Burkholderiales were present at high levels in all atherosclerotic plaques obtained from patients with atherosclerosis with the genus Curvibacter being predominant in all plaque samples. Moreover, unclassified Burkholderiales as well as members of the genera Propionibacterium and Ralstonia were typically the most significant taxa for all atherosclerotic plaques. Other genera such as Burkholderia, Corynebacterium and Sediminibacterium as well as unclassified Comamonadaceae, Oxalobacteraceae, Rhodospirillaceae, Bradyrhizobiaceae and Burkholderiaceae were always found but at low relative abundances of the total 16S rRNA gene population derived from all samples. Also, we found that some bacteria found in plaque samples correlated with some clinical parameters, including total cholesterol, alanine aminotransferase and fibrinogen levels. Finally, our study indicates that some bacterial agents at least partially may be involved in affecting the development of cardiovascular disease through different mechanisms.Atherosclerosis is considered a chronic disease of the arterial wall and is the major cause of severe disease and death among individuals all over the world. Some recent studies have established the presence of bacteria in atherosclerotic plaque samples and suggested their possible contribution to the development of cardiovascular disease. The main objective of this preliminary pilot study was to better understand the bacterial diversity and abundance in human atherosclerotic plaques derived from common carotid arteries of individuals with atherosclerosis (Russian nationwide group) and contribute towards the further identification of a main group of atherosclerotic plaque bacteria by 454 pyrosequencing their 16S ribosomal RNA (16S rRNA) genes. The applied approach enabled the detection of bacterial DNA in all atherosclerotic plaques. We found that distinct members of the order  Atherosclerosis is considered a chronic disease occurring at sites of blood flow disturbance and is the major cause of severe disease, loss of productive life years and death among individuals all over the world. This disease is characterized by accumulation of low-density lipoprotein in the arterial intima, expression of leukocyte adhesion molecules and chemokines by activated endothelium, promoting the recruitment of innate and adaptive immunity cells (monocytes and T cells) to the intima, thus resulting in local inflammation process. Further its intensification may lead to local proteolysis, atherosclerotic plaque disruption and ultimately to thrombus formation, leading to ischemia and myocardial infarction. Therefore, human atherosclerosis can be considered both a metabolic and an inflammatory disease –4.Chlamydia pneumoniae, Helicobacter pylori, some periodontopathogens, including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, as well as various viruses  (0.3–2.1%) and Bacillales (0.1–3.6%) were the important taxa within the phyla Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, respectively. Curvibacter (26.5–47.8%), Propionibacterium (0.1–21.8%), Ralstonia (3.2–14.0%), Burkholderia (0.4–7.9%) as well as unclassified Burkholderiales (11.0–35.9%), unclassified Comamonadaceae (1.0–5.8%) and unclassified Oxalobacteraceae (0.2–4.2%) were typically the most significant taxa for all atherosclerotic plaques, whereas different other taxa were also observed , whereas opposed relationships were found for Granulicatella (ρ = 0.57). Alanine aminotransferase value (ALT) was found to be significant factor for Staphylococcus, Streptococcus and Propionibacterium . Unclassified Rhodospirillaceae showed a positive correlation with the urea level (ρ = 0.51), whereas Burkholderia demonstrated a negative correlation with this parameter level (ρ = –0.51). Unclassified Burkholderiales positively correlated with the fibrinogen level (ρ = 0.52), whereas unclassified Burkholderiaceae negatively correlated with the level of fibrinogen (ρ = –0.67). Moreover, abundance of unclassified Methylobacteriaceae was negatively correlated with the platelets level (ρ = –0.58), abundance of unclassified Microbacteriaceae was positively correlated with the neutrophils counts (ρ = 0.51), and additionally abundance of Streptococcus was negatively correlated with the level of lymphocytes and monocytes   .In the present preliminary pilot study we used a 16S rRNA gene amplicon pyrosequencing approach to comprehensively describe the structure and diversity of the bacterial communities in atherosclerotic plaques derived from common carotid arteries of Russian individuals with atherosclerosis in greater detail. Correlations between bacterial community composition and some clinical values were additionally investigated. The research described herein clearly shows that microbial associations are present in atherosclerotic plaques and, on the one hand, represented by certain bacterial phylotypes in all plaques and, on the other hand, represented by diverse phylotypes across individual patients.Chryseomonas, Wolcott et al. [Flavobacterium sp., Calandrini et al. [Aggregatibacter actinomycetemcomitans as major phylotypes, whereas Mitra et al. [Lactobacillus rhamnosus in various atherosclerotic plaque samples. In the present research work all these bacteria were detected at very low levels or could not be detected at all. Such differences observed within various research works indicate that microbial associations in atherosclerotic plaques are highly diverse and variable between individuals. Therefore, such experiments are extremely necessary to identify key microorganisms that may potentially play roles during the atherosclerotic plaques development in humans depending on previous and current diseases, lifestyle factors and obtained medical treatment. In addition, some researchers analyzed other regions of the bacterial 16S rRNA gene  as wellBurkholderiales were present at high levels in all atherosclerotic plaques studied here with the genus Curvibacter being predominant in all analyzed samples. Bacteria belonging to the order Burkholderiales can be found in various environments including soil and water. Several species of this order have resistance to common antibiotics and are also perilous for different patients, e.g., with chronic lung diseases [Curvibacter includes such recognized species as Curvibacter delicatus, Curvibacter gracilis, Curvibacter lanceolatus [Curvibacter fontanus [Curvibacter in low proportions (below 5%) in some atherosclerotic plaque samples was also reported by Wolcott et al. [Curvibacter was observed in patients with chronic obstructive pulmonary disease compared to healthy individuals [We found that distinct members of the order diseases . At the ceolatus  and Curvfontanus . They art et al. . Furtherividuals .Curvibacter, we also detected Ralstonia and Burkholderia within the order Burkholderiales in all analyzed samples. Species of the aerobic genus Ralstonia can be found in distinct ecological niches, e.g., Ralstonia solanacearum is a soil-borne pathogen causing diseases of many plant species and belonging to one of the most devastating bacterial plant pathogens [Ralstonia eutropha can switch to anaerobic respiration under anoxic conditions, exploiting alternative electron acceptors [Ralstonia pickettii, has been found in distinct clinical environments and recognized as potential nosocomial agent [R. pickettii can cause minor infections as well as invasive infections such as sepsis or meningitis [R. pickettii, Ralstonia insidiosa as well as Ralstonia mannitolilytica have also been recovered from the respiratory tract of cystic fibrosis patients [Ralstonia and Curvibacter, could be potentially transmitted to humans from bottled mineral water. Ralstonia genus was also observed in some atherosclerotic plaques in patients with and without chronic periodontitis using cloning and sequencing of 16S rRNA gene fragments [Besides the dominant genus athogens . Ralstoncceptors . Anotheral agent . In addial agent  and bottal agent . R. pickningitis . R. pickpatients , 40. Falpatients  suggesteragments , 15.Burkholderia phylotypes in all plaque samples. Burkholderia cepacia is resistant to many antibiotics and disinfectants and is mostly associated with infections in patients having lung diseases  as well as in hospitalized patients and individuals having impaired immune systems [Propionibacterium acnes  was specific for atherosclerotic plaques and present in all samples. P. acnes has been described as a member of the normal microflora of the skin, oral cavity and conjunctiva in humans [Burkholderia spp. as well as P. acnes were specific for various atherosclerotic plaques investigated in previous works [P. acnes strains from atherosclerotic plaques of patients with atherosclerosis [Burkholderiales, unclassified Comamonadaceae and unclassified Oxalobacteraceae at significant levels, which limited our ability to describe their potential roles in organisms. Achromobacter species  found at some levels in several our atherosclerotic plaque samples have also been recovered previously, e.g., from lungs of patients with cystic fibrosis [Our molecular analysis additionally revealed the presence of  systems . In addin humans . Howevern humans , 44 as wn humans . Interesus works , 13, 15.clerosis . Except fibrosis  and patifibrosis .Microbacterium, Achromobacter, Pimelobacter as well as unclassified Microbacteriaceae and Alcaligenaceae were negatively correlated with the plasma cholesterol level, whereas the abundance of Granulicatella was positively correlated to the level of this disease marker. Granulicatella spp. belong to normal components of the oral microflora, but have also been associated with different invasive human infections and noted as a cause of bacterial endocarditis [Staphylococcus, Streptococcus and Propionibacterium. All these members are part of the normal flora in humans but may be also considered as major pathogenic bacteria of humans. For example, Streptococcus spp. and Staphylococcus spp. can cause endocarditis, bacteremia, pneumonia, meningitis as well as other infectious diseases [Staphylococcus isolates were also recovered and cultivated from some atherosclerotic plaques of patients with atherosclerosis what was shown in our recent study [Moreover, we found that several groups of microorganisms found in various proportions in atherosclerotic plaque samples correlated with some coagulation, hematological and blood chemistry test analyses. For example, the abundances of the genera carditis . Furtherdiseases , 51. Stant study . HoweverDespite the fact that bacteria were identified in several atherosclerotic plaques in numerous studies –17, 52, In most cases bacteria encased in a biofilm matrix become more resistant to distinct antibiotics and the host immunity system  than are planktonic forms of the same bacteria, potentially increasing the current problems related to medical biofilms , and parFurthermore, Koren et al.  suggesteMechanisms resulting in endothelial erosion  have been unclear; however, recent research works demonstrate an involvement of innate immunity in this process. Ligation of TLR2 expressed by endothelial cells overlying atherosclerotic plaques contributes to endothelial apoptosis and denudation . TLR2 liBurkholderiales were present at significant levels in all atherosclerotic plaques obtained from Russian patients with atherosclerosis. They as well as several other bacteria from 17 bacterial phyla may represent main members of atherosclerotic plaque consortia. Also, we found that several microbes found in atherosclerotic plaque samples correlated with some clinical parameters, including total cholesterol, ALT and fibrinogen levels. Future investigations such as isolation of specific microorganisms from atherosclerotic plaques, their further accurate investigation , metatranscriptome analysis as well as microbial characterization of atherosclerotic plaques of other localization in humans should clarify whether these microorganisms at least partially affect the pathogenesis of atherosclerosis.The human organism is home to various microbial ecosystems which perform distinct functional roles. In the present research, utilizing a 16S rRNA gene amplicon pyrosequencing approach, we revealed that various representatives of the order S1 FigP < 0.01 and **P < 0.001. Abbreviations: alanine aminotransferase (ALT), aspartate aminotransferase (AST), activated partial thromboplastin time (APTT), thrombin time (TT) and international normalized ratio (INR).Correlation analysis is based on the Spearman’s rank correlation coefficients which are shown by color ranging. Negative correlations are displayed in red color while positive correlations are displayed in green color. Significant correlations are marked by *(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


