In human neonatal high pressure hydrocephalus (HPHC), diffuse white matter injury and gliosis predispose to poor neuro-developmental outcome. The underlying mechanism for diffuse white matter damage in neonatal HPHC is still unclear. Analogous to inflammatory white matter damage after neonatal hypoxemia/ischemia, we hypothesized that pro-inflammatory cytokines could be involved in neonatal HPHC. If so, early anti-inflammatory therapy could ameliorate white matter damage in HPHC, before irreversible apoptosis has occurred. In HPHC and control neonates, we therefore aimed to compare cerebrospinal fluid (CSF) concentrations of IL18, IFNγ and sFasL .In neonatal HPHC (n = 30) and controls (n = 15), we compared CSF concentrations of IL18, IFNγ and sFasL using sandwich ELISA. HPHC was grouped according to etiology: spina bifida aperta (n = 20), aqueduct stenosis (n = 4), and fetal intra-cerebral haemorrhage (n = 6). Neonatal control CSF was derived from otherwise healthy neonates (n = 15), who underwent lumbar puncture for exclusion of meningitis.In all three HPHC groups, CSF IL18 concentrations were significantly higher than control values, and the fetal intracranial haemorrhage group was significantly higher than SBA group. Similarly, in all HPHC groups CSF-IFNγ concentrations significantly exceeded the control group. In both HPHC and control neonates, CSF FasL concentrations remained within the range of reference values.Independent of the pathogenesis, neonatal HPHC is associated with the activation of the pro-inflammatory cytokines (IL-18 and IFNγ) in the CSF, whereas CSF apoptosis biomarkers (sFasL) were unchanged. This suggests that anti-inflammatory treatment (in addition to shunting) could be helpful to preserve cerebral white matter. Since the introduction of innovative drainage valves and third ventricular endoscopy, neurosurgical treatment strategies for neonatal HPHC have improved. Nevertheless, HPHC is still associated with irreversible white matter damage and adverse neurological outcome -4. AfterEspecially, the immature central nervous system is vulnerable for inflammatory damage. This is attributed to the specific sensitivity of immature oligodendrocytes for microglial cells, glutamate and free radicals ,10. AlthIn this perspective, we hypothesized that hypoxemia/ischemia-related up-regulation of longer acting cytokines (IL18 and IFNγ) could be involved in neonatal white matter damage by HPHC. If cytokines are involved in ongoing white matter damage, early anti-inflammatory therapy could be beneficial, before irreversible apoptosis has occurred. The apoptosis biomarkers Fas and Fas-ligand (FasL) are members of the tumour-necrosis factor super family. The death Fas (CD95/Apo-1) is located on the cell surface. It plays a pivotal role in transduction of the apoptotic cell death program. Fas and its FasL exist in membrane bound form and soluble forms and can be detected in neonatal CSF . Soluble75, and control patients, this study aimed to determine and compare CSF IL18, IFNγ and sFasL concentrations. We hypothesized that CSF IL-18 and IFNγ concentrations are increased in HPHC, irrespective of underlying etiology. To investigate this, HPHC patients were grouped according to three different aetiologies: spina bifida aperta (SBA), aqueduct stenosis, and hydrocephalus after fetal intra-cranial haemorrhage.In neonatal HPHC characterized by progressive ventriculomegaly and increased head circumference > P75). Since anaesthesia, artificial respiration and internal pressure compensation may quantitatively influence the assessment of intracranial pressure, CSF pressure was not measured routinely during shunt placement. Neonatal HPHC was grouped according to aetiology: SBA , aqueduct stenosis (n = 4); HC after fetal intra-cranial haemorrhage (n = 6). Selection of HC after fetal intra-cranial haemorrhage (i.e. haemorrhage 4–6 weeks before delivery) allowed avoidance of the potentially confounding influence by disintegration of platelets. The diagnosis of fetal post-hemorrhagic hydrocephalus was confirmed by prenatal ultrasound , postnatal ultrasound  and magnetic resonance imaging . Low risk neonates, undergoing lumbar puncture for exclusion of meningitis, served as controls (n = 15). Gestational ages in the three hydrocephalic groups and control group were similar, i.e. between 27–54 and 24–54 weeks, respectively. CSF samples obtained during shunt revisions, neonatal asphyxia and CNS infections were excluded. Cerebral infection was excluded by negative CSF cultures, cellular count, total protein concentration and by assessment of CSF-IL6 concentrations . CSF-IL6 concentrations were measured by commercially available solid-phase enzyme-labelled chemiluminescent sequential immunometric assay on an Immulite analyzer . All CSF IL-6 concentrations were within the normal range 5 pg/ml – 200 pg/ml  according to the manufacturer's instructions. The sensitivity of the assay was 12.5 pg/ml for IL18, 8.0 pg/ml for IFNγ and 0.5 ng/ml for sFasL using Mab for coating and binding (clones 4H9 and 4A5) ,23. The For statistical analysis, Mann-Whitney U test was used with two-sided p values to compare continuous nonparametric group of values, as the distribution of values was non-Gaussian.p < 0.01, and the fetal intracranial haemorrhage group was significantly higher than SBA, p < 0.01; figure p < 0.01; but not significantly different between the groups  pg/ml; aqueduct stenosis: 66 (55–226) pg/ml; fetal intracranial haemorrhage: 223 (103–406) pg/ml; controls: 12.5 (12.5–158) pg/ml. Each group was significantly higher than control, s figure . In all –232 pg/mUnder diverse cerebral pathological circumstances, both astroglial and microglial alterations may be involved in white matter damage and adverse neurological outcome. It is indicated that hydrocephalus-associated brain tissue compression can instigate proliferation of astrocytes and microglial cells resulting in gliosis . This stIn neonatal H-Tx rat , it was shown that shunting could ameliorate gliosis . Since gNeonatal HPHC irrespective of cause, is accompanied by pro-inflammatory cytokine activation (IL-18 and IFNγ) in the CSF. These data suggest that anti-inflammatory treatment (in addition to shunting) could be helpful to preserve cerebral white matter in these patients.CSF: cerebrospinal fluid; GA: gestational age; HPHC: neonatal high pressure hydrocephalus; IFNβ: interferon beta; IFNγ: interferon gamma; IL18: interleukin 18; sFas: soluble Fas ligand; SBA: spina bifida aperta.The authors declare that they have no competing interests.DS contributed to the study design, collection of CSF and writing of the manuscript. UF contributed to the study design, assessment of CSF samples and correction of the manuscript. TS contributed to the assessment of CSF samples and correction of the manuscript. EH contributed to collection of CSF and correction of the manuscript. CS contributed to collection of CSF and correction of the manuscript. AH contributed to the study design, collection of CSF, statistical analysis, and helped drafting and correcting the manuscript. All authors have read and approved the final version of the manuscript.


Angiostrongylus cantonensis, is an emerging infectious disease in America. The objective of this paper was to determine if the intrathecal synthesis of immunoglobulin E is produced during the acute phase of the disease.Eosinophilic meningoencephalitis caused by the helminth Thirteen patients, mean age 4.5 years were studied; a diagnostic lumbar puncture was performed and serum samples taken. Immunoglobulin E (IgE) in serum and in cerebrospinal fluid (CSF) was quantified by nephelometry. Control patients had other infections or other neurological diseases.-6 cells/L and of these 23% were eosinophils. In blood the eosinophils were 13%. The chief symptoms of the patients were migraine, vomiting and fever and 50% presented some meningeal signs. IgE intrathecal synthesis analyzed by the corresponding quotient diagram (Reibergram) was observed in all patients. No intrathecal IgE synthesis was seen in control patients.The mean cell count in the CSF was 500 × 10Intrathecal synthesis of IgE demonstrates the participation of this immunoglobulin in the destruction of the third stage larvae of the parasite in the CSF. The test should be considered in our environment as a tool to aid diagnosis. Angiostrongylus cantonensis. The definitive hosts of this parasite are rats, Rattus rattus and Rattus norvegicus [Eosinophilic meningoencephalitis is a disease caused by the helminth rvegicus -3. Many rvegicus  and are rvegicus -8, is coIgE plays an important role in anaphylactic type 1-hypersensitivity mechanisms, with high values in patients with parasitic infectious diseases accompanied by eosinophilia .Angiostrongylus cantonensis, it was shown that a local IgE synthesis was detected in the first diagnostic lumbar puncture without intrathecal synthesis of immunoglobulin A (IgA), immunoglobulin M (IgM) or immunoglobulin G (IgG). In the present study, we investigated intrathecal IgE synthesis in a larger group of children using CSF/serum quotient diagrams (Reibergrams) and report here an extremely high intrathecal immune response.In an earlier study  on four The novelty of these results is that IgE intrathecal synthesis may be considered in our environment as a diagnostic tool, and as an auxiliary diagnostic method in countries, having other parasites that are involved with the central nervous system.Angiostrongylus cantonensis were studied. The patients were admitted to the Pediatric Hospital of San Miguel Padrón, of the City of Havana between 2002 and 2007 with typical symptoms. CSF and serum samples from 31 patients with other non-neurological infections and other neurological disorders were used as controls. Normally there are no eosinophils in CSF and the blood eosinophil count was expected to be normal in this control group. The project was approved by the hospital ethical committee and written informed consent of the parent or guardian was obtained. CSF lumbar puncture and blood extraction were performed. Because samples were taken for routine analysis, no extra volume was needed for IgE analysis. Samples were collected at the time of admission at the onset of the symptoms, and were kept in small aliquots at -80°C until analysis.Thirteen patients of an average age of 4.5 years with a diagnosis of eosinophilic meningoencephalitis caused by CSF and serum total and differential cell counts were performed by traditional methods. The levels of IgE in serum and CSF were quantified by N Latex IgE Mono assay in a BN Prospec nephelometer . The procedure was standardized for the quantification of IgE in both fluids and enabled the detection of intrathecal synthesis of IgE. Serum albumin was quantified using NOR Partigen radial immunodiffusion plates and in CSF using LC Partigen plates . The data for albumin gave an estimate of the integrity of the blood-CSF barrier.