Complex barriers at the brain's surface, particularly in development, are poorly defined. In the adult, arachnoid blood-cerebrospinal fluid (CSF) barrier separates the fenestrated dural vessels from the CSF by means of a cell layer joined by tight junctions. Outer CSF-brain barrier provides diffusion restriction between brain and subarachnoid CSF through an initial radial glial end feet layer covered with a pial surface layer. To further characterize these interfaces we examined embryonic rat brains from E10 to P0 and forebrains from human embryos and fetuses (6–21st weeks post-conception) and adults using immunohistochemistry and confocal microscopy. Antibodies against claudin-11, BLBP, collagen 1, SSEA-4, MAP2, YKL-40, and its receptor IL-13Rα2 and EAAT1 were used to describe morphological characteristics and functional aspects of the outer brain barriers. Claudin-11 was a reliable marker of the arachnoid blood-CSF barrier. Collagen 1 delineated the subarachnoid space and stained pial surface layer. BLBP defined radial glial end feet layer and SSEA-4 and YKL-40 were present in both leptomeningeal cells and end feet layer, which transformed into glial limitans. IL-13Rα2 and EAAT1 were present in the end feet layer illustrating transporter/receptor presence in the outer CSF-brain barrier. MAP2 immunostaining in adult brain outlined the lower border of glia limitans; remnants of end feet were YKL-40 positive in some areas. We propose that outer brain barriers are composed of at least 3 interfaces: blood-CSF barrier across arachnoid barrier cell layer, blood-CSF barrier across pial microvessels, and outer CSF-brain barrier comprising glial end feet layer/pial surface layer. For decades the meninges have been considered to be no more than a protective shield, but increasing evidence suggests that the complex outer barriers of the brain also function as morphogenic signaling centers  barriers, the arachnoid barrier is a third barrier forming an important interface between blood and CSF in the brain. Dyes injected into the blood tend to stain dura but not the arachnoid suggesting that the arachnoid rather than the dura prevents entry of blood-borne substances into the subarachnoid space Broman, . Later, d Muller  startingd Muller  that inid Muller .Differentiation of the meninges also involves formation of the subarachnoid space. Thus, the spaces that appear in the inner reticulated layer of the primary meninx become confluent and develop into the subarachnoid space and cisterns during late embryonic and early fetal period. Leptomeningeal cells condense at the inner surface of the dura and become arachnoid barrier cells and at the brain surface they form the pial surface layer. In newborn rat and in early fetal human brain the subarachnoid space extends from the arachnoid barrier cell layer to the basement membrane of the pial surface layer and contains CSF and blood vessels bridged by leptomeningeal cells that also form traversing arachnoid trabeculae (see Discussion).Recent investigations have changed the established view of the meninges as merely protective membranes suggesting that they contribute to neural tissue homeostasis by secreting several trophic factors including FGF2, insulin-like growth factor-II and CXCL12/SDF-1  and a CSF-brain barrier (across the ventricular zone) ] and 11 fetuses (38–200 mm CRL) corresponding to 6–21st weeks post-conception (wpc) and two adult brain specimens were examined. The embryos and the fetuses were obtained from legal abortions, as previously described  followed by streptavidin-conjugated DyLight 594 . Finally, a nuclear counterstain with DAPI  was added for 3 min, before sections were coverslipped. Double labeling was performed with the combination of antibodies against BLBP and SSEA-4, YKL-40 and SSEA-4, YKL-40 and IL13-Rα2, and YKL-40 and EAAT1. Details of the primary antibodies including dilutions and suppliers are listed in Table Sections were deparaffinized in xylene and rehydrated using graded alcohols following standard protocols. Endogenous peroxidase was quenched using a 0.5% solution of hydrogen peroxide in methanol for 15 min. Following rinses with TRIS buffered saline , non-specific binding was inhibited by incubation for 30 min with blocking buffer  or 0.2% casein  at room temperature. Prior to overnight incubation at 4°C with primary antibodies diluted in blocking buffer, some antibodies required heat induced epitope retrieval with either citrate buffer (pH6) or TEG buffer (pH9) . At E18 meninges, the subarachnoid space and cisterns were fully developed and immunostaining for claudin-11 demonstrated a strong distinct reactivity of the entire arachnoid blood-CSF barrier Figures . The barThe second trimester human fetal subarachnoid space between the arachnoid blood-CSF barrier and the outer CSF-brain barrier contained numerous bundles of collagen, many blood vessels and a meshwork of leptomeningeal cells forming trabeculae. Immunostaining for collagen 1 clearly delineated the subarachnoid space as shown in a section from a mid-gestational human fetus in Figure In late embryos and early fetuses immunostained for BLBP, SSEA-4, and YKL-40 the entire parenchymal lining of the outer CSF-brain barrier, corresponding to radial glial end feet, exhibited strong membranous immunoreactivity of the conically or deltoid shaped end feet with simple straight membranes. The parenchymal lining in the second trimester showed multiple curly closely apposed delicate end feet membranes best appreciated in a stacked series of confocal images of YKL-40 stained marginal zone Figure . At mid-Slc1a3), were used as representative examples to illustrate dynamic functions of the end feet layer. Double-immunolabeling of brains from human fetuses in the second trimester with antibodies against IL-13Rα2 and YKL-40  The blood-brain barrier proper across the cerebral endothelial cells where YKL-40/SSEA-4 are present in pericytes  lies in the presence of tight junctions between the cerebral endothelial cells of the vasculature of the brain parenchyma. Solute transport mechanisms and restrictions have been studied thoroughly in developing and adult blood-brain barrier and choroid plexuses blood-CSF barrier, discovering distinct, comprehensive and changing transport systems, which indicate that the choroid plexuses blood-CSF and blood-brain barriers are functionally mature with respect to small molecules in early development  was found in neural progenitor cells in forebrains of human embryos and early fetuses  is a member of the lipid-binding protein family that carries small hydrophobic signaling molecules between cellular compartments but also a marker of radial glial cells  an arachnoid blood-CSF barrier across the arachnoid barrier cell layer, (ii) a pial microvessel blood-CSF barrier across the pial microvessels and (iii) an outer CSF-brain barrier across the radial glial end feet layer covered by a pial surface layer and later in development glia limitans with accompanying pial basement membrane. Our findings of claudin-11 in the arachnoid blood-CSF barrier and EAAT1 and IL-13Rα2 in the outer CSF-brain barrier confirm their barrier properties and the spatiotemporal distribution of YKL-40/BLBP/SSEA-4 further establish the necessity of regarding these interfaces as separate entities, since they may play different roles in normal brain development and possibly in several neurological disorders. We therefore propose an elaboration of the model for developing brain barriers as defined by Saunders et al.  and diviThe Review Editor Gavin Clowry declares that, despite having collaborated with author Kjeld Mollgard, the review process was handled objectively. 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.


In particular, there is debate surrounding the route of CSF drainage. Our aim was to quantify CSF flow, volume, and drainage route dynamics ICP was recorded in young (2–5 months) and aged (16 months) SHR. Contrast was administered into the lateral ventricles bilaterally and sequential CT imaging was used to visualize the entire intracranial CSF system and CSF drainage routes. A customized contrast decay software module was used to quantify CSF flow at multiple locations.p = 0.03). Contrast was observed throughout the entire intracranial CSF system and was seen to enter the spinal canal and cross the cribriform plate into the olfactory mucosa within 9.1 ± 6.1 and 22.2 ± 7.1 minutes, respectively. No contrast was observed adjacent to the sagittal sinus. There were no significant differences between young and aged rats in either contrast distribution times or CSF flow rates. Mean flow rates (combined young and aged) were 3.0 ± 1.5 μL/min at the cerebral aqueduct; 3.5 ± 1.4 μL/min at the 3rd ventricle; and 2.8 ± 0.9 μL/min at the 4th ventricle. Intracranial CSF volumes  were 204 ± 97 μL (8.8 ± 4.3%) in the young and 275 ± 35 μL (10.8 ± 1.9%) in the aged animals (NS).ICP was significantly higher in aged rats than in young rats (11.52 ± 2.36 mmHg, versus 7.04 ± 2.89 mmHg, in vivo. These results indicate substantial drainage of CSF via spinal and olfactory routes, but there was little evidence of drainage via sagittal sinus arachnoid granulations in either young or aged animals. The data suggests that spinal and olfactory routes are the primary routes of CSF drainage and that sagittal sinus arachnoid granulations play a minor role, even in aged rats with higher ICP.We have demonstrated a contrast-enhanced CT technique for measuring and visualising CSF dynamics  Cerebrospinal fluid (CSF) dynamics are thought to be altered in many pathological conditions including hydrocephalus ,2, idiopThe role and location of CSF drainage has been studied in both animals and humans. Traditional interpretation has been that most CSF drains into the venous sinuses via arachnoid granulations -15. The in vivo. We chose to compare young and aged spontaneously hypertensive rats (SHR) because their cerebral ventricular volume is thought to increase with age [To investigate CSF dynamics and drainage within the entire rat brain we developed a novel contrast-enhanced computed tomography (CT) technique to image the rat CSF system in three-dimensions. One unique feature of this technique is that the dynamic nature of CSF drainage can be observed with age ,37, whicwith age ,2. UsingAll animal experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by The University of Newcastle Animal Care and Ethics Committee. Experiments were performed on two cohorts of male spontaneously hypertensive rats (SHR) . One group weighed 200-360 g, aged 2–5 months (n = 5); the other group weighed 360-400 g, aged 16 months (n = 5). Due to ethical constraints, we calculated the sample size required to detect a difference in baseline ICP between two different rat strains . Using 2:O2 via a facemask. It has previously been reported in dogs that isoflurane causes no significant change in the rate of CSF production [Animals were anaesthetised with isoflurane  in 50:50%, Noduction . RespiraIntracranial pressure (ICP) was recorded prior to scanning using a SAMBA microcatheter as previously described  with minFollowing baseline arterial pressure and ICP measurement, animals remained on the same base plate and stereotaxic frame, which was positioned on the CT scanner table. All imaging was performed using a 64-slice clinical CT scanner  ,42. The For each animal, serial CT images were loaded into MiStar software . Motion correction was performed, and the resulting images were subtracted from the baseline non-contrast image.A representative 3D reconstruction of the rat CSF system was created by generating a Maximum Intensity Projection (MIP).The MIP was loaded into the MiStar fusion 3D render module. Thresholds were applied to the 3D render to highlight the CSF system.The time taken for injected contrast to reach specific anatomical landmarks within the CSF system was quantified in each animal. The anatomical landmarks included the cerebral aqueduct, 3rd ventricle, 4th ventricle, spinal canal, basal cisterns and cribriform plate. The contrast window range was set to 0–250 Hounsfield Units (HU) for each animal sequence to prevent the false detection of signal noise at each image time-point. The time taken for the contrast to reach each landmark was calculated by analysing each sequential image.Contrast-enhanced CT images were processed using the MiStar software decay module specifically written for rat imaging. CSF flow rate measured at the cerebral aqueduct was used to estimate CSF production. Flow rates