Cerebrospinal fluid (CSF) plays an essential role in early postnatal brain development. Extra-axial CSF (EA-CSF) volume, which is characterized by CSF in the subarachnoid space surrounding the brain, is a promising marker in the early detection of young children at risk for neurodevelopmental disorders. Previous studies have focused on global EA-CSF volume across the entire dorsal extent of the brain, and not regionally-specific EA-CSF measurements, because no tools were previously available for extracting local EA-CSF measures suitable for localized cortical surface analysis. In this paper, we propose a novel framework for the localized, cortical surface-based analysis of EA-CSF. The proposed processing framework combines probabilistic brain tissue segmentation, cortical surface reconstruction, and streamline-based local EA-CSF quantification. The quantitative analysis of local EA-CSF was applied to a dataset of typically developing infants with longitudinal MRI scans from 6 to 24 months of age. There was a high degree of consistency in the spatial patterns of local EA-CSF across age using the proposed methods. Statistical analysis of local EA-CSF revealed several novel findings: several regions of the cerebral cortex showed reductions in EA-CSF from 6 to 24 months of age, and specific regions showed higher local EA-CSF in males compared to females. These age-, sex-, and anatomically-specific patterns of local EA-CSF would not have been observed if only a global EA-CSF measure were utilized. The proposed methods are integrated into a freely available, open-source, cross-platform, user-friendly software tool, allowing neuroimaging labs to quantify local extra-axial CSF in their neuroimaging studies to investigate its role in typical and atypical brain development. Cerebrospinal fluid (CSF) is a clear, colorless fluid that circulates in the brain, providing necessary mechanical and immunological protection to the brain. In addition to its protective purpose, recent findings have shown that CSF circulation plays a crucial role in brain development and function prenatally and during the lifespan , which can serve as indirect markers of abnormal CSF production and absorption. Current findings in lateral ventricles (LV) volume related to ASD have indicated no consistent, significant group differences in children , which utilizes a statistical approach of parametric mapping  included N = 32 males (96 scans) and 19 females (57 scans); see We analyzed 153 structural MR brain images, which were obtained at 3 time points  from 51 typically-developing infants, who were at low risk for ASD . These infants were scanned longitudinally at 6, 12, and 24 months of age as part of a National Institutes of Health-funded, multi-site, Autism Centers of Excellence (ACE) Network study: the Infant Brain Imaging Study (IBIS). Data collection sites had study protocols approval from their Institutional Review Boards (IRB), and all enrolled subjects had informed consent provided by parent/guardian. The MRI scans were acquired at 4 different sites , each equipped with 3T Siemens Tim Trio scanners  brick-based phantom . Five brain masks were utilized, namely FSL-BET Smith, , two in-Different brain tissues were then segmented using a framework of atlas-moderated expectation-maximization (EM) implemented in the AutoSeg toolkit  and a corresponding outer surface (Souter). While the CSF hull surface  that is enclosed between boundaries Sinner and Souter. The Laplace PDE takes the following formFollowing the reconstruction of the cortical surfaces, the next step is to solve a Laplace's equation between the inner surface ( surface  is defin surface  as Sinneu(x) = uL for x ∈ Sinner and u(x) = uH for x ∈ Souter. To correctly measure local EA-CSF, a boundary condition map that defines the Laplace PDE boundary condition must be defined in an anatomically consistent manner. In the solution of the Laplace PDE in the proposed framework, the solution domain is bounded by the Dirichlet condition and the Neumann condition. The Dirichlet condition specifies values of the solution itself on the boundary, while the Neumann boundary condition defines values for the first-order derivative of the solution. The interface with the Dirichlet condition defines Sinner and Souter where streamlines start and arrive, and the Neumann condition defines an open boundary that is parallel to the streamlines  integration method . Choosing the gray matter surface as inner surface would likely exclude minor EA-CSF sections due to segmentations errors of the gray matter boundary.Y, the following linear mixed model was fitted for each subject i:The extracted raw local EA-CSF maps were first mapped to the common MNI surface template space  , were scanned with the same pulse sequence at four different sites using Siemens 3T Tim Trio scanners, at irregular intervals over 2.5 years. This resulted in 35 MRI scans for subject I and 31 MRI scans for subject II. The tissue segmentation, brain surface reconstruction, and computation of local EA-CSF maps were performed independently. Local EA-CSF maps were then analyzed using the local coefficient of variation (CV) as a measure of stability. The CV for a vertex It is noteworthy that the EA-CSF spaces in these two adult subjects were visually smaller than those we observed in our infant dataset. In general, in our experience infants have visually larger EA-CSF spaces than adults. It is thus likely our results yield a conservative estimate of the expected reproducibility in the infant settings.The CV analysis showed excellent stability with mean across-site CV of 1.15 and 1.56% for all cortical regions in Case I and Case II respectively . Higher Q < 0.01) for the age effect resulting from the longitudinal mixed-effects model. Overall 16.9% of the brain showed a significant decrease in local EA-CSF in the first 2 years of infant's life. Local EA-CSF showed a highly negative correlation with age in most of the frontal lobe areas, including bilateral middle frontal gyrus and bilateral superior frontal gyrus. Negative correlation with age was also observed in some temporal regions, such as the bilateral superior temporal gyrus. No cortical areas showed a significantly positive association of age and the local EA-CSF.Consistent with our previous global EA-CSF report , with higher local EA-CSF in males compared to females in regions such as: right middle frontal gyrus, inferior and medial orbital gyri, bilateral inferior frontal gyri, right insula, and right superior temporal gyrus, right supplementary motor cortex, and right superior frontal gyrus. 6.2% of the brain showed more local EA-CSF in males compared to females. There were no regions where females showed larger EA-CSF . All tools developed at the UNC Neuro Image Research and Analysis Laboratory are open source. All raw MRI datasets and associated demographic information employed in this study is available at: NIH/NDA: The studies involving human participants were reviewed and approved by the Institutional Review Boards (IRB) of all participating institutions. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.Method development and computational analysis was mainly performed by MM under guidance by MAS and MDS, with additional guidance and methodological input by GG and SP. Parts of processing was performed by SK and JG. Study design, data acquisition was performed by AE, SD, AE. RM, KB, RS, HH, and JP. Interpretation of results was performed by MM, MAS, and MDS. Document preparation was performed by all co-authors.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.


Investigating muscle activity and coactivation with surface electromyography (sEMG) gives insight into pathological muscle function during activities like walking in people with neuromuscular impairments, such as children with cerebral palsy (CP). There is large variation in the amount of coactivation reported during walking in children with CP, possibly due to the inconsistent handling of sEMG and in calculating the coactivation index. The aim of this study was to evaluate how different approaches of handling sEMG may affect the interpretation of muscle activity and coactivation, by looking at both absolute and normalized sEMG. Twenty-three ambulatory children with CP and 11 typically developing (TD) children participated. We conducted a three-dimensional gait analysis (3DGA) with concurrent sEMG measurements of tibialis anterior, soleus, gastrocnemius medialis, rectus femoris, and hamstring medialis. They walked barefoot at a self-selected, comfortable speed back and forth a 7-m walkway. The gait cycle extracted from the 3DGA was divided into six phases, and for each phase, root mean square sEMG amplitude was calculated (sEMG-RMS-abs), and also normalized to peak amplitude of the linear envelope (50-ms running RMS window) during the gait cycle (sEMG-RMS-norm). The coactivation index was calculated using sEMG-RMS-abs and sEMG-RMS-norm values and by using two different indices. Differences between TD children's legs and the affected legs of children with CP were tested with a mixed model. The between-subject muscle activity variability was more evenly distributed using sEMG-RMS-norm; however, potential physiological variability was eliminated as a result of normalization. Differences between groups in one gait phase using sEMG-RMS-abs showed opposite differences in another phase using sEMG-RMS-norm for three of the five muscles investigated. The CP group showed an increased coactivation index in two out of three muscle pairs using sEMG-RMS-abs and in all three muscle pairs using sEMG-RMS-norm. These results were independent of index calculation method. Moreover, the increased coactivation indices could be explained by either reduced agonist activity or increased antagonist activity. Thus, differences in muscle activity and coactivation index between the groups change after normalization. However, because we do not know the truth, we cannot conclude whether to normalize and recommend incorporating both. Surface electromyography (sEMG) is used to measure muscle activity and may be used clinically to investigate muscle function during activities such as walking in conditions affecting the neuromuscular system . In chilMuscle coactivation, defined as simultaneous activity of agonist and antagonist muscles crossing the same joint, is a normal motor control strategy to increase joint stability and coordination , 7. DuriThe amplitude of the sEMG represents the number of motor units recruited and their firing rate and pattern . HoweverThe different ways of normalizing sEMG data are used interchangeably when investigating muscle coactivation. Although the majority of the studies in the literature usually normalize the sEMG data prior to calculating the coactivation index , 23, 24,Similar to this lack of standardization of sEMG normalization, different indices are used for calculating the coactivation index. For instance, the muscle activity of the antagonist could be compared either to the muscle activity of the agonist only or to the total muscle activity of both the agonist and the antagonist . CompariTherefore, the overall aim of this article was to investigate the effect of sEMG normalization on the interpretation of muscle activity and coactivation. The specific aim was to evaluate the between-subject variability and group differences between the healthy legs of TD children and the affected legs of ambulatory children with CP. In addition, differences between two indices used for calculating the coactivation index were examined.This is a retrospective cross-sectional study, based partly on baseline data from an ongoing randomized controlled trial  and partIn total, 23 ambulatory children diagnosed with unilateral or bilateral spastic CP and classified with Gross Motor Function Classification System level I or II were included in this study. Ages ranged between 6 and 17 years. Exclusion criteria were botulinum toxin A treatment in the lower limb muscles in the preceding last 3 months, and surgery in the legs in the preceding 24 months prior to inclusion. Eleven typical developing (TD) children, within the same age range, were included as reference. Participant characteristics are presented in Walking was assessed using 3DGA . Ten cameras with a sampling frequency of 200 Hz and three AMTI force plates , with a sampling frequency of 1,000 Hz were positioned along a 7-m walkway. Sixteen reflective markers were placed on anatomical landmarks on the lower limbs, according to the Vicon Plug-in-Gait model . ParticiDuring the 3DGA, concurrent sEMG of m. tibialis anterior (TA), m. soleus (SOL), m. gastrocnemius medialis (GM), m. rectus femoris (RF), and m. hamstring medialis (HM), was recorded bilaterally using wireless sEMG . Skin preparation and sEMG electrode placement were done according to the SENIAM (Surface Electromyography for the Non-Invasive Assessment of Muscles) guidelines . The sEMFrom the TD children and the children with bilateral CP, both legs were included in the analysis. From