-3 for Q Albumin and 0.3 - 150 × 10-3 for Q IgE. The reference ranges for the normal CSF/serum ratios are depicted by the thicker upper hyperbolic line (Q lim) and the thinner lower hyperbolic line (Q low). Between these lines is zone of normal biological variation for Q IgE. Intrathecal synthesis of immunoglobulin is detected when the point falls above the thick hyperbolic line marking the Q boundary (Q limit = 0%). Dashed hyperbolic lines show the magnitude of the intrathecally synthesized fraction relative to the Q limit .The results were plotted on a quotient diagram (Reibergram) designed for IgE, which was recently introduced for the quantification of the intrathecal IgE synthesis and constructed on the same principle as quotient diagrams for other CSF immunoglobulins . This foFrom the medical records, the main symptoms were fever and vomiting together with some meningeal signs in 50% of patients. No third stage larvae of the helminth were recovered from the CSF samples. Cerebral angiostrongyliasis is generally diagnosed from the clinical history of the patient and CSF eosinophilia, supported by possible exposure to the infective larvae. The gold standard in the diagnosis remains the detection of the agent that causes the disease, although this is often difficult.Angiostrongylus cantonensis was 500 × 10-6 cells/L with 23% identified as eosinophils. The average proportion of eosinophils in blood was 13%. The quotient diagram, or Reibergram, showed that intrathecal synthesis of IgE was observed for all 13 patients , in which the IgE antibodies bind to the surface of the helminths through receptors, activating the eosinophils to secrete enzymes that destroy the parasites [IgE displays several properties that differentiate it from the other immunoglobulin classes; among them is a cytotropic capacity. This property increases its average half life in the blood by 3 to 4 fold when it is attached to specific receptors on mastocytes and basophils. These cells are involved in the allergic processes, and this is often thought to be their main function. However, macrophages, eosinophils and platelets also have receptors for IgE, which explains IgE participation in the defense against helminths ,17, partarasites ,17. The arasites . There iarasites ,20. ThisAngiostrongylus cantonensis is the immunoglobulin that shows the largest increase compared to IgA, IgG and IgM. Consequently, intrathecal synthesis of this immunoglobulin can contribute to confirmation of the diagnosis, along with clinical symptoms, and can be an early marker of this neuroparasitosis.In our experience, IgE in the CSF and serum in eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis third stage larvae. This method takes into account IgE that enters the CSF from the serum and shows the additional amount found in the CSF due to local production.The advantage of using the Reibergram for demonstrating IgE intrathecal synthesis is that it shows the fraction of IgE produced in the central nervous system, indicating the local response to the In Cuba, there is evidence that no other parasites cause eosinophilic meningoencephalitis, which makes the intrathecal synthesis of IgE a diagnostic tool for research in our environment ,22. FromAngiostrongylus cantonensis and should be considered as a diagnostic tool. In other countries where it is common to find other parasites in the CSF as for example in neurocysticercosis, the diagnostic value of finding IgE intrathecal synthesis is more limited and would only be an indicator of an eosinophilic meningoencephalitis of parasitic origin.IgE synthesis has an essential role in inflammatory processes of the eosinophilic meningoencephalitis caused by The authors declare that they have no competing interests.BPD and AJDC designed the study, performed coordination and drafted the manuscript. RBCF participate in its design and review the clinical profiles of patients. HFH helped to measure protein analysis and to draft the manuscript. JCB contributed to the manuscript. CSM participated in the laboratory coordination and contributed to the manuscript. All authors read and approved the final version of the manuscript.


This review traces the development of our understanding of the anatomy and physiological properties of the two systems responsible for the drainage of cerebrospinal fluid (CSF) into the systemic circulation. The roles of the cranial and spinal arachnoid villi (AV) and the lymphatic outflow systems are evaluated as to the dominance of one over the other in various species and degree of animal maturation. The functional capabilities of the total CSF drainage system are presented, with evidence that the duality of the system is supported by the changes in fluid outflow dynamics in human and sub-human primates in hydrocephalus. The review also reconciles the relative importance and alterations of each of the outflow systems in a variety of clinical pathological conditions. The functional anatomy of the structures responsible for the return of cerebrospinal fluid (CSF) to the general circulation is based on somewhat conflicting evidence as to their location, anatomical features, and functional capabilities. The dual outflow systems for the egress of CSF from the intracranial CSF compartment control the balance between CSF production and drainage that ultimately impacts the constancy of the cerebral environment. This basic information has clinical implications as to the understanding of the basis and clinical consequences of derangements of the CSF outflow systems. Our present understanding of the two major functional CSF drainage systems evolved separately with recognition of the AV route occurring well in advance of our understanding of the importance of the lymphatic outflow pathway.th and 17th century. The detailed dissection of Pacchioni in 1705 revealed for the first time their relationship to the sagittal sinus, which suggested to him a secretory role for these structures. Luschka in the later part of the 19th century again noted the arachnoid structures penetrating the lacuna of the sagittal sinus, which suggested a special function for these arachnoidal projections. He also appreciated that the arachnoid granulations represented enlargement of the normal villi of the arachnoid . The response of the CSF system to elevated rates of CSF formation (over production) has been studied in a patient with hydrocephalus associated with a choroid plexus papilloma by Eisenberg et al , using a0 ml/min .The evidence to date supports the importance of both the cranial arachnoid villi (AV) and lymphatic drainage systems in the egress of CSF from the subarachnoid space. The spinal AV have a lesser but important role especially when there is failure of the primary systems. The anatomy of both systems supports the view that both act as an open unidirectional pathway between the subarachnoid CSF and the vascular circulatory system. The lymphatic outflow system is primarily by way of the olfactory nerve perineural space that traverses the cribiform plate. Other cranial nerves have been implicated but there is little evidence to support an important role in CSF drainage. The cranial arachnoid system appears to allow CSF movement via both intracellular vacuoles and intercellular clefts. In primates they are primarily located along the dura of the superior sagittal sinus near the entrance of the parietal occipital vein, while in lower animals along the transverse sinus near the confluence of the sinuses. The lymphatic system has been shown to develop earlier than that of the AV and therefore appear to be a dominant CSF outflow route in the late fetal and early neonatal period. There is convincing evidence that the AV system loses it efficiency with age, which can influence the total turnover rate of the CSF with possible neurodegenerative consequences. The question of whether the obstruction of one of the systems can alone lead to hydrocephalus is still an open question as little convincing evidence is available that separates the status of the individual components in a clinical pathological settingThe global CSF drainage system in primates becomes operative at about 50 to 70 mm CSF pressure and normally flows out of the subarachnoid system at a differential pressure gradient of 3 to 4 mm Hg. The normal flow rate does vary with age but the average is ca. 0.35 ml/min. The maximum capacity of the global drainage system appears to be ca. 1.0 ml/min above which fluid accumulates in the ventricular system under increasing pressure. It is not possible to determine in primates the percentage of total CSF transport due to the lymphatic and AV outflow systems although it has been observed in humans that there is both a high and low pressure outflow system. It maybe reasonable to assume, based on the sheep studies, that the olfactory perineural lymphatic system is the lower pressure system. There is no experimental data to state with certainty the proportion of total CSF drainage due to either system at resting CSF pressure in primates.The authors declare that they have no competing interests.The sole author Michael Pollay is entirely responsible for the content of the manuscript.


