Here we propose that altered CP/CSF dynamics during neurodevelopment may be considered a risk, causative and/or participating factor for development of schizophrenia.Schizophrenia is a neurodevelopment disorder in which the interplay of genes and environment contributes to disease onset and establishment. The most consistent pathological feature in schizophrenic patients is an enlargement of the brain ventricles. Yet, so far, no study has related this finding with dysfunction of the choroid plexus (CP), the epithelial cell monolayer located within the brain ventricles that is responsible for the production of most of the cerebrospinal fluid (CSF). Enlarged brain ventricles are already present at the time of disease onset (young adulthood) and, of notice, isolated mild ventriculomegaly detected  Schizophrenia is a disabling psychiatric disorder that affects 0.7–1% of the population worldwide. The disease has a heterogeneous presentation, with disorganized positive and negative symptoms that have different levels of intensity/relevance between individuals and across time in the same individual development may interfere with the CP's activity or with the ability of the brain to circulate the CSF, and that this may relate with schizophrenia etiology and pathology, including ventricle enlargement. With this article we intend to bring into the research field the possible role of the CP–CSF nexus in predisposing for/modulating schizophrenia.The CP is a structure highly conserved both phylogenetically and ontogenetically  lie adjacent to the ventricular surface and are bathed by the CSF. In fact, the influence of the CSF content over neural progenitor cells is not restricted to the embryo. In adult mice it was shown that the pattern of CSF flow inside the brain ventricles influences the pattern of subventricular zone (SVZ) neuroblasts migration toward the olfactory bulbs; a process known to be regulated by CP secreted proteins such as Slit 1 . It can be associated with chromosomal abnormalities, congenital infection, cerebral vascular accidents or hemorrhage, and other fetal cerebral and extracerebral abnormalities. The possible mechanisms causing fetal ventriculomegaly are obstruction to CSF flow, hypersecretion of CSF (rare), defective filtration of CSF (rare), and altered development of the intracranial architecture. On this, aqueductal stenosis is the most common reason of obstruction to CSF flow and can be caused by infections, bleeding, or other pathologies that cause gliosis and obliterate the aqueduct , concluded that significant overall effect sizes were consistently demonstrated for total  ventricular volume increase  hydrocephalus, the obstruction of natural CSF flow results in abnormal cell proliferation and decreased number of cells migrating , which contains domains for proteins involved in neurodevelopment, cytoskeletal function, neurite outgrowth, and cAMP signaling . We have recently characterized the CP mouse transcriptome in basal conditions  approach would be to follow newborns, who have been identified with mild ventricle enlargement on fetal ultrasound scan, into adulthood, with a detailed characterization of behavior. The lateral ventricle width is routinely assessed on second trimester ultrasound examination. Furthermore, ventriculomegaly is a relatively common finding (≅1%) of clinical relevance as a marker of a variety of underlying disorders . When ventricle dilatation is mild (10–15 mm), and there are no associated anomalies, the risk of abnormal or delayed neurodevelopment in infancy is still higher than the estimated for the general population. The association between prenatal isolated mild ventriculomegaly and neuropsychiatric disorders remains, however, to be established.A final suggestion would be to monitor ventricular enlargement in the progeny of patients with schizophrenia, as an attempt to identify how early ventricular enlargement is observed in individuals that ultimately develop the disease; and to follow first episode patients on how additional ventricular enlargement may relate with disease progression and response to treatment. It will always be difficult to discriminate between ventricular volume findings and other genetic aspects that may predispose the progeny of schizophrenic patients for disease development, but this will certainly be a piece of information to add to the others proposed this far.The brain barriers have only recently started to be seen as more than mere obstacles for the passage of molecules and cells in and out of the brain. It is now clear that they actively participate in modulating brain homeostasis in health and in disease (Marques et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The significance of amyloid precursor protein (APP) and neuroinflammation in idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer's disease (AD) is unknown.To investigate the role of soluble APP (sAPP) and amyloid beta (Aβ) isoforms, proinflammatory cytokines, and biomarkers of neuronal damage in the cerebrospinal fluid (CSF) in relation to brain biopsy Aβ and hyperphosphorylated tau (HPτ) findings.APOE.The study population comprised 102 patients with possible NPH with cortical brain biopsies, ventricular and lumbar CSF samples, and DNA available. The final clinical diagnoses were: 53 iNPH (91% shunt-responders), 26 AD (10 mixed iNPH+AD), and 23 others. Biopsy samples were immunostained against Aβ and HPτ. CSF levels of AD-related biomarkers , non-AD-related Aβ isoforms , sAPP isoforms , proinflammatory cytokines , interferon-gamma, monocyte chemoattractant protein-1, tumor necrosis factor-alpha) and biomarkers of neuronal damage (neurofilament light and myelin basic protein) were measured. All patients were genotyped for p<0.05) in patients with shunt-responsive iNPH compared to non-iNPH patients. sAPPβ showed a similar trend (p = 0.06). CSF sAPP isoform levels showed no association to Aβ or HPτ in the brain biopsy. Quantified Aβ load in the brain biopsy showed a negative correlation with CSF levels of Aβ42 in ventricular  and lumbar  samples, while the levels of Aβ38 and Aβ40 showed no correlation. CSF levels of proinflammatory cytokines and biomarkers of neuronal damage did not associate to the brain biopsy findings, diagnosis, or shunt response. Higher lumbar/ventricular CSF IL-8 ratios (p<0.001) were seen in lumbar samples collected after ventriculostomy compared to the samples collected before the procedure.Lumbar CSF levels of sAPPα were lower (The role of sAPP isoforms in iNPH seems to be independent from the amyloid cascade. No neuroinflammatory background was observed in iNPH or AD. Idiopathic normal pressure hydrocephalus (iNPH) is a progressive neurodegenerative disorder of unknown etiology in the elderly presenting with gait disorder, cognitive impairment, and urinary incontinence, with enlarged ventricles of the brain but normal or slightly elevated cerebrospinal fluid (CSF) pressure In vivo, Aβ can be detected directly with brain biopsy 11C-labeled Pittsburgh compound B 18F]flutemetamol AD is characterized by the hallmark lesions of amyloid-β (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau (HPτ) in the brain of patients with amnestic cognitive decline Although common pathways for iNPH and AD have been proposed Aβ originates from a cell membrane-spanning protein, amyloid precursor protein (APP), which has diverse roles in normal neuronal function Abnormal levels of proinflammatory cytokines, such as interleukins (IL), interferon-gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-α), in CSF have been noted in various diseases of the nervous system, including AD Elevated levels of neurofilament light protein (NFL) in the CSF, indicating neuronal death and axonal loss, have been found in iNPH and secondary NPH in several studies To our knowledge, studies assessing the association between proinflammatory cytokines and biomarkers of neuronal damage in CSF, and cortical brain biopsy have not been published to date.The objectives of the current study were:1. to determine the levels of AD-related biomarkers , non-AD-related Aβ isoforms , sAPP isoforms , proinflammatory cytokines  and biomarkers of neuronal damage  in lumbar and ventricular CSF, and how they correlate,2. study the relationship between the CSF biomarkers and cortical brain biopsy,3. assess the diagnostic and prognostic value of the CSF biomarkers in iNPH and AD.This study was approved by the Kuopio University Hospital (KUH) Research Ethical Committee, The Finnish National Supervisory Authority for Welfare and Health, and The Finnish Ministry of Social Affairs and Health. All participants or their proxies gave a written, informed consent prior to participation in the study. If the clinician suspected dementia to significantly affect the capacity of the patient to consent, a next of kin, caretakers or guardians consented on the behalf of participants. When a consent was obtained from a participant's proxy, the patients own opinion was inquired and considered, and no patients were recruited against their will.Neurosurgery of KUH solely provides full-time acute and elective neurosurgical services for the KUH catchment population in Middle and Eastern Finland. In addition, the KUH area contains four central hospitals with neurological units and catchment areas of their own www.uef.fi/nph) consists of all evaluated possible NPH patients from the KUH catchment population since 1993 Patients fulfilling the following criteria were further assessed in KUH Neurosurgery as possible NPH patients: (1) primary evaluation and examination by a neurologist indicating NPH; (2) one to three symptoms suggestive of NPH ; and (3) NPH related brain imaging findings (enlarged ventricles (Evans' index>0.3) together with obliterated cortical sulci). The diagnostic workup protocol of KUH Neurosurgery for possible NPH included a clinical examination, CT or MRI scan, and 24 h intraventricular ICP monitoring together with a frontal cortical brain biopsy. Kuopio NPH Registry (The ICP criteria for the shunt treatment in iNPH patients were (1) a basal ICP pressure between 10 and 20 mmHg continuously, or (2) the presence of A-waves or more than 30% B-waves during the 24 h monitoring, when basal pressure was between 5 and 10 mmHg APOE genotype, and a ventricular CSF sample available were included in the study  from the Kuopio NPH Registry with cortical brain biopsy, he study . 