the children with unilateral CP, only the affected leg was included. The healthy legs from TD children and the affected legs from children with CP henceforth will be referred to as TD and CP, respectively.Nexus software  was used to process kinematic data, define gait cycles, detect events, and calculate the spatiotemporal parameters walking speed (m/s), cadence (steps/min) and step length (m), and export c3d-files with data from the 3DGA. Raw sEMG signals were visually inspected for artifacts and noise using Myon ProEMG . A customized MATLAB program , written using the Biomechanical Toolkit , was used for processing the c3d-files.Walking speed, cadence, and step length were normalized to leg length (m) as in Hof , using tThe gait cycle was divided into six phases based on detected events . The firHip flexion/extension, knee flexion/extension, and ankle dorsi/plantarflexion were time-normalized to the gait cycle. The angles at each percentage of the gait cycle were estimated by using a spline fit.The raw sEMG data were band-pass filtered using an eighth-order Butterworth filter with cutoff frequency at 30 and 300 Hz. After visually inspecting the data, for all sEMG channels and each percentage of the gait cycle, an sEMG root mean square (RMS) value was calculated with a window of 50 ms. For each sEMG channel, the highest RMS value (peak RMS) was obtained and used for normalization. In addition, the RMS of each sEMG channel was calculated for each of the six gait phases as defined above and illustrated in After visually inspecting the data and assurance of low intrasubject variability, spatiotemporal gait parameters, kinematics, and sEMG-RMS amplitudes were averaged over the included trials to obtain each leg's mean value.The coactivation index was calculated for all six gait phases, across three muscle pairs  using the following two indices , 8:In coactivation index 1 (CoA1), the antagonist activity was normalized in relation to the mean total muscle activity and multiplied by two to counterbalance the activity of the agonist . In coacThe definitions of agonist and antagonist muscles are often based on the magnitude of the sEMG amplitude, where the higher signal is assigned to the agonist . HoweverStatistical analyses were carried out using MATLAB . From the kinematic data  and sEMG-RMS amplitudes per percentage of the gait cycle, group (TD and CP) averages and 95% confidence interval (CI) were calculated and displayed. For each percentage of the gait cycle, the CP group data were defined as different from TD when the average of the group did not overlap with the 95% CI of the other group.p values from the analysis with non–log-transformed data, henceforth referred to as original analysis, are presented. Mean with 95% CI values for TD and mean with 95% CI deviations of the CP group from TD are retrieved from the original analyses in all cases. Significance was set at p < 0.05, and trends are reported where p < 0.1.Between-group (TD vs. CP) differences in the spatiotemporal gait parameters, sEMG-RMS amplitudes, and coactivation indices for the six different gait phases were tested using linear mixed models. The spatiotemporal gait parameter, muscle or coactivation muscle pair of interest was set as dependent variable, leg as fixed effect, and subject as random effect. Normality of residuals was checked by visual inspection of QQ plots. Where residuals were not normally distributed, analysis was additionally carried out using log-transformed data. In case of similar results, p < 0.01) and shorter normalized step length (p = 0.02) compared to the TD group. The percentage time in single support  was significantly shorter for the CP group (p < 0.04), whereas there was no difference between the groups in percentage time in double support . The relative duration of the different gait phases varied between the groups, where the CP group had significantly longer time in preswing and initial swing compared to TD (p = 0.002 and p < 0.001). The differences in mean timing of the detected events separating the gait phases are illustrated as vertical lines in Twenty-two healthy legs from TD children and 29 affected legs from children with CP were included for the analyses. Spatiotemporal gait characteristics are presented in The residuals from the linear mixed model either were not normally distributed or had an outlier for the majority (26 of 30) of the sEMG-RMS-abs variables and for 13 of 30 variables of the sEMG-RMS-norm. These variables were log transformed, and the results were similar to the original analysis for 35 of the 39 variables in total. The four variables with changed results are marked with α in p = 0.05 and p = 0.001, respectively). Gastrocnemius medialis and SOL were significantly reduced for the CP group during terminal stance  and initial swing (p = 0.03 for both). Rectus femoris was significantly increased for the CP group during weight acceptance (p = 0.04). During midstance, this increase was borderline significant (p = 0.08). Although the average HM amplitude for the CP group was above the TD group during almost the whole gait cycle, no significant group differences were found in this muscle.For sEMG-RMS-abs amplitudes  and significantly reduced during midswing/terminal swing (p = 0.04). During preswing, TA was significantly increased for the CP group (p = 0.05). Gastrocnemius medialis and SOL were significantly increased for the CP group during weight acceptance phase . During terminal stance, GM was reduced for the CP group (p = 0.01), and there was a trend toward a reduction in SOL (p = 0.06). During initial swing, SOL was significantly reduced in the CP group (p = 0.05). Rectus femoris was significantly reduced for the CP group during terminal stance (p = 0.02), preswing (p = 0.003), initial (p = 0.05), and midswing/terminal swing (p = 0.04).For sEMG-RMS-norm , had a trend to be reduced in the CP group during initial swing (p = 0.1), and was similar for both groups in the other four phases. Tibialis anterior/SOL was increased in the CP group during weight acceptance (p = 0.02) and terminal stance (p < 0.01) and was similar between the groups in the other four phases. Rectus femoris/HM showed no significant differences between the TD and CP groups .For CoA1-abs , and midswing/terminal swing (p = 0.002), and there was a trend toward an increase in terminal stance (p = 0.06). In the other three phases, there were no differences between the groups. Tibialis anterior/SOL was increased in the CP group during weight acceptance (p < 0.001), there was a trend towards an increase in terminal stance (p = 0.08), and was similar for the groups in the other four phases. Rectus femoris/HM had a trend to be increased in the CP group during terminal stance (p = 0.05), and the increase was significant during preswing (p = 0.004). In the other four gait phases, there were no differences between the groups.For CoA1-norm , but was similar between the groups in the other five gait phases. Tibialis anterior/SOL was increased in the CP group during weight acceptance phase (p = 0.03) and during terminal stance (p = 0.01) but were similar between the groups in the other four phases. A borderline significant decrease was seen in the CP group for RF/HM during weight acceptance phase (p = 0.08), but no differences between the TD and CP groups were seen for the rest of the gait phases.For CoA2-abs  and during midswing/terminal swing (p = 0.002). In the four gait phases in between, there were no differences between the groups. Tibialis anterior/SOL was increased in the CP group during weight acceptance phase (p < 0.001), and there was a trend toward an increase during terminal stance (p = 0.09). There were no differences between groups in the other four gait phases. Rectus femoris/HM had a borderline significant increase in the CP group during terminal stance (p = 0.05). During preswing, the increase in the CP group was significant (p = 0.004). In the remaining gait phases, there were no differences between the TD and CP groups.For CoA2-norm . A secondary aim was to evaluate differences between two indices for calculating the coactivation index.Our results showed that normalization did not affect the average muscle activity pattern during gait within the TD or CP group but affected the between-subject variability within the groups and the difference in muscle activity between groups. Moreover, muscle activity deviations of the CP group from TD varied across the five investigated muscles.Normalization of sEMG amplitudes is used to reduce between-subject variability caused by nonphysiological factors in order to compare physiological differences between muscles, individuals or sessions , 22, 31.The overall muscle activity pattern in both groups was very similar across the two approaches of handling the sEMG data, because the gait phases with high and low amplitudes barely changed . This isIn the TD group, GM and SOL were especially active during three gait phases: the end of weight acceptance phase, terminal stance, and initial swing . During Despite these alterations in deviating amplitudes of the CP group from TD, in a clinical perspective, evaluating the overall muscle activity pattern may often be more essential in detecting phasic abnormalities rather than relative to TD amplitudes . Our resThere are several approaches for calculating the coactivation index, and in this article, we have looked closer at two indices commonly used. Our results showed statistically increased coactivation indices in the CP group compared to TD in three and four of 18 investigated muscle pairs and gait phases using absolute (CoA1-/CoA2-abs) and normalized (CoA1-/CoA2-norm) sEMG values, respectively. Although the two indices showed the same significant deviations in the CP group from TD, the values in CoA2  were in In phases where there is low agonist activity and already very high coactivation indices in TD, even higher indices in CP cannot be expected. The TD group show clear agonist sEMG burst during weight acceptance, initial and midswing/terminal swing for TA, during terminal stance for SOL and GM and thus low coactivation indices (<75%) for both absolute and normalized values. Our findings indicate increased coactivation index in the CP group in 60% of these gait phases. These increased coactivation indices are in line with expectations and previous studies evaluating coactivation during walking in children with CP , 12.However, there is no general agreement on the role of the coactivation in populations with neurological disorders, beyond abnormal levels that have been reported , 32. PotAdditionally, the interpretation of the coactivation index is closely related to the handling of the sEMG data. Using absolute or normalized sEMG-RMS amplitudes in the calculations gives different pictures of which gait phases the CP group deviate from TD, which emphasizes the complexity of the coactivation index.This article showed that the interpretation of muscle activity and coactivation was affected by normalization approach when evaluating group differences. Although the overall muscle activity pattern did not differ between absolute and normalized sEMG-RMS amplitudes, normalization eliminated variability that could be interpreted as physiological variation within the children and deviating sEMG-RMS amplitudes were found in different phases after normalization. Taken together, these results emphasize the importance of being able to use absolute sEMG-RMS amplitudes in addition to the dynamic peak normalized values and to have knowledge about the underlying physiology in order to interpret sEMG data.When interpreting the coactivation index, it is important to be aware of the methodological approach in order to understand the origin and function, before drawing conclusions on abnormal coactivation levels and making comparisons between different studies. Our findings suggest that increased coactivation index may be explained by other factors than excessive antagonist coactivation, such as the inability to sufficiently activate the agonist.Because we do not know the truth, we cannot conclude whether to normalize the data and recommend considering both absolute and normalized data for a complete interpretation. However, future research should relate to functional outcomes, to better answer whether absolute or normalized sEMG-RMS amplitudes are favorable in the interpretations of altered muscle activity and coactivation index.The datasets generated for this study will not be made publicly available due to Norwegian legislation. Questions regarding the datasets can be sent to the corresponding author.The studies involving human participants were reviewed and approved by Regional Committee for Medical and Health Research Ethics in Middle Norway . Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.YG, SB, and KR contributed conception and design of the study. YG and KR performed the statistical analysis. YG wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.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.