were also calculated elsewhere within the CSF system. A small region-of-interest (ROI) with 6 voxels was positioned within the centre of the aqueduct on the 10 minute time-point scan (post-infusion). The total change in Hounsfield Units for voxels within the ROI was plotted over time, and an exponential decay curve was fitted to the first five decay data points. The exponential decay rate constant is directly proportional to flow within the ROI. This principle was applied to each voxel to generate the Decay Rate Map, measured in ml/min/100 L (which was then converted to μl/min/100 ml). The ROI was then co-registered with the Decay Rate Map to obtain the flow value. Flow was converted into μl/min by multiplication with volume (slice thickness × area of ROI). The process was repeated (with the same fixed ROI) at the 3rd ventricle and 4th ventricle of each animal.Twenty five 0.6 mm coronal CT slices were analysed slice-by-slice to calculate brain and CSF volume. On each slice, a threshold of 100 HU was set and ROI software tools were used to define the area of total brain tissue (using baseline scans) or contrast-enhanced CSF within the cranium. On each slice, the volume was calculated as slice thickness × area of ROI. The combined sum of the ROI volumes was calculated.p < 0.05 level. T-tests are also reported for physiological variables for illustrative purposes, with p-values uncorrected for use of multiple comparisons. Unless otherwise stated, data is expressed as mean ± standard deviation (±SD).Statistical tests were performed using GraphPad Prism Version 6 for Windows . Two-tailed Student’s t-test was used to compare differences in CSF production rate, and CSF volumes between young and aged groups. Significance was accepted at the p = 0.01). Intracranial pressure (ICP) was measured in all animals and traces revealed consistent pulse and respiratory waveforms. A significantly higher ICP was found in aged rats, 11.52 ± 2.36 mmHg, versus 7.04 ± 2.89 mmHg in the younger animals, p = 0.03. Cerebral perfusion pressure  was significantly lower in the young versus the aged animals .Arterial blood pressure (systolic/diastolic) was measured in 3 young animals and 5 aged animals. Mean arterial blood pressure was lower in the young versus the aged animals  and via the olfactory pathway (through the cribriform plate), the optic nerves, and the cervical lymph nodes in all animals Figure . ContrasCSF flow was able to be quantified at multiple sites within the ventricular system, but flow quantification in the extraventricular locations was not possible due to respiratory motion artifact  and contrast dilution, resulting in reduced signal-to-noise ratio and partial volume averaging error caused by the extensive surface area of each drainage route. CSF production rates, as measured by CSF flow rate at the cerebral aqueduct, were 3.14 ± 2.1 μL/min and 2.79 ± 0.57 μL/min in the young and the aged, respectively. CSF flow rates were 3.74 ± 1.93 μL/min and 3.19 ± 0.70 μL/min at the 3rd ventricle and 2.89 ± 0.21 μL/min and 2.70 ± 1.29 μL/min at the 4th ventricle , PhD Candidate (Human Physiology).QY- PhD (Physics).MP- PhD, FRACP (Neurology).CL- MBBS, B Med Sci (Hons), FRACP (Neurology).DB- B. Biomed Sci. (Hons), PhD Candidate (Human Physiology).NS- MBBS, PhD, FRACP (Neurology).DM- PhD (Human Physiology).Contrast-enhanced cerebrospinal fluid flow through the cranium over 60 minutes. Radio-opaque contrast (20 μl) was simultaneously injected into each lateral ventricle at 2 μl/min for 10 min while plain CT images (0.6 mm slice thickness) were taken over 60 minutes. File format: mov.Click here for file3D render reconstruction of cerebrospinal fluid system of the rat. The computed tomography images of one rat were loaded in MiStar software and subtracted from the baseline non-contrast image. A Maximum Instensity Projection was generation and loaded into the MiStar fusion 3D render module. File format: mov.Click here for file


HIV-1 RNA can be found at higher levels in cerebrospinal fluid (CSF) than in plasma, termed CSF/plasma discordance. The clinical significance of CSF/plasma discordance is not known and the degree of discordance considered important varies. We aimed to determine whether a panel of CSF cytokines, chemokines and associated mediators were raised in patients with CSF/plasma discordance at different levels.10) and ‘low discordance’ . CSF mediators significant in univariate analysis went forward to two-way unsupervised hierarchical clustering based on the patterns of relative mediator concentrations.A nested case-control study of 40 CSF samples from the PARTITION study. We used a cytometric bead array to measure CSF mediator concentrations in 19 discordant and 21 non-discordant samples matched for plasma HIV-1 RNA. Discordant samples were subdivided into ‘high discordance’ . In cluster one all mediators had relatively high abundance; this included 18 discordant samples and three non-discordant samples. In cluster two all mediators had relatively low abundance; this included 18 non-discordant samples and one non-discordant sample with ultrasensitive discordance only.10) should also be investigated as mediator profiles were similar to those with discordance >1log10. Sensitive testing may have a role to determine whether ultrasensitive discordance is present in those with low level CSF escape.CSF/plasma discordance is associated with potentially damaging neuroinflammatory process. Patients with discordance at lower levels (ie. 0.5–1log Some studies have defined discordance as CSF HIV-1 RNA 1logSensitive testing can now detect HIV-1 RNA below the traditional threshold of 40 or 50 copies/ml. A proportion of patients have CSF/plasma discordance detected on these assays that would not otherwise be classified as discordant if tested with standard assays This nested case-control study aimed to assess whether CSF/plasma discordance was associated with cytokines, chemokines and accompanying mediators in the CSF, when compared with non-discordant samples. Furthermore we aimed to examine the relationship between CSF mediator profile and degree of CSF/plasma discordance to determine whether discordance at lower levels (ie levels that have been excluded in some published studies) are associated with potentially damaging CNS inflammation.22.1Patients were recruited prospectively through the PARTITION study, which is a multicentre UK study described elsewhere Patients in the LLV group underwent a brief cognitive screen using the International HIV Dementia scale 2.2CSF cell count, protein and glucose measurements, microbiological investigations, and CD4 cell counts were performed by routine methods at the centres of care. As lumbar puncture was performed for research purposes in the LLV group, local CSF testing was only performed in this group if considered clinically indicated by the treating physician. HIV-1 RNA was measured centrally in simultaneous plasma and CSF samples by the Abbott RealTime HIV-1 assay  with a lower limit of quantification of 40 copies/ml, as previously described 2.310 times that measured in a simultaneous plasma sample. In those with unquantifiable plasma HIV-1 RNA, discordance was defined as a CSF HIV-1 RNA >0.5log10 times the lower limit of assay quantification (ie. 40 copies/ml). Samples were categorised as “high discordance” if CSF HIV-1 RNA was >1log10 times that of plasma and “low discordance” if the ratio was 0.5–1log10. Samples with discordance only detected on sensitive assay were termed ‘ultrasensitive discordance’ and analysed within the low discordant group.Discordant samples were selected from the clinical and LLV groups. In the clinical group patients with CNS infection were excluded. Discordance was defined as CSF HIV-1 RNA >0.5logFor controls, we screened all participants in the PARTITION study who did not have discordance, and selected those with a plasma HIV-1 RNA which most closely matched that of samples with discordance. Those with the lowest CSF HIV-1 RNA were selected.CSF viral escape was defined as plasma HIV-1 RNA less than the lower limit of quantification for the standard assay (ie 40 copies/ml), and CSF ⩾40 copies/ml.2.4γ, tumour necrosis factor [TNF]α and its soluble receptors [TNFR1 and TNFR2] and TNFα-related apoptosis inducing ligand [TRAIL], interleukin [IL] 1α, 1β, 2, 4, 6, 8, 10 and 17a, IL17f, and IL1-receptor antagonist [IL1RA]. Analytes were assessed for all patient groups on one plate for MMPs and one plate for all other mediators. Fluorescence intensity was determined using a Bio-Rad platform . Standards and samples were analysed in duplicate and the mean value used in analysis. Standard curves were adjusted at the points of fluorescence intensity saturation, to generate a sigmoid standard curve with 6–8 fluorescence intensity points. To avoid undetectable levels of mediators or missing data biasing the analysis, only mediators that were detected in at least 80% of the cohort were included in the analyses CSF was collected at recruitment and centrifuged to remove cells. Samples were stored at −70 °C and freeze thaw cycles minimised. Thirty-seven mediators  were assessed using a cytometric bead array assay system in accordance with the manufacturers’ instructions  as described previously CSF mediators significantly (p < 0.05) associated with CSF/plasma discordance in univariate analysis were included in a 2-way unsupervised hierarchical cluster analysis based on the patterns of relative mediator concentrations. This offered an opportunity to examine the interactions of mediators and allow samples to be classified by their overall host mediator response. Samples and then mediators were hierarchically clustered using the program Cluster (version 3.0), using the uncentered correlation score as the similarity metric and the average branch linkage method. Results were viewed as heat-maps using Tree View software . Eleven (58%) had high discordance and eight (42%) had low discordance; of which three had ultrasensitive discordance – plasma/CSF HIV-1 RNA 4/40, <7/74 and <10/51 copies/ml . Median Demographic, clinical and laboratory parameters were similar between discordant and non-discordant samples although discordant patients tended to have a lower nadir CD4 . Two of 3.2In over 80% of samples the following mediators had measurements at or below the lower limit of quantification and hence were excluded from further analysis: GCSF, GM-CSF, IFNα2, IFNβ, IL17a, IL17f, IL2, IL4, TNFα, VEGFα, MMP1, 3, 8, 9, 12 and 13. Nineteen (90%) of the remaining 21 CSF mediators were significantly higher in discordant than non-discordant samples . This in3.310 plasma, all with CSF HIV-1 RNA 0.5–1log10 plasma, and two of the three with ultrasensitive discordance. The single discordant sample in cluster two had ultrasensitive discordance . Two of the three samples with CSF viral escape not meeting criteria for CSF/plasma discordance were in cluster two.The samples grouped into two main clusters based on relative mediator concentrations; cluster one with higher concentrations and cluster two with lower concentrations . ClusterThe 19 CSF mediators that were found to be significantly up-regulated in discordant samples in the univariate analysis clustered into two main sets . The fir4In patients receiving ART, the clinical significance of CSF/plasma HIV-1 RNA discordance is poorly understood, and the relevance of low levels of discordance has not been assessed. This study found that CSF/plasma discordance was associated with the increased expression of host inflammatory mediators in the CSF in comparison to non-discordant patients. Moreover, there was no significant difference in the concentration of these mediators between patients with high and low level discordance. In addition, the mediator profiles clustered cases with both high and low discordance together and were distinct from the non-discordant patients. Taken together these findings suggest that discordance of HIV is the CSF is associated with a potentially deleterious host inflammatory response and may therefore be of significance, and that this may have implications for patients even with low levels of discordance. Longitudinal studies assessing these mediators and CSF HIV-1 RNA in conjunction with clinical outcomes are needed to better elucidate the temporal relationship between discordance, host inflammatory response, and clinical outcomes. In addition, more work is needed to examine whether discordance could be a result of an inflammatory process, rather than the cause of it.Whether the