63 patiBiopsy samples representing frontal cortex and subcortical white matter were stained with hematoxylin-eosin and immunostained with monoclonal antibodies directed to Aβ (6F/3D) and HPτ (AT8) as described earlier in detail APOE genotyping DNA was extracted from venous blood using commercial kit according to manufacturer's protocol . A standard PCR method was used in the The ventricular CSF samples were collected immediately after the placement of intraventricular catheter (first 1 mL discarded) in the ICP measurement procedure. In addition, a lumbar CSF sample was available in 49 patients. The lumbar samples were obtained through a lumbar puncture prior to the ICP measurement protocol (12 patients) or 24–48 hours after introducing the ventricular catheter (37 patients).1–42, Innotest Phosphotau(181P), Innotest Tau-Ag, Innogenetics, Ghent, Belgium) according to the manufacturer's protocol at validated laboratory in Neurology (www.uef.fi/neuro), University of Eastern Finland, Kuopio, Finland as described earlier Levels of AD biomarkers  were measured from the CSF samples using commercial ELISA kits , sAPP isoforms , and the proinflammatory cytokines  were analyzed utilizing commercially available multiplexed assays  All clinical, immunohistochemical, and laboratory analyses were performed blinded to the result information of each other.k = 55) was applied.Nonparametric Kruskal–Wallis H and Mann–Whitney U tests were used for comparing CSF levels of measured biomarkers between different groups, and the Wilcoxon signed-rank test for comparisons of two related samples. To define the correlation between different proinflammatory cytokines, Pearson correlation with Bonferroni correction (n = 4) were excluded from the analyses of different biomarkers in relation to biopsy findings, and iNPH patients with co-morbid AD (n = 10) and iNPH patients with negative shunt-response (n = 5) from the analyses comparing true iNPH patients to non-iNPH patients. Some cytokines were below the lower limit of detection of the assays and graded as zero concentration in statistical analyses. One patient had an insufficient CSF sample for the analysis of sAPPs, and another for the analysis of Aβ isoforms Aβ38 and Aβ40.Patients with tauopathy but no amyloid  was used in the statistical analyses. The level of significance was set at p<0.05) in patients with iNPH and a positive shunt reponse compared to non-iNPH patients, while sAPPβ showed a similar tendency  in shunt-responders compared to nonresponders in ventricular and lumbar CSF (Lumbar CSF levels of sAPPα were significantly lower (mbar CSF .p = 0.003) between different brain biopsy groups (p = 0.008), and to the Aβ and HPτ negative group  and lumbar  samples (p = 0.59–0.95) with the presence of Aβ or HPτ in the biopsy (p = 0.84–0.92). There were no statistically significant differences between the levels of CSF Aβ or tau biomarkers in shunt-responding and nonresponding iNPH patients.Ventricular CSF levels of Aβ42 differed . Moreover, lumbar/ventricular CSF IL-8 ratios (p<0.001) were significantly higher in samples collected after the ventriculostomy and ICP measurement compared to those collected before the procedure  towards lower ventricular CSF NFL values was seen in shunt-responders  of these patients showed a favourable response to shunt treatment.As predicted, AD patients had lower Aβ42 and higher p-tau levels in the CSF compared to non-AD patients, although the differences did not reach statistical significance. One explanation to this is that non-AD patients show similar CSF Aβ42 and p-tau findings as biomarkers of comorbid AD tissue pathology without clinical dementia of Alzheimer's type. As no pathological hallmark lesions have been identified in iNPH In contrast to two previous studies In iNPH, reports of abnormal levels of proinflammatory cytokines  in the CSF has been published Previous studies have reported increased levels of CSF NFL in NPH The strengths of the current study include: a large NPH cohort evaluated by cortical brain biopsy, utilization of ventricular and lumbar CSF samples in the analyses, a wide panel of tested CSF biomarkers, and evaluation of clinical outcome and other dementing disorders in the follow-up. The limitations of this study included: a limited number of patients with no shunt response, systematic assessment of shunt response only at 2–3 months, dichotomised shunt response scale, lack of validated objective outcome measures, and lumbar CSF sample available only in half of the cases. It is obvious that at least in prospective research setting shunted patients should be followed-up for a significantly longer time, and validated outcome measures should be utilized. Five patients with diagnostic findings suggesting iNPH did not respond to shunt – possibly due to comorbidities or misdiagnosis, and thus these patients were excluded from the comparisons of (‘true’) iNPH patients and non-iNPH patients in addition to the iNPH patients who were diagnosed with comorbid AD in the follow-up.More basic science and clinical studies evaluating the biology and potential role as diagnostic and prognostic biomarker of sAPPα and β are needed in the future.To conclude, the role of sAPP isoforms in iNPH seems to be unconnected to the Aβ cascade pathway, but rather may be explained by a metabolic failure or ischemia in the brain. No elevations in the levels of proinflammatory cytokines in the CSF were observed in the different diagnostic groups. Consequently, neuroinflammation in iNPH and AD require further study. None of the tested CSF biomarkes showed a potency to discriminate between iNPH and non-iNPH patients or shunt-responders and nonresponders in iNPH in clinical setting.Table S1Correlations of proinflammatory cytokines in lumbar CSF.(PDF)Click here for additional data file.Table S2Correlations of proinflammatory cytokines in ventricular CSF.(PDF)Click here for additional data file.


Antemortem diagnosis of cerebral toxoplasmosis, the second most common opportunistic infection (OI) in HIV-infected individuals in developing countries is a challenge.Toxoplasma gondii (T.gondii) -specific serology and nested polymerase chain reaction (nPCR) were evaluated in sera and ventricular/lumbar cerebrospinal fluid (CSF) of 22 autopsy confirmed cases of cerebral toxoplasmosis with HIV and 17 controls. Frequency of concomitant T.gondii infection was investigated in 17 cases of HIV-associated tuberculous meningitis (TBM).T. gondii IgG on CSF (ventricular and lumbar) and sera was 100% in histology proven cerebral toxoplasmosis ; majority (94%) being high avidity type, suggesting reactivation/reinfection. The sensitivity of B1 nPCR was 100% on ventricular CSF, whereas it was only 77% on lumbar CSF. Based on histology, nPCR, and IgG serology, T. gondii co-infection with TBM was observed in 65% (11/17) of cases.The sensitivity, specificity, and positive and negative predictive values of CSF IgG serology and nPCR are tests with high sensitivity and specificity for the diagnosis of cerebral toxoplasmosis. TBM and cerebral toxoplasmosis can coexist and should be considered in the background of HIV infection in developing countries. Toxoplasma gondii (T.gondii) in the cerebrospinal fluid (CSF) and/or serum in the presence of mass lesions with perilesional edema on cranial CT scan/MRI is considered diagnostic. Neuroimaging facilities are not widely available in all centers or are unaffordable. Hence there is a requirement for alternative tools for the diagnosis of cerebral toxoplasmosis.[Cerebral toxoplasmosis is the most common cause of focal brain lesions in patients with Acquired Immuno Deficiency Syndrome (AIDS). In the U3Sera and CSF samples analyzed in the study were collected at autopsy with informed consent of close relatives of the deceased. At the time of autopsy, sterile lumbar puncture needles and disposable syringes were used for sample collection to prevent cross contamination. As a practice, following clinical autopsies from cases suspected of HIV/AIDS with OIs, the autopsy table, instruments are routinely decontaminated. These autopsies were conducted at a frequency of one per week on an average obviating the possibility of cross contamination. Samples were aliquoted under sterile conditions in a biohazard laminar flow hood and stored at –86°C in Human Brain Tissue Repository for neurobiological studies (Human Brain Bank), in the Department of Neuropathology, National Institute of Mental Health and Neuro Sciences, Bangalore, India. The study was approved by the Institutional ethics committee. The postmortem delay from the time of death to collection and freezing of the samples varied from 3 to 20 h. The study material included CSF  and sera (from heart blood) collected at autopsy. The cases comprised three groups. The clinical and epidemiologic details of all three groups are provided in Table 1Twenty-two HIV-1 seropositive patients (confirmed to be clade C by subtype-specific PCR) who succumbed to cerebral toxoplasmosis manifesting as encephalitis or mass lesions were included. All the cases had histopathologically confirmed toxoplasma lesions in various stages of evolution. HIV-seronegative cerebral toxoplasmosis cases were not available in our center.Mycobacterium tuberculosis (M.tuberculosis) and/or demonstration of acid fast organisms in the smear from the basal exudates in the brain at autopsy with histopathologic evidence of chronic granulomatous meningitis (with or without caseous necrosis) were studied.Seventeen HIV-seropositive cases of TBM confirmed by CSF seropositivity for IgG antibody and immune complexes to lipoarabinomannan antigen of T. gondii antibodies were used as controls. Serum and CSF samples from HIV-positive cases without OI were not available in our cohort and hence could not be included for study.Seventeen HIV-seronegative age- and sex-matched cases who succumbed to road traffic accidents  and whose sera were negative for The investigators carrying out the enzyme-linked immunosorbent assay (ELISA) and nPCR studies were totally blinded to the origin of samples and the diagnosis of cases. Coded samples were provided to them, which were decoded only after completion of the study for analyses of results.T. gondii using commercial ELISA kits  and expressed in International Units (IU)/mL. Sera were tested at a dilution of 1:100 (as recommended in the kit protocol) and CSF dilutions were fixed at 1:10 by titration. Antibody levels greater than 10 IU/mL were considered as positive. Samples positive for IgG antibodies were further tested for their avidity and the results were interpreted as high or low avidity, following manufacturer’s instructions . The mean concentrations of T. gondii-specific IgG in CSF and sera were compared by the Student’s t test.Lumbar and ventricular CSF samples and sera were tested in duplicates for IgG and IgM antibodies to DNA was extracted from aliquots of supernatants of 200 µL CSFs stored at –86°C, using commercial extraction kits .T. gondii.[T. gondii culture DNA . β-Globin PCR was used for house keeping to exclude the presence of polymerase inhibitors and confirm the efficiency of DNA extraction.[T. gondii DNA by nPCR was 100 fg (data not shown). PCR was negative when amplified using DNA from cultures of Aspergillus fumigatus, M. tuberculosis, and Candida albicans.Nested PCR was carried out using primers against the B1 gene of . gondii. The senstraction. The limiT. gondii-specific serology were determined on CSF and sera from groups I and III considering them as true positives and negatives, respectively.The performance characteristics, namely, sensitivity, specificity, and positive and negative predictive values of both nPCR and T. gondii cysts and tachyzoites was confirmed by immunohistochemistry using commercial antibody to P30 antigen .The brains collected at autopsy were fixed in 10% buffered formalin for 4 weeks before sectioning. Lesions identified on gross examination were sampled for paraffin embedding and histologic evaluation. The lesions were mostly located supratentorially and a few in the cerebellum and brain stem, both deep in the parenchyma and close to subarachnoid space. Brain tissue localization of T. gondii were detectable [The mean age of the patients was 31 ± 9 years, (M:F=16:6). Nineteen of the 22 cases clinically manifested with signs of raised intracranial tension and focal neurologic deficits. Neuroimaging studies revealed single or multiple ring-enhancing lesions involving basal ganglia, thalamus, brain stem, and cerebral cortex. Three cases presented with features of encephalitis with headache and seizures. Neuroimaging revealed multiple necrotizing hemorrhagic lesions with diffuse cerebral edema causing mass effect and midline shift . At autotectable , which wtectable .In 14/22 patients with histopathologically proven cerebral toxoplasmosis, all 3 samples  were available. The mean CSF glucose was 53 ± 7 mg/dL; protein: 184 ± 63 mg/dL, and cell count of the lumbar CSF was 21 ± 7/µL.M. tuberculosis- and T. gondii-specific antibodies and protozoal DNA by nPCR.Clinical and epidemiologic details are provided in Serology : The sensitivity, specificity, and positive and negative predictive values were calculated from the data obtained from groups I and III. For T. gondii-specific IgG on all the three samples tested, these were 100% each; whereas for IgM tested on sera, these were 61%, 100%, and 100% and 71%, respectively.T. gondii-specific IgG was present in all CSF  and sera (18/18) tested. The concentrations of IgG in the ventricular and lumbar CSF and sera ranged from 186 to 360 IU/mL (mean 258 ± 50 IU/mL), 182 to 282 IU/mL (mean 231 ± 36 IU/mL), and 196 to 1016 IU/mL (mean 646 243 IU/mL), respectively. While the concentrations in sera were significantly higher (P < 0.05), there was no difference in the mean concentrations of IgG in the ventricular and lumbar CSF samples (P > 0.05). All the patients except one (case 1) had high-avidity IgG antibodies both in the CSF and sera. IgM