Classical swine fever virus (CSFV) causes one of the most critical diseases in the porcine industry worldwide. In Brazil, the first description of the infection was reported in 1888, and the national recognition of the first free zone (FZ) occurred in 2001. Brazil has been recently recognized (2015–2016) by the World Organisation for Animal Health (OIE) with an FZ involving 15 states and the Federal District, corresponding to 95% of the industrial production of pigs in the country, and a non-free zone (NFZ), comprised by the North and Northeast regions of the country, with approximately 18% of the national pig herd and 5% of industrial production. This review aims to describe the history, the control and eradication actions, the recent occurrence of outbreaks in the NFZ, and the results obtained by the surveillance systems’ action in the FZ for CSF in Brazil since its creation. In the passive surveillance system, the notification of the suspect cases of classical swine fever (CSF) is mandatory while in the active surveillance system adopted in the FZ consists of serological monitoring of certified swine breeding farms (CSBFs), intensive pig farming (IPF), non-technified pig herds (NTPig), surveillance in slaughterhouses and monitoring the populations of wild pigs. In this region, the last outbreaks of the disease occurred in 1998, while in the NFZ, 28 outbreaks were detected from 2005 to 2017, with an apparent lethality rate of 93.96% (840/894). However, in 2018 and 2019, 68 new outbreaks were registered with an apparent lethality rate of 75.05% (1095/1459). Therefore, in 2019, the Brazil CSF-Free Strategic Plan was created to eradicate the infection from the country’s NFZ, since outbreaks in this region present a risk of reintroducing the disease FZ. Finally, differences in characteristics between the regions show factors that still need to be considered for the construction of a robust surveillance system in the NFZ and some improvements in the FZ. Thus, the control of CSF throughout the Brazilian territory requires strict sanitary guidelines, promoting animal health and, consequently, the national production chain’s competitiveness. Pestivirus C, is an enveloped RNA virus belonging to the growing genus Pestivirus, within the family Flaviviridae [Classical swine fever (CSF) is one of the most important infectious viral disease of domestic pigs and wild boars, caused by the classical swine fever virus (CSFV), which belongs to the species iviridae ,2,3. CSFiviridae . In the iviridae . Even thiviridae .CSF has a considerable impact on animal health and the swine industry; therefore, it is a mandatory reporting disease for the World Organisation for Animal Health (OIE) . The infControl and prevention are based on reliable diagnosis, elimination of the infected animals, the establishment of restriction zones, movement restrictions, and tracking of possible contacting animals . ProphylCurrently, Brazil is the fourth largest pork producer and exporter in the world. In 2018, with a herd of approximately two million sows and 40 million pigs for slaughter, it was responsible for 3% of the production and 10% of the world exports to more than 70 countries and moving approximately R$150 billion . Still, Currently, Brazil has a CSF free zone (FZ) that comprises approximately 95% of all industrial pig production. On the other hand, there is the occurrence of CSF outbreaks limited to a few States of the North and Northeast regions, classified as CSF non-free zone (NFZ). Regarding territorial extension, the NFZ is expressive and threatens the country’s position in the international market, and the infection compromises local communities with subsistence production . If infeCSF was described in the world in the 19th century , and in In 1946, outbreaks of CSF in Rio de Janeiro, Minas Gerais, and São Paulo led to the death of approximately 2 million pigs directly by the disease, and many others due to the sacrifice determined by the health authorities. Therefore, from 1946 to 1951, the federal government implemented the first CSF Control Program . In 1978, the first record of African swine fever (ASF) occurred in Brazil, in Rio de Janeiro, in pigs fed with food residues from international flights, this time from Portugal. Then, through presidential decree, the adoption of emergency measures to eradicate the disease in the country began [In this health emergency scenario due to ASF, the Ministry of Agriculture, Livestock and Food Supply (MAPA), in 1981, instituted the Program for Combating swine fever (PCSF) to eradicate ASF and control CSF . Brazil After the eradication of ASF, CSF control became the main objective of the PCSF. The program was based on laboratory diagnosis, elimination of positive animals, vaccination against CSFV (Chinese strain), and serological monitoring in slaughterhouses. The actions were focused on identifying seronegative pigs and establishing free zones of the disease. Although this program had already been applied to control the infection, it was impossible to eliminate the virus from the national territory, and many outbreaks of CSF still occurred across the country. Due to the previous program’s failure, in 1992, the official program was reformulated to eradicate the virus, aiming at the progressive fight against the infection, prioritizing the regions based on the epidemiological conditions and the economic importance for the swine production .The new program’s objective was the gradual removal of vaccination against CSFV across the country, and for this, laboratory diagnostic techniques were improved. Because of the further actions, the number of CSF outbreaks has decreased dramatically across the country, especially since the 1990s . This reIn 2000, a seroepidemiological survey was conducted based on scientific sampling methods to survey viral circulation in a wide area of the country, similar to the foot and mouth disease (FMD) free zone (FZ), enabling the use of the structure of existing official services. The results of this survey allowed MAPA, in 2001, to delimit and declare, based on evidence of the absence of viral circulation, a CSF FZ in Brazil . This FZThe first CSF FZ in Brazil covered 51.7% of the national territory, 53.1% of the properties with swine, 78% of the pig herd, 87% of the housed breeding stock, and 93% of the slaughterhouses .The result obtained in the previous phase culminated in the national recognition of a CSF FZ . Then, iCSF surveillance efforts were intensified with the publication of Internal Standards No. 5 in 2009  and No. Passive clinical surveillance from notification by owners, Official Veterinary Service or third parties;Communication of the increase in mortality rates by qualified veterinarians assisting swine breeding establishments;Active and continuous clinical surveillance in swine breeding establishments identified as having the highest risk of reintroducing CSF in the FZ;Serological monitoring of intensive pig farming (IPF) by the collection of samples in slaughterhouses, from breeders, which were sent for disposal;Seroepidemiological monitoring in non-technified pig herds (NTPig) at regular intervals;Semiannual serological monitoring in Certified Swine Breeding Farms (CSBFs);Ante and post-mortem inspection in swine slaughterhouses.Briefly, the components of the CSF surveillance system are described below:If infected, populations of wild pigs may be the primary source for CSF introduction in domestic pig herds . Due to The OIE Terrestrial Animal Health Code  recognizIn 2019, MAPA gathered information from the OVS to evaluate the perception of wild pigs in Brazil . The datThe structuring of the epidemiological surveillance and population management program for wild pigs (Sus scrofa) in CSF FZ aims at the early detection of CSFV in wild pigs; detection of other important diseases; production of data to support the risk analysis processes in pig production; and assistance with the definition of health strategies for national pig production .Passive surveillance based on reports of the presence of sick or dead animals, generating an epidemiological and clinical investigation with a collection of diagnostic material;Active surveillance with an assessment of the biosecurity of farms, mapping the distribution of populations and definition of risk areas, analysis of data from the surveillance system, and collection of serological samples by voluntary management agents;Serological surveillance carried out by fauna managers (hunters) who voluntarily adhere to the program. The legalized hunter must collect serological samples from these wild boars following biosafety measures to prevent the disease’s spread. To this end, the OVS, in partnership with Embrapa Swine and Poultry Research Center, has carried out various courses intending to train these partners, in addition to producing several instructive technical materials, such as the “Necropsy Manual for Swine” .The strategies of the surveillance system for CSF in wild pigs include:Thus, it is important that the abovementioned strategies are implemented correct and constantly checked to have specific data on the wild pig population distributed along the Brazilian Territory, but especially within the CSF free zone.In 2009, there was an expansion of national recognition of the CSF free area, which included the state of Rondônia (RO) . MoreoveSince 2014, the OIE has included the CSF in the list of the diseases evaluated by the Scientific Committee for Animal Disease, intending to recognize the health status of disease-free country or zone. Based on this, MAPA forwarded requests to the OIE for recognition of the CSF free zones, which were officially recognized in 2015 and 2016.This international recognition process prioritized the most relevant regions for producing and exporting pigs and their products. About 82% of the Brazilian pig herds are currently found in the CSF FZ, comprising approximately 50% of the national territory .Brazil currently has a CSF NFZ formed by the States of Alagoas (AL), Amapá (AP), AM (except FZ region), Ceará (CE), Maranhão (MA), Pará (PA), Paraíba (PB), Pernambuco (PE), Piauí (PI), Rio Grande do Norte (RN) and Roraima (RR); and a FZ with recognition , dividedThe NFZ covers about 50% of the Brazilian territory, comprising approximately 18% of the national pig herd, distributed in more than 300 thousand rural establishments, predominantly made up of small family farmers. The NFZ includes 11 Brazilian states , that present diverse productive, social, economic, and environmental characteristics, characteristics that difficult the development of necessary actions to control and eradicate CSF. Although in smaller numbers, this region also has farms with technified production that will benefit directly from reducing the risk of occurrence of CSF and the end of restrictions on trading their products .Pig production in this region represents an essential source of income and animal protein for local populations, especially for small rural producers in a situation of socio-economic vulnerability, who practice, in their majority, the extensive pig production. In this production system, the animals are raised free, without more significant concerns on productivity. Animals of different categories are raised and handled jointly, making health or zoo technical control difficult ,33.As observed in the 1990s, the CSF outbreaks declined significantly in the NFZ. In CE, which is located in the Northeast of Brazil, 12 outbreaks were reported in 2001, zero in 2002, four in 2003, and one in 2004 . BetweenAfter improving the surveillance system in the NFZ, in August 2018, new cases of clinical CSF cases were confirmed in the state of CE. Since then, 68 outbreaks of the clinical disease have been recorded (data until February 2020) in 28 municipalities in Brazil’s Northeast region. The outbreaks emerged in 20 cities in the state of CE, seven cities in the state of PI, and one focus in the state of AL . HoweverDuring the emergence of the outbreaks, all CSF cases were identified, either through notifications to OVS, regarding animals presenting clinical signs that were suggestive of infection, or through OVS active surveillance within or near previously reported outbreak areas. Because it is an NFZ, the containment measures regulated by the Normative Instruction No. 27  have notThe first outbreak was detected in the municipality of Forquilha (CE), in August 2018, by the surveillance actions for the swine hemorrhagic disease syndromes carried out by ADAGRI—Agricultural Defense Agency of the state of CE. However, laboratory analysis confirmed the CSF outbreak two months later, in October 2018 . This foAn intensification on surveillance occurred within the NFZ since the first outbreak in CE, which enabled the detection of new suspicious