neuroinflammation observed in association with discordance led to adverse clinical outcomes in this study is not clear. Neurological symptoms occurred both in those with and without discordance in both groups. Just one patient in the LLV group had an abnormal cognitive screen, however this is an insensitive test for milder forms of cognitive impairment. Cognitive function in HIV positive cohorts is highly variable and impairment is multifactorial, hence to identify insidious neurological damage related to long-term low-grade neuroinflammation would require larger cohorts and more detailed cognitive testing than was performed in our study Surprisingly we did not find an association of CCL2 (MCP-1) with discordance. This chemokine is associated with chemotaxis of monocytes across the blood-brain barrier and has been associated with cognitive impairment in HIV positive populations in the pre and post-combination ART era Samples with higher degrees of CSF/plasma discordance tended to have higher CSF mediator concentrations. This effect was not strong and the difference between low and high discordant groups was not statistically significant for any single mediator. This is consistent with data from the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) study, the largest cohort in this area, which found that although the absolute level of CSF HIV-1 RNA was not related to cognitive performance, the presence of discordant virus was 10 difference and/or <200 copies/ml in CSF) may risk type two error, and the use of these criteria in clinical practice risks missing patients with a potentially significant host inflammatory response 10 should be considered. This is consistent with our previous work demonstrating drug-resistant HIV-1 in CSF in patients with 0.5–1log10 discordance and <200 copies/ml in CSF When taken together the majority of samples with low CSF discordance had raised CSF mediator concentrations clustered in a similar pattern to those with high discordance. Only one low discordant subject had low CSF mediator concentrations and was grouped with the non-discordant cluster. Our data suggests that the criteria for CSF/plasma discordance used in some studies (ie. those excluding patients with <1logReports have suggested that CSF HIV-1 RNA detection at levels below 50 copies/ml may be associated with cognitive impairment Some studies have demonstrated no association between CSF HIV-1 RNA levels and cognitive performance 510) should also be investigated as mediator profiles were similar to those with discordance >1log10. Sensitive testing may have a role to determine whether ultrasensitive discordance is present in those with low level CSF escape.CSF/plasma HIV-1 RNA discordance is associated with a potentially damaging neuroinflammatory process. Patients with discordance at lower levels , ICON, GlaxoSmithKline, AstraZeneca and the Medicines Evaluation Unit. BDM is an NIHR Academic Clinical Lecturer and received funding as part of a NIHR Doctoral Research Fellowship.SN received a research award for this work from the British HIV Association. SN is a MRC Clinical Training Fellow supported by the North West England Medical Research Council Fellowship Scheme in Clinical Pharmacology and Therapeutics, which is funded by the None.


CSF), and the BBB ratio were significantly higher in the ICH than in controls (p < 0.05). Significantly higher levels of CLDN5, OCLN, ZO-1, MMP-9, and VEGF in CSF were observed in the ICH group; these biomarkers were also positively associated with BBB ratio (p < 0.05). Our data revealed that circulating TJs could be considered the potential biomarkers reflecting the integrity of the BBB in ICH.Brain injury after intracranial hemorrhage (ICH) results in significant morbidity and mortality. Blood brain barrier (BBB) disruption is a hallmark of ICH-induced brain injury; however, data mirroring BBB disruption in human ICH are scarce. The aim of this study was to assess the significance of circulating biomarkers in evaluating BBB disruption after ICH. Twenty-two patients with ICH were recruited in this study. Concentrations of the tight junction proteins (TJs) Claudin-5 (CLDN5), Occludin (OCLN), and zonula occludens 1 (ZO-1) and vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were measured by using enzyme-linked immunosorbent assay in serum and cerebrospinal fluid (CSF) samples obtained from patients with ICH. The white blood cell (WBC) count in blood and CSF, albumin (ALB) levels in the CSF (ALB Spontaneous intracranial hemorrhage (ICH) results in significant morbidity and mortality, thirty-day case fatality rates which range from 40% to 50% in most studies –3. HyperThe BBB inhibits transcellular or paracellular passage of molecules across it by an elaborate network of complex tight junctions between the endothelial cells . Tight jThe proteins released from damaged cells into the bloodstream would reveal the BBB disruption, which might be potential biomarkers. Protein S100B appears in plasma after ICH and its levels correlated with ICH outcome . AlbuminBiomarkers reflecting the severity of ICH could increase the discriminative power for outcome evaluation. Two areas of extensive research are neuroimaging and circulating biomarkers. Circulating TJs are currently becoming the hallmark of BBB integrity . A recenComputed tomography (CT) and magnetic resonance imaging (MRI) are the key part of the initial diagnosis, which demonstrate the hematoma size and location of the hemorrhage. Standard CT can detect up to 95% of ICH; however patients presented several days after a small ICH may have little blood visible on a CT scan. MRI is known to overestimate the size of microbleed (the “blooming effect”), with MRI diameter on average more than 150% of pathological lesions , 26. It This prospective study was approved by the Shantou University Medical College; written informed consents were obtained from the patients or their surrogates. Twenty-two patients were recruited during 2013-2014. Patients suspicious of ICH underwent immediately CT scanning or MRI after hospital admission. The diagnosis of ICH was confirmed by CT or MRI. The Digital Imaging and Communications in Medicine (DICOM) format was analyzed centrally for the measurement of hematoma size. As the CSF is very hard to get in health individuals, seventeen patients with disease other than ICH were enrolled as the control for comparison of the biomarkers in CSF. The details of control were shown in The size of the ipsilateral hemisphere and the infarcted area were measured using a standard computer-assisted analysis technique (ImageJ). Calculation of hematoma size was performed using the formula: size of infarcted area/ipsilateral hemisphere × 100 and shown as percentage of the whole hemisphere. The sizes were shown as mean ± SD.Paired serum and CSF samples were obtained on patients admission. Lumbar puncture was used for diagnostic or treatment purposes. CSF sample was collected before treatment. Samples were immediately centrifuged, and supernatants were stored at −80°C until assessment. CSF cytology, total protein, and electrolyte were measured in all of the patients before therapy. The serum biomarkers were measured according to the same protocol as described for the CSF. VEGF and MMP-9 in CSF were measured by ELISA (R&D America Company). CSF and serum TJs levels were measured by ELISAs (ELISA kits from Cusabio (America) company).The assessment of the BBB integrity in the patients and the controls was performed as previously described . We explIndex of OCLN and ZO-1 was calculated with the same equation.U test, Spearman rank correlation, a multivariate regression analysis, and binary logistic regression were applied in the study. A cluster analysis (CA) and a principal component analysis (PCA) were performed to obtain significant principal components. p < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS for Windows version 10.0. All continuous variables were reported as medians with interquartile ranges (IQR). Mann-Whitney Patient demographic and clinical data were summarized in CSF) and blood were significantly higher in the ICH than in the controls (p < 0.05), indicating inflammatory status existed in ICH patients. We did not find significant differences in parameters  between the ICH patients and the controls (p > 0.05). The ALB in CSF (ALBCSF) and BBB ratio in ICH group were significantly higher than in controls (p < 0.05); on the contrary, ALBSerum in ICH was significantly lower than in controls (p > 0.05).The WBC in CSF  observed in ICH than in controls; specifically, CLDN5CSF was 8.28-fold higher than that in control, OCLNCSF was 18.78-fold higher than in control, and ZO-1CSF was 5.16-fold higher than in control (p < 0.05). However, serum levels of CLDN5, OCLN, and ZO-1 had no difference between ICH group and control groups (p > 0.05). Our data revealed that circulating TJs increased in CSF but not in serum of patients suffering from ICH. We did not find significant differences of CLDN5/OCLN or CLDN5/ZO-1 index (both in serum and in CSF) between ICH and controls (p > 0.05)  .CSF, and WBCCSF)  and BBB ratio . No relationship was found between CSF TJs and WBCCSF (p > 0.05), indicating that the inflammatory status may not affect the BBB integrity. The ratio of CLDN5/ZO-1 was positively associated with BBB ratio  and ALBCSF . However, no relationship was found between serum TJs concentrations and the BBB conventional biomarkers (p > 0.05). In addition, no relationship was found between circulating TJs and area of hemorrhage (p > 0.05). Our data revealed that the efficiency of concentrations of CLDN5CSF, OCLNCSF, and ZO-1CSF was higher than serum TJs and other conventional biomarkers in reflecting BBB integrity.A Spearman rank correlation was used to analyze the correlations among circulating CLDN5, OCLN, and ZO-1 and the conventional biomarkers known to be associated with the BBB integrity  . In the CSF and MMP-9CSF were measured in ICH patients and the controls. Our data found that VEGFCSF and MMP-9CSF were significantly higher than those in control (p < 0.05). VEGFCSF was positively associated with the BBB ratio , while MMP-9CSF was positively related to BBB ratio and ALBCSF  . With respect to the VEGFCSF and MMP-9CSF, they had high specificities and sensitivities. Collectively, our data showed that TJs in CSF have high sensitivity and specificity in diagnosis of ICH.Diagnostic value of TJs levels in mirroring the BBB integrity was also evaluated. The sensitivities, specificities, positive predictive value (PPV), and negative predictive value (NPV) of circulating TJs in ICH diagnosis were shown in CSF, CLDN5CSF, OCLDCSF, ZO-1CSF, and MMP-9CSF. The second model included OCLNSerum, ZO-1Serum, DBP, and SBP. The third model included WBCCSF and DBP. However, the AUC value of Component 1 was 0.928, and the sensitivity was 81.8%, and the specificity was 94.1%, not higher than the single parameter from the overall level   to choosl Tables  and 7. TThe need for new therapeutic approaches for ICH has prompted a search for the molecular and cellular mechanisms underlying early or delayed brain damage after ICH . It is dCSF were significantly higher in patients with ICH than the individuals who had no BBB damage. Together with a strong correlation existing between TJs concentration in CSF and the BBB ratio in patients with ICH, our data demonstrate the levels of TJsCSF could be the potential biomarker for mirroring the extent of BBB opening in patients with ICH. In clinical practice, blood biomarkers are the first choice as many of them assist in rapid diagnosis, therapeutic decisions, and easier collection. Unfortunately, we could not find significant elevation of serum TJs nor correlation between CSF and serum's TJs. There may be two reasons to explain; the first one may be due to too small sample size in our study; statistical significance may be gained in a larger cohort. But there may be another reason: no correlation existed between CSF and serum. BBB breakdown is a local event of CNS; TJs elevated in CSF have difficulty inducing significant elevation of TJs in serum, especially considering the CSF volume versus serum volume. Further study is needed to elucidate the details. In conclusion, CSF biomarkers may have high sensitivity and specificity in reflecting CNS pathologic status, which is supported by our data that CSF levels of TJs had higher AUC and high sensitivity and specificity compared to serum TJs and other conventional serum biomarkers studied.In previous study, the