as worst headache of their life. In severe cases patients can present with loss of consciousness (LOC), seizures, cranial nerve palsies and various patterns of motor deficits. It is important to recognize the condition immediately as rebleed in case of aneurysmal cause can be catastrophic. Since headache is a common manifestation of intracranial diseases high index of suspicion is needed. In a recent study, Perry et al have found that following patient factors must lead to suspicion of SAH; age ≥40, witnessed loss of consciousness, complaint of neck pain or stiffness, onset with exertion, arrival by ambulance, vomiting, diastolic blood ≥100 mm Hg or systolic blood ≥160 mm Hg.7Before the advent of CT scanners, LP was the main diagnostic tool for SAH Presently plain CT scan is the initial diagnostic modality. However at times despite clinical suspicion, CT scan may be negative for SAH. CT is considered to be highly sensitive upto 5 days after the SAH. Lumbar puncture is a useful diagnostic test in these cases and is usually done 12 hours after onset of symptoms. LP is indicated if there is high clinical suspicion of SAH, but negative CT. LP is usually done after few hours. The presence of RBCs (More than 1000 cells/ cu mm) or xanthochromia (Level 2 evidence recommendation B) confirms diagnosis of SAH.8 However for the diagnosis of rebleeding of SAH, LP is not useful due to presence of pigments till 3 weeks of first SAH.However traumatic LP can complicate the RBC leading to false positive. To distinguish traumatic tap Vs SAH, CSF is collected in three test tubes and by counting the red cells in the consecutive tubes. A decrease in the red cells would usually indicate a traumatic tap. Another method is to centrifuge the CSF and look for xanthochromia of the supernatant fluid. Spectrophotometry is recommended for xanthochromia detection. Xanthochromia could be seen as early as 12 hours which is due to lysis of RBCs in CSF and release of pigments like oxyhemoglobin, methemoglobin and bilirubin.10 Aseptic meningitis is a condition that needs to be distinguished from other forms of meningitis that need a CSF analysis.11 Presentation of meningitis varies from acute debilitating illness or chronic symptoms as in tuberculosis. Patients suspected to have acute meningitis usually present with altered consciousness, fever and neck stiffness. The classic triad is seen only in 46% of patients. In others one or two of the signs of triad may be present. In addition patients can present with nausea, vomiting, headache and photophobia. In patients with meningoencephalitis additional clinical signs at presentation include altered sensorium, confusion, behavioural changes, seizures, focal neurological deficits.Patients with suspected meningitis is one of the major indication for LP and CSF study. Meningitis can be community acquired or hospital acquired and caused by various micro organisms ranging from bacteria, virus, fungus, protozoa, etc.Once there is high index of suspicion of meningitis usually a single dose of antibiotic is started empirically after taking blood cultures and without waiting for confirmation by LP. A CT scan may be indicated before LP if the patient has suspicion of raised ICP or mass lesion like cerebral edema, or herniation. However CSF analysis is the gold standard for diagnosis of meningitis as well as to identify its etiology .During LP the opening pressure of CSF, samples for routine biochemistry, cell counts are undertaken. In addition CSF samples are used for Gram Stains, microbial culture, antibodies against antigens using polymerase chain reaction (PCR), and immuno histochemistry analysis.Demyelinating diseases are a spectrum of diseases where the patient may present with acute neurological condition to ED. CSF analysis can be very useful in diagnosis of certain diseases and can aid in progress of disease and prognostication following therapy.13 In addition to above diseases, various autoimmune diseases like Guilliain Barrie syndrome, Transverse myelitis, etc can also cause varying degrees of demyelination and degeneration.15 These conditions may result in acute emergencies because of severe inflammatory destruction of CNS tissues or complications thereof.The demyelinating diseases include multiple sclerosis, acute disseminated encephalomyelitis, and neuromyelitis optica (NMO).The pathological findings in these group of diseases includes the presence of focal collections of lymphocytes and monocytes, with varying degrees of demyelination, axonal injury, and astroglial and microglial activation. In addition compliment factors deposition, vasculitis of the blood vessel can be seen. The pathology usually follow a post infectious, or post vaccination or systemic inflammatory response. Leading to autoimmune or inflammatory response to CNS components. Most of these conditions are responsive to therapy and hence early diagnosis and treatment leads to better outcomes for the patient. CSF analysis give valuable information regarding the diagnosis, progression or relapse of the disease as well as effect of treatment .16 After the initial CT scan brain or MRI, centrifuged CSF can be used for presence or absence of malignant cells and various tumor markers like colon, lung, prostate etc.Neoplasms of CNS in which CSF analysis are helpful is usually due to primary CNS lymphoma or secondary leptomeningeal deposits. Patients can present acutely with features of raised intracranial pressure due to mass effect, cerebral edema or herniations. Radiological investigations will help in making the diagnosis. However at times, it may be difficult to distinguish between secondaries and primary tutors or infections like abscess. A CSF analysis may help.Albumino-Cytologic Dissociation: The classic immunologic alteration of CSF is high CSF protein with normal cell count . Pleocytosis may rarely also occur.Neurofilament Light (NFL) Protein Assay: The NFL level correlated with disability in the acute phase of GBS and was strongly predictive of persistent disability. NFL levels were dependent on age in the control group, independent of gender, and correlated with the albuminocytologic dissociation which is indicator of the integrity of the blood-brain barrier in patients.1718Micro RNA (miR), miR-218 depletion in ALS spinal cord tissue is likely due to motor neurons loss because miR-218 levels are mostly maintained within the surviving motor neurons. miR-218 release is related to motor neurons dysfunction and death and occurs in both animal and human models of motor neurons loss and disease.19In cases where the tumor is not surrounded by parenchyma or isolated by arachnoid membranes, tumor-derived cell free DNA (cfDNA) can be detected. Tumor-derived cfDNA is found in the CSF of six of seven tumor patients.20It is important to differentiate infections vs non-infectious spinal cord pathology and CSF analysis will help in diagnosing these conditions.Each cytokine has been identified as potentially useful in the diagnostic discrimination of CNS diseases has been discussed in the literature and is associated with various aspects of these disease processes. The analysis following cytokines plays an important role in the diagnosis of spine pathologies: macrophage derived chemokine (MDC/CCL22), granulocyte macrophage colony-stimulating factor GRO/CXCL10, Interleukins , Platelet-derived growth factor AA (PDGF-AA).20 , neuron specific enolase (NSE), neurofilaments (NF), glial fibrillary proteins, Interlukein (IL6) and cleaved Tau proteins. These represent different components of damage like neurons, axons, glial cells, vascular injury, food spinal cord barrier. Analysis of various biomarkers in the CSF in patients who has sustained injury to spinal cord. Can help in assessing the severity of injury as well as in prognostication of patients.CSF analysis is a very useful investigations in patients presenting with acute neurological conditions. While it is mandatory for acute meningitis to identify the microbial organisms, it will help as a supporting investigation to establish diagnosis, prognosticate and to assess the effect of various therapies to treat the patient conditions. CSF analysis has evolved over years form basic analysis to assessment of various biomarkers to aid the clinician. Hence it is very imperative for the physician to understand the indications for these investigations as these are not widely available and expensive.