cases, confirming another 67 new CSF outbreaks. In October 2019, outbreaks were also detected in the state of PI, in which the focus was more than 300 km away from the border limits of the FZ. Still, new outbreaks in subsistence creation were identified in the state of AL practically one year after the occurrence in the municipality of Forquilha (CE) .To completely eradicate the CSFV from Brazilian territory, the “Brazil CSF-Free Strategic Plan” is being implemented in this NFZ zone. Moreover, the Brazilian government has been intensifying the actions to strengthen the CSF’s NFZ OVS, including training professionals in a veterinary emergency, improving the surveillance system, property re-registration, and animal health education. During the outbreaks, the OVS’s health actions, mainly associated with epidemiological, resulted in the investigation of approximately 2457 properties during the 68 outbreaks in three states .Since the first notification of the disease near the FZ in 2018, the OVS has reinforced the surveillance and prevention actions, inspecting pigs’ traffic and its by-products. It occurred mainly in the states bordering the NFZ, aiming at increasing the protection of the CSF FZ of Brazil.After notification of outbreaks in the CSF NFZ, the OVS has also intensified clinical surveillance on FZ properties. In the state of SE, bordering the state of AL, 1006 properties and approximately 19,000 pigs were inspected and showed no clinical signs of hemorrhagic disease. Likewise, in the state of BA, which borders the states PI, PE, SE, and AL, 4290 properties were inspected, with no evidence of CSFV infection in this area. The monitoring system for swine hemorrhagic diseases in the NFZ is revised continuously to better adapt to the organizational conditions of pig production in the states located in that region, aiming to establish a more effective control/eradication program. The actions adopted in the outbreaks were conducted as recommended by the OIE Terrestrial Animal Health Code  and the Regarding traffic control between the NFZ and the FZ after recognition by the OIE (2015 and 2016), Brazil had already adopted official measures to restrict traffic between zones with different statuses. The last update of the legislation took place in the form of Normative Instruction No. 25 of 19 July 2016, which imposed other traffic restrictions to enhance the previous actions . Most meFurthermore, the disease has been causing significant social and economic impacts and concerns about the possible reintroduction of CSFV in the Brazilian FZ. It is known that until the end of July 2019, the OVS operational costs and the costs related to the compensation to producers exceeded the value of R$1.5 million . HoweverDespite the adoption of health measures to eliminate the outbreaks, the actions were not sufficient to reduce or eliminate the disease’s occurrence due to several factors. The region has diverse, productive, social, economic, and environmental characteristics, which increases the challenge for the development of actions necessary to control and eradicate CSF. Pig production in this region represents an important source of income and animal protein for local populations, especially for small rural producers in a situation of socio-economic vulnerability, who practice, in their majority, the production of pigs extensively ,33. The productive, social, economic, and environmental characteristics increase the challenge of developing the actions necessary to control and eradicate CSF. Therefore, knowing and analyzing aspects of local pig production helps technicians understand health problems, providing support for developing specific strategies to face the challenge. A precise situational diagnosis, including a detailed characterization of the production’s conditions and commercial relations, is essential for effective planning and the direction and management of CSF eradication actions.The quality of the information available is a necessary subsidy for planning and implementing intervention actions. The deficiency in coverage and updating of pig creations is recognized, especially in subsistence production, where the variation between the data is quite expressive . TherefoAnother critical factor, notably in the states of the Northeast Region of Brazil, is the commercialization of live pigs in open markets with other animal aggregations, with the participation of intermediaries ,36. TheyThe lack of passive surveillance activities for swine hemorrhagic syndrome, with the failure of several states in registering these occurrences’ suspicions, contributes to the lack of acknowledgement of the epidemiological situation of the infection ,38. One As a complement to the CSF control measures in the NFZ, by the Normative Instruction No. 10 of 6 April 2020, the use of the CSFV vaccine in the CSF NFZ was authorized, following the Brazil CSF-Free Strategic Plan approved by Ordinance DSA No. 264 of 10 December 2019 . Still, In Brazil, CSF is a disease of mandatory immediate notification to the OVS, which maintains a surveillance system for pigs’ hemorrhagic disease. This system is established by federal regulations aimed at supporting the FZ status of CSF, preventing the reintroduction of the virus, and ensuring the early detection and prompt response to all suspected cases of CSF. To this end, permanent, systematic surveillance actions and standardized strategies are carried out for the entire pig production chain and the elimination and eradication of any outbreaks of the disease in the NFZ. It is important to emphasize that the last occurrence of CSF in the FZ was in January 1998, in the state of São Paulo .The surveillance system for CSF in Brazilian FZ is also based on the actions described above in As part of the actions to maintain the Brazilian CSF FZ status, active and passive surveillance actions were carried out between 1 July 2018, and 30 June 2019, and submitted to the OIE in 2019 to prove the infection-free status in the FZ. During this period, 23,647 commercial and subsistence pigs were inspected by the OVS, and no hemorrhagic disease was found. In the same period, 662 notifications of increased mortality in pigs registered by the OVS were investigated, refuting clinical, epidemiological, or laboratory suspicions of CSF.During the abovementioned period, 78,134 blood serum samples from active serological surveillance in domestic pigs were submitted to ELISA. Of those, 32 samples from 30 herds were reactive, with the suspicion of CSFV being ruled out after negative results on VN and PCR tests, in conjunction with a clinical, epidemiological investigation, confirming the absence of CSFV circulation in the area. Besides, ante- and post-mortem inspections were carried out on 41,867,018 pigs in slaughterhouses, with no clinical signs compatible with CSF. Regarding the surveillance in the wild pigs’ populations carried out by the OVS, 566 blood serum samples were tested for CSFV using ELISA. Of these, 565 samples were negative for CSFV, and only one sample was reagent. Again, in the following complementary molecular and epidemiological investigations, the occurrence of CSF was discarded.In October 2019, an active serological surveillance survey was conducted in subsistence creations located in neighboring international municipalities and CSF NFZ  of Brazil. The results showed an absence of CSFV circulation, agreeing with the information from active surveillance system for CSF used as basis for international certification by veterinary services for the trade of pigs and their products, originated in the FZ. Still, the control of live pigs’ imports, genetic material, and pig products follows the recommendations of the OIE Terrestrial Animal Health Code, and therefore, the traffic of pigs and by-products from NFZ (where outbreaks of CSF have been confirmed since October 2018) to FZ is prohibited. Based on the previous information, Brazil declares that the CSF FZ’s epidemiological situation remains unchanged, maintaining the CSF free zone status with the OIE.The absence of CSF outbreaks in the FZ, since 1998, is a strong indication that the surveillance systems for CSF, together with the control and eradication measures implemented in the FZ, have been effective in controlling the disease. However, the existence of a NFZ within the country and the recent occurrence of outbreaks in the 2018–2019 biennium highlight the potential risk of reintroducing the FZ disease. It is important to highlight that this review includes CSF surveillance data of up to 20 July 2020 and does not include information regarding outbreaks beyond this timepoint.Countries with large territorial extensions and diverse economic realities face greater impasses regarding CSF since control actions are affected by other issues such as regional differences in infrastructure, presence of technically skilled labor, and availability of financial resources. Still, the uneven development of the territories creates situations in which intensive and industrial herds coexist in the same national territory with subsistence pig creations, where animals are raised in a semi-extensive way . In Brazil, states where pig farming is predominantly industrial, and the production substantially participates in the development of the economy, also reported better conditions for the production of epidemiological data, application of prophylactic measures, and compliance with standards possible to monitor the health status of herds. However, in the territories of the NFZ, human development rates are lower in some areas. Non-technified pig herds are predominant, with low participation in the local economy and focusing on the production and fattening of animals, relegating the health focus to the background . Non-technified pig herds have an important social role as an income and feed generator in poor regions where family farming prevails. Still, they are often exposed to severe health problems due to poor infrastructure, low biosafety levels, and lack of regular veterinary medical care . BesidesAnother complicating factor resides in the difficulty of controlling CSF in large territorial extensions. This is mainly due to problems in the effective inspection in traffic of animals, products, and by-products from the NFZ to the FZ, which could reintroduce the CSFV in industrial pig herds. Since the circulation of infected animals or even workers involved in pig farming between disease-free zones and outbreaks was identified as a possible risk factor for introducing the disease , effectiTo this end, active and passive health surveillance actions should continue to be fully applied in the CSF FZ to prevent the reintroduction of the disease and investments and studies to adapt the activities to the productive reality of the NFZ, especially in the border regions. Moreover, further studies on the NFZ aiming to generate data on CSF’s real situation in the regional pig productions are necessary for structuring effective actions, even though the local reality presents possible difficulties for executing such work. Besides, new outbreaks could be avoided by adopting diagnostic techniques based on biological samples that facilitate surveillance activities, such as oral fluid. A recent study showed that the detection of CSFV by PCR in oral fluids showed exciting results in the field, as well as resulting in a 23–40% reduction in the number of sampled animals  and consequently, in the use of materials . Still, Finally, the structuring of a contingency plan for wild pigs, the construction of co-responsibility strategies with the productive sector, as the biosecurity of farms and creating an indemnity fund, are essential factors that must be considered in the control of the disease and the consequent regional socio-economic impact.CSF control and eradication activities in most of the Brazilian territory can be considered successful, taking into account that the last CSF outbreak reported in the FZ occurred in 1998 in the state of SP. This fact indicates the robust surveillance systems for CSF in the FZ. From that period, the free status was granted by the OIE to an extensive area that comprises approximately 95% of the Brazilian industrial swine production. However, outbreaks in parts of the territory characterized as CSF NFZ highlight the potential risk of reintroducing the disease in the FZ. Therefore, considering the epidemiological, productive, and social characteristics of the NFZ, it is essential to intensify surveillance and to carry on the disease control and eradication program. Likewise, localities bordering the FZ need to be prioritized to avoid the reintroduction of CSF in this area, which would negatively affect the pig production chain in Brazil, not only in the health aspect but also regarding international trade.