antibody to T. gondii were less frequent in CSF  compared to serum .In Group I , T. gondT. gondii nPCR was false negative in lumbar CSF, the pathological lesions in the brain were relatively avascular, organizing abscesses with extensive necrosis, edema and focal hemorrhage located in deep parenchyma, away from the subarachnoid space.The sensitivity, specificity, positive and negative predictive values of B1 nPCR on ventricular CSF were 100% each. However, in lumbar CSF, the sensitivity and the negative predictive values were lower at 76.5% and 81% respectively. In four cases where Mean age of the patients (n=17), in this group was 34 ± 6 years, (M:F=16:1). The patients presented with features of chronic meningitis with headache, vomiting, and neck stiffness. Neuroimaging highlighted enhancing exudates in basal cisterns partially entrapping the vessels with occasional ring-enhancing lesions in basal ganglia . The meaT. gondii was negative in all 4 cases, three had parasite-specific IgG antibodies in CSF (>200 IU/mL) (cases 1–3). In one case (case 4), both CSF serology and nPCR were negative, but IgM and low-avidity IgG antibodies were present in the serum.Among the patients with TBM, histologic evidence of cerebral toxoplasmosis was seen in 4 (cases 1–4). Although nPCR for Of the remaining 13 cases without histologic evidence of cerebral toxoplasmosis, three showed nPCR and high titer IgG antibodies in CSF (cases 5–7), three had high IgG levels alone (cases 9–11), and one had both IgM and IgG in CSF (case 8). There were two other cases in this group in which none of the other parameters showed any evidence of cerebral toxoplasmosis except CSF IgM positivity (cases 12–13); the IgM positivity seen in this case could be labeled as false positive, since it was not accompanied by low-avidity IgG antibodies or nPCR positivity.T. gondii in South Indian healthy voluntary blood donors (20.3%).[The incidence of OI of the CNS, including cerebral toxoplasmosis in the HIV-infected individuals has declined considerably with the introduction of HAART in the developed world. However,9 (20.3%). We evaluT. gondii was reported in 69% of AIDS patients with cerebral toxoplasmosis by ELISA[T. gondii serology was 100% on both sera and CSF, suggesting antibody detection to be a useful diagnostic tool even in the HIV-induced immunosuppressive state.[10T. gondii infection, since it is reported to persist for as long as 2 years after primary infection.[918CSF antitoxoplasma serology appears to be remarkably sensitive for the diagnosis of cerebral toxoplasmosis1213 but 12 by ELISA In the pve state. The mean state.10 In the pnfection. Reactivafection.91819 as sT. gondii DNA in the CSF has a sensitivity varying from 0%[T. gondii parasites into the CSF space. Hence repeated testing on sequentially collected samples is recommended.[PCR for g from 0% to 100%,g from 0%20–24 whig from 0% We foundg from 0%27 or intommended. Initiatiommended. Of the fT. gondii with M. tuberculosis was detected by histology/parasite-specific serology/nPCR in 11/17 (65%) of cases. Among these, four (24%) cases had toxoplasma lesions in the brain. A single case with histologically confirmed TBM and cerebral toxoplasmosis, had IgM and low-avidity IgG antibodies present only in the serum [case 4, Co-infection of  case 4, . This pa case 4,  could beT. gondii-specific IgG antibodies in sera and/or CSF appears to be a sensitive and reliable marker for the diagnosis of cerebral toxoplasmosis even in the presence of TBM. Reinfection/reactivation of dormant T. gondii infection appears to be the norm in association with HIV. nPCR for toxoplasma on lumbar CSF has a limited usefulness but could be a useful adjunct to serology. An important message to the treating clinicians in the developing countries is to consider the possibility of concomitant cerebral toxoplasmosis in patients with TBM, especially in the immunocompromised, as this has therapeutic implications.In summary, estimation of


A variety of methods have been used to study inflammatory changes in the acutely injured spinal cord. Recently novel multiplex assays have been used in an attempt to overcome limitations in numbers of available targets studied in a single experiment. Other technical challenges in developing pre-clinical rodent models to investigate biomarkers in cerebrospinal fluid (CSF) include relatively small volumes of sample and low concentrations of target proteins. The primary objective of this study was to characterize the inflammatory profile present in CSF at a subacute time point in a clinically relevant rodent model of traumatic spinal cord injury (SCI). Our other aim was to test a microarray proteomics platform specifically for this application.A 34 cytokine sandwich ELISA microarray was used to study inflammatory changes in CSF samples taken 12 days post-cervical SCI in adult rats. The difference between the median foreground signal and the median background signal was measured. Bonferroni and Benjamini-Hochburg multiple testing corrections were applied to limit the False Discovery Rate (FDR), and a linear mixed model was used to account for repeated measures in the array.We report a novel subacute SCI biomarker, elevated levels of matrix metalloproteinase-8 protein in CSF, and discuss application of statistical models designed for multiplex testing.Major advantages of this assay over conventional methods include high-throughput format, good sensitivity, and reduced sample consumption. This method can be useful for creating comprehensive inflammatory profiles, and biomarkers can be used in the clinic to assess injury severity and to objectively grade response to therapy. Traumatic injury to the spinal cord can result in life-changing neurological deficits. Patients, family members, clinicians, and researchers alike face significant long-term challenges including (but not limited to) pain management, rehabilitation, and functional improvement. According to 2011 estimates, the incidence of spinal cord injury (SCI) is roughly 12,000 cases per year in the USA, 40% resulting from motor vehicle accidents and 27% result from falls, creating a bimodal age distribution in the patient population .The molecular mechanisms underlying inflammation-induced damage and recovery of function at the acute, subacute and chronic stages after SCI are currently being pieced together. Although trauma to the spinal cord results in rapid loss of neurons and glia within gray and white matter and transection of white matter pathways, this acute insult also initiates a robust inflammatory response -6 that cet al. highlight the potential role for biomarkers in assessing initial injury severity and tracking changes in injury status over time. In addition, they emphasize the role of biomarkers in determining therapeutic response more objectively than current highly subjective functional outcome measures like the American Spinal Injury Association (ASIA) Score [With clinical trials testing the effects of new therapies on neurologic outcomes post-SCI already underway (minocycline and erythropoetin to name a few) , and morA) Score . Other iA) Score . This idet al. simultaneously profiled 10 different cytokine responses to H1N1 infection in hospitalized patients using a cytometric bead array system [et al. were able to track serial changes in 17 cytokines using a similar suspension array in patients with Crohn’s disease treated with Infliximab [et al. studied the prognostic value of 30 cytokines in primary myelofibrosis by multiplex sandwich immunoassay [It is becoming increasingly apparent that inflammation plays key roles in both acute and secondary damage, and late-phase recovery after traumatic SCI, and that any novel therapy would almost certainly interact with these processes. In fact, much can be learned from the Immunology field, where researchers have adopted many of the latest technologies to characterize inflammatory processes previously too complex for traditional methods of investigation. Where PCR and standard ELISA were once popular for detecting changes in mRNA and protein respectively, newer multiplex arrays have emerged as the current assays of choice. Recently Lee y system , and Mizunoassay : one of unoassay .et al. studied two commercially available solution-phase microarray platforms against standard ELISA in detecting autoantibodies in human serum [et al. compare strategies like Benjamini-Hochburg [An effort has also been made to validate the reliability of these new assays, focusing mainly on the issues surrounding transition from a uniplex to multiplex format. Pang an serum . They noHochburg  for estiHochburg . FinallyHochburg  are curret al. showed transient increases in IL-1β, TNF-α, IL-6, and M-CSF mRNA by PCR from cord tissue in a rat model of SCI (C8 contusion) [et al. also showed elevated IL-1β mRNA in contused cord, but extended their analysis by showing elevated protein levels in cord by ELISA. Of note, CSF samples also contained low levels of IL-1β [et al. studied 25 cytokines (as well as a number of growth factors) simultaneously in human CSF samples from acute SCI patients [et al. identified 42 possible biomarkers  in CSF from a rat model 24 hours post-injury by mass spectrometry of which 10 correlated with injury severity (ASIA Score) including Ywhaz, Itih4, and Gpx3 [The challenge of characterizing the inflammatory response to acute SCI has been met using a variety of methods from flow cytometry, to PCR, to ELISA. Streit ntusion) . Wang etof IL-1β . These spatients . Lubieniand Gpx3 .P-values using both Benjamini-Hochburg and Bonferroni methods . To account for technical replicates, as well as variation in regional signal intensity within the array, a linear mixed model was applied, which showed only MMP-8 was significantly elevated in injured CSF, a finding subsequently confirmed by western blot.In an effort to characterize inflammatory markers present at subacute time points after traumatic SCI, thought to represent a clinically relevant ‘window of opportunity’ for therapeutic intervention, we have conducted a proteomics analysis of CSF from rats at 12 days after cervical spinal cord contusion for changes in 34 cytokines using a multiplex sandwich ELISA microarray. Initial statistical analysis yielded seven proteins , and tissue inhibitor of metalloproteinase-1 (TIMP-1)), the levels of which were all increased in the CSF from injured animals compared with normal uninjured controls. The FDR was controlled (limit type I errors) by adjusting the Animal care and experimental protocols were carried out according to the guidelines of the National Institutes of Health as well as the policies of our Institutional Animal Care and Use Committee at the University of Colorado Anschutz Medical Campus. Female Sprague–Dawley rats  aged between 3 months and 5 months were used. Animals were anesthetized with an intraperitoneal injection of ketamine and xylazine, shaved and placed in a stereotactic device for small rodent surgery . Lubricating eye gel was applied to prevent drying under anesthesia. After cleaning the surgical site, a midline incision was made extending from the shoulder blades to the base of the skull. Another midline incision at the origin of the trapezius muscles was made, and the paraspinal muscles were scraped from the spinous processes and lamina of C2 to C6 with a small scalpel blade, exposing the spinal column. Hemostasis was achieved using gauze with light pressure. Once the spinal column was exposed, the posterior longitudinal ligament was cut above and below C4. Rongeurs were used to perform a C4 laminectomy, keeping the dura intact.An Infinite Horizon impactor  with a 1.7 mm tip was used to create a unilateral dorsolateral funiculus contusion of the cervical cord. After laminectomy the animal was secured into the impactor by clamping the vertebral bodies of C3 and C5 using Adson stabilizing forceps. Contusion was made by dropping a weight with a preset force of 75 kdynes on the lateral aspect of the cord at the C4 level with a dwell time set at zero. Both displacement and force were recorded to minimize injury variation. The animal was then returned to the stereotactic device. The trapezius muscles were approximated