There is accumulating evidence from immunological, pathological and therapeutic studies that B cells are key components in the pathophysiology of multiple sclerosis (MS).In this prospective study we have for the first time investigated the differences in the inflammatory response between relapsing and progressive MS by comparing cerebrospinal fluid (CSF) cell profiles from patients at the onset of the disease , relapsing-remitting (RR) and chronic progressive (CP) MS by flow cytometry. As controls we have used patients with other neurological diseases. We have found a statistically significant accumulation of CSF mature B cells (CD19+CD138−) and plasma blasts (CD19+CD138+) in CIS and RRMS. Both B cell populations were, however, not significantly increased in CPMS. Further, this accumulation of B cells correlated with acute brain inflammation measured by magnetic resonance imaging and with inflammatory CSF parameters such as the number of CSF leukocytes, intrathecal immunoglobulin M and G synthesis and intrathecal production of matrix metalloproteinase (MMP)-9 and the B cell chemokine CxCL-13.Our data support an important role of CSF B cells in acute brain inflammation in CIS and RRMS. Multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the central nervous system (CNS), is generally assumed to result from an autoimmune attack directed against the myelin sheath Several key findings implicate a pathogenic role of B cells and antibodies in MS. Intrathecal immunoglobulin (Ig) synthesis and oligoclonal bands are found in more than 90% of MS patients B cells are found in the cerebrospinal fluid (CSF) during CNS inflammation, but are largely absent in non-inflammatory conditions Although these studies have clearly shown an important role of CSF B cells in CNS inflammation, their role in MS disease evolution and progression is less clear. New focal white matter lesions appear to develop following new waves of inflammation, involving immune cells which enter the brain from the peripheral blood and cause major blood brain barrier (BBB) leakage mediated by matrix metallproteinases (MMP). MMPs are supposed to be crucially involved in the degradation of the extracellular matrix and to facilitate subsequent leukocyte recruitment across capillary basement membranes to the site of inflammation However, in contrast to early (relapsing-remitting) MS, the further inflammatory reaction in progressive MS seems to be compartmentalized within the CNS We performed this prospective study to analyze CSF B cell phenotypes from patients at the onset of the disease , relapsing-remitting (RR) and chronic progressive (CP) MS with the aim to investigate whether patients/subgroups of patients exhibit certain B cell phenotypes, whether different B cell subpopulations correlate with different MS disease courses and magnetic resonance imaging (MRI) characteristics, and whether we may identify patients for stratification for B cell focused treatment. In order to corroborate the involvement and biological activities of B cells in this study population we also analyzed MMP-9 and CxCL-13 in the CSF.We report a statistically significant increase of CSF mature B cells (CD19+CD138−) and plasma blasts (CD19+CD138+) in CIS and RRMS. Further, this accumulation of B cells correlated with acute brain inflammation measured by MRI and with inflammatory CSF parameters such as the number of CSF leukocytes, intrathecal IgM and IgG synthesis and intrathecal detection of MMP-9 and CxCL-13.MS patients and neurological controls were recruited prospectively from 2004 to 2008 at the Clinical Department of Neurology, Innsbruck Medical University, Austria. This study was approved by the ethical committee of Innsbruck Medical University  and all patients gave written informed consent to the study protocol.The following inclusion criteria had to be fulfilled by the group of CIS and MS patients: (1) clinical symptoms meeting revised “McDonald Criteria” The control group consisted of 30 patients with other neurological diseases (OND). The OND group included 20 patients with noninflammatory OND  and 10 patients with inflammatory OND .CSF (3–8 ml), EDTA-treated whole blood and serum samples  were obtained during standard diagnostic lumbar puncture and peripheral vein puncture, respectively. CSF samples were immediately analyzed for CSF cell populations within 30 minutes after lumbar puncture and cell-free supernatants were stored at −80°C for further diagnostic and scientific purposes. Serum was prepared by centrifugation, 10 min at 3000 rpm, and stored at −20°C until analysis was performed.CSF samples were routinely analyzed for cell counts, quantitative (IgM and IgG indices) and qualitative  analysis of intrathecal Ig-synthesis, and CSF to serum albumin ratio  as indicators of the blood-brain barrier status immediately after lumbar puncture using standard methods .Staining of CSF cells was done as recently described Mature B cells (CD19+CD138−), plasma blasts (CD19+CD138+) and plasma cells (CD19-CD138+): 5 µl CD-45 PerCP (BD 345809) 10 µl CD19-FITC (BD 245776) and 10 µl CD138-PE (BD 347192).1. B cells (CD19+) and T cells (CD3+): 10 µl TriTest CD45-PerCP/CD3-FITC/CD19-PE (BD 342412).2. Only when enough CSF cells were available the following stainings were also included:Monocytes: 5 µl CD-45 PerCP (BD 345809) 5 µl CD14-FITC (BD 345784) and 5 µl HLA-DR-PE (BD 340689).3. Natural killer cells: 10 µl TriTest CD45-PerCP/CD3-FITC/CD16+56-PE (BD 342411).4. Memory B cells: 5 µl CD-45 PerCP (BD 345809) 10 µl CD27-FITC (BD 555440) and 10 µl CD19-PE (BD 345777).5. Erythrocytes were lysed for 10 minutes using 2 ml of lysing solution (BD Biosciences) according to manufacturer's protocol. Then, the tubes were centrifuged and the supernatants discarded. After one additional washing step with 2 ml BD Cell-Wash, CSF lymphocyte subpopulations were analyzed using three-color flow cytometry on a BD FACScan with Cell Quest software (BD Biosciences). Lymphocytes and monocytes were gated according to their forward (FSC) and side scatter (SSC) properties. A minimum number of 1000 events for each CSF staining was acquired for analysis with lyse-wash instrument settings, threshold FL-3 (PerCP) channel at 300 and the gate adjusted SSC versus FL-3. Cell populations are shown in % of total lymphocytes.CSF/MMP-9 Serum.CSF and serum concentrations of matrix metalloproteinase (MMP)-9  and CxCL-13  were measured with commercially available enzyme-linked immunosorbent assays (ELISA) according to manufacturer's protocol. CSF samples were analyzed at a dilution of 1∶51 (for MMP-9) and undiluted (for CxCL-13). Serum samples were analyzed at a dilution of 1∶1501 (for MMP-9) and 1∶3 (for CxCL-13). In order to correct for a passive transfer of MMP-9 and CxCL-13 we calculated a ratio using the following formula: MMP-9 ratio = MMP-9 All MS patients and 26 of 30 OND patients were examined by a standardized brain MRI protocol (duration about 18 min) at the time of diagnostic lumbar puncture. Patients were scanned on a 1.5 T whole-body MR scanner  using the following protocol:Coronal MPRage (repetition time (TR) 1600 ms, Inversion time (TI) 800 ms, echotime (TE) 3.44 ms, matrix 256×224, field of view 230×193 mm, slice thickness of 1.2 mm, number of excitations 1, iPAT factor 2)Transversal diffusion tensor imaging by using a EPI sequence Sagittal SPACE 3D with darkfluid preparation Pause of at least five minutes after administration of contrast agent (Gadolinium-DTPA)Repetition of sequence 1U test, Fisher's exact test and Qui-square test. Correlation of parameters was analyzed with Spearman's non-parametric correlation. Statistical significance was defined as two-sided p-value<0.05 and Bonferroni corrections were made for multiple comparisons when appropriate.Statistical analysis , significance of group differences and linear regression were evaluated using SPSS software  and GraphPad Prism 5 . The distribution of groups was determined via Kolmogorov-Smirnov test. Between-group comparisons were performed with Kruskal-Wallis test, Dunn's multiple comparison post-hoc test, Mann-Whitney CSF samples were analyzed for the presence of B cells by flow cytometry. During acquisition, lymphocytes were gated according to CD45-PerCP staining and SSC properties and subsequent analysis was done using their FSC versus SSC properties .The percentage of T cells and B cells within CSF was measured in each specimen using monoclonal antibodies to CD3 and CD19. Whereas T cells were present in all samples, B cells were barely detectable in the CSF of OND patients, but present in almost all patients with CIS, RRMS and CPMS . CSF B cFrom As mentioned above CD19−CD138+ long-lived plasma cells were nearly absent in all disease groups and no significant differences were found between groups. Further, we found no statistical significances in the frequencies of natural killer cells (n = 83) and monocytes (n = 46) between the groups analyzed.Within MS patients CSF mature B cell, but not plasma blast, counts were significantly higher in CIS and RRMS than in CPMS (p = 0.028). Within CPMS, patients with SPMS (n = 2) had higher CSF CD19+CD138− and CD19+CD138+ counts than PPMS . Further, CSF CD19+CD138− mature B cells were significantly increased (p = 0.01) in patients with an acute relapse  as compared to patients in remission  and progression . No significant differences were found for T cells, plasma blasts and other CSF cell populations. CSF T cells, mature B cells and plasma blasts did not significantly correlate with other clinical parameters such as age, gender, disease duration, EDSS and conversion to clinically definite MS.We next analyzed whether CSF cell populations correlate with MRI activity, defined by the number of lesions on T2-weighted MRI (2 groups: <9 and ≥9 T2 lesions) and the presence of Gd-enhancing lesions on T1-weighted MRI (2 groups: Gd-enhancing lesions present or not) at the time point of CSF sampling. As shown in CSF mature B cells and plasma blasts were also highly significantly correlated with other inflammatory CSF parameters, such as CSF leukocyte counts and intrathecal IgM and IgG synthesis measured by IgM and IgG indices and positive oligoclonal bands . FurtherFrom From Our prospective study was designed to compare the B cell inflammatory response between relapsing and progressive MS by characterizing the CSF cell profiles from patients at the onset of the disease (CIS), RRMS, CPMS and OND. Our data clearly indicate that the cellular B cell response, as measured in the CSF, is clearly more pronounced in acute inflammation, i.e. CIS and RRMS, than in CPMS and OND. The majority of these B cells showed the expression pattern of mature B cells (phenotype: CD19+CD138−), which have the ability to remain in the CNS compartment for years Our finding that CSF B cells are correlated with other paraclinical markers of acute brain inflammation such as high numbers of MRI T2 lesions, the presence of Gd-enhancing MRI lesions, total number of CSF cells, intrathecal IgM and IgG synthesis and intrathecal MMP-9 and CxCL-13 production indicate a direct involvement of B cells in acute brain inflammation. These findings might also explain some of the dramatic effects seen in recent therapeutic trials in RRMS patients. Natalizumab, a recently re-approved drug for MS treatment, has been shown to reduce the numbers of CD19+ B cells and CD138+ plasma cells within the CSF Despite the difference seen in the frequency of CSF B cells between RRMS and CPMS, humoral factors like IgM, IgG and CxCL-13 were found at increased levels in all MS subgroups thus raising the question on their source. Recent studies indicated that the continuous maturation of memory B cells into plasma blasts, driven by disease-associated antigens, might proceed in meningeal germinal centre-like structures within the CNS In our study we found a significant correlation of CSF MMP-9 levels and CSF∶serum ratios with the number of mature B cells and plasma blasts, but not with any other cell type. Further, CSF MMP-9 levels were increased in CIS, in patients with an acute relapse and in patients with Gd-enhancing MRI lesions thus confirming previous results In conclusion the results of our study reveal an accumulation of mature B cells and plasma blasts in the CSF of patients with a CIS and RRMS, which is associated with an increased intrathecal CSF cell count and MMP-9, CxCL-13, IgG and IgM synthesis. Although the specificity of these intrathecal B cells is not known and the role of antibodies in the immunopathogenesis of MS is still unclear, the findings of this study confirm a role of B cells and humoral factors in MS disease activity. Because the core phase of our study has been scheduled for a maximum of 3 years at study initiation, an extension phase is now planned in order to collect follow-up clinical data of the study cohort . These studies will hopefully clarify the implications of different CSF cell subsets for prognosis and potentially add information to the decision process of therapeutic strategies in MS.