While the clinical role of manual therapies in migraine management is unclear, the use of chiropractors for this condition is considerable. The aim of this study is to evaluate the prevalence and characteristics of chiropractors who frequently manage patients with migraine.A national cross-sectional survey of chiropractors collected information on practitioner characteristics, clinical management characteristics and practice settings. A secondary analysis was conducted on 1869 respondents who reported on their migraine caseload to determine the predictors associated with the frequent management of patients with migraine.n = 990; 53.0%). The strongest factors predicting a chiropractor having a HMC include the frequent treatment of patients with axial neck pain , thoracic pain (referred/radicular)  and non-musculoskeletal disorders .A large proportion of chiropractors report having a high migraine caseload (HMC)  contains supplementary material, which is available to authorized users. Migraine is the seventh leading cause of years lived with disability (YLDs) and a common neurological disorder . During Uncertainty remains regarding the mechanisms associated with the initiation of migraine pain. Evidence suggests migraine pain has a central origin involving the cortex and brainstem , 6. IndiChiropractors are one of the most common complementary and alternative medicine (CAM) providers globally –21. The The analyses presented in this paper were drawn from a questionnaire distributed during recruitment for a national practice-based research network (PBRN) titled the Australian Chiropractic Research Network (ACORN) project. This national project is independently designed and conducted by senior researchers at the Australian Research Centre in Complementary and Integrative Medicine (ARCCIM), University of Technology Sydney. The ACORN 21-item questionnaire examining practitioner, practice and clinical management characteristics was distributed to all registered chiropractors across Australia  . The secRecruitment for the ACORN PBRN occurred through a profession-wide recruitment strategy conducted from March through to June 2015. An invitation pack was distributed to all registered Australian chiropractors who were invited to both complete the baseline ACORN questionnaire and to consent to participate in the ACORN PBRN project. Distribution was via post (hard copy), email (survey link) and at several regional profession-based conferences and was also made available through the official ACORN website (SurveyGizmo™). The invitation pack was similarly re-distributed with four reminders starting 4 weeks after the initial invitation .p = 0.065) and gender (p = 0.634). While the ACORN baseline sample is also generally representative of the wider chiropractic population regarding practice location, we found slight differences in terms of the distribution by location with the questionnaire sample slightly over-represented by chiropractors from South Australia, the Australian Capital Territory, Tasmania and the Northern Territory (p < 0.01) [A total of 2005 chiropractors  completed the baseline ACORN practitioner questionnaire. Participants were generally representative of the wider profession with regards to a number of key indicators when compared to registered chiropractors identified by AHPRA  at the time of recruitment  includin < 0.01) .The ACORN questionnaire collected information across three key domains . The dependent variable was the frequency of treatment of patients with migraine; ‘never’, ‘rarely’, ‘sometimes’ or ‘often’, which was dichotomized into those who treat patients with migraine ‘often’ and those who treat patients with migraine ‘less often’ . Data are presented as means and standard deviations, or absolute and relative frequencies.Statistical analyses were conducted using statistical software Stata 13.1 and SPSS 22.0 on those chiropractors who provided an answer to the question on how often they treat patients with migraine (p ≤ 0.25) were included in the regression model. A backward stepwise procedure employing a likelihood ratio test was chosen to determine the independent predictors of chiropractors who treat patients with migraine ‘often’. Statistical significance was set at p < 0.05. Odds ratios were reported with 95% confidence intervals.The bivariate associations between all survey items and the outcome variables were firstly explored using Student’s t-test or chi-square tests, where applicable. Independent predictors of frequency of treating patients with migraine were identified using multiple logistic regression analysis. ACORN survey items with associations from the bivariate analyses . Fewer participants reported managing patients with migraine ‘sometimes’  and only a small percentage reported managing patients with migraine ‘rarely’  or ‘never’ .Of the 1869 chiropractors, 62% were male with a mean (SD) age of 42.1 (12.1) years and most had a Bachelor or Master’s degree qualifications (96%). Participants had worked for an average of 15.8 (11.3) years in practice and worked an average of 27.3 (12.6) patient care hours each week. The majority of chiropractors reported managing patients with migraine ‘often’ (p = 0.001), had more years in practice (p < 0.001), worked a greater number of patient-care hours per week (p < 0.001) and reported a greater number of patient visits per week (p < 0.001) than those chiropractors with a lower migraine caseload (Table p = 0.017) and they less often shared their practice location with a GP (p = 0.046) or psychologist/counsellor (p = 0.043) while more often had a referral relationship with an occupational therapist (p = 0.016), podiatrist (p = 0.016) and/or exercise physiologist (p = 0.031). Additionally, these chiropractors more often used imaging in their practice (p < 0.001) but less often had diagnostic ultrasound on site (p = 0.008) than those chiropractors with a lower migraine caseload (Table Chiropractors with a high migraine caseload (‘often’ group) were more often older p = 0.00, had morad Table .Table 1Dp < 0.001), smoking/drugs/alcohol (p < 0.001), physical activity (p = 0.005), occupational health and safety (p < 0.001), pain counselling (p < 0.001), nutritional supplements (p < 0.001) and medications (including for pain/inflammation) (p < 0.001) than those chiropractors who less often managed patients with migraine. In addition, those chiropractors with a high migraine caseload more often treated patients presenting with neck, thoracic and low back pain, upper and lower limb disorders, postural disorders, degenerative conditions , non-musculoskeletal conditions (p < 0.001), other headache disorders (excluding migraine) including cervicogenic and tension type headaches (p < 0.001) and spine health maintenance/prevention (p < 0.001) than chiropractors with a lower migraine caseload. In addition, they were more likely to treat pregnant women (p < 0.001), athletes/sports people (p < 0.001), Aboriginal and Torres Strait Islander people (ATSI) (p < 0.012), patients with work injuries (p < 0.001) and traffic injuries (p < 0.001), patients from non-English speaking ethnic groups (p < 0.035), people receiving post-surgical rehabilitation (p < 0.001), and younger and older patients  than those chiropractors with a lower migraine caseload. The treatment techniques/methods more often used by chiropractors with a high migraine caseload were high velocity, low amplitude (HVLA) spinal manipulation (p = 0.023), drop-piece techniques (p = 0.015), sacro-occipital techniques (p < 0.001), instrument adjusting (p = 0.001), biophysics (p = 0.040), applied kinesiology (p = 0.001), functional neurology (p < 0.001), dry needling (p = 0.006), heat/cryotherapy (p = 0.002), orthotics (p < 0.001) and extremity joint manipulation methods (p < 0.001).Table Logistic regression analysis identified a range of factors independently associated with the likelihood of a chiropractor having a high migraine caseload. These factors included the chiropractor often discussing medications with their patients (including for pain/inflammation) , treating patients with neck pain  , neck pain (referred/radicular) , thoracic pain (referred/radicular) , low back pain (referred/radicular) , upper limb musculoskeletal disorders  , providing spinal health maintenance/prevention , treating non-musculoskeletal disorders , treating athletes/sports people , employing functional neurology methods in their patient management  and less often having a psychologist/counsellor located in the same practice as the chiropractor   –32 and aFor instance, there is a need to better understand all of the relevant patient management approaches included within chiropractic migraine management and whether these approaches vary from those reported in routine Australian chiropractic practice which favours spinal manipulation, soft tissue therapy and exercise prescription . For insOur analyses did not identify any practitioner characteristics  that were associated with a high migraine caseload, suggesting that a broad cross-section of the Australian chiropractors are frequently managing those with migraine. However, our research highlights several practice-setting and clinical management characteristics associated with chiropractors managing a high migraine caseload and which raise valuable questions about the therapeutic or philosophical approaches that may be common to chiropractic migraine management.Our study found chiropractors with a high migraine caseload were associated with treating spine regions  including referred and radicular spine symptoms associated with noxious stimulation of nerve endings and direct nerve root compression respectively , as wellOur analyses identified chiropractors with a high migraine caseload as more likely to provide treatment of patients with non-musculoskeletal conditions. While migraine itself is classified as a neurological disorder, the classification of migraine as a non-musculoskeletal condition is less straight forward when considering evidence of an association with neck pain and the potential role of neck pain in migraine pathophysiology , 47, 48.Our study also found chiropractors with a high migraine caseload are associated with providing spinal health maintenance and prevention. While there is limited research to identify a universal evidence-based definition of chiropractic maintenance care , 60, theOur analyses identified chiropractors with a high migraine caseload as more likely to not have a psychologist/counsellor practicing at the same practice location. While psychologists can be a key healthcare provider for those with headache , 66, 67 ‘without the use of drugs or surgery’, a defining therapeutic and philosophical approach to patient care encouraged by chiropractic political bodies [The association with discussing medications (including for pain/inflammation) by chiropractors who often manage migraine raises valuable questions about the nature of these patient discussions. These discussions may reflect the practitioners aim to assist migraine patients to manage their health l bodies , 72 proml bodies , 74. As l bodies .Our secondary analysis of the ACORN cross-sectional survey provides an opportunity to answer a number of questions and identify further pertinent questions for future enquiry regarding chiropractic migraine management. Drawing strong conclusions from our research may be limited due to our analysis being secondary and the quality and fit of existing data to our research. As such, it cannot be concluded that the associations drawn from this secondary analysis are unique to the management of migraine patients. Our findings rely on practitioners understanding the classification criteria for migraine headache and the retrospective recall of practitioners when answering the original ACORN questionnaire. The Likert categories provided in the ACORN questionnaire  for the frequency of migraine management are also subject to practitioner interpretation of these terms. There would also be a risk of selection bias if the features of the practitioners responding to the ACORN survey are less than representative of the wider profession. While the associations reported from our secondary analysis of the ACORN cross-sectional survey are preliminary, the findings nevertheless are valuable in helping to generate hypotheses to further explore the management and effectiveness of headache and migraine management by chiropractors.Migraine appears to be a significant component of chiropractic caseload. There is a need for more high-quality research to better understand how chiropractors manage this patient population and to understand the prevalence, burden and comorbidities associated with migraine patients who seek help from these providers. Such information is important in helping to inform safe, effective and coordinated care for migraine sufferers within the wider health system.