using simple interrupted stitches (4–0 size vicryl suture). Skin was closed with surgical staples.Animals were again anesthetized at 12 days following contusion and were placed in the stereotactic device. The old incision was extended rostrally between the ears to expose the occipital bone of the skull. Subcutaneous tissue was scraped off the scull with a scalpel, and a small burr hole (from 1 mm to 2 mm diameter) was drilled through the occipital bone, exposing the meninges. A slit was then made in the dura and a small diameter cannula  was carefully guided under the dura  along the internal surface of the occipital bone and into the cisterna magna (depth of 7 mm). A syringe was used to draw approximately 200 μL of CSF from each animal, making sure the sample was not bloody. All grossly bloody taps were excluded from the analysis . Protease inhibitor cocktail  was quickly added and samples were placed on dry ice. Samples were stored at −80°C until further use. Animals were then sacrificed by intrahepatic injection of ketamine and xylazine. Control CSF was obtained from age-matched animals (no contusion) in a similar fashion. Figure n = 4) using the 34 cytokine preconfigured sandwich ELISA Rat Cytokine Array G2 . To reduce batch variability100 μL of each CSF sample  occupied a single array such that only one chip was used for the experiment. CSF samples were not diluted. The chip was read using a GenePix 4000B Microarray Scanner . Of note, the array was arranged such that each antibody was spotted twice, creating two technical replicates per protein of interest. Any remaining CSF was aliquoted and placed back at −80°C. Specific protocol details can be found at the website of RayBiotech Inc [http://www.raybiotech.com). A schematic of the chip format and image of representative control and injured arrays in the Cy3 channel are shown in Figure Samples were removed from −80°C, thawed on ice, and briefly centrifuged. CSF from injured animals was compared with control CSF  from two injured and two control animals were run on a 12.5% polyacrylamide gel , and transferred overnight onto PVDF. Westerns were run in duplicate and probed with a polyclonal antibody to MMP-8 . Blots were also probed for Transferrin , previously shown to be unaffected by SCI , as a loCSF from injured and control animals  were in the same way as MMP-8 western blots (above). Lysate from 293 T cells over-expressing TIMP-1 (Santa Cruz) and rat whole brain lysate (Abcam) were used for positive controls. The blot was cut and probed separately for transferrin and TIMP-1 (Santa Cruz Biotechnology).P-values. IL-1α, Leptin, B7-2/CD86, GM-CSF, IL-1β, MMP-8, and TIMP-1 were all increased in CSF of injured animals compared with controls. Of these results, only IL-1α, MMP-8, and TIMP-1 showed significant increases across both replicate sets after Benjamini-Hochburg adjustment. As expected, there were no statistically significant differences among control datasets (data not shown). Bonferroni adjusted P-values are shown for comparison.Out of 96 probe sets tested (48 spots with replicates), seven proteins showed a statistically significant difference in median signal intensity (background subtracted) in at least one technical replicate (the response). Proteins with significant results at the 5% level are reported in Table P-values are highlighted. In this model, only MMP-8 and Thymus Chemokine-1 showed statistically significant changes based on raw P-values. MMP-8 was elevated in injured animals: the same conclusion as from the probe set level tests (simple model). Thymus Chemokine-1 did not reach significance in the simple model, but showed a statistically significant decrease in the linear mixed model based on the raw P-value; a result that did not hold up after Benjamini-Hochburg adjustment. This discrepancy is the result of significant variability within the Thymus Chemokine-1 dataset combined with large differences in the magnitude of signal between injured and control animals. After Benjamini-Hochburg correction of mixed model data, MMP-8 was the only cytokine with a significant change between injured and non-injured animals (P < 0.0001). Figure n = 8) from the MMP-8 and TIMP-1 datasets in box plot format.To account for technical replicates, as well as regional signal variability across the array as a whole, a linear mixed model was applied to the data. Table To confirm that microarray samples contained a protein with the correct molecular weight for MMP-8, CSF from injured and control animals were run and probed for MMP-8. A probe for Transferrin was used as a loading control. Westerns for both MMP-8 and Transferrin were aligned to create Figure Western blot for TIMP-1 was also performed to test for type II errors in the statistical analysis. Specifically we were interested in testing whether there were hits present in the simple model that did not reach significance in the mixed model. The western blot for TIMP-1 along with Transferrin loading controls are shown in Figure The primary objective of this study was to characterize the inflammatory profile present in CSF at a subacute time point in a clinically relevant rodent model of traumatic SCI. Our other aim was to test a microarray proteomics platform specifically for this application. Studying inflammation at 12 days post-SCI is highly relevant in that it falls within a clinical ‘window of opportunity’ where patients have been stabilized and are generally more receptive to therapy. We also agree with the SCI field in general that CSF is an important source of potential biomarkers given that it is safe to obtain, readily available, and in close proximity to neuronal tissue of interest. Using small volumes of CSF we were able to study 34 cytokines/growth factors simultaneously, detecting significant changes in seven inflammatory markers. After applying statistical models designed for multiplex testing, we also found robust increases in MMP-8 within CSF, which to our knowledge represents a novel subacute phase biomarker for SCI.et al.  were transient, returning to normal levels by 24 hours post-injury, and remaining at these levels for the remainder of their analysis (10 days) [et al. showed elevated IL-1β protein in CSF at one hour [et al. in that protein levels remained elevated at 7 days, but fits nicely with our IL-1β simple model result. Although changes in IL-1β did not hold up to mixed model analysis, it is quite possible that this result is real, and that we are observing a lull in protein concentration between early- and late-phase inflammation. Further analysis is needed to confirm this result. Yang et al. showed elevated levels of IL-1β, IL-6, and TNF-α by western blot from cord lysates, a result that peaked at 6 hours, returned to normal by 24 hours, and was dependent on injury severity [As with any new technology, comparison of results to previous data is crucial in assessing the reliability of the new system. It is generally accepted that a multiphasic inflammatory response occurs after SCI: an early phase peaking at 24 hours and extending from 9 to 10 days, and a later phase between 14 and 180 days post-injury . Studiesone hour . Their rseverity   that these MMPs are released by infiltrating neutrophils responding to inflammation [et al. performed a comprehensive MMP/TIMP mRNA profile in an acute murine model of SCI. They showed an increase in MMP-3, 7, 10, 11, 19, and 20 at 24 hours post-SCI, and elevated levels of MMP-2, 12, and 13, but not MMP-8 at 5 days, colocalizing MMP-12 mRNA to Iba1 positive macrophages in the lesion epicenter [et al. who showed an increase in MMP-8 mRNA following SCI between 3 and 21 days post-injury in mouse cord lysate samples [et al.[et al.[MMPs and their endogenous inhibitors (TIMPs) have been studied extensively in the setting of SCI. de Castro ammation . Wells epicenter .Using mi samples . Increass [et al., as wells [et al.. In the s [et al.. Finallyl.[et al., despitel.[et al. have beeComparing MMP-8 and TIMP-1 signals in both microarray statistical models illustrates the importance of data interpretation in multiplex testing. MMP-8, by far the greatest protein change detected, was significant across both models, suggesting the ability of the array and mixed statistical model combination to correctly identify true changes in the dataset. We would like to reiterate the importance of methods like western blot in verifying protein size when using an ELISA-based system because other immunoreactive proteins may disrupt the analysis.TIMP-1, by contrast, only reached statistical significance in our simple model. Lack of significance in the linear mixed model indicates that this result is more likely to be a type 1 error than a true positive. Western blot was performed to test this hypothesis. Although it is possible that our inability to detect TIMP by western blot reflects the ability of the mixed model to control the FDR, it is also possible that TIMP-1 is truly elevated following SCI and, as discussed above, the microarray was just more sensitive at identifying biologically significant changes in this molecule.Clearly, uncertainty exists when interpreting the results of multiplex testing. Different statistical treatment of the data gives us clues, but follow-up testing is often required by conventional methods. This is especially true when using the Benjamini-Hochburg correction, accepting a certain number of false positives per assay. However, there are ways, within the confines of the multiplex format, to reduce uncertainty. One obvious option would be to increase the sample size of each dataset. If we were to run two chips instead of one would we not increase our power to detect differences in the data? This brings up the concept of batch variability: the observation of significant variation between batches (chips in our experiment). This variability commonly offsets any statistical ‘gain’ from the corresponding increase in sample size. A better alternative would be to run sequential arrays, one for screening, as presented here, and another ‘confirmation array’, where only hits from the screening array are spotted, in greater numbers. This sequential approach, although more expensive and time consuming, avoids the problem of batch variation, and prevents controls for the FDR from making costly type II errors.To our knowledge our study is the first to demonstrate significant increases in MMP-8 protein in CSF after traumatic injury to the spinal cord. We propose that MMP-8 is a novel subacute-phase CSF biomarker for SCI. In addition, our study supports the use of sandwich microarray proteomics as a valid approach for studying CSF in the setting of SCI in rats, and potentially human SCI patients. This assay reliably detects low levels of CSF protein, and has several advantages over conventional methods including quick and easy protocol, high-throughput format, and use of relatively small amounts of precious sample. Disadvantages include relative cost compared with traditional methods, and the need to confirm hits . Moreover our study stresses the importance of controlling the FDR given the multiplex format. In summary, this assay has the potential to detect new target molecules, characterize complex time-dependent pathophysiologic responses to SCI (like inflammation) and, as more therapies reach the clinic, identify biomarkers with which to assess injury severity and grade response to treatment.ASIA, American Spinal Injury Association; BH, Benjamini-Hochburg multiple testing correction; BSCB, blood-spinal cord barrier; CNS, central nervous system; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; FDR, false discovery rate; IL, interleukin; MMP, matrix metalloproteinase; mRNA, messenger RNA; PCR, polymerase chain reaction; SCI, spinal cord injury; TIMP, tissue inhibitor of metalloproteinase; TNF, tumor necrosis factor.The authors declare that they have no competing interests.ML, KM, and SD designed the study. KM conducted all animal injuries and KM and ML performed CSF extractions. ML and KJ performed the microarray and western blot analysis. PD performed all statistical analysis. Manuscript was written by ML, PD, KM and SD. All authors have read and approved the final manuscript.