Both taurine, an inhibitory neurotransmitter and granulocyte colony-stimulating factor (G-CSF), a growth factor, possess neuroprotective and neurotrophic properties in vitro. However, the mechanisms of their underlying neuroprotective effects are not fully understood.In the present study, we investigated the potential protective benefits of taurine, G-CSF and the combination of taurine and G-CSF against excitotoxicity induced by glutamate in primary cortical neuronal cultures.25 mM taurine, 25 ng/ml G-CSF and the combination of 25 mM taurine and 25 ng/ml G-CSF showed a protective effect reaching 75%, 75% and 88%, respectively. Furthermore, taurine exerted its protective effect through down-regulation of expression of GRP 78, CHOP, Bim and caspase 12.The results showed that all of these treatments, taurine, G-CSF and the combination of taurine and G-CSF, protected primary cortical neurons against excitotoxicity induced by glutamate. ER stress is suppressed by taurine after glutamate toxicity. Taurihe brain -13. Taurhe brain -16. In ahe brain -19. Morehe brain ,21.G-CSF is one of the few growth factors currently approved for clinical use for routine treatment of neutropenia . It primAlthough it is established that taurine and G-CSF have many beneficial effects under a variety of conditions of cell damage, the protective mechanisms are still unclear. We have recently demonstrated that taurine protects PC12 cells against ER stress induced by oxidative stress . Here, wBasal medium-Eagle, fetal bovine serum, poly-D-lysine, taurine, Penicillin-Streptomycin and other chemicals were purchased from Sigma . Mouse anti-actin, rabbit anti-GRP78, rabbit anti-CHOP/GADD153, rabbit anti-caspase-12 antibodies and secondary mouse and rabbit antibodies were purchased from Santa Cruz Biotechnology . Rabbit anti-Bim antibody was purchased from Assay Designs . Adenosine 5’-triphosphate (ATP) Bioluminescent assay kit was purchased from Promega . RIPA buffer was purchased from Thermo Scientific . Pregnant Sprague Dawley rats were purchased from Harlan  and housed in the animal care facility at Florida Atlantic University. The procedures for the care and use of rats, in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, were approved by the Institutional Animal Care and Use Committee of Florida Atlantic University.oC. The resulting pellet was resuspended in GME and plated on appropriate tissue culture plates precoated with 5 ug/ml of poly-D-lysine. Cells were maintained for 1 hour in a humidified incubator  before the incubation medium was replaced with serum-free neurobasalmedium (GIBCO) supplemented with B27 and 500 uM glutamine. The cultures were maintained in an incubator for 14 -18 days.Primary cortical neuronal cell cultures were prepared using a previously described protocol . BrieflyCells were measured by ATP assay. Neurons at 14 days in vitro were preincubated with 25 mM taurine for 1 hour. Then the neurons were treated with 100 uM glutamate for 4 hours. ATP solution was added to each well and cells were incubated for 10 minutes, after which levels of ATP were quantified in a luciferase reaction. The luminescent intensity was measured using a luminometer  after transferring the lysate to a standard opaque walled multi-well plate.Primary cortical neuron cultures were lysed in RIPA buffer  containing 1% (v/v) mammalian protease inhibitor cocktail from Sigma and separated on a SDS-PAGE. After proteins were transferred to a nitrocellulose membrane, the membrane was then blocked in blocking buffer  for 1.5 hours at room temperature. After blocking, the corresponding primary antibody was incubated for one hour, followed by one hour incubation with the corresponding HRP-conjugated secondary antibody at room temperature. Extensive washes with blocking buffer were performed between each step. The protein immuno-complex was visualized using ECL detection reagents.All data shown were expressed as the mean ± SEM. The Student’s t-test or one-way ANOVA was used to compare means between groups. Differences of P<0.05 were considered statistically significant.Excessive levels of the neurotransmitter glutamate trigger excitotoxic processes in neurons that result in cell death .To identPreviously, we found that 25 mM taurine resulted in the optimal neuroprotection against glutamate induced excitotoxicity . For thiG-CSF has been widely investigated in terms of protection of neurons in stroke, as shown in numerous papers ,26,33-36To test whether the combination of taurine and G-CSF promotes protection against glutamate induced toxicity, we treated primary neurons with 25 mM taurine plus 25 ng/ml G-CSF for 1 hour, followed by glutamate treatment for 4 hours. The results are shown in Fig. To investigate if ER stress can be induced by glutamate and then suppressed by taurine, specific ER stress effector proteins were analyzed by western blot. Glucose regulated protein-78 (GRP78) is an ER-associated chaperone, which facilitates protein folding in ER . The expin vitro model of primary cortical neuronal cell death induced by glutamate. Taurine and G-CSF protected primary cortical neurons against glutamate-induced neurotoxicity as determined by measuring cell viability using the ATP assay. On the other hand, we found that the combination of taurine and G-CSF gave a synergistic enhancement of protection against glutamate in primary cortical neurons. We have further shown that the suppression of ER stress is an essential underlying mechanism for taurine-induced neuroprotection. Our investigation of the intracellular mechanisms downstream of ER stress demonstrated a reversal by taurine of glutamate-induced increases in GRP78, CHOP, Caspase-12 and Bim levels.In the present study, we have demonstrated the potent protection by taurine and by G-CSF in an A previous paper reported that taurine and basic fibroblast growth factor (bFGF) in combination gave an enhanced neuroprotection in granule neurons against glutamate induced excitotoxicity . They shMany neurological disorders such as Alzheimer’s disease, stroke and Parkinson’s disease have been linked to the overactivation of glutamatergic transmission and excitotoxicity as a common pathway of neuronal injury -48. Prev2O2 in PC12 cells was prevented by taurine treatment [Although taurine has been investigated and applied to treat many diseases, the protective mechanism is still not fully understood. We have already demonstrated that ER stress induced by Hreatment . In the In the present study, we demonstrated that both taurine and G-CSF protect primary cortical neurons against glutamate-induced cell death. Interestingly, we found that the combination of taurine and G-CSF results in an enhanced protective effect. Because both taurine and G-CSF are neuroprotective agents that are approved for clinical use, the combined administration of these two factors may constitute a viable therapy with potentially enhanced therapeutic efficacy. Moreover, taurine suppressed the ER stress induced by glutamate. Further investigation will be performed to examine the specific pathway responsible for ER stress induced by glutamate and to identify molecular targets in the ER stress pathway that are specifically inhibited by taurine, G-CSF and their combination.The authors declare that they have no competing interests.


Technological advances have made it possible to examine the human cerebrospinal fluid (CSF) in a manner that was previously impossible. CSF provides a window into the changes that occur in the central nervous system (CNS) in health and disease. Through analysis of the CSF, we discern indirectly the state of health of the CNS, and correctly or incorrectly, draw conclusions regarding mechanisms of CNS injury and repair.To review the current state of knowledge of changes in the CSF in multiple sclerosis.Establishing CSF markers that permit evaluation of the various biological processes in multiple sclerosis remains a challenge. Of all the biological processes, inflammatory markers are probably the best identified. Detection of oligoclonal immunoglobulin bands in the CSF is now established as the single most useful laboratory marker in the CSF to aid in the diagnosis of multiple sclerosis. Markers of demyelination, remyelination, neuro-axonal loss, neural repair and regeneration, and astrogliosis are only now being recognized. A good surrogate for any of these pathophysiological processes has not been defined to date.The goal of future research is not only to define surrogate markers in the CSF for each of the above functions, but also to extend it to other more readily accessible body fluids like blood and urine. A synopsis of the current literature in most of these areas of CSF evaluation pertaining to multiple sclerosis is presented in this article. The cerebrospinal fluid (CSF) has been the focus of attention in multiple sclerosis for a very long time. The “colloidal gold curve” was used in the diagnosis of multiple sclerosis (MS) before the advent of modern techniques of protein separation and evaluation.–2 A “par3. What has become clear over the years is the fact that CSF IgG levels or IgG index are consistently elevated, 24 h intrathecal IgG synthesis is abnormally increased, and the IgG produced in the CNS has a restricted charge pattern, resulting in an abnormal electrophoretic profile known as oligoclonal bands (OCBs). In addition to typical large and abundant proteins like prealbumin, albumin, transferrin, and immunoglobulins that can be identified by standard electrophoresis, many other proteins have been identified in the CSF of MS patients by using advanced sensitive techniques. Today, there are > 400 proteins that have been detected in normal CSF, and some of these proteins show promise as markers for the disease process when expressed in abnormal amounts in the CSF. Additionally, investigation has extended changes in the CSF to lipids and nucleic acids. The goal of this communication is to provide an overview of most of the recent advances in our understanding of changes in the CSF in MS. The reader is referred to reviews on specific topics for additional information, as an in-depth discussion on all these topics is beyond the scope of this limited review.The CSF is clear and colorless in all patients with MS, and most patients have normal cell counts and total protein levels. Even during an acute exacerbation, total CSF protein and cell counts remain normal, although sometimes a modest mononuclear pleocytosis can be identified. Protein levels of over 100 mg/dL are distinctly unusual in MS and should alert the physician to an alternate diagnosis as also pleocytosis of over 100 cell mmIgG Index, also known as the “Link Index”, was defined by Hans Link and colleagues as the ratio of CSF IgG to CSF albumin to the ratio of serum IgG to serum albumin.