Here, using electrophysiology, optogenetics and pharmacological tools, we compared in adult rats M1wk and S1BF in vivo responses in the whisker areas of the motor and primary somatosensory cortices to passive multi-whisker deflection, their dependence on Po activity, and their changes after a brief intense activation of Po axons. We report that the latencies of the first component of tactile-evoked local field potentials in M1wk and S1BF are similar. The evoked potentials decrease markedly in M1wk, but not in S1BF, by injection in Po of the GABAA agonist muscimol. A brief high-frequency electrical stimulation of Po decreases the responsivity of M1wk and S1BF cells to subsequent whisker stimulation. This effect is prevented by the local application of omega-agatoxin, suggesting that it may in part depend on GABA release by fast-spiking parvalbumin (PV)-expressing cortical interneurons. Local optogenetic activation of Po synapses in different cortical layers also diminishes M1wk and S1BF responses. This effect is most pronounced in the superficial layers of both areas, known to be the main source and target of their reciprocal cortico-cortical connections.Rodents move rhythmically their facial whiskers and compute differences between signals predicted and those resulting from the movement to infer information about objects near their head. These computations are carried out by a large network of forebrain structures that includes the thalamus and the primary somatosensory (S1BF) and motor (M1wk) cortices. Spatially and temporally precise mechanorreceptive whisker information reaches the S1BF cortex via the ventroposterior medial thalamic nucleus (VPM). Other whisker-related information may reach both M1wk and S1BF via the axons from the posterior thalamic nucleus (Po). However, Po axons may convey, in addition to direct sensory signals, the dynamic output of computations between whisker signals and descending motor commands. It has been proposed that this input may be relevant for adjusting cortical responses to predicted vs. unpredicted whisker signals, but the effects of Po input on M1wk and S1BF function have not been directly tested or compared  Rodents move their whiskers to sense their nearby environment. Extracting sensory information from whisker movement crucially depends on feedback-based, precise comparisons of the intended and resulting motion. These computations are believed to be mainly carried out in the “barrel” field of the primary somatosensory cortex (S1BF) and the whisker region of the primary motor cortex  weighing 250–300 g. No apparent differences in neuronal responses were noticed between animals of different sex under our experimental conditions. We checked that all animals had normal, intact whisker sets at the beginning of the experiments. The animals were housed under standard colony conditions; food and water were supplied All recording sessions were terminal experiments. The anesthetic applied for the recording experiments was urethane (1.6 g/kg i.p.), which induced deep .The animal was placed on a water-heated pad  set at 37°C to keep body temperature stable, and its head positioned in a rodent stereotaxic frame . After a midline skin incision, the periosteum and muscle were retracted, to expose the skull. Local anesthetic (Lidocaine 1%) was applied to all skin incisions. A craniotomy was then drilled and enlarged using mini-rongeurs, and the duramater over the target areas was opened.Tungsten macroelectrodes (<1 MΩ) or microelectrodes  were lowered stereotaxically in order to obtain field potentials and unit recordings, respectively, in whisker motor cortex  or deep (D: 900–1500 μm) cortical layers. In each experiment, we recorded responses in different cortical depths in both S1BF and M1wk areas. Later, we grouped all responses recorded at depths of 200–600 μm within the “superficial layer” category, and those recorded between 900–1500 μm in the “deep layers” category. In every animal used, we obtained 2–3 superficial layer datasets and 2–3 deep layer datasets. The 300 μm gap between the superficial and deep layers was devised as a safeguard against potential errors in laminar localization due to small misalignments or drift of the recording electrode from the intended cortical depth. The recording signals were filtered  and amplified using a DAM80 preamplifier (WPI). These data were recorded continuously, sampled at 1 or 10 kHz, respectively, via an analog-to-digital converter built in to the Power 1401 data acquisition unit, and fed into a PC computer for off-line analysis with Spike2 software .in vivo the Po thalamocortical axon terminals in either M1wk or S1BF. To this end, we first injected the adeno-associated viral vector AAV5-CaMKIIα::ChR2(H134R)-eYFP.WPRE.hGH  into Po. This vector drives high levels of expression of the light-activated cation channel, channelrhodopsin-2, tagged with the enhanced version of the yellow moiety of the Aqueoria victoria fluorescent protein (ChR2-eYFP) in the Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα)-expressing cells, ensuring that thalamo-cortical cells transport the protein product to the terminal arborizations in the cortex , attached to a Picospritzer II precision electrovalve system . The micropipette tip was then left in place for 10 min before removing it from the brain. Finally, the bone defect was covered with a layer of hemostatic sponge  soaked in saline, and the muscle and skin sutured over it. Animals were allowed to recover, and returned to their cages.We stereotaxically injected this vector into Po, in both cerebral hemispheres in 11 rats (22 hemispheres). Animals were positioned in a David Kopf stereotaxic apparatus and placed on a water-heated pad set to 37°C. The midline of the scalp was sectioned and retracted, and a small craniotomy was drilled over Po  delivered either through an optical fiber (core diameter 120 μm) or through an optrode, composed of a tungsten macroelectrode 0.5–0.8 MΩ attached to an optical fiber . To selectively activate the ChR2-eYFP-expressing Po neuron axons, the optical fiber tip or optrode were inserted directly into different layers of M1wk or S1BF using a micromanipulator (WPI).2, which is below the damage threshold of ~100 mW/mm2 for blue light . Illumination intensity was <30 mW/mmElectrical stimulation of Po thalamocortical neurons was applied using 120 μm diameter stainless steel bipolar electrodes (WPI) stereotaxically positioned into the nucleus. Current intensity (20–100 μA) was adjusted in each experimental case to the double of the minimum required to elicit responses in the recorded area. Single square pulses (0.3 ms duration) were applied . Pulses were also applied as single trains at 50 Hz for 1 s. We selected these parameters to resemble the effect of the sustained multiple vibrissae stimulation likely to occur during whisking episodes. High-frequency electrical stimulation evokes highly synchronous thalamic firing and facilitates the study of long-lasting cortical effects. As shown by Middleton et al. , sensory2, 20 ms, released with Picosprizer II electrovalve system) and delivered through a polyethylene tube  of a small group (2–3) posterior whiskers were then produced with air pressure pulses  following the electrical stimulation of Po , or two times following blue-light activation of Po axonal arborizations in the cortex (a single long-lasting light pulse of 300 ms).A) receptors, muscimol  was stereotaxically injected in Po to reduce neuronal activity in the nucleus.To gain further insight onto the neuronal circuits involved in M1wk cortex responses to passive whisker movement and their modulation by Po activation, we reduced neuronal activity in either S1BF or Po using locally applied pharmacological agents. In a set of experiments, lidocaine  was injected into S1BF cortex to block neuronal activity in this area. In other experiments, a selective agonist for gamma-aminobutyric acid receptor-A , was applied to M1wk. This toxin selectively blocks GABA release in the GABAergic terminals of PV interneuron axons, as well as glutamate in the glutamatergic terminals of some pyramidal neurons  over a 1-min period. Recordings started 10 min after drug delivery.After electrophysiological recordings were completed, all animals were euthanized (see above for details) and their brains were serially sectioned for histological verification of the stimulation and recording sites. Though a cannula inserted in the left ventricle, 100 ml of saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer  were infused. The brains were then stereotaxically blocked and removed from the skull, postfixed for 24 h at 4°C in the same fixative and cryoprotected in a 30% solution of sucrose in PB during for 2–3 days at 4°C. Brains were serially sectioned (50 μm thickness) in the coronal plane on a freezing microtome .In the brains of the animals tested with electrical stimulation, two serial sections were collected. They were alternately stained with Nissl or cytochrome oxidase histochemistry .Sections were mounted on glass slides, dehydrated through passage in ascending grades of alcohol, defatted in xylene for 30–60 min and finally coverslipped with DePeX .Following rinses in hydrogen peroxide  and Triton X-100 , sections were incubated in a purified rabbit antiserum against GFP that also recognizes eYFP  + 2% Triton X-100 + 3% normal goat serum (NGS) + 1% bovine serum albumin (BSA) in PB . After several rinses in PB, sections were incubated in biotinylated goat anti-rabbit IgG  + 2% Triton X-100 + 3% NGS + 1% BSA in PB . Following new rinses in PB, the sections were incubated in avidin-biotin-peroxidase complex solution  + 2% Triton X-100 . Peroxidase activity was visualized using a glucose oxidase-DAB-nickel protocol  + purified mouse anti-PV antiserum  + 2% Triton X-100 + 2% normal donkey serum (NDS) in PB . Following repeated rinses in PB, the sections were then incubated simultaneously with secondary AlexaFluor 488-conjugated anti-rabbit donkey IgG , and secondary AlexaFluor 647-conjugated anti-mouse donkey IgG  in PB containing 2% Triton X-100 and 2% NDS .Somatosensory-evoked potentials (SEPs) were extracted in the M1wk and S1BF cortices by calculating the average from responses to 100 whisker stimuli. The mean of the peak latency, peak amplitude and the curve area were calculated from the evoked potentials. The area was measured from the negative slope beginning to the same voltage level with positive slope of the first negative wave. Response magnitude variation of area SEP (%) was calculated using as control (100%) the same stimulation protocol in which Po was not stimulated.Single-unit activity was extracted with the aid of Spike2 software  for spike waveform identification and analysis. The sorted spikes were stored at a 1-ms resolution and isolated single-units were analyzed and quantified. The mean tactile response was measured from the peristimulus time histogram  as the number of spikes evoked in the 0–50-ms time window after the stimulus onset divided by the number of stimuli. Neuronal responses larger than two times the mean activity in the 0–50 ms time window before the stimulus onset were considered in this study. The PSTH was calculated from groups of 100 air-puff stimuli in control condition, or after Po thalamocortical pathway stimulation . We measured latencies as the time elapsed between stimulus onset and the largest peak in the PSTH. Unitary response magnitude variation (%) was calculated using as control (100%) the first whisker stimulation train before Po stimulation.t-test (STT). Non-normally distributed data were compared with Wilcoxon-matched pairs test (WT) or with Mann-Whitney U test (M-WUT) for independent samples. For multiple comparisons after electrical stimulation or blue-light channelrodopsin activation, we used one-way analysis of variance (ANOVA) plus T3 Dunnet’s multiple comparisons as a post hoc test. Data are