observed BBB opening after SAH was related to the damage of tight junction . The molCSF in ICH patients demonstrated that damaged BBB integrity leads to peripheral WBC transferred into the CNS. The TJs' completeness in BBB strictly depends on signals provided by the CNS microenvironment [CSF change the permeability of the BBB, participating in the opening of the BBB in ICH patients [BBB disruption increases cerebrovascular permeability, allowing the entrance of leukocytes into the brain parenchyma which can in turn cause edema formation . Higher ironment . Increasironment . There iironment  and invoironment . Neutropironment . In our ironment . MMP-9 dironment –44. Elevpatients , 46. OurCSF in ICH patients is significantly higher than that in control, which is positively associated with the BBB ratio, revealing that higher concentration of VEGFCSF contributes to the BBB permeability in ICH. VEGF perturbs TJ integrity by decreasing OCLN and ZO-1 expression and causing CLDN5 and ZO-1 protein disruption [CSF, indicating that higher VEGFCSF may be a crucial factor in BBB high permeability in ICH [Both BBB disruption and consequent vasogenic edema determine the clinical course of ICH , 47. It sruption . Positivy in ICH .In some cases, the diameter of brain parenchyma blood vessels affected by ICH is too small to be observed by conventional imaging techniques. A recently proposed model of cerebral microscopic hemorrhage suggested that transient loss of endothelial barrier function might be an underlying process . HyperteCSF, VEGFCSF, and TJsCSF could be sensitive biomarkers as their expressions in some patients were upregulated just after ICH (data not shown). TJs have been considered attractive targets for transient breakdown of the BBB in therapies for various CNS disorders [CSF, CLDN5CSF, OCLDCSF, ZO-1CSF, and MMP-9CSF, which could explain 81.76% of total variance in the data set. To find out whether the panel TJs in our study can reflect more accurately the BBB disruption and early brain injury after ICH than conventional biomarkers, larger sample size is needed.Although early diagnosis and intervention may be paramount to improving patient prognosis , little isorders . A panelisorders . When weThe main limitation of TJs in CSF as the diagnostic biomarker is that LPs are not to be routinely done in some patients with ICH. Under this circumstance, circulating biomarkers and image detection should be carefully used according to patient's status. Another limitation of our study was that we could not measure the TJs at the different time points. Also, we have enrolled relatively small numbers of subjects that mainly have significantly large hematoma size. Our study will have better clinical significance if studying subject includes the patients with microbleeding; however, the patients' choice remains a challenge. Given that the CSF TJs serve diagnostic purposes for BBB disruption after ICH, further study will be needed to identify that the biomarkers could be translated into routine clinical practice. The sensitivity, specificity, PPV, and NPV for CSF TJs in diagnosis of ICH also need to be evaluated.


To study if diet is associated with human plaque stability, atherosclerotic plaques from carotid endarterectomy on 56 patients (21 Portuguese and 35 Swedish) were analysed by IRMS and histology. Plaque components affecting rupture risk were measured. Swedish plaques had more apoptosis, lipids and larger cores, as well as fewer proliferating cells and SMC than the Portuguese, conferring the Swedish a more rupture-prone phenotype. Portuguese plaques contained higher δ13C and δ15N than the Swedish, indicating that Portuguese plaques were more often derived from marine food. Plaque δ13C correlated with SMC and proliferating cells, and inversely with lipids, core size, apoptosis. Plaque δ15N correlated with SMC and inversely with lipids, core size and apoptosis. This is the first observational study showing that diet is reflected in plaque components associated with its vulnerability. The Portuguese plaques composition is consistent with an increased marine food intake and those plaques are more stable than those from Swedish patients. Marine-derived food is associated with plaque stability.Mediterranean diet has been suggested to explain why coronary heart disease mortality is lower in southern than northern Europe. Dietary habits can be revealed by isotope ratio mass spectrometry (IRMS) measurement of carbon (δ Coronary heart disease (CHD) mortality differs markedly across Europe and is generally lower in the southern than in the northern and eastern parts of the continent2346789101113C) and nitrogen (δ15N) can be used to differentiate between different types of terrestrial and marine-derived food (To what extent dietary habits can influence CHD risk by direct effects on atherosclerotic plaque structure is not known. Using isotope ratio mass spectrometry (IRMS) it is possible to estimate the dietary origin of molecular components incorporated into biological tissues by analysing the composition of certain isotopes13ved food .To understand the role of diet in CHD mortality, we investigated the dietary origin of the nitrogen and carbon molecules present in atherosclerotic plaques obtained from Portuguese and Swedish carotid surgery patients. We also analysed how the dietary origin of the plaque nitrogen and carbon molecules related to structural components associated with plaque stability.There were no significant differences in clinical characteristics between Portuguese and Swedish patients, except that the use of statins was more common in Swedish patients and the time between the clinical event and operation that was shorter in the Portuguese patients .We first analysed the structural components important for plaque vulnerability in both Portuguese and Swedish plaques. Swedish plaques were found to have more apoptotic cells, increased levels of lipids and larger cores than Portuguese plaques . The SweIn 13C and δ15N isotopes in Swedish (in blue) and Portuguese (in red) plaques demonstrated markedly different patterns  and maize , than the Swedish diet, which in general contains a significant amount of potatoes , as well as other C3 foodstuffs . The δ13C and δ15N values now measured in the plaque samples further support this epidemiologic data.There is a wide range of possible speculations for the differences in mortality between southern and northern EuropeIt is well documented that the majority of acute cardiovascular events are caused by rupture of atherosclerotic plaques with a thin fibrous cap. Morphological examinations have demonstrated these plaques are characterized by inflammatory infiltrates (primarily macrophages), large necrotic cores, extensive intra- and extra-cellular lipid accumulation, cell death and reduced smooth muscle repair capacity789101225There are some differences between the patient cohorts that could affect our findings. (1) The time of collection (Portuguese plaques 2000–2001 vs. Swedish plaques 2005–2011), (2) the time between the clinical event and surgery  and (3) the use of statins (more common in Swedish patients). Data from the AtheroExpress biobank have shown that atherosclerotic plaques obtained in 2010–2011 had a more stable phenotype than plaques obtained in 2001–200213C and δ15N values do not only reflect the diet, but also depend on the type of tissue17Our results using samples from Portugal and Sweden should be interpreted with caution, as they cannot be directly extrapolated to other regions of Europe. Large multinational studies ideally evaluating the characteristics of the plaques in asymptomatic patients through Europe are now required. Our study cannot provide exact amounts of dietary elements or even exact types of maritime/seafood dietary sources. The IRMS results also have to be interpreted with some caution, as the δTaken together, these data indicate that the atherosclerotic plaque composition is consistent with the intake of certain dietary atoms. The composition of the Portuguese atherosclerotic plaques is associated with the increased intake of seafood and those plaques are more stable than those from Swedish patients.This study is pioneer in showing that marine-derived food is associated with plaque stability. Despite the need for further studies, the ultimate clinical implication of this knowledge is to encourage a simple and cheap strategy, as marine dietary intake in prevention of atherosclerosis, to stabilize rupture-prone plaques that ultimately lead to myocardial infarction and stroke.We studied human atherosclerotic plaques obtained by carotid endarterectomy from 56 patients: 21 Portuguese and 35 Swedish. The Swedish patients underwent carotid surgery at the Skåne University Hospital, Malmö, Sweden, during 2005 to 2011 and the Portuguese were operated in 2000–2001 at the Cardiovascular Institute of Lisbon, Lisbon, Portugal. The patients’ characteristics are described in All methods were carried out in accordance with the approved guidelines. The study was approved by the local ethical committee . All patients gave informed consent.13C/δ15N analysis and one for histology. The plaque samples for the isotope fractionation analysis were dissected into different regions .All plaques were snap-frozen in liquid nitrogen at endarterectomy. Two consecutive 1-mm-thick transverse sections of the most stenotic region were sliced from each sample, one for δ13C, δ15N) analysis was performed at IRMS facility at the Environmental Isotope Laboratory (EIL) at University of Waterloo, Ontario, Canada. The required weight of each sample was 0.25-0.30 mg. The ratios of the samples were calibrated against several different standards of known isotopic composition. The analytical precision obtained for the standards was <0.3‰ for N and <0.2‰ for C (1σ)The samples were dried and prepared as previously describedIn Situ Cell Death detection kit POD  was used, according to manufacturer’s instructions. Proliferation was assessed by staining with mouse monoclonal [PC10] anti-human proliferating-cell nuclear antigen (PCNA) proliferation marker (ab29) . Sections were subsequently incubated with biotinylated polyclonal rabbit anti-mouse F(ab)2  for 30 minutes and then with peroxidase-labelled streptavidin . Measurements of the area of plaque (% area) for the lipids, macrophages, smooth muscle cells, apoptosis and proliferation, as well as the core region were quantified blindly using Biopix Q 2.1.8  after scanning with ScanScope Console Version 8.2 .The fragments were cryosectioned in transversal 8 μm sections, fixed with Histochoice , dipped in 60% isopropanol and in 0.4% Oil Red O in (60%) isopropanol (for 20 min) to stain lipids. For macrophage assessment, primary monoclonal antibody mouse anti-human CD68, clone KP1 , diluted in 10% rabbit serum 1:100, and secondary antibody biotinylated polyclonal rabbit anti-mouse, rabbit F(ab´)2 , dilution 1:200 in 10% of rabbit serum, were used. For smooth muscle cells , primary antibody monoclonal mouse anti-human smooth muscle actin clone 1A4 , diluted in 10% rabbit serum 1:50, and secondary antibody biotin rabbit anti-mouse Ig , dilution 1:200 in 10% of rabbit serum, were used. To visualize apoptotic cells in plaques, TUNEL  13C and δ15N was approximately normal (skewness <0.33) and no log transformation was applied. Values are presented as mean . Two-group comparisons were performed with Chi-square or Mann-Whitney test. Spearman’s rho test was used for correlation analysis. Multiple linear regressions, with δ13C or δ15N as dependent variables, were used to adjust the relationship between histologic plaque components and δ13C and δ15N, for potential confounding factors. Age, gender, current smoker, diabetes, country of origin and histological plaque component were entered into the regression model. We also investigated whether presence of symptoms, time between symptom and operation, statin use, degree of stenosis and date of the operation, could act as confounding factors for the relationship between isotope pattern and histological plaque components. However, these factors showed no substantial effect on this relationship and were therefore not included in the final regression model. Differences were considered statistically significant at P < 0.05. SPSS 21  has been used for statistical analysis.The distribution of δHow to cite this article: Gonçalves, I. et al. Direct association between diet and the stability of human atherosclerotic plaque. Sci. Rep.5, 15524; doi: 10.1038/srep15524 (2015).