[Abnormal elevated intrathecal IgG synthesis is the basis of the OCBs in MS. The elevated  albumin.4 This ra albumin.6 Althoug albumin. While at albumin. They des albumin. that was albumin. The init albumin. As silve albumin. Silver e albumin.After developing the immunoblot analysis of CSF in 1986, we have routinely employed this technique at our institution for examination of CSF for OCBs in over 10,000 samples (∼ 600 samples per year). The silver staining techniques for agarose gels and the current technique of immunoblot analysis of CSF are described below:Agarose isoelectric focusing was carried out by using standard techniques. Briefly, isoelectric focusing of unconcentrated CSF was carried out at constant current using 1% zero-EEO agarose containing 10% ampholines, pH 3.5 to 10. At completion of the procedure, the gel was fixed in 60% methanol and 4% acetic acid, washed liberally in deionized water, dried using a hair dryer, and stained using silver as described below.All reagents used for staining are described in Agarose isoelectric focusing was carried out as described above. Paired samples of CSF and serum (diluted 1:300) were run such that the IgG profile in the CSF could be compared to that of the serum. Application paper on which was loaded approximately 20 μL of CSF or diluted serum, was subjected to isoelectric focusing. Electrode solutions of 0.5 M acetic acid and 0.5 M sodium hydroxide were used as anodalyte and cathodalyte respectively. Focusing should be done in constant current mode for ideal results. The run was interrupted after the first 15 minutes  to remove the strips. Additional focusing was carried out for 15 or 20 minutes until the voltage reached around 1,500 volts. The electrode strips were removed at the completion of the run and the gel was briefly washed in PBS. A suitably sized nitrocellulose paper soaked in PBS was used for the transfer of proteins from the gel to the nitrocellulose paper. Although we previously used PBS with 20% methanol to prepare the nitrocellulose for transfer, the use of methanol was not noted to add any advantage for the protein transfer and is no longer used. By simple contact and adsorption, all the proteins can be successfully transferred to the nitrocellulose paper after firm contact for 30 minutes. The gel was removed at the end of this period and the paper washed in PBS for five minutes for three changes. Quenching was accomplished by soaking the nitrocellulose in 5% horse serum for 15 minutes. After treatment of the gel for one hour with a suitable antiserum conjugated to alkaline phosphatase or peroxidase, the staining was completed using the appropriate substrate for color development. The nitrocellulose paper was dried and subjected to examination to detect oligoclonal bands that were present in the CSF but not in the serum. Known positive and negative samples were run in every sample to assure quality standards.A comparison of the silver staining and immunoblot techniques was undertaken in identical CSF samples from MS and nonMS patients. Reproducibility and improved resolution were consistently observed with the immunoblot analysis . FurtherPresence of OCBs in the CSF is indicative of abnormally increased synthesis of intrathecal IgG. Often, but not always, this finding is associated with an elevation of IgG index and 24-hour IgG synthesis. While the latter two measures are quantitative, definition of IgG bands is a qualitative measure of the IgG produced, namely, an evaluation of the charge heterogeneity of the intrathecal immunoglobulins. Although quantitation of IgG bands can be quite readily accomplished, there is little value in this exercise as the number of bands seldom correlates with the degree of disability. Instead, it may be an indirect measure of the duration of the disease. Bands that appear seldom disappear; new bands appear over time and as the disease endures, the number of bands also increases. Although there is evidence that the number of bands seldom correlates with disability, there is some evidence to suggest that the disease is milder in band-negative patients, who comprise < 3% of all MS patients.–16Is CSF in early onset or childhood multiple sclerosis and acute disseminated encephalomyelitis (ADEM) different from that seen in adults? In a study that examined CSF from 136 patients with multiple sclerosis and disease onset before the age of 16 years, oligoclonal bands were seen in 92% of the patients. Unlike i18In a recent study that examined OCBs in the CSF of 441 patients with MS and 77 patients with ADEM, OCBs unique to CSF were seen more consistently in MS (89%) but not in ADEM (10%). ApproximAntigenic specificity of oligoclonal IgG has been the subject of intense investigations. Reactivity to antigens that are universal to MS patients but not to controls, is yet to be defined in CSF or serum. Reactivities of OCBs were examined using CSF from 14 MS and 14 normal individuals by using random hexamers expressed in a bacteriophage library. The aminIdentification of OCBs is invaluable for the diagnosis of MS. According to the most recent McDonald criteria, if OCBs are present or an elevated IgG index is identified, this finding can be used with lesser stringent criteria on MRI to satisfy dissemination in space. OCBs areTwo-dimensional electrophoresis followed by mass spectrometry of candidate “spots” resulted in the identification of numerous proteins in the human CSF. The technique termed “proteomics”, allows detailed separation of proteins in biological fluids. Analysis of the “proteome” of cells and body fluids offers promise to define pathogenesis of various disorders, and analysis of CSF from MS patients has been the focus of such attention. Detection of proteins unique for multiple sclerosis offered an opportunity to identify disease-specific proteins and opened up possibilities of identifying causative agents if such agents were expressed in the CSF. Pooled MS CSF from three patients and three inflammatory disorders of the CNS was examined in one such study. CSF was In a subsequent study, MS CSF was examined using a different technique that did not require isoelectric focusing followed by SDS PAGE. Instead,30It should be noted that potential biomarkers of MS can be proteins that are preferentially expressed in the CSF of MS patients, or can be proteins expressed in both MS and nonMS groups. The significance of proteins exclusively seen in the control but not the MS group is unclear. It should also be remembered that the techniques of proteomics are qualitative and not quantitative and do not differentiate proteins expressed preferentially in larger quantities in MS patients and therefore, probably bear significance from the standpoint of pathogenesis.Phospholipids, ceramides, sphingomyelin, cerebrosides, cholesterol, and their derivatives constitute the bulk of the dry mass of the CNS. Analysis of lipids in the CSF from MS patients33 did noTranscriptomics is the study of “expression profiling” in the brain and consequently in the CSF, at a given point in time. Collection of the CSF under ideal conditions that maintain the integrity of mRNA is crucial in such studies as mRNA is very unstable. Once collected and appropriate cDNA molecules are produced, gene array technologies help to identify upregulated as well as downregulated genes. This fledgling technology is currently in a state of development for CSF and may serve to define changes that characterize the various stages of multiple sclerosis, inflammation, demyelination, remyelination, and neural genesis. In a recent study, the transcriptome from the B cells in CSF in MS was correlated to the proteome identified by mass spectrometry. Complete correspondence was identified between the V(D)J recombination and somatic hypermutation of the variable region of the immunoglobulin gene and sequences in the variable region of the IgG, indicating that B cells are indeed the source of some of the components of the proteome seen in the CSF in MS patients.Soluble mediators of inflammation, namely, lymphokines, prostaglandins, chemokines, and C5b-9 complement membrane attack complex have been examined in CSF in limited studies of multiple sclerosis.–39 IL1, How well does the CSF reflect demyelination in the CNS? Pioneering studies of electron microscopy of sediments from ultracentrifuged CSF from MS patients identified the typical multilamellar myelin fragments with distinct major dense line and interperiod lines as extracellular fragments during clinical exacerbations. These ob53Is there any clinical utility in the measurement of CSF MBP? Measurement of CSF MBP levels in multiple sclerosis is sometimes used to confirm a clinical exacerbation as elevated levels are generally not seen in patients during periods of remission. Elevated levels usually remain elevated for 4–6 weeks and therefore can be used as a marker for recent disease activity. A normal value however does not imply disease quiescence for the reasons identified above. However, it should be noted that the definition of disease activity used in most of these studies predates the MRI era of investigations in MS, and therefore does not reflect true disease activity as defined by gadolinium-enhancing lesions. The limitations outweigh the usefulness of evaluating MBP levels in the CSF due to which measurement of CSF MBP levels is not the standard of care in routine analysis of CSF of patients with multiple sclerosis.Remyelination is not the primary mechanism of recovery of function in multiple sclerosis. In fact, most lesions remain demyelinated at the time when return of function occurs. This is best seen in optic neuritis where the optic nerves, which are commonly affected, show severe demyelination at the site of the original optic neuritis at autopsy, although during life, the patient reported normal or near-normal vision. Often, this is the sentinel event that led to the future occurrence of MS, an event that may have occurred some 30–50 years prior to death. Many patients have trouble remembering which eye was affected during the sentinel optic neuritis although return of myelin did not occur. In the nonhuman primate model of demyelination with lysolecithin, remyelination was found to be slow in the optic nerve as compared to the spinal cord. AlthoughCellular and molecular substrates of progression and disability in MS are only beginning to be understood. Axonal injury is without doubt a significant contributor to disability as also astrogliosis of the demyelinated plaque, which may impede repair and regeneration. There are several markers in the CSF that reflect these changes and show promise as surrogate markers in the CSF for injury that correlate with disability and progression.Phosphorylated forms of neurofilament proteins maintain the integrity of the axon and their repulsion of negative charges keep the axons bulky and cylindrical. With axonal injury, the neurofilaments become nonphosphorylated and the axons collapse and can no longer sustain normal function. Antibodies to nonphosphorylated neurofilaments have been useful in detection of injured axons in confocal microscopy. Recently, these antibodies have been used to detect the extent of axonal injury characterized by the presence of nonphosphorylated neurofilaments in the CSF. The studThree groups of proteins constitute normal neurofilaments; light, medium, and heavy chains. A number of studies have identified that neurofilament light chains are increased in the CSF in the majority of patients with relapsing, remitting multiple sclerosis.–62 ThereGlial fibrillary acid protein (GFAP) and protein S100B are proteins that are unique to the glia. GFAP, the major protein of the glial intermediate filament, was first isolated from an MS plaque from the fibrous astrocytes that densely populate a typical plaque. Protein 63In conclusion, CSF evaluation permits the examination of changes in the CNS that occurs in health and disease. With the advent of refined techniques that generate large amounts of data and better storage and analysis of data using bioinformatics, information is becoming available from CSF that aid in the diagnosis, treatment, and research in multiple sclerosis. Advances from these areas will surely change the face of multiple sclerosis and empower the patient and physician to understand and treat this disorder better in the years ahead.


