Extracellular matrix metalloproteinase inducer  is an inducer of matrix metalloproteinases and has roles in leukocyte activation and migration. We reported previously that in MS and its animal model, experimental autoimmune encephalomyelitis, cell surface-associated EMMPRIN was significantly elevated in leukocytes around inflammatory perivascular cuffs in the CNS. In this study we report that activated T-cells can secrete soluble form of EMMPRIN (sEMMPRIN) upon activation. As sEMMPRIN is also present in biological fluids, we determined whether sEMMPRIN is altered in the CSF and sera of MS subjects. Sera from individuals without neurological conditions served as controls, while CSFs collected from subjects undergoing discectomy, and without evidence of CNS pathology, were used as a comparator group. We found that serum levels of sEMMPRIN from clinically stable MS patients or other inflammatory conditions did not differ from control subjects. Paired serum and CSF samples demonstrated poor correlation of sEMMPRIN. Interestingly, sEMMPRIN levels were approximately 60% higher in CSFs compared to sera. sEMMPRIN CSF levels were significantly higher in secondary progressive compared to primary progressive subjects. Thus we conclude that measurement of sEMMPRIN in serum is not informative for disease activity in MS. The differential expression of sEMMPRIN in the CSF of primary and secondary progressive MS invites hypotheses of the still undefined roles of EMMPRIN in the CNS. Multiple sclerosis (MS) involves the influx of leukocytes into the central nervous system (CNS) leading to demyelination and axonal degeneration. Relapsing-remitting MS (RRMS) is the most common form of MS characterized by relapses interspersed with periods of clinical stability. A large number of RRMS patients eventually transition to secondary progressive MS (SPMS) where disability accumulates without obvious clinical relapses. Another progressive form of MS, primary progressive MS (PPMS), is diagnosed in about 10–15% of patients and is characterized by steady loss of function from onset without obvious relapse activity.Extracellular Matrix Metalloproteinase Inducer , a type I transmembrane glycoprotein of the immunoglobulin (Ig) superfamily, seems crucial as it can interact with adhesion molecules including integrins [Among the various indicators of disease activity in the MS brain , adhesiontegrins , 6. Therntegrins . EMMPRINntegrins . The uprntegrins , 10.In addition to its expression on the cell membrane, EMMPRIN can be secreted by cells and released into the extracellular space. This form of soluble EMMPRIN (referred to as sEMMPRIN hereafter) appears as a smaller fragment by virtue of processing by transmembrane type 1 (MT1)-MMP  or as a ™ PLUS  centrifugation. Briefly, heparinized blood was diluted 1:1 with phosphate-buffered saline (PBS) and carefully layered over Ficoll and centrifuged. The cell interface layer was collected, washed twice in PBS and re-suspended in serum-free AIM-V media  [Blood was drawn from healthy adult human volunteers and PBMCs were purified using Ficoll-Plaque Canada) .Wells of a 96-well round bottom plate were coated with 1 μg/ml anti-human CD3 (BD Pharmingen) for 3h at 37°C. For non-activated T-cell conditioned media, wells were left uncoated. Isolated PBMCs were plated at 200,000 cells/well in a final volume of 100 μl. One μg/ml of soluble anti-human CD28 (BD Pharmingen) was also added to the anti-CD3 coated wells upon plating the PBMCs. This stimulation condition resulted in an enrichment of activated T-cells above 90% (data not shown). Conditioned media from each condition were harvested for EMMPRIN ELISA after 48h and 72h of activation.2 value of 0.99 or higher was consistently observed. We note that the manufacturer was unable to provide information on the specific epitope that the antibody to EMMPRIN binds to, other than that the ELISA antibody is generated against the full-length form of EMMPRIN.sEMMPRIN was analyzed from 5 μl of sera or CSF and 20 μl of conditioned media from in vitro cultures of PBMCs using the solid phase sandwich human EMMPRIN ELISA kit  according to the manufacturer’s protocol. The amount of sEMMPRIN was read off a standard curve generated from known quantities of EMMPRIN provided by the manufacturer. The standard curve was generated using the 4-parametric logistic curve-fit and the RFor IgG measurement, a 2000-fold dilution of CSF samples was analyzed using total human IgG ELISA kit . All serum and CSF samples were measured in duplicate wells.To address the reliability of sEMMPRIN measurements, we subjected aliquots of CSFs from 5 subjects to ELISA measurements conducted at 2 time periods spaced 20 months apart. The results