The suboccipital muscles are connected to the upper cervical spinal dura mater via the myodural bridges (MDBs). Recently, it was suggested that they might work as a pump to provide power for cerebrospinal fluid (CSF) circulation. The purpose of this study was to investigate effects of the suboccipital muscles contractions on the CSF flow. Forty healthy adult volunteers were subjected to cine phase-contrast MR imaging. Each volunteer was scanned twice, once before and once after one-minute-head-rotation period. CSF flow waveform parameters at craniocervical junction were analyzed. The results showed that, after the head rotations, the maximum and average CSF flow rates during ventricular diastole were significantly increased, and the CSF stroke volumes during diastole and during entire cardiac cycle were significantly increased. This suggested that the CSF flow was significantly promoted by head movements. Among the muscles related with head movements, only three suboccipital muscles are connected to the upper cervical spinal dura mater via MDBs. It was believed that MDBs might transform powers of the muscles to CSF. The present results suggested that the head movements served as an important contributor to CSF dynamics and the MDBs might be involved in this mechanism. The suboccipital area consists of complex anatomical structures. The myodural bridge (MDB) as dense soft tissue communication extends from the suboccipital musculatures to the cervical spinal dura mater123456789101112131412345789101345787810714The cardiac cycle-related CSF flow pulsations were depicted as CSF flow waveforms. Based on the waveforms, the CSF flow quantitative parameters of the craniocervical junction were calculated. And the results of both scans and the comparison between them are presented in The temporal CSF dynamics parameters were presented in the The CSF dynamics amplitude parameters were presented in the The CSF dynamics volumetric parameters included the mean values of the systolic CSF flow volume (VS), the diastolic CSF flow volume (VD) and the net-flow volume   and werep value, 0.002).Furthermore, the detailed CSF stroke volume variations after the one-minute-head-rotation period were analyzed and results were presented in p value 0.320).In the above results, among forty cases, a small number of measurements were found as outliers based on Box and Whisker Plots analysis. It was impossible to re-measure the subjects with outliers, so these outliers were not included in T-test. Additionally, the intervals between the one-minute-head-rotation and the second cine-PC scan ranged from 1.0–3.6 minutes, with a mean value of 1.9 ± 0.57 minutes, because of the process of re-positioning MRI scan ahead of the second cine-PC scan. During the scanning procedure, the heart beat of each subject was monitored. The mean heart rate values before and after the head rotation period were of 70.7 ± 8.8 and 69.9 ± 7.3 beats per minute, respectively, showing no significant change in heart rate after the head rotation period , including most researchers involved in this study, was subjected to MR imaging . Informed consent was obtained from all of them and none of them had any history of cardiovascular, neurological, endocrine and cervical disorders.2; section thickness, 5 mm; and 2 signal intensity average. The encoding direction was in the head-to-foot orientation for all volunteers. PC- images were obtained at each time points, for a total of 25 measurements equally distributed over the cardiac cycle. The imaging time duration varied between 2 to 4 minutes, depending on the volunteer’s heart rate.A cine-PC MR imaging methodBefore the cine-PC scan, sagittal T2 weighted images of head and neck were obtained by a quick MR scan to provide anatomic details. Based on the median sagittal images, a transverse plane for the cine-PC scan was designated at the level of the atlas upper border a.Each volunteer was scanned twice. The first scanning was performed after a resting period. After that, each volunteer was required to perform continuous head rotations for a period of 60 seconds, while lying on their back on the MRI bed and their heads against the bed. The head rotation speed was set at around 0.5 cycles per second. One head rotation cycle was counted when the head was rotated from median position to one side and next by another side and then return. Volunteers turned their heads according to the researcher’s instructions for normalization of the one-minute-head-rotation. A scan for re-positioning underwent immediately after the one-minute-head-rotation period and then the second cine-PC scan followed.Cine-PC images were transferred to an independent workstation (aw46mr) for CSF flow analysis using the Analyze (Report Card 4.0) software. Using the region-of-interest function of the Analyze software, an irregular contour was drawn manually to encompass the entire cross-sectional area of the subarachnoid space at the level of the atlas upper border . The regp value of less than 0.05 indicated a statistically significant difference.The range, mean, and standard deviation were calculated for each parameter. All parameters were compared between both scans acquired before and after the one-minute head rotation period. Statistical significance was calculated using a paired sample t-test or Chi-Square test. A How to cite this article: Xu, Q. et al. Head movement, an important contributor to human cerebrospinal fluid circulation. Sci. Rep.6, 31787; doi: 10.1038/srep31787 (2016).


Acute ischemic stroke is common and disabling, but there remains a paucity of acute treatment options and available treatment (thrombolysis) is underutilized. Advanced brain imaging, designed to identify viable hypoperfused tissue (penumbra), could target treatment to a wider population. Existing magnetic resonance imaging and computed tomography-based technologies are not widely used pending validation in ongoing clinical trials. T2* oxygen challenge magnetic resonance imaging, by providing a more direct readout of tissue viability, has the potential to identify more patients likely to benefit from thrombolysis – irrespective of time from stroke onset – and patients within and beyond the 4·5 h thrombolysis treatment window who are unlikely to benefit and are at an increased risk of hemorrhage.This study employs serial multimodal imaging and voxel-based analysis to develop optimal data processing for T2* oxygen challenge penumbra assessment. Tissue in the ischemic hemisphere is compartmentalized into penumbra, ischemic core, or normal using T2* oxygen challenge (single threshold) or T2* oxygen challenge plus cerebral blood flow  data. Penumbra defined by perfusion imaging/apparent diffusion coefficient mismatch  is included for comparison.n = 6) prior to serial multimodal imaging: T2* oxygen challenge, diffusion-weighted and perfusion imaging .Permanent middle cerebral artery occlusion was induced in male Sprague-Dawley rats ; 55 ± 41 to 37 ± 12 mm3 ; 84 ± 64 to 42 ± 18 mm3 , as ischemic core grew: 155 ± 37 to 211 ± 36 mm3 (single apparent diffusion coefficient threshold); 178 ± 56 to 205 ± 33 mm3 ; 139 ± 30 to 168 ± 38 mm3 . There was evidence of further lesion growth beyond four-hours .Across the different methods evaluated, T2* oxygen challenge combined with perfusion imaging most closely predicted 24 h infarct volume. Penumbra volume declined from one to four-hours post-stroke: mean ± SD, 77 ± 44 to 49 ± 37 mmIn conclusion, T2* oxygen challenge combined with perfusion imaging has advantages over alternative magnetic resonance imaging techniques for penumbra detection by providing serial assessment of available penumbra based on tissue viability. Following an acute ischemic stroke, the ischemic penumbra represents electrically inactive and metabolically compromised but potentially viable tissue which is the target for acute stroke therapies. It ultimately becomes incorporated into final infarct unless salvaged by reperfusion. The thrombolytic agent alteplase  is the only drug currently licensed to treat stroke. It may be administered within 4·5 h of stroke onset based on clinical symptoms and a noncontrast computed tomography (CT) scan to exclude intracerebral hemorrhage or major established hypodensity Thrombolysis decisions may soon be informed by more advanced imaging, identifying a potential responder population based on individual pathophysiology. Magnetic resonance imaging (MRI) perfusion imaging (PI)/diffusion-weighted imaging (DWI) mismatch is widely accepted as a marker of penumbral tissue, while DWI/FLAIR mismatch may have utility in predicting lesion age and is under investigation as a selection marker for thrombolysis when stroke onset time is unknown We have developed such an alternative approach for penumbra detection. T2* oxygen challenge (T2*OC) detects compensatory mechanisms in response to reduced cerebral blood flow (CBF), which may identify a distinct MRI tissue signature of penumbra. Penumbra displays a higher venous deoxyhemoglobin concentration than surrounding tissue due to increased oxygen extraction fraction (OEF) T2*OC may provide insight into the dynamic behavior of hypoperfused tissue, and better prediction of tissue viability compared with the PI/DWI paradigm. A small proof of concept study in acute stroke patients Compartmentalizing penumbra using two parameters  rather than using a single T2* threshold may improve the technique further and provide a more accurate assessment of penumbra. We sought to compare single- and dual-threshold analysis for penumbra quantification using T2*OC and to evaluate its utility for serial scanning to track penumbra fate over the first four-hours in a rat permanent middle cerebral artery occlusion (MCAO) model. Serial PI and DWI (zero to four-hours), and 24 h T2-weighted imaging provided information on ischemia, acute tissue injury, and infarct, respectively, and alternative assessments of penumbra from PI/apparent diffusion coefficient (ADC) mismatch and ADC lesion growth.All experiments were performed under a UK Home Office license, were subject to the Animals (Scientific Procedures) Act 1986) and approved by the local University Ethical Review Panel. Figure 986 and an = 6, 290–350 g, Harlan, Bicester, UK) with 5% isoflurane and maintained at 2% in medical air, enriched with oxygen (30%).Anesthesia was induced in male Sprague-Dawley rats  and blood gas analysis. Permanent MCAO (pMCAO) was achieved with a 5-0 silicon rubber-coated intraluminal monofilament advanced to block the origin of the middle cerebral artery MRI data were acquired on a Bruker Biospec  7-T/30-cm system equipped with an inserted gradient coil  and a 72-mm birdcage resonator.http://rsb.info.nih.gov/ij/). The average baseline signal (over five-minutes) was subtracted from the peak signal during OC, then divided by the average baseline signal and multiplied by 100.A single-shot, gradient echo EPI sequence  was used to assess T2*-weighted signal change during OC. The paradigm for the T2*-weighted OC sequence was five-minutes breathing air, followed by five-minutes breathing 100% oxygen, and then 10 mins breathing air. Four coronal MRI slices throughout MCA territory, selected to minimize the effect of susceptibility distortion around the ear canals, were used to generate T2*-weighted signal change maps using ImageJ  TE: 43 ms, TR: 4000·3 ms, matrix 96 × 96, FOV 25 × 25 mmA coronal RARE T2 sequence  was used for T2-derived infarct measurement at 24 h post-stroke.Volumes of tissue with abnormal T2*OC response were determined using two approaches: (1) applying a single threshold  to T2*OC maps in ImageJ to derive tissue volumes 2/s) were calculated using Paravision  and subsequently analyzed using ImageJ. CBF maps were also generated using ImageJ. The ADC lesion was thresholded using an in-house absolute ADC value of 0·59 × 10−3 mm2/s, which when applied to data at four-hours post-stroke generated an ADC lesion which matched 24 h (brain swelling corrected) infarct following permanent MCAO in Sprague-Dawley rats.Quantitative ADC maps . Following coregistration, T2*, ADC and CBF thresholds were applied to relevant images  for voxel-based analysis and volumetric measurements of tissue compartments.Voxel-based analysis, generated using codes written in Matlab  allowed unbiased analysis of the spatial, volumetric, and dynamic evolution of penumbra and ischemic core. Tissue was compartmentalized using thresholds set for two parameters: (1) T2* signal change and CBF for T2*OC data sets; and (2) ADC and CBF data sets for PI/ADC mismatch. Color-coded images were derived based on T2*OC signal change and CBF values, or ADC and CBF values enabling analysis of temporal changes in voxel distribution across ischemic core, penumbra, and nonischemic tissue.2) and then a volume by multiplying the areas by slice thickness (1·5 mm) and adding the volumes for the four consecutive slices. Group data were generated for each tissue compartment at each acute time-point poststroke for T2*and CBF, ADC/CBF, and from the single T2* threshold data sets.The number of voxels within each tissue compartment was counted and converted to an area by multiplying the number of pixels by the pixel area (0·0678 mm−3 mm2/sec).Large veins and venous sinuses were segregated from the main tissue compartments by excluding tissue with elevated T2*  and CBF . Ventricles were excluded from the ADC/CBF data sets by applying an ADC threshold value  and summing the data for either four or eight slices. Penumbral volume was derived by subtracting the ADC lesion volume at each time-point from the swelling-corrected 24 h infarct volume and was quantified over four slices, for direct comparison with T2*OC and PI/ADC mismatch and for all eight slices available from DWI.The absolute ADC threshold of 0·59 × 10Infarct was calculated from regions of hyperintensity on the RARE T2 scans and corrected for ipsilateral brain swelling and contralateral compression Ischemic core and penumbra volumes were summed and subtracted from edema-corrected infarct to calculate a prediction error for each method.t-test and Bonferroni correction. A paired t-test was used to analyze ADC-derived lesion volume at four-hours and T2-defined infarct at 24 h.Data are presented as mean ± SD. Comparison of physiological variables, lesion evolution, and volumetric differences in ischemic core and penumbra across the four methods were analyzed using one-way ANOVA with Student's paired 2) during OCs] were maintained within physiological limits. Recordings collected immediately before each OC (Table 2) or PaO2. A small increase in blood pH was recorded at four-hours compared with one-hour and 2·5 h time-points (P < 0·0001).Physiological variables [apart from partial pressure of oxygen in arterial blood  from the 24-h T2-defined (edema-corrected) infarct is presented in Table Volumetric Table  and spatThe sum of penumbra and ischemic core volumes, for the different methods, was compared with (edema-corrected) infarct volume to assess their predictive value Fig. . Dual thPositive identification of penumbra is not employed in the current imaging selection for intravenous rt-PA administration, which is based on exclusion of intracerebral hemorrhage or extensive established ischemic damage on noncontrast CT. While several observational clinical studies support perfusion/diffusion mismatch as a penumbral marker that may identify a ‘responder’ population, both within three-hours of onset and at times up to nine-hours after onset This study provides evidence for the utility of T2*OC serial imaging in preclinical acute stroke research, and improved predictive value when T2*OC is combined with CBF data using voxel-based analysis. The maintenance of the increased T2* signal during OC and its return to baseline following OC is consistent with T2*-weighted signal change reflecting differences either in penumbral oxygen consumption or vasculature response to OC compared with surrounding tissue per se may have supported penumbra tissue and attenuated ADC-lesion growth during the scanning session. In previous studies using this rat strain and stroke model, where no OCs were applied, less than 10 mm3 of mismatch remained by three to four-hours post-stroke. There is evidence in the literature supporting the potential of oxygen to prevent penumbra loss. In a published study where PaO2 was maintained at 235–240 mmHg, no deterioration in mismatch volume was apparent over the first four-hours post-stroke 2 attenuated ADC lesion growth without altering CBF, thereby preserving CBF/ADC mismatch, and reducing infarct volume in permanent and transient MCAO models As expected, a measureable volume of penumbra was detected with all four analysis methods at one-hour post-stroke and decreased in size with time, as tissue became incorporated into the ischemic core. Because a significant volume of penumbra was detected using all four methods at four-hour post-stroke, the OCs This pilot study was not designed to establish the superiority of one method over another, but to explore the relationship between T2*OC responsiveness and blood flow in the evolving stroke lesion: as such, it was not powered to detect statistical differences between techniques. The data demonstrate a trend to improved prediction of final infarct volume by T2*OC combined with perfusion imaging, which needs to be confirmed in a larger study. The potential for translation of T2*OC as a clinical imaging method has been established Finally, improvements in penumbra detection may be possible with susceptibility-weighted imaging. These sequences measure magnitude information, but they also exploit phase information, thereby producing T2*-weighted sequences which are exquisitely sensitive to the dephasing effects of paramagnetic substances such as deoxyhemoglobin, and provide enhanced contrast over conventional gradient echo sequences.Dual threshold T2*OC/CBF voxel-based analysis identified tissue with characteristics of penumbra within anterior cerebral artery/MCA collateral supply territory, which decreased in size over time. When compared with alternative methods, T2*OC combined with CBF produced the smallest error in predicting subsequent infarct volume. The additional information it delivers, complementary to current MRI techniques for stroke diagnosis, supports its potential use within the routine stroke imaging protocol and adds to evidence promoting T2*OC as a valuable adjunct to PI/DWI imaging for penumbra detection. There was some evidence that repeat OCs delayed the evolution of stroke-induced damage, but more prolonged normobaric hyperoxia and strategies (e.g. intravenous oxygen carriers) to improve oxygen delivery to penumbra are required to influence final outcome.


Lepidium meyenii Walpers (L. meyenii) and Lepidium peruvianum Chacon (L. peruvianum) are presented. The two isotypes correspond to two holotypes of Peruvian medicinal herb known under the same common name “Maca”, as originally deposited in the Herbarium of San Marcos University in Lima, Peru dated back to 1843 and 1990 respectively.Using Liquid Chromatography-Mass Spectrometry (LCMS) , profiles of the two isotypes labelled under the same common name Maca deposited in the Medicinal Plant Herbarium, in Australia and Poland, but identified under two different scientific names synonymous” to L. meyenii and L. peruvianum may be misleading. On the basis of presented results the scientific name L. meyenii, used since 1843 up-today for cultivated Peruvian Maca by numerous reference sources worldwide, including Regulatory Bodies in the USA, EU, Australia and most lately in China, appears to be used in error and should be formally revised.The results demonstrate distinct differences in taxonomy, visual appearance, phytochemical profiles and DNA sequences of the two researched Maca isotypes, suggesting that the two Maca specimens are dissimilar and formal use of the term “Lepidium peruvianum Chacon, provides all the characteristics peculiar to this historically-documented herb grown in Andean highlands, which may be linked to its traditional use and accepted functionality, confirmed in recent clinical study to be relevant to its present day use for expected dietary, therapeutic and health benefits.It is concluded, that the isotype of cultivated Peruvian Maca labelled under its scientific name  Peruvian Maca – a centuries-old South American medicinal herb restricted in distribution to high Andean plateaus above 4,000 m a.s.l. – has been traditionally used by indigenous Peruvians as a vital dietary supplement and an important staple food component in their diet -6. This Lepidium meyenii Walpers , which is usually formed after being fused with the taproot, resulting in a turnip- or reddish-like tuberous body  and Lepidium peruvianum Chacon (L. peruvianum) is presented.Therefore, in this paper, based on comprehensive Glucosinolate profiles in both aerial and subterranean parts, a comparison of phytochemical characteristics in specimens of the two isotypes deposited in the Australian and Polish Herbarium of Medicinal Plants under the same common name “Maca” of Peruvian origin, but labelled under two different scientific names - L. meyenii, nor any available holotype of L. peruvianum as deposited in the same Herbarium by Chacon , where Chacon has sourced the Maca holotype deposited in the USM Herbarium (Holotype #89129), the location, from where Maca was also sourced for previously reported clinical study  Herbarium of Medicinal