Background. Patients afflicted with Alzheimer's disease (AD) exhibit a decrease in the cerebrospinal fluid (CSF) concentration of the 42 amino acid form of β-amyloid (Aβ42). However, a high discrepancy between different centers in measured Aβ42 levels reduces the utility of this biomarker as a diagnostic tool and in monitoring the effect of disease modifying drugs. Preanalytical and analytical confounding factors were examined with respect to their effect on the measured Aβ42 level. Methods. Aliquots of CSF samples were either treated differently prior to Aβ42 measurement or analyzed using different commercially available xMAP or ELISA assays. Results. Confounding factors affecting CSF Aβ42 levels were storage in different types of test tubes, dilution with detergent-containing buffer, plasma contamination, heat treatment, and the origin of the immunoassays used for quantification. Conclusion. In order to conduct multicenter studies, a standardized protocol to minimize preanalytical and analytical confounding factors is warranted. The amyloid cascade hypothesis proposes that an imbalance in Aβ production and clearance leads to an increase in Aβ load and that this initiates taupathology and neuronal degeneration which ultimately causes dementia [APP) gene or in the presenilin-encoding (PSEN1 and PSEN2) genes, which are involved in metabolizing the APP protein, invariably lead to AD pathology. The 42 amino acid long form of Aβ (Aβ42) has also proven to be the best established CSF biomarker for amyloid pathology in the brain. Aβ has therefore become the primary target of many clinical trials in their search for novel treatment strategies as well as a core biomarker candidate for monitoring disease-modifying effects [By the year of 2000, it was estimated that more than 25 million people suffered from dementia, with Alzheimer's disease (AD) being the most common subtype accounting for around 50 percent of all cases , 2. Hist plaques –5. The cis of AD . Not onlis of AD , but Aβ dementia . The hyp effects . β (Aβ42), total tau (T-tau), and tau phosphorylated at threonine 181 (P-tau181) in identifying subjects with incipient AD among patients with mild cognitive impairment (MCI) and they were found to provide good accuracy [β42 levels while tau levels are normal [β42 [β42 levels. Recently a large multicenter study assessed the diagnostic value of the 42 amino acid long form of Aaccuracy . The neuaccuracy , are seeaccuracy –15, it he normal , 17. Howal [β42 . This tyal [β42 . This paAll CSF samples were obtained by lumbar puncture (LP) between the L3/L4 and L4/L5 intervertebral space. Except when otherwise noted, a volume of 10–12 mL CSF was collected in polypropylene tubes followed by centrifugation  and storage in smaller aliquots at −80°C. Plasma was obtained by the centrifugation  of whole blood in EDTA tubes . The plasma was aliquoted into polypropylene tubes and stored at −80°C pending analysis. All samples were thawed at room temperature (RT), if nothing else is declared. The samples used for evaluation of confounding factors were aliquots from samples sent for routine diagnostic purposes. All samples were decoded so that no information could be linked to an individual patient.β42 levels due to differences in pretreatment, the first study comprised 15 AD and 15 control samples and the second comprised 20 AD and 20 control samples. The patients who received the diagnosis of AD fulfilled the DSM-III-R criteria of dementia [Two case control studies assessed the differences in CSF Adementia . Healthy The study was conducted according to the provisions of the Helsinki Declaration and was approved by the ethics committee of the Universities of Gothenburg and Lund and the Karolinska Institute, Sweden.β42 concentrations were obtained using the Innogenetics NV INNO-BIA xMAP technology (INNO-BIA AlzBio3) [β-amyloid1–42) [β42 levels, using different assays should pose no problems. In a case control study, these assays were compared with four other commercially available Aβ42 immunoassays from Innogenetics (INNO-BIA plasma Aβ forms) [β42 ELISA) [Unless otherwise stated, the CSF AAlzBio3) . For praoid1–42) , using aoid1–42) , which hoid1–42) . Since pβ forms) , Meso-Scβ forms) , and The2 ELISA) , 28, to U-test was employed for unpaired comparisons. The data is presented as median and percentiles (5th and 95th). Correlation analyses were performed using the Spearman correlation coefficient (rho). Receiver operating characteristic (ROC) analyses were performed to evaluate the discrimination power of the different assays using the area under the curve (AUC). SPSS 15.0 was employed for all univariate analyses.Since several variables were found to be skewed the nonparametric Friedman's or Wilcoxon tests were used for pairwise comparisons while the Mann-Whitney Freshly collected CSF from ten different subjects was tapped into glass , polypropylene , and polystyrene  tubes. The tubes were incubated at RT for one hour on a Boule mixer and thereafter stored in a freezer (−80°C) pending analysis.  Six samples of CSF were divided into three aliquots of which one was run through a lumbar catheter , one was run through a lumbar pressure meter catheter , while the third aliquot was analyzed without any pretreatment.β42 is sensitive to handling at RT, eight freshly collected samples, within 3 hours after withdrawal, were divided into two aliquots. The first aliquot was analyzed immediately, while the other was analyzed after 24 hours of incubation at RT. Six control samples were set on each plate to adjust the levels according to the interassay variability. To test whether Aβ42 is sensitive to freezing eight samples of CSF were collected freshly and divided into two aliquots. The first aliquot was analyzed immediately while the second was stored at −80°C for at least one week pending analysis. Two control samples, each analyzed in two duplicates, were used to adjust for interassay variability. The samples were analyzed using Innogenetics ELISA.  In order to test if Aβ42 in CSF at −80°C were retrieved from repeated analyses (N = 214) of an internal quality control sample  during 26 months. The control sample was analyzed in duplicates at each occasion using the Innogenetics ELISA.  Data on the long-term storage stability of A To test for the impact of incubation at RT in combination with freezing, twelve samples of CSF were collected freshly and divided into seven aliquots of which one was immediately frozen. The remaining six aliquots were stored at either +4°C or RT for four hours, 24 hours or three days and thereafter frozen pending simultaneous analysis using Innogenetics ELISA.  To test for the sensitivity of different processes of freezing, different freezing temperatures, and different thawing conditions freshly collected CSF from ten subjects was divided into eight aliquots and analyzed. Two aliquots were tested for the process of freezing; one was frozen on dry ice/ethanol and thereafter stored at −80°C for three days while the other was immediately stored at −80°C. Three aliquots were frozen at −20°C at stable temperature, −20°C in an auto-defrosting freezer, or −80°C for three days. Three aliquots were stored at −80°C for three days and thereafter tested for thawing in a fridge (+4°C), at RT and in a water bath at +20°C.  To test whether the initial LP conditions influence the biomarker level, freshly collected CSF from nine subjects was either collected at RT or in tubes placed on ice. The samples were left to incubate for three hours and thereafter frozen at −80°C.β42 CSF spinal chord gradient was assessed by sequentially withdrawing four fractions of 10 mL CSF from seven patients; fraction 1 (0–10 mL), 2 (11–20 mL), 3 (21–30 mL) and 4 (31–40 mL).The AμL (0.004–0.1% v/v) was then added to 10 different CSF samples (95% v/v) and compared with the corresponding water-diluted CSF (95% v/v). The samples were analyzed using Innogenetics ELISA. To examine if blood contamination influence CSF biomarkers a blood sample was diluted by water and freeze-thawn in dry ice/ethanol in order to lyse erythrocytes. Serially diluted blood equivalent to an erythrocyte level of 200, 1000 and 5000 / To test the influence of blood-brain barrier (BBB) dysfunction on CSF biomarkers a plasma sample was serially diluted by water to an albumin level corresponding of approximately 0.25, 0.50, 1.0 and 2.0 g/L (0.625–5% v/v) when added to 8 CSF samples (90% v/v) with an albumin level of approximately 0.20 g/L. The samples were compared with corresponding water-diluted CSF (90% v/v). This series represents different degrees of BBB permeability with a CSF/serum albumin ratio of 5, 11, 18, 30 and 55, respectively ) × 1000)). The samples were analyzed using the Innogenetics ELISA.β42 levels. Plasma was preferred as opposed to CSF due to the inconvenience of repeated fluid sampling by LP on healthy subjects. The baseline blood sample was withdrawn at nine o'clock in the morning following a nonstandardized breakfast, the follow-up sample after three weeks at the same time in the morning following an overnight fast and the postprandial sample one hour after a standardized breakfast the same day. The samples were analyzed using Innogenetics INNO-BIA plasma Aβ forms.Blood was withdrawn at three different occasions from nine cognitively healthy subjects to test whether fasting or subsequent food intake influences the plasma baseline ACSF from 14 psychiatrically and neurologically healthy subjects undergoing knee prothesis surgery was serially collected by LP with an 18-Gauge Portex epidural needle at baseline, after four to six hours (mean 5.3 hours) and after 24 hours, for further details on this procedure see Anckarsäter et al. . The samβ precipitation during centrifugation. Furthermore, 18 samples of CSF were divided into two aliquots of which one was boiled at 100°C in a heating block for ten minutes. Both samples were thereafter centrifuged at +4°C for ten minutes (2000 × g). CSF from ten subjects was divided into three aliquots each of which one was analyzed without any pretreatment. The other two aliquots were centrifuged for ten minutes (2000 × g), at RT or at +4°C to evaluate the effect of Ag) at +4°C for ten minutes and analyzed using the Innogenetics ELISA. CSF from 15 AD patients and 15 controls were divided into two aliquots of which one was preincubated for 15 minutes at 100°C in a heating block. Both aliquots were centrifuged . The addition of 0.1% v/v Tween 20, 0.05% v/v Triton X100 and 0.5 mg/mL BSA to 50 mM PBS (pH7.4) was used to further assess the improvement of Aβ42 detection. A CSF pool was serially diluted  in different buffers as well as in the provided assay buffer. Different buffer concentrations  (pH 7.4)), and different buffer substances ), were tested for the effect on the measured Aβ concentration levels between different commercially available Aβ42 assays were evaluated and the assays were compared with respect to how well they perform in discriminating between AD patients and healthy controls. CSF from patients with AD (n = 20) and healthy controls (n = 20) was analyzed using assays from Innogenetics , Meso-Scale Discovery (Triplex), and The Genetics (ELISA). The commercial Innogenetics ELISA was also tested by replacing the detection antibody with the monoclonal antibody 4G8. Neat and threefold diluted (0.05% Tween 20 in PBS) CSF samples were analyzed according to the instructions from the manufacturers.Divergences in measured Aβ42 levels were significantly altered by storage in different test tubes. The Aβ42 levels significantly decreased when CSF was stored in polystyrene tubes (208 ng/L (126–467)) compared with polypropylene (271 ng/L (152–478)), as previously shown by others [β42 level of as much as 35% . The Aβ42 level also decreased when CSF was stored in glass tubes compared with