presented as mean ± standard error of the mean (SEM). The threshold level of significance was set at P < 0.05, indicating this as (*), P < 0.01 as (**) and P < 0.001 as (***).Statistical analysis was computed using GraphPad Prism 5 software . For single comparisons and normally distributed data , we used the paired Student’s two-tailed Sections stained with Nissl cytochrome oxidase or immunostained for eYFP with the DAB-nickel method were examined under bright-field optics at 10× using a Nikon Eclipse E600 microscope to delineate the precise location of the electrode tracks and vector deposits. Only cases with correct electrode positions in Po were included in the electrophysiological data analysis. Likewise only cerebral hemispheres in which the ChR2-eYFP transfected neurons were confined to Po , and panoramic, high-resolution mosaic images were produced using a precision motorized stage  driven by the AnalySIS image software package . Images illustrated in the figures were adjusted in brightness and contrast using Canvas X software .Double-labeling analysis of eYFP and PV was carried using a Spectral Leica TCS SP5 confocal microscope by sequentially applying argon (488 nm) and helium–neon (543 nm) laser lines to ensure complete channel separation. Image stacks were obtained moving the sample in the Z-axis in increments of 0.5 μm. Regions of interest were imaged using a 63× oil objective . Image stacks and maximal projections were analyzed both in separate and merged channels. Orthogonal views were generated using ImageJ open access software .The field potentials evoked by the application of air puffs to a small zone of the whisker pad  were recorded separately in the contralateral M1wk and S1BF Figure . In bothn = 14) than with M1wk . This difference is shown in overlapped SEP1s in Figure P = 0.684), while SEP1 peak in the deep layers of M1wk is delayed compared to that in the deep layers of S1BF .Mean SEP1 peak latencies of this first component were only slightly shorter in S1BF .The SEP1 peak amplitude evoked by whisker pad stimulation, was slightly larger in the superficial layers of both areas than in their deep layers, although these differences were not statistically significant . In contrast, SEP1 area in the M1wk remained essentially unchanged , indicating that the first component of the SEP in M1wk does not require S1BF cortex activity.The observation that SEP1 latencies in the superficial layers of S1BF are only marginally shorter (~1 ms) than in M1wk, raised the question whether the whisker pad inputs driving M1wk SEP1 reach this area via a relay in S1BF, or through a direct ascending pathway. It is well established that S1BF projects heavily to M1wk. Thus, the whisker pad inputs driving M1wk might reach this area via a relay in S1BF but also through a direct ascending pathway from the Po nucleus. To investigate this latter possibility, we applied lidocaine in S1BF cortex to reduce its activity, particularly that of its superficial layers, which originate most of the cortico-cortical projections to M1wk  to reduce neuronal firing . In contrast, SEP1 in S1BF was not affected by increased Po inhibition . The preservation of whisker-evoked responses in S1BF is consistent with the fact that, unlike M1wk this area receives ascending whisker pad inputs from Po as well as from the VPM, which was spared by the muscimol injections. As an additional control, we confirmed that the dose injected abolished responses recorded in Po to whisker stimulation but preserved normal responsivity in the rostrally-adjacent ventral lateral nucleus to motor cortex stimulation .To test the ability thalamocortical Po input to modify the global neuronal activity of M1wk and/or S1BF cortices, we compared the responses in these cortices to tactile whisker pad stimulation before and after a brief electrical stimulation of Po. Specifically, we compared responses to a control train of air-puffs (100 puffs at 1 Hz) delivered before Po stimulation with those produced by identical air-puff trains applied 10–110 s (“1st train”), 120–220 s (“2nd train”) and 230–330 s (“3rd train”) after the application of electrical stimuli , returning to control levels only in the 3rd air-puff train. In the deep layers of M1wk, although SEP1 amplitude was also reduced, here the effect was smaller and briefer (85.3 ± 6.1% of control in the 1st air-puff train after Po stimulation) and differences were not significant . Differences in peak latency in superficial or deep M1wk layers after Po activation were also non-significant .Electric Po stimulation produced a decrease in M1wk cortex SEP1 compared to control . Peak latencies also increased after Po activation in the superficial S1BF layers , but this increase reached statistical significance only in the deep S1BF layers .Confirming recent observations by Castejon et al. , we obseTaken together, the above observations show that in M1wk, as in S1BF, the repeated activation of Po thalamocortical synapses produces a protracted decrease of the cortical response to subsequent sensory stimulation.Because fast-spiking PV-expressing GABAergic interneurons control cortical excitability and are a major synaptic target of thalamocortical axons  and deep cortical layers . Under omega-agatoxin, Po electrical stimulation did not reduce SEP1 area in either superficial M1wk layers  or deep layers .Compared to the saline-injected condition, successive trains of air-puff stimulation applied 10 min after injecting of omega-agatoxin into M1wk  the non-stimulated (spontaneous) condition; (b) during a simple air-puff stimulation on the whisker pad; (c) after a single pulse of electrical Po stimulation; and (d) during three sequential air-puff trains delivered following a single electrical stimulation train of Po.n = 23; and 0.3 ± 0.1 spikes/s; n = 16, respectively). Simple air-puff stimulation of the whisker pad increased spiking in both superficial  and deep layer cells  of M1wk cortex . Electrical stimulation of Po with single pulses  induced an orthodromic spike response in both superficial and deep M1wk layer cells . While all neurons recorded in M1wk responded to air-puff stimulation, we found that only 72% in superficial layers and 64% in deep layers of recorded neurons fired in response to the electrical activation of Po neurons.Unit recordings revealed a low spontaneous activity in both superficial and deep cortical M1wk layer neurons , and then more markedly during subsequent air puff trains, consistent with our previous SEP1 analysis . Specifically, superficial M1wk layer cells lowered their spiking to 92.3 ± 3.6% during the 1st air puff train , and kept a lowered responsivity during the 2nd  and 3rd  air-puff trains , and was back to normal by the 3rd air-puff train.Following a single electrical stimulation train of Po, M1wk unit activity was diminished during the 1st air-puff train  in Po axons innervating M1wk and S1BF cortices by means of stereotaxically-guided injections of adenoviral vectors in this thalamic nucleus Figure  was deliAt the end of the recording sessions, animals were sacrificed and perfusion-fixed, and their brains processed for microscope analysis. As the viral vector we used drives the co-expression of ChR2 and eYFP, we ascertained the selective transfection of Po cells and their axons by immunolabeling the brain sections for eYFP. A dense population of neuronal cell somata and dendrites was observed at the injection site in the thalamus Figure . In 15 oLabeled Po axons were visible along their path in the white matter and terminal fields in the cerebral cortex. In S1BF, axonal arborizations were most profuse in L5a and L1a Figures . In M1wkP > 0.05).In the ChR2-transfected animals, we first analyzed cortical activity  to trains of whisker pad air-puff stimulation prior to and following a blue-light stimulation pulse applied to the tissue surrounding the recording site. To this end, we inserted an optrode at different depths within M1wk or S1BF cortex Figure . An equiF = 3.432, P = 0.037; n = 25); however, when light was applied to the deep cortical layers, no significant changes were observed in the SEP1 recorded in these layers .Ten seconds after we light-activated the Po axons in the superficial layers of M1wk, SEP1 area to a 1st air-puff train in the same layers went down . Similarly to M1wk, blue-light application did not modify SEP peak latencies in S1BF .Blue-light activation of Po axons in S1BF significantly reduced subsequent responses in this area to whisker pad air-puff stimulation. In the superficial S1BF layers, both SEP1 and unit responses decreased .We took advantage of the fact that Po terminals are largely confined to particular cortical layers , at 500 μm  or 800 μm under the pia surface . In contrast, SEP1 area remained basically unchanged in deeper recording sites . Light stimulation did not change SEP1 peak latencies at any depth .In M1wk, an optic fiber tip was lowered into the cortex at a depth of ~300 μm pointing downwards, directly illuminating L3b, where Po axons form abundant terminal arborizations Figure . Blue-liP = 0.013; n = 10), and their reduction persisted even 4 min later, during the second air puff train . By contrast, unit responses in the deep layers did not decrease  indicating that activation of Po terminal axons in L2/3 influences subsequent sensory processing in both L2/3 and L5a.Likewise, unit responses to whisker pad stimulation Figure  decreaseP = 0.031; n = 7). More marked changes were observed at 500 μm (which roughly corresponds to L4 in S1BF) during the air puff train administered 10–110 s afterwards , and even during a second air-puff train delivered 130–230 s after the light pulse . SEP area did not significantly change in deeper recording sites . SEP peak latencies did not change at any depth .In a final set of experiments, the fiber optic placed on the pial surface of S1BF, and SEPs and unit recordings were made underneath, at different depths Figure . Blue-liP = 0.001; n = 8; Figure F = 1.025, P = 0.368; n = 13). These results indicate that the activation of L1 Po terminal axons can decrease cortical neuron responsivity to tactile whisker pad stimulation across of S1BF cortical columns, except in L6.Unit responses were also reduced in superficial S1BF layers  electrical stimulation of Po or a light-pulse stimulation (300 ms) of optogenetically transfected thalamocortical Po terminals lessens the responsiveness of cortical cells to subsequent tactile whisker pad stimulation for several minutes  have been recently investigated Depression of responsiveness following thalamocortical activation is believed to involve both a history-dependent depression of thalamocortical synapses and an engagement of cortical inhibitory neurons  the Po-mediated inhibition that we normally observed in the cortex decreased after the toxin application  the amplitude of whisker-evoked cortical potentials n Figure .Additional indication in favor of the participation of PV-expressing interneurons is provided by our confocal microscopic observation of frequent appositions between PV-immunolabeled cells with terminal varicosities of ChR2-eYFP transfected Po axons in brain sections of the animals recorded in the optogenetic assays (Supplementary Figure Whisking rodents infer the position, size and texture of objects around their head by computing relationships between motor commands to whisker pad muscles and mechanoreceptive signals from the whisker pad. These computations are carried out by multi-level neuronal loops including thalamic nuclei VPM and Po and cortical areas M1wk and S1BF (Ahissar and Zacksenhouse, Besides, it is interesting to consider that M1wk and S1BF are not only cortico-cortically connected to each other (Mao et al., DC-T and ÁN designed the experiments, performed the experiments and analyzed the data; DC-T, FC and ÁN prepared the figures and wrote the article.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.