In the further course of time, the difference even increased (p < 0.001). This study confirmed the key association between early signs of brain damage evidenced as an elevated AST activity and the prediction of the final patient’s clinical outcome. The study showed that the level of AST in the CSF could be the relevant diagnostic biomarker of the presence and intensity of brain tissue damage.Brain ischemia after central nervous system (CNS) bleeding significantly influences the final outcome of patients. Catalytic activities of aspartate aminotransferase (AST) in the cerebrospinal fluid (CSF) to detect brain ischemia were determined in this study. The principal aim of our study was to compare the dynamics of AST in 1956 CSF samples collected from 215 patients within a 3-week period after CNS hemorrhage. We compared concentrations of the AST catalytic activities in the CSF of two patient groups: survivors (Glasgow Outcome Score (GOS) 5–3) and patients in a vegetative state or dead (GOS 2–1). All statistical evaluations were performed using mixed models and the F-test adjusted by Kenward and Roger and the Bonferroni adjustment for multiple tests. The significantly higher catalytic activities of AST in the CSF from patients with the GOS of 2–1 when compared to those who survived (GOS 5–3,  Haemorrhage of the central nervous system (CNS) is associated with a high risk of severe brain deficit or even death. Intracerebral haemorrhage is immediately followed by an increase in intracranial pressure, acute blood microvessel constrictions and decreased cerebral blood flow. All of these pathologies can contribute to the development of initial global cerebral ischemia. Furthermore, cerebral vasospasm, which develops three days after subarachnoid haemorrhage (SAH), leads to delayed cerebral ischemia (DCI) ,2,3,4,5.Current analysis of the cerebrospinal fluid (CSF), especially for scientific purposes, is based on the determination of several more specific parameters to assess the presence and extent of CNS damage. Among these parameters, the levels of neuron-specific enolase, S100B protein, 14-3-3 protein, τ-protein or neurofilaments are preferred ,10,11,12In our hospital ICU (intensive care units), both for acute cases and for patient’s monitoring, the measurement of aspartate aminotransferase (AST) catalytic activities in the CSF is used with good results. Some studies using this parameter have been published over the years ,17,18,19Approximately 30% of the AST is localized in cell cytoplasma and 70% in the mitochondria of cells of different origin. The function of this enzyme is the reversible transport of amines from aspartate to α-ketoglutarate . Cell daThe aim of this study was to compare AST catalytic activities in the CSF of two groups of patients: survivors (GOS 5–3) and patients either in a vegetative state or dead (GOS 2–1) over the 3-week observational period following CNS haemorrhage. The diagnostic value of the AST biomarker is evaluated in this study. In total, we investigated 1956 samples of CSF from 215 patients over a 3-week period after a CNS haemorrhage. The patients were clinically evaluated during hospital discharge using the Glasgow Outcome Score (GOS), which enables division of the patients into 5 categories; the GOS 5 score being patients without any neurological deficit, GOS 4 being patients with a minor neurological deficit, GOS 3 being patients with a major neurological deficit, GOS 2 being patients in the vegetative state, and GOS 1 being patients who died. We divided our patients into 2 subgroups—patients with GOS 5–3 , 24.0% with intracerebral haemorrhage (ICH), 6.0% with simultaneous SAH and ICH) and patients with GOS 2–1 .In the subgroup of patients who survived with GOS 5–3, we registered 61.7% females and 38.3% males. The age of females was from 20 to 88 years with a median of 61 years. The age of males was from 23 to 86 years with a median of 57 years.In the subgroup of patients in a vegetative state or who died, we registered 41.7% females and 58.3% males. The age of females was from 50 to 83 years with a median of 73 years. The age of males was from 9 to 86 years with a median of 62 years.All CSF samples were taken via permanent external CSF drainage into a test tube without anticoagulants and immediately transported to the laboratory. The cell count was measured and microscopic specimens were prepared immediately after receiving the sample. Another part of the sample was centrifuged and evaluated for the concentrations of total protein, glucose, lactate and catalytic activities of AST. The rest of the supernatant was temporarily stored at a temperature range from +4 °C to +8 °C for potential future analysis.We followed concentrations of AST catalytic activities in the CSF and in the blood using the IFCC  method with a Cobas 6000 analyzer . Hemolytic samples of CSF were excluded to eliminate the risk of falsely increased concentrations of AST catalytic activities in the CSF.r = 0.07; p ˃ 0.05).The influence of serum AST concentrations on catalytic activities of the AST in the CSF were excluded using the Pearson correlation coefficient  9 software . Values In order to test the difference in catalytic activities of AST in the CSF between living and dead patients or vegetative state patients (variable Patient Outcome—GOS 5–3 and GOS 2–1), the mixed models—a general procedure, which allows taking repeated measurements—were used. Fixed models were represented by the variable Patient Outcome. Random models were first represented by the variable Patient  and the interaction Patient*Day of treatment. The estimated value of the random component Patient*Day of treatment was in all cases either zero or negligible. Based on these results, this random effect was excluded and the simplified random models were further utilized. All final random models were thus represented only by the variable Patient. In order to model the development in time of catalytic activities of AST in the CSF in the two groups of patients with different Patient Outcome, mixed models with variable Patient Outcome and the interaction Patient Outcome*Day of Treatment as fixed effects and variable Patient as a random effect were used. The variance–covariance structure was diagonal. The significance of fixed components was tested using an F-test adjusted by Kenward and Roger .The tests were performed at a significance level of 0.05. The Bonferroni adjustment for multiple tests was utilized in an appropriate circumstanceWe found predominantly higher AST catalytic activities in the CSF of both subgroups of patients after the CNS haemorrhage compared with control group of patients without the CNS injury. Furthermore, the tendency to significantly higher AST catalytic activities in the CSF was observed in patients with poor outcome (GOS 2–1) compared with survivors (GOS 5–3) .Immediately after a CNS haemorrhage, AST catalytic activities in the CSF were increased in both groups of patients compared with normal value 18.0 IU/L  . In patiDuring a long period of the CSF analysis, we set the useful basic schema for the framework mapping of the CSF compartment ,25. An iOur aim was to present our positive experiences with the measurement of AST catalytic activities in the CSF using the assessment of their development in the group of our patients after the CNS haemorrhage. This impairment allows us to determine the accurate time of its initiation. We only used results of the former clinical indicated standard CSF analysis for this retrospective study. We were limited by the number of patients and by different numbers and timings of CSF analysis in individual cases. Therefore, we unified all patients with different types of the CNS haemorrhage and different ways of the taking the CSF and compared only two subgroups—those with poor outcome (GOS 2–1), and survivors (GOS 5–3).Contrary to these facts, our results show a significant tendency towards higher AST catalytic activities in the CSF of patients in vegetative states or dead (GOS 2–1) compared with survivors (GOS 5–3) . DiffereDuring a long period, we gained practical experiences with different CSF parameters of the CNS tissue injury. We finally preferred AST catalytic activities as the significant part of the CSF basic analysis. Its advantages are common availability, reliable analysis, fast gain of results and low price. The disadvantage of this method is that it is useless in cases of haemolytic CSF samples.We present our good experiences with the AST catalytic activities in the CSF using the comparison of their development in two subgroups of patients after the CNS haemorrhage—those with poor outcome (GOS 2–1), and survivors (GOS 5–3). We consider significantly increased values in patients in a vegetative state or dead (GOS 2–1) as a direct relation between the extent and seriousness of the CNS injury and the patient outcome. Especially high values of AST catalytic activities in the CSF predict high risk of poor outcome. Low values are usually favourable.


Costesia belongs to the Gigaspermaceae, one of the most basal groups of arthrodontous mosses. While none of the species in this family has a peristome, earlier stages of sporophyte development often disclose its basic structure. The study of Costesia sporophytes at the early stages of development was conducted to identify possible similarities with Diphyscium, the genus sister to Gigaspermaceae plus all other arthrodontous mosses in the moss phylogenetic tree. Diphyscium shares a strongly unequal cell division pattern with the Dicranidae. In groups more closely related to Diphyscium, as it is the case of Costesia, this pattern is not known. Our study of Costesia found only irregular presence of slightly unequal cell divisions that may then be considered as a plesiomorphic state in peristomate mosses. The most frequently present pattern revealed in Costesia is common with the Polytrichaceae, a more basal moss group with nematodontous peristomes.The Chilean endemic genus  Costesia, one of the most basal lineages of arthrodontous mosses. Although found at the earliest stages, unequal cell divisions are not stably exhibited throughout the division process. The main developmental pattern in this genus unexpectedly links it to the nematodontous peristome type of the Polytrichaceae.We looked for the presence of unequal cell divisions in peristomial layers of The peristome is a structure at the mouth of moss capsules, which enhances the process of spore release by means of hygroscopic movements. The role of the peristome for Bryophyta classification is as important as that of flower and fruit structure for Magnoliophyta. Molecular phylogenetic data in general coincide with the classification based on the peristome structure, defining main subclasses . Recent Diphyscium  Broth., was also studied for sporophyte development by Searching for an answer to this question, we addressed the Gigaspermaceae which has an intermediate phylogenetic position between eristome . The num results . One speGigaspermum and Lorentziella, and a second clade including Oedipodiella, Chamaebryum, and Costesia, the two latter having elongate setae, i.e., with less reduced sporophytes. Costesia was found in the basal position to the two other genera of the second clade, and therefore is the most promising for peristome studies. Two alternative hypotheses were tested: the first one implies that Costesia possesses a 4:2:3 pattern, inherited from the Diphysciales, which is the closest ancestral group in the phylogenetic tree  Cuvertino, Miserere & Buffa (= C. spongiosa Thér.) with young sporophytes were collected from Lago Peñuelas National Reserve, in Valparaíso Region, central Chile .Plants of -2 • s-1), thus enabling various stages of sporophyte development to be studied.Some living plants were subsequently cultivated in the testing chamber MLR32 Sanyo (temperature + 7°С/+ 12°С (night/day), light period 10 h, PPFD - 14μmol • mAtrichum undulatum (Hedw.) P. Beauv. were included for comparison. Specimens were fixed and observed with the same protocols outlined below .Additionally, the sections of young sporophytes of All material collected in the field was fixed shortly after collecting in 2.5% glutaraldehyde in 0.05M PBS. Further steps were performed after several weeks or a few months. Specimens were post-fixed with 1% osmium tetroxide in PBS, pH 6.8, for 6 h. The material was then dehydrated through an ascending ethanol-acetone series to 100% acetone. Next, samples were embedded in Araldite 6005 medium, according to the manufacturer’s protocol. Sections were cut 2 µm thick with glass knives, put on glass slides without mounting medium, stained with 0.01% berberine or in combination with DAPI, and scanned under LSCM Olympus FV-1000 based on Olympus BX61, using 473 nm or combination of 405 and 473 nm lasers. Z-stacks of several scans were usually obtained and are presented here.Atrichum undulatum was done for comparison after a pattern resembling Polytrichaceae was noticed in Costesia. It included serial sections for four young capsules; razor blade sectioning of fresh material was also made in order to confirm that the pattern seen in serial sections can readily be found in other populations.We made a complete series of sections 2 µm thick, allowing measure the distance from the section where sporophyte apical cell first appears within the ring of calyptra. Sectioning was stopped when patterning of peristomial layers became irregular. The series show different stages of sporophyte development, as can be assumed from the number of cells in the peristomial layers, especially in IPL, which however do not necessarily correspond to the length and width of sporophytes. Altogether twenty series of transverse sections and sixteen series of longitudinal sections were done, of which eleven series of transverse sections represent specific patterns discussed below  are shown in Atrichum we observed the {4}:4:2 formula along a considerable distance at early stages of peristome development  asymmetric divisions occur in IPL in most distal part of the amphithecium , field work organization (JL). Field work conducting , plant fixation and embedding (US), anatomy study , microscopy , manuscript preparation .Russia Foundation for Basic Research, 19-04-00976 and MBG Institutional research project 18-118021490111-5.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.