Plant in Lismore, Australia and in Herbarium of Medicinal Plants at the Research Institute of Medicinal Plants (RIMP) in Poznan, Poland. Sub-samples of both specimens L. meyenii and L. peruvianum:Chromatographic (HPLC) spectra:  The glucosinolate profiles in the aerial and subterranean parts were comparatively examined by HPLC method. The method described by Piacente et al.  derived from black mustard was used as a reference substance.L. peruvianum and L. meyenii were ground in a Waring blender and extracted in methanol with 15 minutes sonication. The extracts were centrifuged at 3,500 rpm for 10 minutes and the supernatant sampled for analysis of Glucosinolates (as Benzyl glucosinolates and derivatives thereof) determined by HPLC using UV-Vis detection within the range 230-300 nm and between 2.1 and 4.0 min detection scan.Plant specimens were prepared for analysis by HPLC as follows: the dried sub-samples of aerial or subterranean parts of each Comprehensive phytochemical profiling was performed on a Phenomenex Luna C18, 4.6 × 100 mm, 3 μm HPLC column (ARL-TM125). An Agilent 1100 HPLC was equipped with a PDA detector and coupled to an Agilent 1100 Mass Selective detector with an APCI interface. Detection was attained between 190–600 nm and APCI Pos, 150eV respectively. The mobile phase employed was A: water 0.05% TFA, B: Acetonitrile 0.05% TFA from 10% B - 95% B over 20 mins, flow rate 0.75 mL/min.2PO4 over 6 mins followed by a gradient elution to 0.05 M KH2PO4: ACN over 19 mins. The column oven temperature was 40°C with a flow rate of 1.0 mL/min and a 10 μL injection volume. Glucosinolates were detected at 225 nm.Specific glucosinolate analysis was performed on a Phenomenex Synergi 4 μm, 250mm × 4.6mm HPLC column (ARL-TM125). However, in this part of the study, the column was eluted isocratically with 0.05M KHL. meyenii and L. peruvianum were then both used as “fingerprints” characterizing each analyzed subterranean part of Maca isotype. In one of several analytical HPLC separation cycles for subterranean parts of both isotypes, differences in resultant spectra were extended into 3D Maca spheroids.The resultant HPLC spectra and resolution profiles of Preliminary DNA identification: In order to extend HPLC to potential genetic profiling, an attempt has been made to compare the two isotypes from co-depositions in the Herbarium of Medicinal Plants located in the SCU Lismore, Australia and RIMP in Poznan, Poland, by applying routine DNA sequencing technique. Extraction of viable protein and isolated genomic DNA was performed on one gram of grated and pulverised under liquid nitrogen samples of dry subterranean parts of L. meyenii and L. peruvianum using procedure developed in Molecular Plant Breeding Laboratory, the Royal Melbourne Institute of Technology (RMIT). After confirmation of DNA presence amplification and DNA sequence detection was performed using a Hybaid thermocycler.Lepidium species, the following available sequences belonging to Lepidium draba  are absent or present in trace quantities in L. peruvianum (lower trace), while very distinctive flavonoid compound in L. peruvianum was not detected in L. meyenii. Those differences between the two Maca isotypes were further confirmed , it will be possible to express observed obvious differences in HPLC traces statistically and determine a degree of significance in selected section(s) of traces (say between 2.1 and 4 min detection scan) for the two tested isotypes.L. meyenii Walpers (LP-W) and hypocotyl of L. peruvianum Chacon (LP-C) demonstrate that lines numbered 1, 3, 7 and 9 representing LP-C show more bands as compared to corresponding lines 2, 4, 8 and 10 with the use of the same primer , the term also adopted by Chacon , 21.L. peruvianum and use the term root (root proper) in reference to the underground part of L. meyenii. The term “Maca hypocotyl” has been adopted and consistently used in many Maca-related publications , which have not as yet been described, indicates an existence of species level differentiation between L. meyenii and L. peruvianum. MS fragmentation spectra of those compounds are consistent with kaempferol and quercetin diglycosides , contained no Glucosinolates but typically contain flavonoids which are used by plants to adapt to light conditions. Plants of the same genus can be expected to have more secondary metabolite compositional commonality than plants of a different genus. In the case of s Figure , which hfoliatum . Some con Figure  would bem Figure , 4 and 5n the EU  and Austn the EU , 39 as “et al.  and p-methoxybenzylglucosinolate. Therefore, it is reasonable to assume, that the distinctive major peaks on HPLC trace for the hypocotyl of L. peruvianum may be assigned to those compounds. The HPLC trace for subterranean part of L. meyenii has essentially different profile from the one of L. peruvianum in terms of shape and retention time for its key detected components , it is worthwhile to demonstrate an appearance of different bands (with distinctive-smears) representing the two isotypes,which may indicate an existence of genetic differences and genetic polymorphism. Distinctive “smears” of DNA . It appears that subterranean part of cultivated Peruvian Maca, referred to in research publications under the two scientific terms – L. peruvianum and L. meyenii, is the only plant from Lepidium genus, which is confirmed in analytical and clinical work, as displaying wide range of therapeutic properties. It contains the nutrients and other active compounds which appear to be responsible for traditionally known since the times of Incas, medicinal and beneficial health effects linked to the use of this herb by natives of Peru and users in other countries throughout the world, where the herb is exported  started an investigative procedure to clarify the issue of an error in the scientific name used for Maca and some 3 years later, adopted Lepidium peruvianum Chacon as the scientific name representing cultivated Peruvian Maca, with official publication in Peruvian Normas Legales-375606  and who have been accused of improper motives, recommend and defend continued use of the name L. meyenii as the valid name for Maca in the USA. The ongoing public debate over the ‘correct’ botanical name for Maca is an interesting case where taxonomic and nomenclatural disagreements get in the way of the functioning of supply chains.It appears that, irrespective the formal adoption of the ian Maca , its oldian Maca . Here itian Maca  in theirL. meyenii and L. peruvianum are currently accepted for use as “synonymous”. In Australia, Food Standards Australia and New Zealand (FSANZ) accepts the use of Maca powder as non-traditional and not novel food under the scientific name Lepidium meyenii, although Lepidium peruvianum is considered as the synonym ’ medicines, without restriction (Lepidium meyenii” is also accepted by the ACCM (Lepidium peruvianum Chacon for Maca plant used as traditional Peruvian food. However, on the advice from the ACCM, TGA does not accept L. meyenii and L. peruvianum as separate species, due to the fact that the data provided to the ACCM are still inadequate to support the safety and quality of the L. meyenii as a new complementary medicine substance for use in listed medicines in Australia (In the EU 2007 Catalogue  both nam synonym . In 2006triction . The namthe ACCM  as an Auustralia .L. meyenii (L. meyenii is referred to Maca plant of Peruvian origin grown in Andean Highlands or to “Chinese Maca” cultivated in Qinghai-Tibet Plateau, Yunnan, where Maca plantations were apparently established in China back in 2003.In China, after initial approval for import of Maca products to China in 2002, Chinese National Ministry of Health has approved the use of Maca powder in 2011 as the new resource food under the scientific name  meyenii . HoweverLepidium peruvianum Chacon for cultivated Peruvian Maca – as the only plant, which was verified in clinical trials, as capable to induce its traditional functionalities for expected health benefits.It is reasonable to assume, that as with most changes, it will take time for academics, commerce, relevant Government and international Legislative Bodies to adopt correct scientific name Lepidium peruvianum Chacon should be reserved exclusively to cultivated Peruvian Maca plants with their distinctively-formed subterranean part tuberous roots - hypocotyls utilised for food and/or dietary supplement.Taking into account botanic, taxonomic and biochemical facts presented in this paper, it is concluded that the scientific name Lepidium peruvianum Chacon.The conclusion as presented in this paper is in line with a consensus of opinion reached by the panel of Peruvian scientific specialists and experts in taxonomy on Maca and other native plant species, who have corrected scientific name for cultivated Peruvian Maca, being now formally referred to by Peruvian Normas Legales 375606 as synonymous” should be reviewed for further definite research confirmation, formal acknowledgement and – if justified - corresponding correction in formal and practical terms internationally.The question still remains as to validity of conflicting international use of different scientific terminology attached to the common name “cultivated Peruvian Maca”, whether both isotypes represent different species under two alternative names and if yes - then, the use of the term “


HIV can spread into the central nervous system (CNS) early in the course of infection and this turns into intrathecal inflammation and neuronal damage. We aimed to investigate clinical and immunological parameters associated with elevated CSF VL in HIV-infected ART-naïve patients.HIV+ ART-naïve patients underwent a comprehensive battery of neurocognitive (NC) tests and lumbar puncture (LP) for CSF HIV-RNA detection. Plasma HIV-RNA and peripheral T-cell immune-phenotypes (CD38/CD45RA/CD45R0/CD127 on CD4/CD8) were also assessed (flow cytometry). High-CSF HIV RNA was defined as≥10000cp/mL (H-CSF), while CSF HIV RNA<10000cp/mL characterized low VL patients (L-CSF). Chi-square and Mann-Whitney tests were used. Parameters independently associated with CSF VL were explored by multivariate regression.A total of 131 patients were retrospectively enrolled. Forty-two patients (32%) had CSF VL >10000 cp/mL.We hereby describe a 32% prevalence of H-CSF in a cohort of HIV+ ART-naïve patients. Subjects with high-CSF viral replication are mostly with higher systemic immune activation, in particular the percentage of naïve CD8 T-cell is positively associated with CSF VL, irrespective of plasma VL. In HIV+ ART-naïve patients, especially if featuring a hyperactivated T-cell immune-phenotype, lumbar puncture should be considered to further guide CNS-targeted cART.