Broadly defined, chemical information science (CIS) covers chemical structure and data analysis including biological activity data as well as processing, organization, and retrieval of any form of chemical information. The CIS Gateway (CISG) of F1000Research was created to communicate research involving the entire spectrum of chemical information, including chem(o)informatics. CISG provides a forum for high-quality publications and a meaningful alternative to conventional journals. This gateway is supported by leading experts in the field recognizing the need for open science and a flexible publication platform enabling off-the-beaten path contributions. This editorial aims to further rationalize the scope of CISG, position it within its scientific environment, and open it up to a wider audience. Chemical information science is an interdisciplinary field with high potential to interface with experimental work. Chem(o)informatics, which organizes and extracts knowledge from functional data associated with small molecules, analyzes structure-activity relationships, and predicts active compounds, is considered to be a part of CIS2. In addition, CIS encompasses ‘big data’ analysis in chemistry, which is an emerging field3. Hence, CIS represents a wide spectrum of computational efforts and also interfaces with experimental chemistry, especially synthetic and medicinal chemistry, and interdisciplinary drug discovery research. The derivation of experimentally testable hypotheses, design of experiments, and large-scale analysis of experimental data are among the central tasks of CIS, in addition to information retrieval from chemical data and the development of computational methods and infrastructures.Chemical information science (CIS) comprises any form of chemical structure or data analysis and chemical information retrieval2 with open post-publication peer review4. It has been the first –and continues to be the only– open review publication platform for CIS including chem(o)informatics.The CIS Gateway (CISG) of F1000Research was conceptualized and initiated in 2015 to provide a venue for open access publication of high-quality papersTo put emphasis on high-quality publications, CISG has assembled an international advisory board of leading experts and implemented a two-stage review process that is coordinated by gateway advisors. Submissions to CISG are first forwarded to the advisory board to conduct a pre-review (stage 1) and assess whether or not a manuscript fits the scope of CISG and has scientific potential to advance the field. This assessment does not restrict the type of contributions that are submitted, as further discussed below, but serves as an initial checkpoint for scientific quality. Conclusions of the advisory board are forwarded to the authors. If a manuscript is considered to be relevant and of sufficient scientific potential, it is published in CISG and subjected to open peer review (stage 2); if not, authors still have the option to publish their work in F1000Research outside CISG. Members of the advisory board help with reviewer suggestions for CISG publications and frequently participate in the open peer review process.CISG is open to submission of the entire portfolio of F1000Research manuscript categories, except clinical practice articles and editorials (by invitation only). Original research and methods articles, data notes, and software tool descriptions are encouraged and so are opinion articles. The latter offer investigators the opportunity to provide personal accounts of CIS-related topics, articulate their views, and initiate open discussions in the field utilizing the CISG platform. Moreover, CISG strongly encourages off-the-beaten-path contributions including the publication of negative results, demonstration of methodological failures or conceptual flaws, and discussion of new or visionary CIS concepts, even if perceived controversially.Also welcome are case studies presenting individual projects. Case studies provide excellent opportunities to report on interdisciplinary projects combining computational and experimental approaches, for example, in drug discovery research, evaluate computational predictions, and showcase the interplay between computations and experiments.Although CISG has been devised in the spirit of open science and data sharing, it is well understood that researchers from the pharmaceutical or other industries usually are not at liberty to disclose their data such as structures of novel chemical entities. This should not prevent the publication of scientifically stimulating case studies or other contributions in CISG. It is a strength of CISG’s two-stage review process that partly confidential studies can be evaluated by the advisory board and approved for publication if they represent compelling scientific insights and stories, even if not all the data reported can be freely shared.3. New methodological developments are required to handle big data and data note publications communicating related issues will play an important role going forward.The advent of the ‘big data’ era in chemistry offers additional opportunities. Rapidly growing volumes of increasingly complex and heterogeneous chemical data challenge database structures as much as data analysis and knowledge extraction5 or introducing a mathematical framework for the study of polypharmacology6 and data notes reporting curated sets of kinase inhibitors7 for computational research or the collection of all two- and three-dimensional activity cliffs that could be isolated from public chemical structure and activity data sources8. CISG already contains publications covering rather diverse research topics and it is sincerely hoped that there will be more to come.Among others, current CISG publications include research articles describing the analysis of biologically relevant molecular similarity valuesIn summary, CISG offers a flexible publication platform for an unprecedentedly diverse array of contributions that should be attractive to many investigators in the field.CISG is positioned in a core area where it competes with conventional journals such as the Journal of Chemical Information and Modeling , Journal of Cheminformatics , or Molecular Informatics . Among these, CISG is the only open peer review publication platform and hence fills a void. In addition, J. Chemif. and CISG are open access publications. Historically, J. Chem. Inf. Model. and its precursor, the Journal of Chemical Information and Computer Sciences (J. Chem. Inf. Comput. Sci.) have been flagship journals for the CIS and chem(o)informatics community. However, over the past few years, J. Chem. Inf. Model. has been changing its focus, departing from CIS, and increasingly concentrating on molecular simulations and modeling. As a consequence, currently there is no longer a clear hierarchy among conventional journals in the CIS arena and new opportunities for publication of CIS research are desirable. Clearly, the possibility to accommodate off-the-beaten-path contributions, which CISG strongly encourages, sets it apart from conventional journals and provides further motivation to consider CISG for dissemination of first-class research. Efficient processing of submissions and much shorter publication times of CISG are additional benefits.9. Hence, CISG should also be of considerable interest to a wider scientific community beyond computational experts. This expansion is further supported by the diversity of contributions CISG is able to accommodate, as described above.CIS itself is an interdisciplinary field that spans a wide range of computational sub-disciplines, as discussed above. However, there are ample opportunities to further increase its interdisciplinary nature by exploring interfaces of CIS with experimental work involving chemistry. A good example is the still evolving field of chemical biology, which benefits from the integration of computational approachesCISG strives for publishing top-level research including method development and applications and will put further emphasis on off-the-beaten-path contributions, which set it apart from conventional journals. The ultimate goal of CISG is becoming a primary venue for publication of research in the broadly defined area of CIS, also involving interdisciplinary work with experimental components from the life sciences. Each CISG publication should be regarded as a must-read in the field, an ambitious but feasible goal. Last but not least, a noted strength of CISG is support by leading investigators recognizing the need for open science. Going forward, this culture will be fostered, focusing on contributions from a premier advisory board.


Schizophrenia is a heterogeneous group of disorders. The familial-sporadic distinction has been considered under a range of genetic models. Research supports a strong association of de novo copy mutations with sporadic schizophrenia. The aim of the study was to determine a more homogenous phenotype for genetic research via comparison of various clinical and socio-demographic variables in familial and sporadic schizophrenia.A cross-sectional observational design was used. This study included 384 participants with schizophrenia/schizoaffective disorder from an Afrikaner founder population in South Africa that previously participated in genetic research. A comprehensive data capturing sheet was completed from a pre-existing database that contains information obtained from the Diagnostic Interview for Genetic Studies, chronological clinical summary reports and additional sources of information. The study protocol was approved by the Research Ethics Committee from the Faculty of Health Sciences at the University of Pretoria. Logit models were fitted using the backward elimination procedure to investigate relationships where the dependent variable was binary. Odds ratio estimates were calculated for independent variables that were significant in the model. The Kruskal-Wallis test was conducted to compare the means of groups. For cases where there were significant differences a post-hoc test with a Bonferroni correction was done to determine which groups differ significantly.There were 214 familial and 170 sporadic subjects. 279 had a diagnosis of schizophrenia, 66 schizoaffective, bipolar type and 39 schizoaffective disorder, depressive type. 242 were male and 142 female. The age at onset of the primary psychiatric diagnosis, season of birth, co-morbid diagnoses, symptomatology, suicidality history and marital status weren’t significantly different when considering the combined schizophrenia/schizoaffective disorder group and its relationship to familiality. Early deviant behaviour was however decreased in the sporadic group. These findings were replicated when analysing schizophrenia independently from schizoaffective disorder. The sporadic schizoaffective disorder, bipolar type did however have a significantly lower age at onset (mean 20.18 versus 25.07 years), 8.8 times more hallucinations, 6.6 times more odd behaviour before the age of 10 and were 2.8 times more likely to be single. The bipolar type also had 2.9 times more suicide attempts as opposed to ideation. This finding wasn’t statistically significant. The sporadic schizoaffective group overall was 2.2 times more likely to abuse substances. The depressive type didn’t differ significantly with regards to age at onset, season of birth, co-morbidities, early deviant behaviour, symptomatology, suicidality or marital status.The combined schizophrenia/schizoaffective group didn’t differ significantly, nor did schizophrenia and schizoaffective disorder depressive type when analysed independently. The sporadic schizoaffective bipolar type differed significantly on multiple important variables that suggest a poorer prognosis and increased disease severity. The sporadic schizoaffective bipolar type forms a more homogenous group, with genetic studies hinting at relatively specific genetic risk factors. Studies elucidating the genetic architecture of this group could prove invaluable in clarifying the aetiology of schizophrenia.