We aimed to investigate A2A receptors in the basal ganglia of a DYT1 mouse model of dystonia. A2A was studied in control Tor1a+/+ and Tor1a+/− knock-out mice. A2A expression was assessed by anti-A2A antibody immunofluorescence and Western blotting. The co-localization of A2A was studied in striatal cholinergic interneurons identified by anti-choline-acetyltransferase (ChAT) antibody. A2A mRNA and cyclic adenosine monophosphate (cAMP) contents were also assessed. In Tor1a+/+, Western blotting detected an A2A 45 kDa band, which was stronger in the striatum and the globus pallidus than in the entopeduncular nucleus. Moreover, in Tor1a+/+, immunofluorescence showed A2A roundish aggregates, 0.3–0.4 μm in diameter, denser in the neuropil of the striatum and the globus pallidus than in the entopeduncular nucleus. In Tor1a+/−, A2A Western blotting expression and immunofluorescence aggregates appeared either increased in the striatum and the globus pallidus, or reduced in the entopeduncular nucleus. Moreover, in Tor1a+/−, A2A aggregates appeared increased in number on ChAT positive interneurons compared to Tor1a+/+. Finally, in Tor1a+/−, an increased content of cAMP signal was detected in the striatum, while significant levels of A2A mRNA were neo-expressed in the globus pallidus. In Tor1a+/−, opposite changes of A2A receptors’ expression in the striatal-pallidal complex and the entopeduncular nucleus suggest that the pathophysiology of dystonia is critically dependent on a composite functional imbalance of the indirect over the direct pathway in basal ganglia. The clinical features of dystonia are relatively well-defined, produced by the ill-timed activation of agonist-antagonist muscles, and presenting either as focal or generalized. Conversely, the pathological mechanisms underlying such a heterogeneous group of movement disorders remain elusive ,2,3. UnlA common form of primary early onset generalized dystonia is caused by 3 bp deletion (GAG) in the coding region of the TOR1A (DYT1) gene, which results in a defective protein called torsinA, whose role in dystonia pathology is unclear . In animNoteworthy, the deficit in D2 receptor-mediated transmission can be counteracted by A2A receptor pharmacological antagonism in the striatum of a genetic mouse model of DYT1, and also of DYT11 and DYT25 dystonia, suggesting that an imbalance in D2/A2A functions may represent a convergent pathogenetic mechanism ,20. MoreTherefore, besides the dysfunction of D2 receptors, A2A receptors may be of interest in dystonia pathophysiology for several reasons. In this paper, we aimed to investigate whether the D2 receptor dysfunction is coupled with any change in the A2A receptor expression in the basal ganglia of a DYT1 mouse model. We report that opposite to the downregulation of D2 receptors in the striatum, an up-regulation of the A2A receptors occurs in the striatal-pallidal complex, whereas A2A down-regulation occurs in the entopeduncular nucleus of a DYT1 dystonia mouse model.p < 0.001). Moreover, Western blotting analysis revealed a significant increase in A2A receptor levels in the striatum  and globus pallidus , but a significant decrease in A2A expression in the entopeduncular nucleus  of mutant Tor1a+/− compared to control Tor1a+/+ mice , but was unchanged in the globus pallidus  and in the entopeduncular nucleus  of Tor1a+/− mice (n = 7) compared with Tor1a+/+ (n = 7)  .n = 6) is much higher than in the globus pallidus (n = 8) (n = 8) were similar compared to the caudate–putamen of Tor1a+/+ mice (n = 8) compared to Tor1a+/+ mice , have shown that the A2A mRNA expression level in the caudate–putamen of Tor1a+/+ ( (n = 8) A, wherea+/+ mice B. Surpri+/+ mice C.To allow a precise morphological definition of the A2A receptor aggregates on the striatal-pallidal complex in control and mutant mice, we performed a fluorescence staining, followed by detection with confocal microscopy. A better understanding of A2A receptors’ subcellular distribution came out in images acquired using 63× oil immersion objective  with an additional digital zoom factor (1×–1.5×–2×). The immuno-fluorescent signal appeared extremely specific without background staining, showing A2A positive small grains with elliptical or roundish shape about 0.3–0.4 μm in diameter, isolated or contiguous, composing irregular clusters of size variable in the different areas of the basal ganglia of Tor1a+/+ mice A,C,E.Noteworthy, in Tor1a+/+ mice, A2A small grains appeared to be diffusely covering the neuronal compartments of the striatum, globus pallidus and entopeduncular nucleus, uniformly distributed in the neuropil, whereas grains were rare and almost absent on the cell nuclei and in striatal axonal bundles A,C,E. MoIn Tor1a+/− mice, A2A receptor grains appeared either increased in number in the striatum and globus pallidus, or reduced in number in the entopeduncular nucleus B,D,F, cop = 0.013) and in the globus pallidus , and their lower density in the entopeduncular nucleus  of Tor1a+/− (n = 4), compared with correspondent basal ganglia areas of the Tor1a+/+ mice (n = 4)  .n = 13, t = 2.077 df = 24, p = 0.04); in the globus pallidus, 2.342 ± 0.14 > 1.378 ± 0.06 SEM μm ; and in the entopeduncular nucleus, 4.938 ± 0.39 > 1.61 ± 0.08 SEM μm . However, the fluorescence intensity of the A2A positive grains was comparable in the correspondent areas of TOR1a+/+ versus TOR1a+/− (data not shown).While the numbers of A2A receptors were either increased in the striatal-pallidal complex or decreased in the entopeduncular nucleus, their size appeared smaller in all the basal ganglia structures of TOR1a+/−. A representative number of A2A positive grains were randomly selected from the basal ganglia of control and mutant mice: the mean perimeters per A2A positive grain in TOR1a+/+ versus TOR1a+/− were, respectively, (two tailed T test): in the striatum, 2.06 ± 0.19 > 1.62 ± 0.08 SEM μm  positive cholinergic interneurons of mutant Tor1a+/− mice. Double-labeling fluorescence was obtained, with ChAT reactive large neuronal bodies and thick primary dendrites marked in red B,E, and 2  in Tor1a+/+ mice, and 47.44 + 2.98 SEM μm2  in Tor1a+/− mice .The overlapping areas of A2A receptors on striatal ChAT neuronal bodies were 36.37 + 2.92 SEM μmIn our study, different techniques demonstrate specific changes in A2A receptors in the basal ganglia circuits of Tor1a+/− mice, with increased or decreased expression, respectively, in the striatal-pallidal complex and entopeduncular nucleus. We briefly discuss the limits of our morpho-chemical study, and the possible relevance of A2A changes to the pathophysiology of dystonia in this DYT1 animal model.The high specificity of the polyclonal antibody was demonstrated in the Western blot analysis, with a selective band corresponding to the 45 kDa migration level of A2A receptor monomers , and by In previous studies with electron microscopy, the A2A labeling in the striatum was most commonly localized in dendrites and dendritic spines, in fewer amounts in axon terminals and in very low levels in neuronal soma and glia, whereas in the external globus pallidus (GP), the A2A labeling was mainly presynaptic ,29. In oThe high distribution of adenosine A2A receptors in the striatum and globus pallidus of control Tor1a+/+ mice and their selective increase in the same areas of Tor1a+/− mice suggest a major role of A2A within the indirect pathway for basal ganglia physiology and dystonia pathophysiology. However, it is worth noting that besides the selective postsynaptic localization of A2A receptors on striatal medium spiny enkephalin neurons in the indirect pathway , a morphOur immuno-fluorescence technique and confocal microscopy with high power objective demonstrate the increased distribution density of A2A in Tor1a+/− mice in the neuropil, likely targeting dendrites and spines in the striatum and globus pallidus, as well as on the soma of the striatal projecting neurons, striatal cholinergic interneurons, and neurons in the globus pallidus. The widespread increase in A2A receptors in the striatal-pallidal complex may have functional relevance, affecting acetylcholine release in the striatum and regulating neuronal excitability and plasticity on cortico-striatal glutamatergic terminals, on GABAergic projecting neurons, and on GABAergic terminals in the globus pallidus ,25,31,32Moreover, the light A2A expression generally observed in the entopeduncular nucleus of Tor1a+/+ mice  and its To verify whether the tissue levels of cAMP are influenced by the changes in A2A expression, we evaluated the basal content of cAMP in tissue homogenates of the different basal ganglia nuclei from Tor1a+/+ and Tor1a+/− knock-out mice. Surprisingly, we found significantly increased cAMP levels only in the striatum of Tor1a+/−, whereas in the globus pallidus and in the entopeduncular nucleus, cAMP levels were similar in Tor1a+/+ and in Tor1a+/− mice. The cAMP synthesis in striatal neurons is stimulated by dopamine D1 receptors, which are significantly decreased in DYT1 transgenic mice , and by To explain the discrepancy between the higher levels of cAMP in the Tor1a+/− striatum and the unchanged cAMP levels in the globus pallidus and entopeduncular nucleus, we should consider the interference of other related factors in addition to the increased or decreased stimulating action of the A2A receptors. We can hypothesize that the A2A receptor dependent cAMP synthesis is out of the inhibitory control of the deficient D2 receptors, but the D2 receptors appear to be functionally impaired both in the striatum as well as in the globus pallidus ,32.In a previous work, we reported that PDE10A-dependent cAMP hydrolyzing activity as well as total PDE activity are unaffected in the striatum of a mutant mouse model of DYT1 dystonia, wherein at the same time the PDE10A-dependent cAMP hydrolyzing activity was increased in the globus pallidus and decreased in the entopeduncular nucleus . TherefoIndeed, PDE10A is selectively expressed in medium spiny neurons, but not in striatal interneurons, and in particular it is absent in cholinergic