Amyloid-beta plaque deposition represents a major neuropathological hallmark of Alzheimer’s disease. While numerous studies have described dendritic spine loss in proximity to plaques, much less is known about the kinetics of these processes. In particular, the question as to whether synapse loss precedes or follows plaque formation remains unanswered. To address this question, and to learn more about the underlying kinetics, we simultaneously imaged amyloid plaque deposition and dendritic spine loss by applying two-photon in vivo microscopy through a cranial window in double transgenic APPPS1 mice. As a result, we first observed that the rate of dendritic spine loss in proximity to plaques is the same in both young and aged animals. However, plaque size only increased significantly in the young cohort, indicating that spine loss persists even many months after initial plaque appearance. Tracking the fate of individual spines revealed that net spine loss is caused by increased spine elimination, with the rate of spine formation remaining constant. Imaging of dendritic spines before and during plaque formation demonstrated that spine loss around plaques commences at least 4 weeks after initial plaque formation. In conclusion, spine loss occurs, shortly but with a significant time delay, after the birth of new plaques, and persists in the vicinity of amyloid plaques over many months. These findings hence give further hope to the possibility that there is a therapeutic window between initial amyloid plaque deposition and the onset of structural damage at spines.The online version of this article (doi:10.1007/s00401-012-1047-8) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is the most common form of dementia, which is characterized by two neuropathological hallmarks, namely β-amyloid plaque deposition and intracellular neurofibrillary tangles. The progression of disease pathology is further accompanied by a substantial loss of neurons and synapses. Controversial findings have, however, been reported regarding the relationship of synapse loss and plaque load , 33. ForKM670/671NL and PS1L166P mutations [APPPS1 mice are double transgenic for APPutations . We wantutations  (B6.Cg-T−1 body weight). Additionally, dexamethasone (0.02 ml at 4 mg ml−1) was intraperitoneally administered immediately before surgery [−1). Imaging began following a 21-day recovery period after surgery.A cranial window over the right cortical hemisphere was surgically implanted as previously described , 17. The surgery . A circuz-stacks of 230 × 230 × 150 μm3 with 2 μm z-resolution and 1024 × 1024 pixels per image frame (0.22 μm/pixel) were taken with a 40× IR-Achroplan water immersion objective  to analyze amyloid plaques. For higher-resolution images to count dendritic spines, the same objective was used with 1 μm z-resolution and 512 × 512 pixels per image frame (0.11 μm/pixel). The high-resolution (2048 × 2048 pixels with 0.14 μm/pixel) and large volume (283 × 283 × 300 μm3) images with 2 μm z-resolution to monitor dendritic spines before and after plaque deposition were taken with a 20× W Plan-Apochromat water immersion objective .Long-term two-photon in vivo imaging was performed as previously described , 18, 29.z-stacks by manually scrolling through the images of subsequent time points of the same position. The spine scoring method has previously been described [t test. Multiple group comparison was done by one-way ANOVA followed by Tukey–Kramer post hoc test. The slope from a linear regression was tested for statistical difference from zero by F test. Distributions of morphological subtypes of dendritic spines were tested for differences by Chi-square test. All statistical analysis and graphs were done using Prism 5.04 . Figures were arranged using Adobe Illustrator CS4.All images were deconvolved using the adaptive blind 3D deconvolution algorithm of AutoDeblur with ten iterations . The images were maximum intensity projected . In some figures, distracting neighboring dendritic elements were removed. Spines were counted in escribed , 18, 25.escribed . Filopodescribed . The volescribed . All dat−1 control, 0.511 ± 0.075 μm−1 > 50 μm from plaque, 0.264 ± 0.092 μm−1 < 50 μm from plaque, p < 0.05; Fig. −1 control, 0.466 ± 0.034 μm−1 > 50 μm from plaque, 0.238 ± 0.086 μm−1 < 50 μm from plaque, p < 0.01, one-way ANOVA with Tukey–Kramer post hoc test, errors represent SD). The dendritic spine density in the vicinity of amyloid plaques did not significantly differ between age groups . It is important to mention that we were actively looking for spine-bearing dendrites to analyze in the surrounding volume of amyloid plaques, which were subjectively more difficult to find, yet still present, in the older cohort. During the same time interval, plaque size increased by almost threefold, from a mean radius of 5.413 μm at 3 months to 13.410 μm at 18 months .In the present study, we used the APPPS1 mouse model, which co-expresses human amyloid precursor protein with the Swedish mutation (KM 670/671 NL) and L166P mutated presenilin 1 . In thesp < 0.001, repeated measures ANOVA with Tukey–Kramer post hoc test). Over the same time period, no spine loss was detected >50 μm distant from plaques or in wild-type animals having no plaques at all. Notably, spine loss kinetics were not significantly different between both age groups . Long-term in vivo imaging makes it possible to follow individual spines over time and to determine the fraction of lost and gained spines. Consequently, we could identify the factor responsible for a net decrease in spine density. Such an analysis revealed that the overall decline in spine density could be attributed to a relative increase in the fraction of lost spines over 4 weeks. The fraction of gained spines, on the other hand, remained the same when compared to areas distant to plaques or in control animals in both age groups . In other words, the observed loss of spines at pre-existing plaques is caused by an increase in spine elimination rather than by a malfunction in the process to form new spines.After this single time-point analysis, we focused on the kinetics of amyloid plaque growth and spine loss over 4 weeks. Therefore, the spine density at mature plaques and at nascent plaques was analyzed in young and old cohorts. Within the peri-plaque region (<50 μm around plaques) we found a significant reduction in spine density in both young and aged mice . In contrast, plaque radius in aged mice remained unchanged . Lack of plaque growth is supported by a similar finding in aged Tg2576 mice, another mouse model of AD [Plaque growth was quantified by converting the cubic root from volumes in 3D images to yield a linear measure, analogous to the radius . In the l et al.   were imaged over a period of 3 months. In these large volumes of high resolution, we were able to detect the exceptional event of plaque birth in close proximity to dendrites seven times over. Imaging was started at 2–3 months of age when few amyloid plaques were present, with new plaques expected to form during the following months [n = 247 dendritic spines). A significant reduction in spine density did however occur 4.5 weeks after initial amyloid plaque formation . Over the same time period, plaque volume increased significantly immediately after the plaque’s appearance . Thus, there seems to be a latency of about 4 weeks between plaque formation and the onset of dendritic spine elimination in the vicinity of plaques.Finally, we analyzed the kinetics of individual dendritic spines before and during the de novo formation of new plaques. The observation of plaque birth is a very rare event , 41, 61 g months . To the We co-investigated and correlated amyloid plaque growth with dendritic spine kinetics in APPPS1 mice by long-term two-photon in vivo imaging. Several studies previously reported dendritic spine loss around amyloid plaques in a range of different mouse models and brain regions by applying both ex vivo and in vivo approaches , 54, 59.−1, Fig. −1). Hefendehl et al. imaged mice up to an age of 10 months and observed no decline in plaque growth kinetics by that age. However, when we extended the observation period to an age of 18–19 months, we found that plaque growth declined essentially to zero  compared to areas farther away from plaques and in age-matched control animals. Interestingly, similar results were found for both age cohorts. Thus, when monitoring the relative spine density over 4 weeks at already-existing plaques, we found a reduction to 57.9 ± 28.3 % in young mice and 67.8 ± 20.8 % in aged mice , fraction of lost spines (b), and spine density kinetics (c) at 3 and 18 months of age for dendrites less than 50 µm away from amyloid plaques. Error bars indicate SD for a and b and SEM for c. d 3D volume rendered view from the two-photon in vivo image from figure 5a. YFP-labeled dendrites are shown in green and methoxy-X04 stained amyloid plaques in blue. The xz- and yz-view highlights that the investigated dendrite from figure 5a does not directly pass through the plaque. The black rectangle marks the picture detail from figure 5a and the scale represent 10 µmSupplementary material 1 (TIFF 1762 kb) Direct comparison of spine densities (Below is the link to the electronic supplementary material.