Signal hyperintensity on unenhanced MRI in certain brain regions has been reported after multiple administrations of some, but not all, gadolinium-based contrast agents (GBCAs). One potential initial pathway of GBCA entry into the brain, infiltration from blood into the cerebrospinal fluid (CSF), was systematically evaluated in this preclinical study.GBCA infiltration and distribution in the CSF were investigated in healthy rats using repeated fluid-attenuated MRI up to 4 h after high-dose (1.8 mmol/kg) administration of six marketed and one experimental GBCA. Additionally, gadolinium measurements in CSF, blood and brain tissue samples (after 24 h) were performed using inductively coupled plasma mass spectrometry.Enhanced MRI signals in the CSF spaces with similar distribution kinetics were observed for all GBCAs. No substantial differences in the gadolinium concentrations among the marketed GBCAs were found in the CSF, blood or brain tissue. After 4.5 h, the concentration in the CSF was clearly higher than in blood but was almost completely cleared and lower than the brain tissue concentration after 24 h.In contrast to the brain signal hyperintensities, no differences in penetration and distribution into the CSF of healthy rats exist among the marketed GBCAs.Gadolinium-based contrast agents can cross the blood-CSF barrier.• Fluid-attenuated MRI shows GBCA distribution with CSF flow.• GBCA structure and physicochemical properties do not impact CSF penetration and distribution.• GBCA clearance from CSF was almost complete within 24 h in rats.• CSF is a potential pathway of GBCA entry into the brain.•  Gadolinium-based contrast agents (GBCAs) are frequently used in MRI examinations and are generally considered to have an excellent safety profile . FollowiIn order to study this more systematically and especially under controlled and reproducible conditions, animal models were established and confirmed prospectively the brain signal hyperintensities seen in the retrospective patient studies –28. The The mechanism of GBCA accumulation in the brain is unknown and raises several questions. This study focuses on the initial pathway of GBCA entry into the brain. In the brain, two barrier systems exist, the BBB and the blood-cerebrospinal fluid (CSF) barrier . RegardiThe aim of this preclinical study was to evaluate the penetration of GBCAs from the blood into the CSF and the distribution kinetics within different CSF cavities in a systematic manner. Therefore, GBCAs with linear and macrocyclic structures and different physicochemical properties  were evaluated by FLAIR MRI up to 4 h post injection (p.i.). In addition to the marketed GBCAs, the experimental macromolecular agent gadomer was used to assess the effect of molecular size. CSF and blood samples were obtained at 4.5 and 24 h after GBCA administration for inductively coupled plasma-mass spectrometry (ICP-MS)-based gadolinium quantification. Additionally, samples from the cerebellum and pons were analysed to determine gadolinium concentration 24 h p.i.One hundred and two healthy Han-Wistar rats  were obtained from Charles River . The animals were kept under standard laboratory conditions and standard rat chow and water were provided ad libitum. The animals were handled and treated according to German animal regulations. The MRI and CSF sampling were performed under anaesthesia with 1.5 % isoflurane .The study was performed in two parts: First, an MRI-based evaluation of GBCA infiltration and distribution within the CSF up to 4 h after the administration, and second, the quantification of CSF, blood, cerebellum and pons gadolinium concentration 24 h p.i. Fig. . Ninety-MR imaging was performed using clinical 1.5-T MRI  and a dedicated two-channel rat head coil . For the evaluation of the fluid space, T2-weighted MRC for anatomical references of the CSF space was used. For MRC, a variable flip angle 3D-TSE sequence  with an initial refocusing flip angle of 180° (decreased to 120° for the refocusing echo train) and a turbo factor of 79 were used. The spatial resolution was 0.3 × 0.3 × 0.6 mm (field of view (FOV) = 100 × 48 mm; 30 transversal slices). MRC was followed by pre- and post-contrast heavily T2-weighted FLAIR sequence with identical parameters. However, a non-selective inversion recovery pulse with an inversion time of 2,250 ms was included, and TR was extended to 9,000 ms. The first FLAIR sequence was started 1 min after GBCA injection immediately followed by a second scan. The middle of the scan time (16:14 min) was set as the time point for the temporal evaluation . Another MRC and FLAIR measurement was performed 4 h p.i.For CSF sampling, the anaesthetized animals were positioned in a stereotactic frame. The head was flexed, and a small incision was made inferior to the occiput to expose the dura mater of the cisterna magna. A catheter capillary tube was inserted into the cisterna magna, and the CSF was collected into the tube. The animals were euthanised by exsanguination, and a blood sample was collected. In the second part of the study, the brain was removed and dissected to sample the cerebellum and pons. The gadolinium concentrations of the CSF, blood, cerebellum and pons were measured by ICP-MS .Image analysis was performed by two experienced readers who were blinded to the experimental groups. In the MRC images, manually defined regions of interest (ROIs) were drawn in two adjacent slices around the third and fourth ventricles, the aqueduct and in the subarachnoid space at different anatomical locations . Examples of the ROI placement are shown in Fig. Signal enhancement of the CSF spaces was observed in all FLAIR images after GBCA administration. Figure For quantitative image analysis ROIs were placed at different CSF spaces to evaluate the distribution pattern and kinetics of the GBCAs over 240 min Fig.  due to aCSF signal enhancement was detected for all GBCAs at comparable levels. A rapid signal enhancement was found immediately after administration in the inner CSF cavities , which was followed by successively declining CSF signals. After 240 min the SI reached almost baseline level. By comparison, the CSF signal in the subarachnoid space  increased at a slower rate with a peak at 25 min p.i. Subsequently, decreasing SIs were observed until 240 min p.i., declining more rapidly and rigorously at the level of the spinal cord than on the lateral and cerebral level.Analytical determinations of the gadolinium concentrations in the CSF and blood were conducted 4.5 and 24 h post GBCA injection by ICP-MS  to 1.06 ± 0.05 nmol/g (gadodiamide). In the pons, slightly lower gadolinium concentrations were detected. In both structures analysed, the lowest concentrations were found for gadomer  and subarachnoid space. The faster signal increase in the inner cavities demonstrates that the primary location of GBCA infiltration is most likely the choroid plexus located in the ventricles. The fenestrated capillaries of the choroid plexus are relatively permeable to smaller substances, such as GBCAs, which can pass into the choroid plexus interstitium. The choroid plexus continuously secretes CSF, and the choroid plexus epithelium forms the blood-CSF barrier. Importantly, the blood-CSF barrier is known to be physiologically more leaky than the BBB , 35.This study demonstrated that all marketed GBCAs cross the blood-CSF barrier to an almost identical extent. However, the ability to cross this barrier seems to depend on the molecular size as demonstrated by the considerably lower CSF gadolinium concentration for the experimental gadomer which is significantly larger (17 kDa) than the marketed GBCAs (<1 kDa). In contrast to the analytical gadolinium quantification, the reduction was not observed with FLAIR MRI as the r1 relaxivity of gadomer is about a factor of five higher than that of the other GBCAs . After pFor the marketed GBCAs, the averaged CSF gadolinium concentrations are about a factor of 7.4 higher than the respective blood concentrations at 4.5 h p.i. However, after 24 h GBCAs are almost completely cleared from the CSF, and the respective gadolinium concentrations are much lower than those in the blood. This is in contrast to gadolinium concentrations in the cerebellum and pons that are higher than those in the CSF and blood at 24 h p.i. This demonstrates that all GBCAs can be found in the brains of rats 24 h after the administration. Slight quantitative differences between the agents seem to exist. In the cerebellum of animals administered gadodiamide, the gadolinium concentration was higher than that of the other GBCAs Fig. .3+ at physiological conditions with linear than with macrocyclic GBCAs [GBCA kinetics of distribution and excretion from the cerebellum and pons at later time points cannot be described with this study. However, in a recently published mouse study, the brain gadolinium concentration decreased between 3 and 45 days p.i., indicating a persistent GBCA elimination from the brain tissue during this period . Clinicaic GBCAs . HoweverAlthough we demonstrated the infiltration and distribution of GBCAs into the CSF, the further GBCA distribution into the brain parenchyma is not conclusive. Assuming that the GBCA in the CSF represents the source of the gadolinium found in the cerebellum and pons, different pathways of distribution might exist. In the classical view, the penetration of substances from the CSF into the brain parenchyma is mediated by diffusion, a process that is much slower than CSF convection and flow. Hence, this process is not very effective and restricted to the upper surface of the brain parenchyma . The perThis study has certain limitations. It was performed on healthy rats without known BBB disorders. This can approach but cannot fully replicate the clinical conditions in patients, especially regarding BBB integrity and function. Gadolinium was quantified in the cerebellum and pons; however, no histological evaluation of these structures was performed. Other limitations arose from the used MRI method. The altered SI levels after MRI scanner servicing do not allow a direct comparison of the two subgroups. In addition, the SI of the heavily T2-weighted FLAIR sequence depends on the T1 and T2 relaxation times of the CSF in a complex manner. Since GBCAs possess different r1 and r2 relaxivites in water , a correIn summary, this study shows that GBCAs can penetrate from blood into the CSF independent of their chemical structure or physicochemical properties. Only the molecular size seems to be an important parameter as demonstrated by a lower CSF gadolinium concentration after administration of the macromolecular agent gadomer. Dynamic FLAIR MRI demonstrates a kinetic from the inner CSF spaces to the subarachnoid space and suggests a passive distribution and wash-out driven by convection and CSF flow. Twenty-four hours after injection, GBCA clearance from the CSF was almost complete, whereas slightly higher gadolinium concentrations were found in the cerebellum and pons, suggesting delayed excretion from these structures. To date, the mechanism of final distribution from the CSF into the brain and specifically to the DN and the GP could not be evaluated in this experiment and needs further study.