polypropylene, however it did not reach statistical significance. Some of the previously reported differences in absolute values of Aβ42 might thus be due to adsorption to different test tubes probably caused by the hydrophobic nature of this analyte. In consequence, standardization of collection tubes is necessary in order to be able to compare absolute concentration values among different centers. The Ay others , 30, 31,β42 to the lumbar catheter walls during LP might also render a difference in analyte concentration and would urge for standardization. However, two different catheters were tested of which none significantly altered the concentration of Aβ42 as compared with the baseline level. Adhesion of Aβ42 levels suggesting that this biomarker is stable when left for at least a day at RT. Furthermore, no significant alteration in the level of Aβ42, as previously described [β42 levels of freshly frozen samples and samples frozen after incubation at RT or at +4°C for four hours, 24 hours or three days. These results are in contrast to one study, which found Aβ42 to be decrease by 20% after two days incubation at RT, while no difference was found when comparing fresh CSF to frozen/thawed CSF [β42 implemented as a baseline value was decreased and thus also the Aβ42 level in the frozen CSF. Furthermore, the study had a very limited sample size which may contribute to the divergent results. Another study showed, contradictory to ours, that the Aβ42 concentration was increased after 24 hours of incubation at RT [It is essential for reliable biochemical analysis that the stability of a biomarker is thoroughly investigated in order to implement the appropriate preanalytical handling. Eight CSF samples were analyzed within three hours from withdrawal and after 24 hours of incubation at RT. No significant difference between the paired samples was detected in the Aescribed , was fouawed CSF . Howeveron at RT . Howeverβ42 at −80°C was assessed through an internal quality control sample which was analyzed on a weekly basis during a time period of 26 months. The coefficient of variation (CV) on 214 different runs was 7.5%, which is less than the inter assay CV (7.7%) reported by the manufacturer (Innogenetics NV) and consequently signify the storage stability of Aβ42 during the accounted time period  was seen for neither of the various routes for freezing, the different storage temperatures, nor the diverse thawing procedures. Moreover, no difference in Atrometry , 37. Theβ42 when successively withdrawing four 10 mL portions CSF; that is, the four portions did not significantly differ in their Aβ42 level. Although there was no gradient for CSF Aβ42 along the spinal chord, it is still recommended to take a standardized volume of CSF at LP since other proteins such as albumin [Since CSF proteins originating from brain cells may have a decreasing rostro-caudal concentration gradient, while proteins released from the leptomeninges and blood derived proteins have a lower ventricular than lumbar CSF concentration, withdrawal of different CSF volumes might affect the outcome of biochemical analysis . By with albumin  and espe albumin  will be μL) of blood to CSF should provide reliable information on the impact of contamination, unnoticeable to the eye, on the Aβ42 levels. However, no significant changes accounting for larger differences than the intraindividual assay CV was seen when comparing CSF contaminated with blood to neat CSF (It is not uncommon that the CSF gets contaminated by blood during the LP procedure . Since tneat CSF . β42 concentration in the diluted CSF was significantly (P = .008) decreased by as much as 49% (228 ng/L (165–378)) compared with the neat CSF Aβ42 concentration (433 ng/L (291–851)) , and the A91–851)) . One exp albumin , α2-macrglobulin  and low-globulin , and it omarkers , 47, foromarkers . It may β42 values have diverged among different centers the decreased Aβ42 levels in AD compared with controls have been consistent. The possible influence of an overnight fast or food intake on Aβ42 levels has been brought forward mainly due to inconsistencies in studies concerning the plasma levels of Aβ [β42 level compared with either fasting or postprandial levels. Furthermore, it would thus seem unlikely that the CSF Aβ levels would be affected when the plasma levels were not.Even though the absolute CSF Als of Aβ . Howeverβ levels would give cause for a standardized sampling time for everyday clinical routine. In a previous study, wherein 6 mL of CSF was withdrawn each hour, it was shown that Aβ had a large diurnal variability [β levels peaked at 12 hours and 23 hours with troughs at baseline and 25 hours with significant fluctuations of more than 50% within 6 hours. However, no complete return to baseline values was seen for Aβ. Our data showed more stable levels with a slight but significant decrease of 9.3% (P < .001) in CSF Aβ42 after 4–6 hours (mean 5.3 hours), which tended to return to baseline levels after 24 hours . Diurnal variability in CSF Aiability . During β42 level does not seem to be influenced by circadian rhythms to any greater extent, other analytes might be which would support a standardized time interval during the day for CSF withdrawal. In this study, our attempt was to reflect the variation in CSF withdrawal time that might be a reality in some clinical settings. One explanation to the difference between our results and the study by Bateman et al.  might beP = .002) decrease in the concentration of Aβ42 in both of the centrifuged samples (RT 203 ng/L (138–340); +4°C 203 ng/L (139–341)) when compared with the noncentrifuged samples (228 ng/L (147–354)). This indicates that a portion of Aβ42 in CSF might originate from cells that have undergone lysis that would precipitate together with the cells during the process of centrifugation. Furthermore, the Aβ fraction accessible to the antibody was further addressed by exposing CSF to heat denaturation. Herein, 18 CSF samples were divided into one heat exposed aliquot versus one unexposed aliquot and both were submitted to centrifugation prior to analysis. The Aβ42 concentration increased significantly (P < .001) in the heat treated samples (278 ng/L (170–471)) as compared with the untreated samples (210 ng/L (127–357)). This result was replicated in a case control study were the increase of Aβ42 was larger in the AD patient group  compared with the control group  , when comparing untreated versus treated samples, indicating methodological stability.Ten samples were divided into three aliquots in order to test for precipitation during centrifugation, with and without cooling during the process. There was a significant ( < .001) . Consequβ42 measurement performance compared with the provided assay buffer. The pH of CSF was investigated and found to increase rapidly in RT from a starting value of pH 7.9 and reaching a plateau at pH 8.7 already after five hours. To test if the Aβ42 antibody binding capacity is altered due to differences in pH during analysis, which could be a confounding factor if employing a buffer with a low buffer capacity, pooled CSF was tested in three different pH systems . Compared with the levels of Aβ42 obtained in provided assay buffer system, the different pH tested did not alter the Aβ42 levels. Furthermore, BSA (0.5 mg/mL) and two different detergents (0.05% Triton100 and 0.1% Tween20) were added to test if the signal of Aβ42 could be improved by possibly decreasing the negative effects of protein interaction with the solid surface of the beads and/or the air-liquid interface. The signal was equally improved for all three additives (data not shown) as compared with the assay buffer system and the detergent effect was further assessed, what follows.Factors known to affect the solubility and stability of proteins were investigated for its cofounding effects during analysis. Different buffer concentrations  and different buffer substances  did not affect the CSF Aβ42 in CSF from AD patients is that plaques in the brain act as sinks for Aβ42, preventing it from reaching the CSF. In CSF Aβ may either exist as a free soluble peptide, as oligomers [α-2-macroglobulin [ε4 allele is the strongest known genetic risk factor for AD [β clearance and an altered clearance effect, in this case, is thought to be allele specific due to a decreased binding efficiency between Aβ and ApoE4 as compared with the other isoforms [One hypothesis for the decreased level of Aligomers , or bounglobulin , apolipoglobulin , apolipoglobulin , 55, albglobulin , low-denglobulin , and traglobulin . The APOr for AD , 59. Carisoforms . β42 measureable level [β42 in proportion to the free Aβ42 in untreated samples and whether the total amount of Aβ42 could further improve the differentiation between AD and controls. The measured concentration of Aβ42 increased after the threefold dilution with the detergent containing buffer. The most striking increases were found for the xMAP assays AlzBio3 and Aβ forms. The Aβ42 median concentration for the neat CSF samples varied by more than a factor of eight, between the different assays tested . Herein, it is clearly shown that divergences in absolute Aβ42 levels between different centers could be explained by the fact that different ELISAs are utilized with different protocols as well as assay methodologies . Whether the divergences in the increase of Aβ42 levels between the two differently treated CSF samples of the controls and the discriminating power between neat/detergent CSF compared with native/denatured samples could be explained by the differences in study sample, methodological reasons or differences in complex stability needs to be addressed by further studies. Detergent and heat treatments give rise to a similar increase in the measured level of Aβ42 levels in CSF, possible confounding factors were assessed in relation to the CSF Aβ42 levels. The confounding factors found to influence the preanalytical procedures for CSF collection and sample processing, analytical procedures and techniques ultimately leading to altered Aβ42 concentrations are summarized below.Due to the high between-center variability  of reported Aβ42 concentration in noncentrifuged CSF samples possibly due to a release of the analyte caused by cell lysis—it is important to centrifuged CSF within a standardized time interval after LP. (i) Increased Aβ42 levels due to adsorption of analyte to different types of test tubes—standardization of test tubes used for CSF sampling that is, polypropylene.(ii) Decreased Aβ42 levels—probably due to dissociation of Aβ bound to proteins or release of Aβ from oligomers. For these reasons a standardization of dilution factors, buffer additives and sample processing is necessary prior to analysis. (iii) Pretreatment of CSF with detergent-containing buffers or heat denaturation lead to an increase in Aβ42 concentration decreased at the addition of plasma corresponding to a CSF/serum albumin ratio of 11–55—probably due to the binding of free Aβ to plasma proteins.(iv) The CSF Aβ42 concentration—between center comparisons cannot be made when employing different assays. This problem cannot be solved until an international Aβ golden standard is available. (i) Different immuno-assays employing various antibodies and possibly dissimilar sources for the calibrator peptides lead to divergences in the absolute Aβ42 does not seem to be affected by a spinal chord gradient, circadian rhythms, blood contamination or by storage/thawing conditions other proteins may be. It is necessary to use a standardized protocol to allow for between-center comparisons, for a detailed protocol see Blennow et al. [Even though the CSF concentration of Aw et al. .K. Blennow has participated in an advisory board for the Innogenetics. The other authors have nothing to disclose.