interneurons ,39. A2A Anyway, the relevant impact of cAMP on the membrane excitability of striatal medium-sized spiny neurons and cholinergic interneurons has been widely described ,42,43. OFinally, to evaluate whether the changes in A2A expression in Tor1a+/− mice are regulated at translational or post-translational levels, we used the sensitive real-time PCR technique analysis of A2A mRNA in basal ganglia. According to previous studies, the A2A mRNA can be found only on the striatum in cell bodies of medium spiny striatal neurons and occasionally in dendrites, coding for the synthesis of A2A receptors, which would be transported to the terminals of striatal-pallidal efferent neurons till the globus pallidus . Within Instead, a surprising A2A mRNA significant increase was observed in the globus pallidus of Tor1a+/−, whereas A2A mRNA was detected just in a very low amount in the globus pallidus of Tor1a+/+. Therefore, in Tor1a+/− it is likely that the globus pallidus is coding for its own A2A receptors, and such A2A neo-expression may change, at least in part, its dependent role from the striatal A2A inputs. The A2A mRNA changes in the basal ganglia of Tor1a+/− appear to be specific, since in Parkinson’s disease A2A mRNA appears either decreased in the striatum or unchanged in the globus pallidus . Future Tor1a mutation [C57BL/6 Tor1a+/− knock-out mice, which mimic the loss of function of the DYT1 dystonia mutation , were brmutation . All theFor biochemical studies, the animals were killed by cervical dislocation, and the brains were removed rapidly and placed on an ice-cold plate. Thick brain sections were cut with an Oxford vibratome, and the caudate–putamen, globus pallidus and entopeduncular nucleus were dissected out rapidly from both hemispheres under a stereomicroscope, and promptly frozen in liquid nitrogen and stored at −80 °C .For morphological studies, the animals were deeply anesthetized with tiletamine/zolazepam (80 mg/kg) and xylazine (10 mg/kg) and perfused trans-cardially with 1% heparin in 50 mL 0.1 M sodium phosphate buffer (PBS), and with 250 mL 4% para-formaldehyde in 0.1 M in PBS (pH 7.4). The brains were removed immediately and post-fixed in the same fixative solution overnight at 4 °C; then they were equilibrated with 30% sucrose overnight, and finally frozen in liquid nitrogen and stored at −80 °C .v/v Triton X-100, 5 mM β–mercaptoethanol, 1 mM PMSF and 2% v/v antiprotease cocktail . Thirty micrograms of proteins were loaded on a 9% SDS polyacrylamide gel and subjected to electrophoresis under a reducing condition. The proteins were then transferred to a nitrocellulose membrane . The blots were incubated overnight at 4 °C with a rabbit polyclonal anti–A2A receptor antibody , or mouse anti-β-actin  as a reference standard. A2A receptors-reactive bands were revealed by horseradish peroxidase-conjugated secondary antibodies , incubated in a lumi-light-enhanced chemiluminescence substrate , and exposed to Chemidoc . Densitometric analysis of scanned blots was performed using the NIH ImageJ version l.29 program .The quantitative analysis of A2A expressions in basal ganglia was assessed by Western blotting. Tissues were lysated in 20 mM Tris-HCl buffer pH 7.2 containing 0.2 mM EGTA, 5 mM MgCl2, 0.1% g for 30 min at room temperature and the supernatants were acetylated, according to the instructions of the commercial kit manufacturer . The kit uses a polyclonal antibody to cAMP, which in a competitive manner binds the cyclic nucleotides in the sample, or cyclic nucleotides conjugated with alkaline phosphatase molecules added to the incubation medium. The samples were incubated for 2 h at room temperature, and thereafter the substrate p-nitrophenyl phosphate (pNPP) was added for 1 h to reveal the residual alkaline phosphatase activity in the medium, which generates a yellow product, readable on a microplate reader  at 405 nm. The intensity of the yellow color in the medium is inversely proportional to the concentration of cyclic nucleotides in the samples and in comparative standards.The frozen samples were immediately submerged in 0.1 M HCl, and rapidly homogenized. The homogenates were centrifuged at 13,000× −ΔΔCt method was used to evaluate the relative expression ratio for A2A receptors compared with the internal control gene β–actin. The following sequences of primers were used: A2A receptors (a) forward 5′–TCTTCTTCGCCTGCTTTGTCC–3′, (b) reverse 5′–GCCCTCATACCCGTCACCA–3′; β-actin (a) forward 5′–GCGCAAGTACTCTGTGTGGA–3′, (b) reverse 5′–AAGGGTGTAAAACGCAGCT–3′.Total RNA was prepared using the TRI Reagent , according to the manufacturer’s instructions, and quantified with a NanoDrop 1000 spectrophotometer . One microgram of RNA was reverse-transcribed using the QuantiTect reverse transcription kit . Real-time PCR was performed on reverse-transcription products with the PowerUp™ SYBR™ Green Master Mix in the QuantStudio 3 Real-Time PCR System . Thermal cycling conditions consisted of an initial denaturation step at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 1 min. The threshold cycle (Ct)  was used for comparison analysis. The 2Coronal brain sections (40 μm thick) were cut with a freezing microtome. Tissue sections were then incubated for 1 h at room temperature in 10% donkey serum solution in PBS 0.25%–Triton X-100 (PBS-Tx). The sections were incubated with the primary rabbit anti-A2A antibody  1:200, 3 days at 4 °C; thereafter, the sections were incubated with cyanine 3 (cy3) conjugated secondary antibody  1:200, 2 h at room temperature. For negative controls, representative sections were processed with the omission of the primary or of the secondary antibody. Cell nuclei were detected with a blue-fluorescent DNA stain by 4′,6–diamidino–2–phenylindole (DAPI) . After washout, tissue sections were mounted on plus polarized glass slides with Vectashield mounting medium  and cover-slipped.z-stack projections in the z-dimension  were collected. Z-stack images were acquired to analyze the whole neuronal soma, which spans multiple confocal planes. The confocal pinhole was kept at 1, the gain and the offset were adjusted to prevent saturation of the brightest signal and sequential scanning for each channel was performed. The confocal settings, including laser power, photomultiplier gain, and offset, were kept constant for each marker. Fluorescent images were acquired with a LSM700 Zeiss confocal laser scanning microscope , with a 5×, a 20× objective, or a 63× oil immersion lens  with an additional digital zoom factor (1×–1.5×–2×) under no saturation conditions. Single-section images (1024 × 1024) or A negative staining was obtained omitting the primary or the secondary antibody. The 5× and 20× objectives were used to define areas of interest in the dorsolateral striatum, globus pallidus, and entopeduncular nucleus; the distribution of A2A receptors was first acquired using 20× objective with an additional digital zoom factor (1×–1.5×–2×), and thereafter 63× oil immersion objective .n ≥ 4 mice/genotype), and exported for analysis with ImageJ software . Software background subtraction was utilized to reduce noise.For quantitative analysis of the number of A2A positive spots per microscopic field and of their perimeters, images were collected from at least 3–4 slices per animal, processed simultaneously from each basal ganglia nucleus (A double immunofluorescent staining for choline-acetyltransferase (ChAT) and A2A was used to evaluate the colocalization of A2A receptors in striatal cholinergic interneurons. Brain sections were incubated with goat anti-ChAT  and rabbit anti-A2A receptors . Both primary antibodies were used at a 1:200 dilution, in 0.1 M PBSS containing 0.3% Triton X–100 for 72 h at 4 °C. Sections were then rinsed three times for 5 min at room temperature and subsequently incubated with cyanine 2 (Cy2) and cyanine 3 (cy3) conjugated secondary antibodies  for 2 h at room temperature at 1:200 dilution in a 0.1 M PB solution containing 0.3% Triton X–100. For negative controls, representative sections were processed with the omission of the primary or of the secondary antibodies. Cell nuclei were detected with a blue-fluorescent DNA stain DAPI . Subsequently, sections were rinsed in PBS, mounted on plus polarized glass slides with Vectashield mounting medium  and cover-slipped.The sections were preliminary examined under an epi-illumination fluorescence microscope . Confocal laser scanner microscopy  was used to acquire immunofluorescent images as reported in the previous subsection. Immunofluorescence intensity and colocalization analysis were evaluated by using the Java image processing and plugin analysis program included in Fiji ImageJ . To quantify the density of the A2A specific marker on a defined area, the “overlapping signal” of the A2A signal on ChAT neuronal bodies was calculated.p value of < 0.05 was considered statistically significant.Biological samples and microscopic fields were randomly selected from the basal ganglia areas of the two experimental groups. All data were initially entered into an Excel database  and the analysis was performed using the Statistical Package for the Social Sciences Windows, version 15.0 . Descriptive statistics consisted of the mean ± standard deviation (SD) for parameters with gaussian distributions (after confirmation with histograms and the Kolgomorov–Smirnov test), or of the mean ± standard error mean (SEM). Comparisons were performed in compliance with data characteristics either with the Paired Samples Test, or ANOVA one-way or ANOVA two factors and multiple comparisons by Bonferroni test. A The increased expression of A2A receptors in the striatal-pallidal complex of Tor1a+/− knock-out mice may suggest a gaining of function of such receptors, overwhelming the D2 loss of function in the indirect pathway; moreover, the associated decrease in A2A expression in the entopeduncular nucleus may be functionally significative. Future studies should clarify the relevance of A2A changes in the direct and indirect pathway in dysregulating the basal ganglia network in dystonia pathophysiology.