Hyperglycemia and insulin resistance are well-known features of critical illness and impact the mortality rate, especially in sepsis. Retinol binding protein 4 (RBP4) promotes insulin resistance in mice and is systemically elevated in patients with obesity and type 2 diabetes. We investigated the potential role of RBP4 in critically ill patients.We conducted a prospective single-center study of serum RBP4 concentrations in critically ill patients. One hundred twenty-three patients  were studied at admission to a medical intensive care unit (ICU) before initiation of specific intensive care treatment measures and compared to 42 healthy nondiabetic controls. Clinical data, various laboratory parameters and metabolic and endocrine functions were assessed. Patients were followed for approximately 3 years.P < 0.001). Patients with liver cirrhosis as the primary underlying diagnosis for ICU admission had significantly lower RBP4 levels as compared with other ICU patients. Accordingly, in all ICU patients, serum RBP4 closely correlated with liver function and increased with renal failure. No significant differences of serum RBP4 concentrations in septic patients with pulmonary or other origins of sepsis or nonseptic patients could be revealed. Acute phase proteins were inversely correlated with RBP4 in sepsis patients. RBP4 did not differ between patients with or without obesity or preexisting diabetes. However, serum RBP4 levels correlated with endogenous insulin secretion (C-peptide) and insulin resistance (HOMA index). Low serum RBP4 upon admission was an adverse predictor of short-term survival in the ICU, but was not associated with overall survival during long-term follow-up.Serum RBP4 was significantly reduced in ICU patients, independently of sepsis, as compared to healthy controls (Serum RBP4 concentrations are significantly reduced in critically ill patients. The strong associations with hepatic and renal function, insulin resistance and acute mortality collectively suggest a role of RBP4 in the pathogenesis of critical illness, possibly as a negative acute phase reactant, and allow a proposition as a potential novel biomarker for ICU patients. Hyperglycemia, glucose intolerance and insulin resistance are common features of critically ill patients, especially in patients with sepsis or septic shock and even in those without a history of diabetes -3. MaintRetinol binding protein 4 (RBP4), a 21-kDa protein synthesized in the liver and adipose tissue, has recently been described as an adipokine involved in the development of insulin resistance in mice and humans ,10. In tRecently, low levels of serum RBP4 have been reported in critical ill patients with sepsis of pulmonary origin compared to nonseptic patients . HoweverOur study investigated serum RBP4 concentrations in a large cohort of "untouched," treatment-naive critically ill patients (septic and nonseptic patients) from a medical ICU at the moment of admission to the ICU. We aimed at understanding the potential involvement of RBP4 in the pathogenesis of insulin resistance in critical illness, its regulation in severe systemic inflammation and its potential clinical use as a biomarker in ICU patients. We demonstrate that serum RBP4 levels were significantly reduced in ICU patients as compared to controls, independent of etiology of critical illness or origin of sepsis (pulmonary versus nonpulmonary), and they were closely related to liver and kidney function. The dysregulation of serum RBP4 in ICU patients was not associated with preexisting diabetes or obesity, but might contribute to the insulin-resistant state of the critically ill patients. High RBP4 levels were indicative of a beneficial short-term course of disease, but did not predict the overall survival of the patients in a 3-year follow-up period.sepsis, according to the definitions in current guidelines [nonsepsis. Sepsis patients were subdivided into cohorts of sepsis of pulmonary and nonpulmonary origin on the basis of the clinical and radiological classification performed upon admission.From the medical ICU at the University Hospital Aachen, Germany, a tertiary care university hospital, patients were prospectively enrolled at the time of ICU admission. Patients who were expected to stay < 72 hours at the ICU  were not asked to participate in this study . Writtenidelines , or as bForty-two healthy, nondiabetic blood donors  who had normal blood counts, normal liver enzymes and normal C-reactive protein levels served as controls.The ICU patients were compared by age, sex and severity of illness using the score on the Acute Physiology and Chronic Health Evaluation (APACHE) II obtained at admission and the simplified acute physiology score (SAPS) 2 . LaboratPeripheral venous blood samples were obtained at admission before therapeutic intervention, immediately placed on ice, centrifuged and stored at -80°C. All measurements were performed in a blinded fashion by the same investigator. RBP4 serum concentrations were measured using an enzyme-linked immunosorbent assay according to the manufacturer's instructions  . Serum rU test or between more than two groups by Kruskal-Wallis analysis of variance and the Mann-Whitney U test for post hoc analysis [P < 0.05 were considered statistically significant. The prognostic value of the variables was tested by univariate and multivariate analyses in the Cox regression model. After significant results from the uni- and multivariate Cox regression analyses, Kaplan-Meier curves and log-rank test calculations were performed subsequently for different cutoff values for RBP4 . The threshold of 12 mg/L RBP4 yielded the highest log-rank values. Kaplan-Meier curves were plotted to display the impact on survival [Analyses were performed with SPSS 12.0 software . Owing to skewed distributions of most variables, the median and range are given. Differences between two groups were assessed by the Mann-Whitney analysis . Comparisurvival .P < 0.001; Figure The median serum concentration of RBP4 in healthy controls was 28.1 mg/L  as anticipated from previous studies using the same assay ,18. In on = 85) with patients without sepsis (n = 38). In the sepsis group, the focus of infection was either pulmonary (n = 45), abdominal (n = 19) or other . In the nonsepsis group, the etiology of critical illness was decompensated liver cirrhosis (n = 11), cardiopulmonary (n = 16) or other diseases (n = 11). Interestingly, serum RBP4 did not differ significantly between patients with sepsis (median 15.2 mg/L) and without sepsis , pseudocholinesterase activity , prothrombin time  and inversely with bilirubin . Of note, RBP4 serum concentrations remained significantly  elevated in controls  compared to ICU patients without cirrhosis .Interestingly, ICU patients with liver cirrhosis displayed the lowest RBP4 serum levels among all subgroups Figure . Liver fr = 0.493, P < 0.001; Figure r = 0.389, P < 0.001), cystatin C  and decreasing glomerular filtration rate .Serum RBP4 concentrations were also correlated with markers of renal failure, specifically increasing creatinine  or interleukin-6 levels . RBP4 thereby displayed characteristics of a negative acute phase reactant [Although there was no significant difference between septic and nonseptic patients, systemic inflammatory markers were associated with serum RBP4 concentrations. Classical biomarkers of systemic inflammatory responses were inversely associated with serum RBP4, such as C-reactive protein (reactant . Howevern = 42) did not differ from patients without diabetes  did not show higher serum RBP4 than patients without severe obesity . By multivariate Cox regression analysis, the effect of RBP4 on ICU survival was independent of age, BMI, renal function  and CRP, but indistinguishable from liver function. Two models were tested in multivariate Cox regression analyses, using ICU outcome  as the endpoint. The first model included RBP4, age, BMI, creatinine, GFR (cystatin C-based) and CRP, and RBP4 was the only parameter that remained independently significant (P = 0.041) to predict ICU outcome in this combination. The second model combined RBP4 and markers of hepatic dysfunction . In this model, pseudocholinesterase remained the only independently significant (P < 0.001) parameter predicting ICU survival. If all parameters were combined, pseudocholinesterase was the dominant predictor for ICU outcome.We next analyzed whether serum RBP4 concentrations upon ICU admission may predict clinical outcome and mortality. Interestingly, patients who died during ICU hospitalization (33/123) displayed significantly lower serum RBP4 concentrations than the patients who were surviving Figure . The benP = 0.0009). On the other hand, serum RBP4 levels did not predict the overall survival of the ICU patients in the approximately 3-year follow-up . The source of circulating RBP4 in critically ill patients appears to be primarily a combination of two factors: hepatic synthesis and renal clearance.It is well established that the liver is the main source of RBP4 in humans ,12, althIn addition, it is well established that serum RBP4 is increased in patients with chronic renal failure, making renal clearance a relevant confounding factor for serum RBP4 concentrations in patients with diabetes or renal disease ,29. As aOur results further indicate that the elevated serum RBP4 might be involved in the development of insulin resistance in ICU patients. Hyperglycemia and insulin resistance are well-known features of critical illness and affect the mortality rate, especially in septic patients ,30,31. PThe reduction of serum RBP4 in critically ill patients compared to controls was also of direct prognostic relevance. Our data revealed an association between low RBP4 and higher short-term mortality on ICU, whereas long-term survival was not affected. The underlying pathomechanisms are currently unclear. One explanation could be that this is a simple epiphenomenon as high(er) RBP4 levels may indicate preserved liver function; along this line, high "classical" biomarkers of liver function are also associated with a beneficial outcome (data not shown), and the predictive value of serum RBP4 for ICU survival was statistically indistinguishable from liver function in multivariate Cox regression analysis.On the other hand, other explanations should be considered. Interestingly, serum RBP4 levels were negatively correlated with markers of inflammation. This raises the possibility that RBP4 could be an important component of the so-called anti-acute phase proteins , at leasOur study has demonstrated reduced serum RBP4 concentrations in critically ill patients prior to intensive care treatment measures. RBP4 serum levels at ICU admission appeared independent of underlying disease etiology, but were strongly associated with hepatic and renal function. The correlation with insulin resistance and the relation with the acute mortality collectively suggest that RBP4 may have implications for the pathogenesis of critical illness and could serve a novel biomarker for ICU patients. Future studies should consider including RBP4 as a factor for multivariate analyses and aim to unravel the kinetics of RBP4 levels in the time course of critical illness.• Retinol-binding protein 4 (RBP4) has been suggested to contribute to insulin resistance, a common characteristic of critically ill patients with implications for adverse outcome.• RBP4 serum levels are significantly reduced in critically ill patients as compared to healthy controls.• RBP4 serum levels at ICU admission prior to intensive care measures appeared independent of underlying disease etiology.• Serum RBP4 concentrations are closely related to liver and kidney function and correlate with endogenous insulin secretion and resistance.• The negative association of serum RBP4 with inflammatory markers suggests a potential role in the anti-acute phase response in critical illness.• Low RBP4 levels are an adverse predictor of short-term mortality at the ICU.P: P value; PCHE: pseudocholinesterase; r: correlation coefficient; RBP4: retinol binding protein 4; SAPS: Simplified Acute Physiology Score; SIRS: systemic inflammatory response syndrome.APACHE II: Acute Physiology and Chronic Health Evaluation; BMI: body mass index; CRP: C-reactive protein; ELISA: enzyme-linked immunosorbent assay; GFR: glomerular filtration rate; HOMA-IR: homeostasis model assessment index of insulin resistance; ICU: Intensive Care Unit; The authors declare that they have no competing interests.AK, FT and CT designed the study, analyzed data and wrote the manuscript, RW performed RBP4 and adipokine measurements, and ES, HZ, HD and SV collected data and assisted in patient recruitment.