The percentage of sperm motility obtained from the remaining testis was significantly decreased (p < 0.05). Histological examination of the testis in open surgery (group I) (where the scrotum was left opened) revealed severe hemorrhages, edema and fibrosis. The test is showed degenerative changes in the seminiferous tubules and interstitial orchitis. Histological examination of the testes removed using a closed technique, (in groups II and III) where the scrotum wound was sutured, revealed hyperplasia of spermatogenic series and Leydig cells. In conclusion, unilateral orchidectomy had compensatory effects on the weight and volume of remaining testis. Adverse effects on sperm motility and viability can affect the fertility of the animal. Unilateral orchidectomy (UO) is required when further breeding potential is important. It is sometimes necessary to remove a single testis in a matured stallion for therapeutic reasons. In this study, twelve donkeys were used to evaluate three techniques of unilateral castration, histological and morphological changes on the remaining testis. Results of the study showed that each of the surgical techniques used had its advantages and disadvantages in comparison with the other two techniques. Therefore the selection among the three techniques depends on the surgeon preferences and the environment in which the unilateral orchidectomy is performed. The volume of the remaining testis recorded at the end of the study was significantly greater than that estimated at the start of the study ( It is sometimes necessary to remove a single testis in a matured stallion for therapeutic reasons, such as testicular tumors, varicocele, hydrocele, testicular trauma, orchitis, periorchitis, spermatic cord torsion and most cases of scrotal hernia.When performing orchidectomy, the scrotal or inguinal incision can be sutured or it can be left open to heal by second intention. The decision is based on many factors. The purpose of this study was to evaluate three different surgical techniques of UO in donkeys, as well as the histological and morphological changes on the remaining testis. Animals. Twelve donkeys, 4-10 years old weighing 100 to 230 kg, were included in this study. The animals were kept in a clean, well ventilated, warm, lightened stable, and had free access to food and water. Careful palpation of the scrotum and testes of each donkey confirmed the presence of two normally descended testes. The scrotum and the testes were normal in size and the testes were freely movable inside the scrotum. Ethical approval used for all procedures was obtained from a local ethics committee acting under guidance from Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt.Anesthesia and operation protocols. Aseptically, castration was carried out under the effect of intramuscular xylazine HCl  0.5 mg kg-1 and local infiltration anesthesia using 2% lidocaine HCl  injected at the site of incision inside the scrotum subcutaneously. The animals were divided into three groups each of four animals. The donkeys were restrained in lateral recumbency and the upward hind limb was held in cranially fixed position. In group I, the scrotal incision was left open after castration to heal by second intension. In group II, the scrotal incision was sutured to allow primary wound closure of the scrotal skin, and in group III, unilateral scrotal ablation was performed and the remaining scrotal incision was sutured. First operation Technique. On the day of surgery the donkeys were kept away from food and water from the previous night. They were restrained in lateral recumbency. The upward hind limb was held in cranially fixed position to allow sufficient working area in the inguinal region. Scrotal circumference and length of each testis were recorded using a caliper. The scrotum was prepared for aseptic surgery using chlorhexidine scrub solution. With 10 mL of 2% lidocaine hydrochloride a ring block was given at the proximal third of the scrotal neck, followed by injection of 5 mL as a linear infiltration at the site of incision. 7 The testis was prolapsed through this incision and pulled away from the body. A fenestration was made through the mesorchium and the vascular portion of the cord. For crushing of the blood vessels, an equine emasculator was placed on the spermatic cord for 5 min on a distance approximately 5 cm proximal to the testis; the vascular portion of the cord was ligated with placement of catgut No. 1 . Ligature into the crushed zone of the spermatic cord. After ligation, the testis was removed with a scissor. The vaginal tunic was then closed with the same suture material. The scrotal incision was left open to facilitate drainage from the wound site.In group I: An incision parallel to the median raphe was made on the cranial aspect of the scrotum of the left testis. The incision was passed through the whole layers of the scrotum and the parietal tunic of the testis based on technique described by Cox.: The testis was removed in a same manner to group I, but after removal of the testis, the dead space of the scrotal sac in this side was obliterated with a simple continuous suture pattern using No. 3 vicryl . The subcutaneous tissue was also closed in the same technique. The skin was sutured with simple interrupted sutures using No. 0 vicryl. These were not removed but were left to resorb. A piece of gauze was applied through the distal end of the scrotal wound to allow drainage of the exudate; this drain was changed daily for the first three days following operation  intramuscularly and procaine benzylpenicillin  18 mg kg-1 intramuscularly. The scrotal incision of donkeys in group I was rinsed to remove excessive crusted exudates and then digitally reopened and explored. The drained scrotal cavity was then rinsed massively. The changes in donkey behavior and the size of the swelling at the site of surgery were monitored daily. In group II and III the drain was changed daily for the first week post castration. Measurements. The removed testis was used as a control for the corresponding one for certain parameters; the removed testis was undergone the following examinations: Testicular weight (TW) with and without the epididymis, epididymal semen parameters measure for the percent of sperm motility, and the percent of live sperms. Liberated sperms from minced epididymal tail in physiological saline solution were examined. Motility percentage was tested using phase contrast microscope. Percentage of abnormal sperm and percentage of live sperm were estimated from a film stained by eosin and nigrosin. Second operation. After 120 days, the donkeys of all groups were subjected to orchidectomy of the remained testis. The dimensions of the remained testis were measured by caliper; weight of the removed testis and epididymis was estimated, and semen liberated from the epididymal tail was treated as the first one. Post-operative care. Management procedures for the next few days included hosing the outside of the scrotum to remove obvious contamination. Chlorhexidine ointment 1%  was applied topically to the inside of both hind legs to prevent skin scalding from the scrotal discharge.Measurements. The volume of the testis was calculated from ellipsoid equation. The difference between the volume of right and left testes was compared. The volume of removed testis in the second stage of study was compared with its volume at the start of the experiment. The difference in weights between the two testes was statistically studied. The characteristic of sperm from each testis were investigated. Histology. Sections from each testis were fixed in 10% neutral buffered formalin, dehydrated in a graded alcohol series, cleared with methyl benzoate and embedded in paraffin wax. Sections of 5 μm were cut and stained with hematoxylin and eosin (H & E).8 Stained sections were examined under light microscope  and photographed using digital camera .Computerized morphometric analysis. Spermatogonia, spermatocytes, spermatid, spermatozoa and Sertoli cells were counted in 10 cross section of the test is using transmitted light, bright-field microscope . Statistical analysis: Statistical analysis was carried out by analysis of variance (ANOVA) and the results were compared using Student t-test. The level of significant difference was 5%, probability value of less than 0.05 was considered statistically significant. A p-value of less than 0.01 was considered highly significant (p < 0.01), p-value of less than 0.001 was considered extremely significant (p < 0.001). All statistical tests were performed using Graphpad Software Package .Anesthesia. The local anesthesia provided by lidocaine in combination with xylazine was enough in performing the castration without any anesthetic risk or complications and the animals were able to stand directly after the operation. No anesthetic complications were recorded during or after surgery. Operative and post-operative complicationsGroup I. Slight swelling of the scrotum was apparent the day after each orchidectomy but began to subside after three days. Then the swelling was subsided gradually. Scrotal discharge continued for one week after castration and the wound began to decrease gradually in size at the end of the first week of castration. Daily flushing of the scrotal wound was very important to remove debris and flies accumulated inside the wound cavity. Hemorrhage occurred in two animals in this group within the 48 hr post operation, and was completely controlled with gauze packing the scrotal cavity for 24 hr. Two animals in group I and one animal in group III showed cautious gait during the first week of operation. Healing period required for the wound in this group was about three weeks.Groups II and III. Severe (group II) and mild (group III) swellings of the scrotum and the surrounding area were observed in all animals of these two groups. The swelling was extended forward on the ventral aspect of the abdomen and reached the umbilicus. It resulted in pressure on the prepuce and urethra causing difficulties in urination for the first three days following orchidectomy in three animals in group II  was significantly more than that of estimated at the start of study (79.86 ± 5.95 mL) for the left one (p < 0.05). Volume of left testis and right one at time of removal were significantly different (p < 0.05). The weight of the removed testes was 105.36 ± 7.70 g and 134.50 ± 13.82 g for the right and left ones, respectively. The dissected left and right epididymis weights were 20.20 ± 1.87 g and 23.93 ± 2.53 g, respectively. The motility percentages of sperm obtained from the remained right epididymis tail (84.00 ± 2.91) were significantly lower (p < 0.05) than that obtained from the left one (73.00 ± 3.39). The total abnormalities percentages were 23.26 ± 1.34 for sperm obtained from left epididymal tail and 29.36 ± 1.22 for the right one and the difference was significant (p < 0.01).Histology. Histological examination of the testis in group I revealed severe hemorrhages  when compared with the control group. The mean concentration of Sertoli cells and spermatogonia in the closed surgery groups (Group II and III) was highly significantly increased (p < 0.01) when compared with control group. In addition, there was no significance (p > 0.05) of the mean concentration of spermatid and spermatozoa when compared with the control group , closed  and total ablation (complete excision of the scrotum) were used for unilateral orchidectomy, the time to healing and the postoperative complications were similar in the last two groups . Each of the three techniques showed its advantages and disadvantages as mentioned in results, therefore the selection among the three techniques could be referred to the preference of the surgeon and the field where the castration will be performed because performing of un-sutured castration in the field or in hot areas is followed usually with complications of contamination of the scrotal wound with flies and debris and this technique will be more suitable for animals kept in clinic for a period of time. Suturing the scrotum after castration resulted in rapid healing process of the scrotal wound even with the swelling occurred after castration. This swelling was usually avoided with careful suturing of all underlying tissues in the scrotal wound, and subsided in few days following castration by massage of the swollen area, applying of a drain in the wound at the end of the suture line and with walking of the animal regularly in the days following castration. Total ablation of the testis and its surrounding tissues on its side was better when the surgeon intended to close the scrotal wound. The removal of the remaining skin and the underlying tissues in the scrotal wound decreased the size of the wound cavity to a large extent and the swelling occurred after castration was usually less than that with using the sutured technique. Techniques used to castrate horses include open, half-closed or modified open and closed.19-21 Except for peritonitis and evisceration, complications of castration are not considered life-threatening. In the present study, complications following UO varied according to the technique used but all complications were easily treated and controlled during the first week following castration except for a scrotal swelling that occurred in group II and III and needed longer time to subside. The removal of left testis resulted in compensatory effect on the right one which was removed after twelve weeks where its volume increased significantly. This might be due to increase in serum LH and FSH concentrations and perhaps, higher intratesticular testosterone.4 The reason could be explained because the UO increased the mean diameter of seminiferous tubules by 21% (p < 0.01) and of their lumina by 51%.22Complications that can arise following surgical castration include scrotal swelling or edema, excessive hemorrhage; septic funiculitis formation, septic peritonitis, omental herniation, incisional infections, hydrocele, penile trauma and evisceration (eventration).et al.,23 In the present study, epididymal weight was significantly increased by UO. This result was also recorded by Barnes et al.,23 The decrease of sperm motility and increase in abnormality percentages may be due to inflammatory reaction after removal of first testes.24,25The compensatory effect was also reflected on testis where the weight of the remaining testis increased significantly, this was similar to the result obtained by Barnes 26Histological examination of the testes of donkeys whose scrotum was sutured after unilateral orchidectomy (i.e. groups II and III) revealed hyperplasia of spermatogenic series and Leydig cells. Similarly, Putra and Blackshaw reported that UO was correlated with an increase in the number of Sertoli cells and germ cells and germ occupying the seminiferous epithelium. The compensatory hypertrophy primarily involved proliferation of Leydig cells and Sertoli cells.27 Unilateral orchidectomy increased the mean diameter of seminiferous tubules by 21% and of their lumina by 51% but did not significantly increase mean height of seminiferous epithelium or estimated length of seminiferous tubules per testis.28 The increase in the testicular weight of unilaterally castrated pigs was correlated with an increase in the number of Sertoli and germ cells at three months of age and germ cells at four months of age occupying the seminiferous epithelium. This was correlated with increased total seminiferous tubule length and larger cross-sectional area of the tubule. Sertoli cell occupancy did not differ significantly between unilaterally castrated and intact boars.27Testis and epididymal weight were increased linearly with age and after UO. Tubule diameter and epithelial height were greater in UO than in intact stallions. Testes of stallions underwent compensatory hypertrophy after UO.In conclusion, unilateral orchidectomy could be performed successfully using different surgical techniques either closed or open. The surgical approach and technique used for UO depends on the clinical indications for surgery and the nature of the case. Primary closure technique is recommended to minimize local inflammation and swelling and thereby minimizing any effect they may have on the remaining testis. In addition, field castration is better performed using the suture technique or total ablation while for animals are kept in the clinic the three techniques could not show significant difference in advantages and disadvantages. Although UO results in compensatory effects on weight and volume of the remaining testis, its adverse effects on sperm values can affect the fertility of the animal.