Cerebrospinal fluid (CSF) is the liquid that fills the brain ventricles. CSF represents not only a mechanical brain protection but also a rich source of signalling factors modulating diverse processes during brain development and adulthood. The choroid plexus (CP) is a major source of CSF and as such it has recently emerged as an important mediator of extracellular signalling within the brain. Growing interest in the CP revealed its capacity to release a broad variety of bioactive molecules that, via CSF, regulate processes across the whole central nervous system (CNS). Moreover, CP has been also recognized as a sensor, responding to altered composition of CSF associated with changes in the patterns of CNS activity. In this review, we summarize the recent advances in our understanding of the CP as a signalling centre that mediates long-range communication in the CNS. By providing a detailed account of the CP secretory repertoire, we describe how the CP contributes to the regulation of the extracellular environment—in the context of both the embryonal as well as the adult CNS. We highlight the role of the CP as an important regulator of CNS function that acts via CSF-mediated signalling. Further studies of CP–CSF signalling hold the potential to provide key insights into the biology of the CNS, with implications for better understanding and treatment of neuropathological conditions. Drosophila [A singular feature of the CNS, crucial to its function following neural tube closure and homeostasis throughout adulthood, is the presence ofCSF, which represents the key signalling interface between various distant regions of the CNS. CSF occupies an intricate and interconnected network of ventricles and cavities spanning the whole CNS that collectively give rise to the brain ventricular system. This complex component of CNS architecture consists of four brain ventricles—the paired lateral ventricles, 3rd ventricle, and 4th ventricle, that are connected to the central canal of the spinal cord and the subarachnoid space . PreservFor most of history, CSF has been assumed to primarily act as a fluid cushion providing mechanistic protection to the brain, an osmotic buffer system, and a route for clearance of metabolic waste and toxic compounds from the brain. This rather narrow view has been recently challenged by the growing evidence pointing to the expanded role of CSF as a conduit for delivery of instructive cues involved in the regulation of multiple aspects of CNS embryogenesis, adult neurogenesis, and modulation of adult brain function . These fThe CP is a secretory tissue located within each of the brain ventricles that is present in all vertebrates . ConsideIn this review, we aim to provide an overview of recent advances regarding the composition of CSF from the standpoint of CP secretome and its multifaceted impact on the regulation of various aspects of CNS embryogenesis and maintenance in adulthood.Specific aspects of neural tube development in vertebrates permit early and complete separation of CSF from the surrounding environment, thus allowing for precise regulation of its content early on during embryogenesis . The impApart from other functions related to the mechanical protection and metabolic turnover, CSF constitutes a signalling environment indispensable for proper growth and functional maturation of the CNS. eCSF has been demonstrated to be crucial for proper execution of genetic programs underlying embryogenesis of various CNS regions as well as control of adult neurogenesis ,26. A grIn addition to its role as a vehicle for distribution of bioactive molecules, hydrodynamic forces associated with CSF flow establish instructive cues that are able to activate ion channels expressed by adult neural stem cells (NSCs) in direct contact with CSF, which can act as mechanosensory receptors, regulating adult neurogenesis . InteresThe ability of the4 CSF to reach and affect processes in various regions of the CNS is not limited to the cell populations in direct contact with the ventricular space. Instead of simply being drained into the blood stream via arachnoid villi granulations , it has Given the function of CSF as an essential route for the long-range trafficking of factors across the CNS, CSF has been also explored as a potential source for biomarkers providing information allowing for early detection and diagnosis of distinct pathological conditions such as neurodegenerative diseases or various types of brain cancer ,40.Taken together, CSF constitutes crucial element of the CNS extracellular microenvironment with increasingly appreciated roles that extend beyond being a simple mechanic buffer or drainage system for the brain metabolism. A flurry of recent discoveries revealed CSF as a signalling nexus distributing and integrating signals, consisting of a wide array of bioactive compounds, within the whole CNS.The choroid plexus (CP) is a secretory tissue protruding into the lumen of all brain ventricles, namely the lateral ventricle CP (LV CP), the 3rd ventricle CP, and 4th ventricle CP (4V CP), in the form of a sheet of epithelial cells that are in direct contact with the CSF and encapsulate richly-vascularized stroma . Unlike As such, the CP represents a complex tissue that fulfils distinct roles essential to the CNS function. Several lines of evidence clearly established the CP as the major site for CSF production , despiteHowever, the key functional feature of the CP, conferred by the presence of junction proteins in the epithelium ,45, is tSod3 gene expression, encoding a metabolic enzyme, is limited to the adult 4V CP, whereas the expression pattern for protein kinase encoded by the Penk gene, is completely reversed [Despite shared morphology and function, embryonic CPs preserve their specific domain identities. They reflect position of the CP along the midline axis and underlie distinct transcription signatures and heterogeneous proteomic profiles observed between different embryonic CPs ,60. Intrreversed ,52, revereversed ,60. Therreversed ,67. OverBma1, Per1, and Per2, which allow the CP to influence activity of the key hypothalamic centre involved in the sleep/wake rhythmicity via secreted signals carried by CSF [Another emerging aspect of CP function is the intrinsic ability to sense and respond to changes in the CSF as well as broader physiological changes. It has been recently shown that the CP expresses genes encoding components of circadian clock machinery, such as d by CSF ,69. Remad by CSF , which id by CSF . It is nd by CSF ,73. Reced by CSF .Due to the expression of specific receptors, the CP has been also shown to respond to the presence of neurotransmitters present in CSF such as serotonin or nicotine, which are able to elicit robust changes in the CP metabolism and transcriptome ,76. In aCSF plays an important role as the key modulator of neuroinflammation. CSF contains distinct pools of activated immune cells, which can be enriched in various neurodegenerative diseases such as Alzheimer’s disease (AD) ,79. MoreBy virtue of its strategic location at the centre of the brain ventricular system, possession of barrier-like properties enabling tight control over CSF content, close contact with blood-borne signals, robust secretory capacity, and ability to sense changes in the local environment, the CP is uniquely poised to act as a master regulator of long-range signalling in the CNS. The CP thus acts as a principal nexus for integration and transmission of signals along the brain–body axis.Deciphering the identity of molecular components underlying the diversity of CSF-mediated signalling along with the identification of the key sources of these factors has become undoubtedly one of the most exciting directions pursued in the field of the CSF research ,92, withEmerging knowledge of CP transcriptome and proteome profiles together with growing insight into the CSF content brought into the spotlight the CP as an important source for a plethora of biologically-active compounds found in the CSF at different developmental stages and physiological states ,27,60. EWhile the roles for most of these compounds remains unknown, some initial findings indicate their involvement in diverse biological processes. We have previously shown that Wnt-5a, secreted by embryonic 4V CP, is able to influence the morphogenesis of the developing cerebellum . LikewisRbp4 or Wisp1, or endothelial cells producing NT3, raising an interesting possibility that these cell subpopulation actively contribute to the spectrum of signalling molecules released from the CP into CSF [Further expanding the array of secreted substances, the CP has been recently established as the major source of another important class of regulatory molecules contained in the CSF - microRNAs . Given iinto CSF ,107.The CP is also involved in active transport of various blood-borne molecules, including cerebral transport of various micronutrients such as folate or iron, both essential for proper brain development ,120. In Further exploration of the ability of the CP to shape CSF content led to recent findings that have established the CP as the key producer of binding proteins and multimolecular complexes regulating the extracellular transport of various signalling molecules via CSF ,123. TheExosomes represent a novel class of membranous extracellular vesicles increasingly recognized for their role as messengers involved in the long-range distribution of various compounds with regulatory functions , thus beInterestingly, exosomes’ cargo varies over time, a process that may contribute to the age-dependent changes in the CNS function . ExosomeLipoproteins represent another important group of extracellular particles produced by the CP . LipoproUnderscoring the signalling potential of lipoproteins, low-density lipoproteins isolated from eCSF are responsible, to a significant extent, for neurogenic activity of the eCSF . On the Similarly to exosomes, lipoproteins were also shown to incorporate and actively transport miRNAs . Further2+ influx into neuronal cells probably as a function of the ability of ApoJ to directly bind to Aβ in the CSF [Aside from serving solely as scaffolding proteins for lipoproteins, apolipoproteins recently emerged as regulatory molecules in their own right with importance for the brain function . Interac the CSF ,165. Mor the CSF . On the  the CSF . Moreove the CSF , thus id the CSF .CSF-mediated signalling and neurogenesis are intimately linked processes with fundamental role in the development and homeostasis of the CNS. This is highlighted by the fact that neural precursors remain in close contact with CSF throughout life . NeuroepAdult qNSCs, reside in two main anatomically-restricted germinal regions, the subventricular zone (SVZ), localized in the wall of the lateral ventricles, and the SGZ in the hippocampus ,175. qNSDirect contact between neurogenic brain regions and CSF is the hallmark of CNS biology throughout life. CSF is the chief source of trophic factors and instructive cues underlying the key aspects of embryonic development and CNS patterning. The importance of CSF is also conserved in adulthood, when it plays a key role in the regulation of adult neurogenesis and the perturbation of its content is involved in numerous pathological conditions. Considering major breakthroughs in our understanding of CP function, it is becoming increasingly evident that the CP is the major player regulating signalling properties of CSF. This view is further emphasized by a growing list of signalling factors and transporting vesicles either directly produced in the CP or actively transferred from blood across the CP, which acts as a selective barrier between blood circulation and CSF (summarized in