Cerebrospinal fluid shunt infection can be recalcitrant. Recurrence is common despite appropriate therapy for the pathogens identified by culture. Improved diagnostic and therapeutic approaches are required, and culture-independent molecular approaches to cerebrospinal fluid shunt infections have not been described.To identify the bacteria and fungi present in cerebrospinal fluid from children with cerebrospinal fluid shunt infection using a high-throughput sequencing approach, and to compare those results to those from negative controls and conventional culture.This descriptive study included eight children ≤18 years old undergoing treatment for culture-identified cerebrospinal fluid shunt infection. After routine aerobic culture of each cerebrospinal fluid sample, deoxyribonucleic acid (DNA) extraction was followed by amplification of the bacterial 16S rRNA gene and the fungal ITS DNA region tag-encoded FLX-Titanium amplicon pyrosequencing and microbial phylogenetic analysis.The microbiota analyses for the initial cerebrospinal fluid samples from all eight infections identified a variety of bacteria and fungi, many of which did not grow in conventional culture. Detection by conventional culture did not predict the relative abundance of an organism by pyrosequencing, but in all cases, at least one bacterial taxon was detected by both conventional culture and pyrosequencing. Individual bacterial species fluctuated in relative abundance but remained above the limits of detection during infection treatment.Numerous bacterial and fungal organisms were detected in these cerebrospinal fluid shunt infections, even during and after treatment, indicating diverse and recalcitrant shunt microbiota. In evaluating cerebrospinal fluid shunt infection, fungal and anaerobic bacterial cultures should be considered in addition to aerobic bacterial cultures, and culture-independent approaches offer a promising alternative diagnostic approach. More effective treatment of cerebrospinal fluid shunt infections is needed to reduce unacceptably high rates of reinfection, and this work suggests that one effective strategy may be reduction of the diverse microbiota present in infection. Cerebrospinal fluid (CSF) shunt placement has been the mainstay of treatment for hydrocephalus for over 50 years. For children with first CSF shunt infection, prognosis for clearance is poor, with re-infection rates ranging from 12–26 percent. in vitro exist in a largely dormant, antibiotic-resistant mode, This recalcitrance of CSF shunt infections to treatment may be related to complex, adherent assemblages of microbes, known as biofilms, on the shunt catheter surface. Given the recalcitrant nature of CSF shunt infection, the emerging evidence of a role for biofilms, and the increased application of culture-independent molecular approaches to medical microbiology, we sought to better characterize the diversity and natural history of the microbial communities  in CSF shunt infections using molecular approaches. The study received Institutional Review Board approval from the Seattle Children’s Research Institute and the University of Utah, as well as approval from the Primary Children’s Medical Center (PCMC) Privacy Board. For all study subjects, except those from PCMC prior to March 18, 2010, consent was obtained for additional CSF to be collected on each occasion that regular CSF samples were obtained during treatment for CSF shunt infection. Prior to March 18, 2010 at PCMC, we used CSF remaining for after routine processing and testing in the PCMC Microbiology Laboratory.Children ≤18 years old undergoing treatment for conventional culture-confirmed CSF shunt infection at either Seattle Children’s Hospital (SCH) or PCMC were eligible for enrollment in this study. A CSF shunt infection was defined as identification of organisms on microbiological culture of CSF fluid obtained from a partial or complete CSF shunt system. CSF shunt system(s) included ventriculoperitoneal, ventriculoatrial, ventriculopleural, arachnoid cyst shunts, subdural shunts, and lumboperitoneal shunts; temporary devices only such as external ventricular drain(s), Ommaya reservoir(s), ventricular access devices (reservoirs) and subgaleal shunts were excluded. Negative controls included phosphate buffered saline (PBS) and five sets of both stock CSF from donors without CSF shunts. Donors included adult research participants with brain tumors, and commercially available sterile CSF from adults. Negative controls underwent concurrent deoxyribonucleic (DNA) extraction along with either one or two infection episode samples, followed by subsequent testing as described below.Sterile conditions were standard practice throughout recovery and storage of CSF. The first CSF sample for diagnosis of infection was usually obtained from needle aspiration of the shunt reservoir under sterile conditions outside the operating room in a bedside “shunt tap”. The initial CSF sample analyzed in this study either was leftover from this first diagnostic sample or was obtained in the operating room under sterile conditions from the system being removed during the first surgery to treat infection. Subsequent CSF samples, including those at the end of the infection, were generally obtained under sterile bedside conditions through a sampling port within sterile extension tubing attached to the external ventricular drain.After CSF was obtained for the study, samples were stored at 4°C for up to a week. CSF was then aliquoted into vials of ∼100 µl for the study and stored at −70°C; PCMC samples were shipped overnight to Seattle on dry ice for analysis. After December 9, 2011, 200 µl of CSF was stored in 450 µl MO-BIO CB1 solution at the time of sample collection.All CSF samples were tested using routine aerobic culture techniques in hospital-certified laboratories at both SCH and PCMC. Conventional cultures are the traditional diagnostic approach used to detect typical pathogens in infectious diseases and were performed in a clinical microbiology laboratory following Clinical and Laboratory Standards Institute guidelines; however they do not detect all bacteria present in human disease For each CSF sample, bacterial DNA was extracted from either 100 µl of CSF or 200 µl of 650 µl of CSF/buffer using the BiOstic Bacteremia DNA isolation kit (MO-BIO). DNA was eluted in 100 µl of buffer. At a minimum, the initial CSF sample from the infection episode was analyzed, and in one instance the entire infection course was also analyzed.Partial ribosomal amplification was performed as previously described.  CCC TGT GTG CCT TGG CAG TCT CAG TCC TCC GCT TAT TGA TAT GC -3′).The fungal primers ITS1F, 5′- CTT GGT CAT TTA GAG GAA GTA A-3′ and ITS4R, 5′- TCC TCC GCT TAT TGA TAT GC-3′ were used for amplifying a 500 bp region of the fungal internal transcribed spacer (ITS) DNA sequence. The primer sets used for FLX-Titanium amplicon pyrosequencing included the addition of linker A and 8 base pair subject-specific barcode sequence at the 5′ end of forward primer ITS1F-A (5′-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-barcode- CTT GGT CAT TTA GAG GAA GTA A-3′) and the biotin and linker B sequence at the 5′ end of reverse primer ITS4R-B  was used for polymerase chain reaction (PCR) under the following conditions: 95°C for 5 min followed by 35 cycles of 95°C for 30 s; 54°C for 40 s and 72°C for 1 min, a final elongation step at 72°C for 10 min was also included. The PCR products were cleaned by using Diffinity Rapid Tip  and pooled. The small fragments were removed by Agencourt Ampure Beads .Tag-encoded FLX-Titanium amplicon pyrosequencing (TEFAP) was performed as described previously. Pseudomonas aeruginosa PAO1 (ATCC number: BAA-47) was grown in tryptic soy broth  medium at 37°C with shaking for 16 hours. An aliquot was serially diluted in TSB broth. The diluted bacteria were plated out by using a Whitley automatic spiral plater , which was used to count colony-forming units (cfu). The bacteria broth was further diluted from 1010 to 104 cfu ml−1. Total DNA extractions were performed using 1 ml of each diluted samples.5′-CCA TGA AGT CGG AAT CGC TAG-3′; 16S-R, 5′-GCT TGA CGG GCG GTG T-3′; 16S-Probe, 5′-6-FAM-TAC AAG GCC CGG GAA CGT ATT CAC CG-3′. Roche LightCycler 480 Probes Master was also used for the assay. The PCR reactions were as follows: 50°C for 2 min, 95°C for 10 min, and 35 cycles of 95°C for 15 sec and 60°C for 1 min. The DNAs extracted from the series dilutions were used to set up a standard curve, the Ct values of the quantitative real-time PCR assay (qPCR) assay are compared with the standard curve. The qPCR assay using probe and primers showed that a concentration as low as 106 cfu/ml can be detected on the DNA samples.The CSF samples’ DNAs were detected using a Roche LightCycler 480. The specific primers and probe for bacteria 16S rRNA gene were as follows: 16S-F, The proportion of individual bacterial species detected in each initial CSF sample was summarized descriptively using graphical displays. Bacterial species that comprised less than 1% of reads within a sample were grouped together as “other”. Individual species or taxa identified by sequencing were compared with findings from bacterial culture. Changes in proportion of bacterial species for a single subject over the course of CSF shunt infection treatment were tabulated for species or taxa that comprised over 2% of sequenced reads; this threshold was selected for ease of interpretation.Paenibacillus species, Bacillus species, and Methylobacterium jeotgali. bTEFAP of several of the samples  identified a large variety of additional bacterial species, each generally at relatively low abundance, and many of which are not considered typical CSF shunt infection pathogens.Bacterial species identified by 16S rRNA bTEFAP in the initial samples from the CSF shunt infections in eight different children are shown in Bacterial culture results are compared to 16S bTEFAP findings in Fungi were also identified by ITS fTEFAP at the time of initial CSF samples for the same eight CSF shunt infections Table 2.S. epidermidis, which was identified by bTEFAP along with a substantial proportion of Paenibacillus species. Bacterial quantitation by qPCR of the samples detected 1.15×107 CFU/ml equivalents on day 2, followed by no detectable bacterial DNA above the assay’s limits of detection  thereafter. After day 2, bTEFAP identified a large variety of other species at relatively low abundances throughout the course of infection treatment. bTEFAP did not identify any 16S reads among CSF and PBS negative controls. The proportions of some bacteria fluctuated, such as Streptococcus species; others, such as Bacillus and Pseudomonas species, which are not targets of antimicrobial therapy, even rose over the course of infection treatment. Intravenous antibiotics that were used in this infection and that may have driven the observed microbial abundance changes are shown in Finally, the proportion of bacterial species detected by bTEFAP over the course of CSF shunt infection A is shown in To our knowledge, this is the first study that describes the microbiota present in CSF shunt infection. Using 16S and ITS TEFAP, we have demonstrated that numerous bacterial species, including anaerobes, as well as fungal organisms were detected in the CSF of children with CSF shunt infections. With the exception of fungal counts in two samples, these organisms were detected at TEFAP counts well above those in negative controls. The organisms recovered by TEFAP, including many beyond those recovered by standard aerobic bacterial culture, suggest heterogeneous microbiota. The composition of the microbiota changed over the course of infection treatment, probably in response to antibiotic use and surgical removal of the shunt. Future studies of a larger patient population will be needed to determine the relationships between the diverse CSF microbiota and clinical outcomes, particularly the response to treatment and occurrence of reinfection.Paenibacillus species, Bacillus species, and Methylobacterium jeotgali. Paenibacillus species are particularly intriguing in this context, as they have been reported to have biomedical applications. Bacterial organisms that comprised substantial proportions of the 16S TEFAP findings included Staphylococcus aureus; however, the proportion or burden of S. aureus at the time of shunt replacement is unknown. Study of additional, longitudinal samples from children who have developed serial CSF shunt infections is needed to determine if bacterial species identified at the first infection are related to those seen at the second infection. Further investigations such as these will hopefully inform more effective CSF shunt infection treatment.16S bTEFAP analysis suggested that CSF bacterial microbiota changed over the course of infection treatment. The results in There are several limitations to this work. The selection of appropriate negative controls is difficult. Since the negative controls we selected were from adults, they may not be physiologically comparable to the infection samples. While we considered the use of CSF from children with shunt obstruction without clinical evidence of infection, we were concerned about the potential role of microbial biofilms in this clinical scenario and felt it warrants a separate, independent line of inquiry. CSF from children with functioning shunts may be an ideal negative control, but challenging to obtain. Moreover, while the ideal negative control would be CSF from uninfected children whose shunts have been in-situ for a similar period to the infected cases, this would not be ethically feasible since sampling of CSF from the shunt introduces risk of infection. We anticipate future work examining bacteria and fungal sequences will need to incorporate negative controls from several sources, including but not limited to: donor CSF, children undergoing shunt revision surgery for shunt failure, and children undergoing initial CSF shunt placement.In addition, there are several opportunities for contamination of CSF samples during recovery, storage, and experimentation. However, sterile conditions were sought throughout recovery and storage of CSF samples, and contamination during experimentation is improbable given low recovery from negative controls that were handled identically to and concurrently with the CSF samples. Therefore, contamination is unlikely to have a major contribution to the bacterial and fungal diversity observed. The low numbers of infection episodes analyzed renders this study preliminary in nature. Testing from the hospital-certified laboratories was limited to routine aerobic cultures, practices that are subject to individual laboratory practices, including policies affecting identification of all organisms on a plate, incubation for extended periods of time, and enrichment for slow growing and fastidious organisms. We did not have information about variability or density of colonies on the original plates. In addition, fungal and anaerobic cultures were not obtained. Finally, detection of microbial DNA, which could be from either viable or nonviable cells, by PCR does not guarantee that living bacteria or fungi were present at the time of sample collection. However, the differences observed between infection and control samples indicates that these microbes were present at some point up to collection.While the clinical importance of these CSF microbiota remains to be determined, the current findings provide additional support to the emerging evidence of a role for biofilms in CSF shunt infection. Biofilms are often comprised of multiple microbial taxa held together by an extracellular matrix, Numerous bacterial and fungal organisms appear to be present in the microbiota that comprises CSF shunt infection. In evaluating CSF shunt infection, fungal and anaerobic bacterial cultures should be considered in addition to aerobic bacterial cultures, and culture-independent approaches may prove to be useful as a primary or adjunctive diagnostic modality. Furthermore, better understanding of the diverse microbial environment in active CSF shunt infection is needed. The persistence of these microbiota despite antibiotics may suggest the presence of biofilm. More effective treatment of CSF shunt infection is badly needed to reduce unacceptably high rates of reinfection, and this early work suggests that one effective target may be the reduction of the diverse microbiota present in infection. Future work must focus on defining the relationships between the diverse CSF microbiota found in this study with clinical outcomes, including response to treatment and reinfection; as well as on attainment of optimal negative control subjects.