Surgery for skull base lesions has advanced considerably in the past few years. The improvement in surgical results could be attributed to the availability of refined imaging modalities, modern technological advances and multidisciplinary team approach. In this report, we present our personal experience in the surgical management of 45 children with a variety of skull base lesions treated over 10 years. This article includes a retrospective analysis of the surgical approaches used and their results with a review of the literature. Anterior sphenoidectomy provides access to the middle and posterior skull base as well. The loss of supportive structures in the nasal cavity to hold fat grafts and titanium mesh in place can be a drawback of this approach when anterior skull base is reconstructed. The paraseptal and middle meatal approaches are used mainly for lesions on the contralateral side and they are preferred more than the middle turbinectomy approach when reconstruction with a fat graft is expected. Endoscopic endonasal transsphenoidal surgery had been proved safe and effective, in experienced hands, not only for adults but even in children. The minimal invasiveness of this procedure, compared with the transcranial or transsphenoidal microsurgical approaches, makes it ideal for the treatment of several pediatric pathologic conditions such as pituitary adenoma, in which it is essential to preserve the anatomic and functional integrity of hypothalamic pituitary axis, both to assure a normal growth and to maintain good sinonasal viability. This endMost often, tumors involving the cavernous sinus have been surgically treated through radical surgical approaches such as face-splitting maxillectomy, transcranial lateral skull base approaches, or petrosal approaches. With the use of the endoscopic endonasal transsphenoidal approach, we can manage tumors of anterior skull base and cavernous sinus  that are not adherent to the carotid artery or dura. The ideal head positioning for the endoscopic endonasal transsphenoidal approach is 15 flexion of the forehead *chin line.17 EndoscKnowledge of the layers of the scalp and its vascular supply is essential in planning surgical incisions that preserve potential reconstructive flaps and maximize primary wound healing. The three most important layers are the pericranium, the galea, and the temporo parietal fascia. The blooThe other scalp-derived flap for reconstruction of cranial-base defects is the temporoparietal fascial flap. Unlike the temporalis muscle flap, which is a separate entity and is based on the deep temporal vascular system, the temporoparietal fascial flap receives its blood supply from the superficial temporal vessels. Both a temporalis muscle flap and a temporoparietal fascial flap can be raised simultaneously from the same side of the head for reconstruction. The temporoparietal flap is elevated off the subcutaneous tissue of the lateral scalp over the temporal and parietal regions. This flap cannot be used if that area of the scalp has been exposed to therapeutic radiation because of the high probability of scalp loss after removal of the underlying temporoparietal fascial blood supply from the overlying cutaneous tissues. This flap is about 10 cm wide and as long as 16 cm long. It can be tunneled into the nasopharynx, the orbit, the posterior anterior fossa, the middle fossa, and mastoid or temporal defects. The thin, supple nature of the flap coupled with its robust blood supply and minimal donor site morbidity makes it valuable for reconstruction of lateral defects of the cranial base. Unlike tRadiotherapy is most frequently considered an adjunctive after resection of malignant tumors of the cranial base. Most patients undergoing cranial-base surgery for recurrent malignant disease have received a full course of external beam radiation therapy. In this situation, brachytherapy often is used as an effective method of delivering an additional therapeutic dose to the site of recurrent disease. Brachytherapy relies on implanted percutaneous catheters temporarily loaded with a radioactive source, such as iridium, several days after surgery. Gamma knChemotherapy has not proved to be useful in the management of solid tumors of the base of the skull. Pediatric rhabdomyosarcoma, however, has proved chemosensitive. Chemotherapy along with radiation therapy often is used when this tumor occurs at the base of the skull. Biologic modifiers and immunotherapy are under investigation.Variety of complications have been described in the literature .26] Com Com26] CPostoperative leakage of CSF and the risk of meningitis are the most common complications after cranial-base surgery. As many as 20% of extensive resections of the cranial base entail a CSF leak. To deterHigh-flow CSF leaks usually are managed by means of direct closure as soon as possible. If the location of the leak is not evident, CT cisternography is performed to localize the source. If the leak is low flow, nonoperative measures such as lumbar spinal drain can be tried. If a low-flow leak has not stopped within 5 days, reoperation usually is necessary.Sometimes it is difficult to determine whether the fluid collected is CSF. This can occur when CSF is leaking into the nasal cavity through the eustachian tubes or through a defect in the floor of the anterior fossa. Fluid that contains glucose probably is CSF because nasal secretions do not contain glucose as does CSF. This test is not specific, however, because serous exudate from the surgical wound often is mixed with nasal mucus in the same area as the suspected leak. The presence of a CSF leak can be most specifically confirmed by means of testing for the protein *≤2-transferrin. Only three body fluids contain this protein: CSF, perilymph, and the vitreous fluid of the eye. The presence of *≤2-transferrin almost confirms the presence of CSF.Meningitis is a complication that can follow CSF leak. We always provide prophylactic antibiotics to all patients. The antibiotics will be continued if there is a leak. In the case of meningitis, appropriate antibiotics based on the culture are used. Recurrent meningitis can be a rare complication in undetected CSF leaks.Unlike spontaneous or posttraumatic CSF fistula, postoperative leaks almost always necessitate surgical closure. Persistent CSF leak after a reasonable wait warrants re-exploration. The technical options for closure depend on factors such as the exact site of the leak, a history of radiation therapy, the size of the defect, the relation of the defect with mucosa-lined cavities, the available reconstructive flaps, and the technical ability of the surgeon.Although endoscopic closure of CSF fistula is becoming popular, it may not be feasible in acute postoperative leaks. Proper exploration of the resection site and closure or reclosure of areas may be necessary. They often necessitate application of vascularized tissue. The exception is isolated sphenoidal sinus defects usually caused by transsphenoidal hypophysectomy. Artificial dural substances and fibrin glue are of immense help.In the case of sphenoidal sinus leaks, endoscopic closure with sealing agents such as fibrin glue or hydroxyapatite cement or autografts such as fat and fascia are useful. Fibrin glue is particularly effective in sealing low-flow CSF leaks. High-flow leaks are managed with lumbar drainage before placement of fibrin glue. The fibrin glue immediately seals the leak, and the fat ensures long-term obliteration of the sinus to prevent delayed leakage after resorption of the fibrin glue. Fibrin glue was first used as a hemostatic agent in the early 1900s. It was applied as an adhesive in the 1940s. Since 1980s, highly concentrated forms have been used as a tissue sealant and adhesive. Fibrinolysis can occur when fibrin glue is exposed to excess amounts of plasma. Because the thin layer of scar tissue generated by fibrin glue alone can be eroded by the constant pressure of the CSF pulsations, it is best used with an autogenous fat or fascial graft to enhance long-term stability.Hydroxyapatite cement can be applied to the small bony defects; it forms a hard barrier to future CSF leakage. Several types of hydroxyapatite cement materials are mixed with a dilute acid aqueous solution that causes setting of the calcium phosphate reactants. Setting usually occurs in 8–15 minutes, depending on the preparation. Once set, these materials stay water sensitive for 4–6 hours and can be dissolved during this period by excess blood or CSF. Hemostasis and temporary (12 hours) use of a lumbar drain is recommended when these materials are applied, regardless of the type of cement. The material must be applied directly to the surface of viable bone after removal of all the surrounding mucosa. This may not be successful for leaks within the ethmoidal sinuses as entrapment of mucosa is unavoidable and success is doubtful.In addition, repair of dura is equally important. A number of new materials are available such as preserved bovine pericardium and acellular dermis as dural substitutes.Postoperative cerebral edema, hematoma, vascular emergencies, air embolism, and seizures need immediate attention and treatment. These children require long term follow-up and good rehabilitation especially after reconstructive surgery to enhance their functional capacity.Vascular complications needs special mention and can cause hypotension, worsening of neurological state. Early recognition can avert the danger. Identification of source is of utmost importance. Direct surgical methods or endovascular methods can be applied to stop the bleeding.Skull base surgery is evolving as a specialty through microscopic and endoscopic approaches. Proper imaging, planning, preparation with meticulous surgical technique, and standard intensive care management are mandatory to achieve best results. With the available modern gadgets, majority of skull base lesions can be successfully treated. Team approach and attention to special problems are necessary for pediatric population.


In the experiments, there may be some errors on the reads which affect the performance of the DNA assembly algorithms. Existing algorithms, e.g. ECINDEL and SRCorr, correct the error reads by considering the number of times each length-k substring of the reads appear in the input. They treat those length-k substrings appear at least M times as correct substring and correct the error reads based on these substrings. However, since the threshold M is chosen without any solid theoretical analysis, these algorithms cannot guarantee their performances on error correction.DNA assembling is the problem of determining the nucleotide sequence of a genome from its substrings, called k substring is correct using threshold M. Based on this optimal threshold M that minimizes the total errors . Experimental results on both real data and simulated data showed that our calculation is correct and we can reduce the total error substrings by 77.6% and 65.1% when compared to ECINDEL and SRCorr respectively.In this paper, we propose a method to calculate the probabilities of false positive and false negative when determining whether a length-k substring using different thresholds. Based on this calculation, we found the optimal threshold to minimize the total error of false positive plus false negative.We introduced a method to calculate the probability of false positives and false negatives of the length- Since DNA assembling is the first step in bioinformatics research, there are many different technologies, e.g. BAC-by-BAC approach  is a variant Tt of T, isGiven a length-G is generated by other models, e.g. Markov Chain, pocc can also be calculated easily. However, this part has little effect on the overall result, as the number can be cut in the later calculation.Genome sequences, especially the non-coding sequences, are highly heterogeneous in composition. The i.i.d. model cannot reflect the real situation of the genome sequences well. On the other hand, genome Tt be a k-length substring obtained at position i of G. Since only those reads containing the substring from position i to i - k + 1 can contribute Tt, copy of Tt can be obtained from at most l - k + 1 different reads, exactly l - k + 1 reads in most cases when l - k + 1 ≤ i ≤ g - l + 1. When g >> l, the probability that Tt is sampled s' times at can be approximated byLet g is large, psam can be approximated by the normal distribution with mean equalsWhen and variance equalspsam from training data.In some real experimental data where each position is not sampled with the same probability, we may estimate the mean and variance of Tt of T is sampled, the probability pt that we get the k-tuple T instead of Tt as input because of error isWhen a read with variant perr is the probability of single nucleotide error occurrence. Note that as perr is usually very small, pt can be ignored and assumed zero when t ≥ 3 in practice. Here we assume when there is error, the occurrence probability of each nucleotide (three possible nucleotides) is the same. Similar as pocc, the formula for pt can be modified when genome G is generated by other models. The probability that the k-tuple G [i...i + k - 1] is sampled s' times and r'(r' ≤ s') of them is T because of errors iswhere G [i...i + k - 1] = Tt', 0 ≤ t' ≤ t. In order to get r samples of T from the n reads, d variants of T appear in different positions of G and rj samples of T are getting from the j-th variants such that Σrj = r. Therefore, the probability that T appears exactly r times in the input is approximatelywhere d variants of T and the samples in the remaining positions. This approximation is fine when g >> d and n is large, which is valid for most experimental data.Equation (1) is an approximation because we have not considered the interdependence of the positions of the Pr(Yr), we can calculate the probability of true positive as follows:Once we calculate P(Yr) can be calculated from Equation (1). Pr(¬ X0) = )n-k+1 and Pr(Yr|¬ X0) can be calculated from Equation (1) by considering that the probability that T appearing in G is zero, i.e. set g, 0) = 0 and g, t) = pocc /).The probability of false positive one minus the probability of true positiveThe probability of false negative can also be calculated similarly:M, once we calculate the probabilities of true positive, false positive and false negative for all possible thresholds M for a particular substring length k. We can then find the optimal threshold M* for that particular k which minimizes the total errors FP + FN or maximizes the total accuracy.Since we only consider integer threshold The authors declare that they have no competing interests.FC and SMY studied the read correction problem, suggested the main idea of calculating the optimal threshold M. HL and WLL worked out the details of calculation, worked out the experiments and drafted the manuscript. All authors read and approved the final manuscript.