ST-elevation myocardial infarction (STEMI), caused by acute occlusion of the infarct-related coronary artery, is an emergency condition. The primary therapy is restoration of full antegrade flow by either percutaneus coronary intervention (PCI) or thrombolytic therapy (TT). Although primary PCI is superior to TT in patients with STEMI, there are many limitations in clinical practice. TT decreases mortality in STEMI patients, but as experience with thrombolytic agents grows, the potential risks of serious side effects become more apparent. The major complications are bleeding, hypotension and skin rash.We report on a case of cerebrovascular accident (CVA) caused by cerebral emboli following TT. We concluded that the fact that the patient was in arterial fibrillation (AF) was a major contributing factor to her CVA. This is an extremely rare condition, and our case appears to be the second one reported on in the literature. One case of cerebrovascular accident due to cerebral emboli following thrombolytic therapy (TT) has previously been published.2Primary percutaneus coronary intervention (PCI) is superior to TT but there are many limitations to PCI. TT is therefore an effective treatment of choice in STEMI, as it is easy to perform anywhere and at any time.All thrombolytic agents share a common mechanism of activating plasminogen into plasmin, which in turn activates the fibrin degradation pathway. The efficacy and safety of various thrombolytic agents have been well documented in large clinical trials.5The most feared complication of fibrinolytic treatment is intracranial haemorrhage. Risk factors for haemorrhagic complications include increasing age, elevated pulse pressure, uncontrolled hypertension, recent stroke or surgery, the presence of a bleeding diathesis, and severe congestive heart failure. Although the haemorrhagic complications of TT are well documented, this type of embolic cerebral infarction has not been documented in the literature. Hence we report on our case.An 80-year-old female patient presented to the emergency department with severe chest pain. The duration between the onset of pain and presentation to hospital was 60 minutes. Electrocardiography revealed atrial fibrillation (AF) and ST-segment elevation in leads D2, D3 and AVF . PhysicaLaboratory findings were as follows: creatinine kinase-MB: 25 ng/ml, troponin I: 18 ng/ml, and normal liver function tests. The patient had been seeing a cardiologist (MB) with regular outpatient visits. The patient had left ventricular (LV) failure and the last cardiac echocardiograph had been performed one month earlier. In that evaluation, no trombus was detected. The chest pain was very severe on admission, but a new echocardiograph could not be done before TT. Her orthopnoeic breathing was assumed to be due to her LV failure.Acute inferior myocardial infarction was diagnosed and she was hospitalised in the coronary care unit for intravenous TT. Streptokinase 1 500 000 U was administered over 60 min in the fourth hour of chest pain. Two-dimensional echocardiographic examination at the bedside (Vivid 3 Pro) was performed following the therapy. It revealed left ventricular, left atrial and right-sided dilatations, third-degree mitral and second-degree tricuspid insufficiency, ejection fraction of 40 to 45% and 36 mmHg pulmonary artery pressure. No thrombus was detected in any cavity.The patient had deteriorated after two hours of intravenous TT. Motor aphasia and right hemiparesis then developed. The Babinski sign was positive in the right lower extremity. Immediate cerebral tomography (CT) was performed and showed no pathological abnormality. The patient then consulted a neurologist. In the second CT 12 hours later, extensive infarction of the left frontal area was seen . On the STEMI is an urgent condition caused by acute thrombotic occlusion of the coronary arteries. Angiographic studies have shown that coronary arterial thrombosis is present in about 85% of patients with STEMI.PCI is the more favourable approach, but it presents many technical and scientific limitations. In our case, because of the respiratory insufficiency, the patient could not lie down so we could not do PCI. We decided to carry out intravenous TT with streptokinase because of its lower incidence of cerebral haemorrhage.TT is an effective and easy therapeutic method that can be used anywhere and at any time. As in all therapeutic options, TT has certain limitations and complications. The most important and feared complication of a fibrinolytic agent is bleeding, especially intracranial bleeding. It occurs in 0.9% of patients treated with tPA.7TT for STEMI has reduced mortality at the expense of additional intracranial haemorrhage. The proof of efficacy of thrombolysis for STEMI comes from nine randomised placebocontrolled trials in a total of 58 511 patients. The meta-analysis of these trials showed an overall survival advantage of about 2% (11.5 vs 9.6%) in favour of thrombolysis.8Two randomised trials compared low-molecular weight heparin (LMWH), enoxaparin, with unfractionated heparin in patients with UA or non-STEMI.With the introduction of fibrinolytic therapy and, importantly, after the publication of the ISIS-2 trial,11It is now clear that fibrinolysis recanalises thrombotic occlusions associated with STEMI, restores coronary flow, reduces infarct size and improves myocardial function and survival over both the short and long term.In the present case, neurological signs were noted two hours after TT. It was assumed that the patient had had an intracranial haemorrhage. A cranial CT scan was immediately obtained, but haemorrhage was excluded and no abnormality was detected. In the second cranial CT scan, which was performed 12 hours later, a left frontal embolic infarction was detected. This is a rare condition. We continued to treat the STEMI and cerebral infarction, together with a neurologist/neurosurgeon consultant. On the follow up, the neurological problems progressed, congestive heart failure developed, clinical deterioration occurred, and subsequently the patient died.In this case, although a thrombus was not detected during echocardiographic examination, it is likely that the TT induced lysis and fragmentation of an undetected microthrombus and the subsequent dislodging and embolisation of pre-existing cardiac microthrombi, which caused the cerebral infarction. Distal embolisation secondary to lysis of arterial thrombi in an aortic graft occlusion has been reported. It was speculated that this complication occurred when a combination of fresh and old thrombus was present.There is only one other case report in the literature describing embolic cerebral infarction following TT for STEMI.1This case represents an extremely rare clinical condition, which we report on to show the importance of the treatment of STEMI with TT. We deduced that the fact that the patient was in AF was a major contributing factor to her CVA. In conclusion, patients receiving TT for the treatment of STEMI should have constant neurological and cardiovascular re-evaluation and clinicians must be prepared to handle such complications in a timely manner.


Microglial cell-associated neuroinflammation is considered as a potential contributor to the pathophysiology of sporadic amyotrophic lateral sclerosis. However, the specific role of microglia in the disease pathogenesis remains to be elucidated.We studied the activation profiles of the microglial cultures exposed to the cerebrospinal fluid from these patients which recapitulates the neurodegeneration seen in sporadic amyotrophic lateral sclerosis. This was done by investigating the morphological and functional changes including the expression levels of prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), TNF-α, IL-6, IFN-γ, IL-10, inducible nitric oxide synthase (iNOS), arginase, and trophic factors. We also studied the effect of chitotriosidase, the inflammatory protein found upregulated in the cerebrospinal fluid from amyotrophic lateral sclerosis patients, on these cultures.We report that the cerebrospinal fluid from amyotrophic lateral sclerosis patients could induce an early and potent response in the form of microglial activation, skewed primarily towards a pro-inflammatory profile. It was seen in the form of upregulation of the pro-inflammatory cytokines and factors including IL-6, TNF-α, iNOS, COX-2, and PGE2. Concomitantly, a downregulation of beneficial trophic factors and anti-inflammatory markers including VEGF, glial cell line-derived neurotrophic factor, and IFN-γ was seen. In addition, chitotriosidase-1 appeared to act specifically via the microglial cells.Our findings demonstrate that the cerebrospinal fluid from amyotrophic lateral sclerosis patients holds enough cues to induce microglial inflammatory processes as an early event, which may contribute to the neurodegeneration seen in the sporadic amyotrophic lateral sclerosis. These findings highlight the dynamic role of microglial cells in the pathogenesis of the disease, thus suggesting the need for a multidimensional and temporally guarded therapeutic approach targeting the inflammatory pathways for its treatment.The online version of this article (10.1186/s12974-017-1028-x) contains supplementary material, which is available to authorized users. The exact pathomechanism of neurodegeneration in amyotrophic lateral sclerosis (ALS), esp. the sporadic forms of ALS, remains poorly understood, thus leading to the lack of an effective therapeutic intervention for the disease , 2. SeveActivated microglia have previously been reported in the autopsy samples and animal models of familial ALS (FALS), as well as throughout the symptomatic stages as demonstrated by neuroimaging of the ALS patients –11. WhilTherefore, we studied the effect of the disease-related factors circulated in the cerebrospinal fluid of ALS patients (ALS-CSF) on microglia to understand the direct nature of the insult in the SALS pathogenesis. Our hypothesis was based on the assumption that ALS-CSF may provide a mirror to the molecular effectors being produced and propagated in the diseased CNS, which may lead to progression of the disease. The neurodegenerative potential of ALS-CSF has already been documented in a plethora of molecular, electrophysiological, and behavioral studies, and the efficacy of the model in recapitulating the ALS pathology has been suggested –32. Our Diagnosis of ALS was based on the revised El Escorial criteria . CSF samv/v CSF in Dulbecco’s modified Eagle’s medium (DMEM) and the effects were studied in duplicates or triplicates for individual samples. The study consisted of the following experimental groups:Normal control (NC): cultures propagated in DMEM alonev/v in DMEM)NALS (disease control): cultures exposed to CSF of the NALS (10% v/v in DMEM)ALS: cultures exposed to ALS-CSF , the groups were as follows:NC: cultures propagated in DMEMv/v of DMEM)Buffer control (buffer): cultures exposed to buffer CHIT-1: cultures exposed to CHIT-1 in a dose equivalent to that found in ALS-CSF   were dissected, freed of meninges, and mechanically triturated in DMEM. The single cell suspension thus obtained was propagated in DMEM with 10% FBS (Gibco BRL), with a seeding density of 2.5 × 10protocol . Both thEnriched microglial cultures were obtained using a method by Saura and colleagues to ensure the purity and yield . Briefly4 cells/ml and allowed to reach 70–80% confluence. The cultures were then subjected to different experimental conditions.The NSC-34 motor neuronal cell line was procured  and maintained in accordance with the published protocol . To studl-lysine-coated 13-mm circular coverslips (0.1 mg/ml). The cultures were then exposed to the ALS-CSF or CHIT-1 experimental groups and fixed with 4% paraformaldehyde (PFA) for 15 min at RT. Following blocking with 3% bovine serum albumin (BSA), the cultures were subsequently incubated with primary antibodies of interest followed by fluorescently labeled appropriate secondary antibodies (FITC or Cy3)  were then compared between the study groups.We quantified the cellular expression of proteins of interest in terms of mean fluorescence intensity of individual cells on the images captured by a confocal microscope for each immunolabeled protein, using the inbuilt software of Leica Microsystems , 38, 39.For the measurement of soma area, the contour of cells was carefully drawn using the polyline profile in