Cerebrospinal fluid absorption (CSF) at the cribriform plate is mediated by direct extracranial connections to the lymphatic system. Given the accessibility of these pharmacologically responsive vessels we hypothesized that the rate of CSF outflow can be modulated via the intranasal delivery of drugs known to affect lymphatic contractile activity.2O, r = 0.42, p = 0.0058). Specifically, the delivery of NG-monomethyl L-arginine (L-NMMA) significantly increased CSF absorption by 2.29 fold over no treatment (2.29 ± 0.34 mL/min), while the thromboxane A2 analogue U46619 resulted in a 2.44 fold increase in CSF absorption over no treatment (2.44 ± 0.55 mL/min). Saline delivery did not significantly increase CSF absorption (0.88 ± 0.097 mL/min). A trend of increased CSF absorption upon noradrenaline delivery was observed: however, this did not reach statistical significance. Increasing drug concentrations inversely correlated with CSF outflow resistance across all drug classes .Fluid was infused into the lateral ventricle of anesthetized sheep and inflow rate and CSF pressure measured during intranasal administration of pharmacological agents. CSF absorption was calculated at steady-state CSF pressures. The ability of a pharmacological agent to alter CSF absorption was related to the steady-state intracranial pressure (ICP), the concentration and the class of pharmacological agent delivered. An increase in drug concentration correlated with an increase in CSF absorption at high ICP (45 cm HThe administration of nebulized pharmacological agents intranasally has the potential to provide an alternate method to non-invasively modulate CSF absorption and outflow resistance. The regulation of intracranial homeostasis is strictly controlled due to the finite space offered by the fixed volume of the skull. As defined by the Monroe-Kellie hypothesis, tight volumetric regulation of the cerebrospinal fluid (CSF), blood and tissue spaces is critical in maintaining fluid balance; deflections in any of these volumes outside of the autoregulatory capacity of the brain can result in cerebral edema, high intracranial pressures, and compromised cerebral perfusion. The primary mechanism to control intracranial pressure (ICP) is via regulation of CSF production and absorption within the ventricular and subarachnoid spaces . Strong -6 M to α-adrenergic receptors can increase phasic contractions and increase flow on quiescent vessels, however NA stimulation of vessels that display spontaneous rhythmic activity results in reduced contraction force and outflow [3) and augment smooth muscle contractions in a concentration-dependent fashion; higher concentrations lead to vessel spasm and attenuate flow [Given the direct connection between the CSF compartment and the pharmacologically responsive lymphatic vasculature within the turbinate region, the potential exists to modulate lymphatic pumping and thus CSF absorption. Potential drugs that have been found to modulate lymphatic contractile activity include noradrenaline (NA). Indeed, NA binding at <10 outflow -12. Simiate flow -17. In cate flow .Utilizing these pharmacological agents, the aim of these experiments was to test whether the intranasal delivery of these drugs can alter CSF absorption. We have found that indeed this is the case and provide a proof-of-concept for the possibility to influence CSF absorption in a non-invasive fashion.After at least 48 hours for acclimatization, sheep (15–45 kg) were fasted 12 hours prior to surgery. All sheep experiments were acute followed by euthanasia with an IV injection of sodium pentobarbital into the catheterized cephalic vein using a dose of 1 mL/5 kg of Euthanyl Forte. Thirty-six animals were used in the study with 22 included in subsequent data analysis; 14 were excluded due to surgical and/or technical issues. Experiments were approved by the ethics committee at Sunnybrook and Women’s College Health Sciences Centre and conformed to the guidelines set by the Canadian Council on Animal Care and the Animals for Research Act of Ontario.Sodium pentothal (15–25 mg/kg) was given intravenously to induce anesthesia. Sheep were set in a ventral position, intubated, ventilated and surgical anesthesia was maintained with 2-3% Isoflurane/oxygen. An 18–20 Ga catheter was placed in the cephalic vein for venous pressure measurements. An IV line provided hydration using lactated Ringers solution at 5 mL/kg/hr. The opposite cephalic vein was connected to a fluid-filled transducer to record venous pressure. Midsagittal and coronal incisions were made in the scalp to expose the coronal suture and the scalp and posterium pulled aside. A hole was drilled in the skull lateral to the sagittal suture and at approximately 10 degrees to the sagittal plane. Two 18 or 20 Ga angiocatheters were inserted through the hole into each lateral ventricle of the brain and sealed in place.Using a modified protocol described in , a reser2O and 45 cmH2O. All analysis utilized raw outflow numbers derived from the linear regression equations.Using the linear equations derived from each single experiment, we calculated theoretical CSF outflow rates at preset intracranial pressures of 25 cmHAt steady-state pressure, ventricular inflow rate equals CSF outflow rate. From the regression analysis of steady-state ICP versus flow, the CSF outflow resistance can be calculated from the slope of the line. All analysis utilized slopes derived from the linear regression equations.t-test and Pearson Correlation test. Results were deemed statistically significant at p ≤ 0.05.The data was analyzed using repeated measures ANOVA, post-hoc Dunnett’s, Students paired Given the direct connection between the CSF compartment and the extracranial lymphatics within the nasal turbinate region, we hypothesized that the nebulization of pharmacological agents into the nasal cavity may increase CSF absorption Figure . While t2O. In contrast, traumatic brain injury or intracranial hemorrhage have been found to result in ICP reaching above 40 cmH2O, and aggressive interventions are typically initiated when pressures exceed 30 cmH2O [2O and also by extrapolation of the data, at 45 cmH2O. At a steady state pressure of 25 cmH2O there was no statistically significant drug effect on CSF absorption regardless of drug concentrations or drug class   , CSF outflow rates were increased significantly relative to no treatment upon delivery of 100 μM L-NMMA and 1 μM U46619. Saline delivery did not significantly increase CSF absorption (0.88 ± 0.097 mL/min)   Figure .Expansion of the ventricular space due to a deficit in CSF absorption can lead to an increase in ICP and a decrease in cerebral perfusion pressure, these scenarios being commonly seen in traumatic brain injury and hydrocephalus. With regards to the latter, the most common solution is either CSF diversion with a shunt or endoscopic third ventriculostomy (ETV), both being highly invasive with potential long-term negative consequences. From a theoretical perspective, the intranasal delivery route for pharmacological agents may provide a means to modulate CSF outflow in a non-invasive manner by targeting the lymphatic vasculature at the cribriform plate. In this report we provide proof-of-concept that supports the above hypothesis using well-characterized albeit not necessarily clinically-applicable pharmacological agents that modulate the lymphatic vasculature resulting in a change in CSF outflow.in vitro experiments using isolated lymphatic vessels support our in vivo analysis [Our data highlights a number of important considerations when modulating CSF absorption intranasally. Indeed, clear differences exist between certain classes of agents in their ability to promote CSF absorption. For example, NA appears to be less efficient in inducing CSF absorption when compared to the thromboxaneA2 analogue U46619. Consistent with this observation, analysis ,23, and analysis . AdditioWhile our hypothesis focuses on modulating the turbinate lymphatic vasculature in order to influence CSF absorption, other pathways may play a role in augmenting CSF outflow with the most obvious being the uptake of drug into the blood vasculature. Indeed, it has been demonstrated that the intravenous delivery of catecholamines can influence lymphatic contractile activity and flow from efferent lymphatic vessels in sheep ,25. HoweWe have demonstrated the utility of delivering pharmacological agents intranasally to modulate CSF absorption non-invasively. This has the potential to complement the current surgical interventions that are used to treat pathologies characterized by a deficit in CSF absorption such as hydrocephalus. Indeed, procedures to treat hydrocephalus such as ETV result in an increase in CSF drainage from the ventricles into the subarachnoid space; the augmentation of CSF absorption via the nasal lymphatics coupled with other interventions may aid in CSF clearance.The authors declare that a patent is currently under review relating to the content of the manuscript.HK participated in the design of the study, data analysis and interpretation, and drafted the manuscript. SAM carried out the surgical procedures. MGJ conceived of the study and its design. All authors have read and approved the final version of the manuscript.2O ICP. At a constant ICP of 30 cmH2O, drug concentrations were plotted against CSF outflow for (A) saline delivery, (B) noradrenaline (NA) at 50 μM and 500 μM (n = 5), (C) L-NMMA at 10 μM and 100 μM (n = 4) and (D) U46619 at 0.1 μM and 1.0 μM (n = 5). Asterisk denotes significance relative to NT (ANOVA/Dunnett’s test).Increasing drug dose results in a significant increase in CSF outflow at 30 cmHClick here for file