the same manner as described above. The report was generated for the soma area for each ROI, and the mean area per image was analyzed and compared within the groups.After 48 h of ALS-CSF exposure, microglial cultures were observed and quantified for the morphological diversity ranging from ramified/resting to amoeboid/activated cells in randomly chosen fields, by phase contrast, as well as by Iba-1 immunofluorescence. Ten such fields were analyzed in duplicates for each study group, and the results were compared.For a temporal analysis, the cultures were exposed to CSF for 12, 24, and 48 h, respectively, and the decanted media from the cultures were used for the estimation. Quantitative analysis of the secreted IL-6, IL-10, IFN-γ, and TNF-α levels was performed using specific rat ELISA kits and according to the manufacturer’s instructions .For glutamate estimation, the medium from the normal controls and each of the subsets exposed to ALS-CSF for 12, 24, and 48 h was collected and centrifuged at 14,000 rpm to remove the cellular debris. Glutamate levels were measured using the glutamate assay kit . The measurement was based on enzymatic conversion, and the absorbance was read at 450 nm using a colorimetric ELISA microplate plate reader .After CSF exposure for 48 h, the cultures were treated with dichlorofluorescin diacetate (DCFDA), harvested, and lysed and the resultant fluorescence release was measured at the excitation/emission spectrum of 480/530 nm in the 96-well plate reader . Fluorescence/cell was calculated  and statistically analyzed. Another set of cultures grown on 13-mm coverslips was incubated with DCFDA at 37 °C for 30 min and directly viewed  under a confocal laser microscope.Media from different experimental groups were collected as previously discussed. In addition, lysates were prepared from the microglial cultures after 48 h of exposure to ALS-CSF. The level of nitrates was assayed in the samples using a nitric oxide assay kit  at 540 nm using the colorimetric ELISA microplate reader.Microglial cultures and NSC-34 cells seeded on 96-well plates (500 cells/well) were subjected to different experimental conditions, and MTT -2,5-diphenyltetrazolium bromide) and/or lactate dehydrogenase (LDH) assays were performed on these cultures and/or culture media according to the previously published protocols .Following exposure to CSF for 48 h, total RNA was extracted from the microglial cultures using the RNeasy Plus Mini Kit  and quantified using 1 μl of RNA in a NanoDrop 2000 spectrophotometer . Total RNA (10 ng) was reverse transcribed (RT) with random primers using high-capacity cDNA reverse transcription kit . Amplification was carried out in triplicates using specific primers and hydrolysis probes for the target genes  microglia in response to ALS-CSF . This coght bias  towards ght bias  at the ll-Glutamate release from activated microglia downregulates astroglial glutamate transporter expression [2+ wave propagation [pression , a pathopression . Apart fpagation  or inducpagation , 38. Altpagation , PGE2 [6pagation , and NO pagation , which wpagation , 66. Recpagation , 67, 68.Apart from a reduced trophic support, downregulation of microglial VEGF has been shown to correspond with a phagocytic microglial phenotype . In a siThese observations clearly suggest that following exposure to ALS-CSF, microglial cells are activated and adopt a toxic phenotype. Additionally, we have provided compelling evidence inflammatory response of the spinal cord-derived microglia to ALS-CSF-mediated insult. Interestingly, in contrast to the early microglial activation, the maximal degenerative changes in the neurons exposed to ALS-CSF were observed at 48 h . SimilarIn addition, the present study attempts to explain the continued failure of anti-inflammatory therapeutic approaches in the clinical trials, despite obvious pathological contribution of inflammation in ALS , 72. SinCHIT-1, the protein primarily upregulated in the ALS-CSF, is considered as a marker of chronic activation of macrophages in the peripheral system . Althougpush-forward mechanism, where microglia are the first to strike, possibly accompanied by astrocytes, thus setting up a neuroinflammatory cascade and eventually leading to accentuated neurodegeneration. In the light of the above, a combinatorial therapeutic approach is warranted, by suppressing the pro-inflammatory pathways and promoting the anti-inflammatory role of microglia.Based on our findings, we propose that microglial pathology in SALS is morphologically and functionally dynamic, leading to the neurodegeneration through myriad pathways Fig. . It appe


Background: Analysis of cerebrospinal fluid (CSF) is a frequently used diagnostic tool in a variety of neurological diseases. Recent studies suggested that investigating membrane particles enriched with the stem cell marker CD133 may offer new avenues for studying neurological disease. In this study, we evaluated the amount of membrane particle-associated CD133 in human CSF in neuroinflammatory and degenerative diseases.Methods: We compared the amount of membrane particle-associated CD133 in CSF samples collected from 45 patients with normal pressure hydrocephalus, parkinsonism, dementia, and cognitive impairment, chronic inflammatory diseases and 10 healthy adult individuals as controls. After ultracentrifugation of CSF, gel electrophoresis and immunoblotting using anti-CD133 monoclonal antibody 80B258 were performed. Antigen-antibody complexes were detected using chemiluminescence.Results: The amount of membrane particle-associated CD133 was significantly increased in patients with normal pressure hydrocephalus (p < 0.001), parkinsonism (p = 0.011) as well as in patients with chronic inflammatory disease (p = 0.008). Analysis of CSF of patients with dementia and cognitive impairment revealed no significant change compared with healthy individuals. Furthermore, subgroup analysis of patients with chronic inflammatory diseases demonstrated significantly elevated levels in individuals with relapsing-remitting multiple sclerosis (p = 0.023) and secondary progressive multiple sclerosis .Conclusion: Collectively, our study revealed elevated levels of membrane particle-associated CD133 in patients with normal pressure hydrocephalus, parkinsonism as well as relapsing-remitting and SPMS. Membrane glycoprotein CD133 may be of clinical value for several neurological diseases. The cerebrospinal fluid (CSF) is frequently analyzed for diagnosing a variety of neurological diseases. The basic CSF-analysis includes cell count, cytological findings and total amount of protein . SpecifiFigure 1). Although the biological function of this glycoprotein remains to be demonstrated, it gained attention in the past few years. CD133 labels stem and progenitor cells in various organs notably in the neural and hematopoietic system  and was approved by the local institutional review board. CSF samples were collected in inpatients who received a diagnostic lumbar puncture, indicated to exclude or verify neurological disease, and consented for research-based CSF analysis.n = 6), parkinsonism (n = 6), dementia and cognitive impairment (n = 8) and chronic inflammatory central nervous system disease (n = 25). Clinical data and medical information were collected from our institutional database. In patients with chronic inflammatory central nervous system disease, the Expanded Disability Status Scale (EDSS) score was assessed during the stay (We enrolled (i) 10 patients who received CSF analysis to exclude subarachnoid hemorrhage or inflammatory disease showing normal CSF analysis and these patients are in the following referred to as healthy subjects, (ii) patients with normal pressure hydrocephalus   from the collected CSF was frozen at -80°C immediately after adding protease inhibitor according to the manufacturer’s instruction . CSF samples (1 ml each) were thawed on ice and sequentially centrifuged for 30 min at 10.000 × e below) . Extracte below) . Caco-2 escribed  and the Probes and positive controls were subjected to Tris-Glycine gels  and transferred to polyvinylidene difluoride membranes  as published , 2012. At-test). Other data are shown as median and interquartile ranges (compared using Mann–Whitney U-test). Absorbance values of each group are calculated referring to healthy subjects. A logistic regression model was used to investigate the relationship between CD133-deviation and basic CSF analysis as well as EDSS-score and lesion load. All statistical tests were two-sided with a significance level of α = 0.05 without adjustment for multiple comparisons.Data were processed using Office 2013 software package . Statistical analysis was performed with SPSS statistical software package 21.0 . Distribution of the data was established using the Kolmogorov–Smirnov test. Normal distributed data are presented as mean ± standard deviation (SD) as well as standard error of the mean (SEM)   . The bloTables 1, 2. Basic parameters of CSF-analysis is provided in the Supplementary Table SD) or 42% (SEM); p < 0.001] and six others with parkinsonism . In the latter cases, the increase of CD133 was observed in patients with atypical parkinsonism , patients with parkinson disease showed only a trend toward increased CD133 . CSF-associated CD133 levels were also elevated in eight patients with dementia/cognitive impairment [190% ± 150% (SD) or 52% (SEM)] but the increase is not significant by comparison to healthy subjects (p = 0.237). Lastly, the analysis of 25 patients with chronic inflammatory diseases revealed an elevated amount of CD133 in their CSF  . Regression analysis revealed no association between CD133-deviation and parameters of basic CSF-analysis  .The characteristics of all included patients with normal pressure hydrocephalus, Parkinson’s disease, dementia and cognitive impairment, and chronic inflammatory disease are summarized in Figure 2), we performed a sub-analysis and grouped these 25 patients into relapsing-remitting multiple sclerosis , secondary progressive multiple sclerosis , primary progressive multiple sclerosis  and patients with isolated myelitis (n = 3). Baseline characteristics, clinical and radiological parameters are shown in Table 2.Given that patients with chronic inflammatory diseases showed the highest increase in CSF-associated CD133 (SD) or 59% (SEM); p = 0.768; and 222% ± 134% (SD) or 109% (SEM); p = 0.273, see Figure 4]. In contrast, patients with RRMS and SPMS showed increased CD133-levels compared to healthy controls , and 434% ± 135% (SD) or 111% (SEM); p = 0.010, respectively. Finally, further analysis revealed no correlation between radiological parameters   and CD133-deviation, however, a statistical trend was seen when comparing EDSS-score with CD133 .Compared to healthy controls, levels of CD133 immunoreactivity in CSF revealed no increase in PPMS and myelitis-patients [155% ± 90%  of the membrane particles and their origin (plasma membrane versus multivesicular bodies), we would need an exhaustive characterization  of CD133-positive membrane particles isolated from each patient group . These aOur study revealed elevated levels of membrane particle-associated CD133 in patients with normal pressure hydrocephalus, parkinsonism, relapsing-remitting and secondary-progressive multiple sclerosis. Thus, CD133 may be of clinical value for several neurological diseases.All data supporting this study are included in this published article and its supplementary information files.The study was approved by the local institutional review board and informed consent was obtained from the participant.TB: study concept, data acquisition, analysis and interpretation of data, writing the manuscript. LM: data acquisition, analysis and interpretation of data. HL: data acquisition, analysis and interpretation of data, critical revision of manuscript. SK: data acquisition, analysis and interpretation of data, critical revision of manuscript. PB: data acquisition, interpretation of data, critical revision of manuscript. PS: data acquisition, interpretation of data, critical revision of manuscript. JM: data acquisition, interpretation of data, critical revision of manuscript. DC: data acquisition, interpretation of data, critical revision of manuscript. HH: study concept, data acquisition, analysis and interpretation of data, writing and revision of manuscript.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.