in Serum samples from subjects of the following health conditions were: healthy controls (HC), Crohn’s disease (CD), ulcerative colitis (UC), type 1 diabetes (T1D), and RRMS. The ages (years ± SD) of these groups were, respectively, 31 ± 12, 25 ± 8, 33 ± 11, 26 ± 8, and 37 ± 12. All subjects were deemed clinically stable at the time of their blood collection.For the paired serum and CSF study, these were collected from the same individuals considered “active”, with demyelinating event within the past 3 months, or “inactive”, without apparent demyelinating event the previous 3 months. Subjects had the following diagnosis: Subject 1 (S1)—other neurological disease ; S2—clinically isolated syndrome (CIS), active; S3—CIS, inactive; S4—CIS, active; S5- RRMS, active; S6—RRMS, active; S7- RRMS, active; S8- RRMS, active; and S9- RRMS, inactive. Subject S6 was in the midst of an acute relapse when specimens were obtained.CSFs were collected by traditional lumbar punctures from 64 patients with RRMS (25), SPMS (21) and PPMS (10) and ALS (8) and stored at -80°C until further use. The age of most but not all patients was recorded at the time of the collection, and the known average ages (years ± SD) were 39 ± 11 (RRMS), 48 ± 7 (SPMS), 48 ± 7 (PPMS) and 58 ± 16 (ALS). Control CSF samples were collected by lumbar puncture under general anesthesia prior to microdiscectomy surgery (discectomy group). These 7 subjects were symptomatic with degenerative disc disease refractory to non-surgical management for >12 weeks but lacked evidence of CNS pathology on history and physical examination by a neurosurgeon. Their mean ages were 56 years ±17. Unlike the paired serum and CSF that were available from some individuals with MS, we did not have access to paired serum and CSF from the ALS subjects; thus, only CSFs were analyzed for the ALS group.All the samples were collected from adult participants who provided their written informed consent to participate in this study, which was approved by local institutional ethics committee {The Conjoint Health Research Ethics Board (CHREB) of the University of Calgary}.± SD and sEMMPRIN as well as IgG values are expressed in pg/ml. p-values <0.05 were considered statistically significant. The minimal/main data set underlying the findings in our study is presented as S1 TableDifferences in variables between groups were tested by one-way ANOVA with Tukey’s post hoc test or Student’s t-test using Graphpad prism software. Correlation analysis was performed by Pearson correlation. Data is represented as mean We addressed whether sEMMPRIN levels alter upon activation of T-cells. Compared to non-activated conditions, we found that sEMMPRIN levels in conditioned media of T-cells activated with anti-CD3 and anti-CD28 were significantly elevated . These fEarlier studies of ALS and systemic sclerosis had shown higher serum sEMMPRIN levels compared to healthy controls . HoweverWe analyzed sEMMPRIN levels in paired sera and CSF samples collected at the same draw from subjects along with unpaired sera and CSF samples from other MS collections. For the latter, we found that the CSF had statistically significant higher levels of sEMMPRIN over content found in serum samples .Further, in the majority of the paired samples collected from both the CIS and RRMS subjects, sEMMPRIN levels in the CSF was higher than in the corresponding serum samples . HoweverWhen we measured sEMMPRIN in the CSF of individuals with different forms of MS, we observed large variability between the groups . NotablyFurthermore, sEMMPRIN levels from all the MS subjects did not associate with their CSF IgG levels . Also, sEMMPRIN levels did not differ significantly between male and female MS subjects (p = 0.07) as well as between different age groups .CSF samples from 8 ALS patients were analyzed for differences in sEMMPRIN levels. In contrast to reports that sera sEMMPRIN is significantly higher in ALS subjects over healthy controls , we obseEMMPRIN is highly expressed in the brain capillary endothelium  and neurWe observed that serum sEMMPRIN levels were poorly correlated with CSF sEMMPRIN and that sEMMPRIN levels were markedly higher in the CSF compared to serum This suggests important roles of EMMPRIN in the blood-brain barrier interface or within the CNS. EMMPRIN can interact with different adhesion molecules on endothelial linings including integrins , 6, and Our current findings show that sEMMPRIN is elevated in SPMS CSF over PPMS CSF. Downstream molecules of EMMPRIN include the MMPs. Serum and CSF MMPs including MMP-9  and MMP-The presence of high sEMMPRIN levels in the CSFs of the discectomy group and MS subjects suggest that EMMPRIN is a molecule of high turnover in the CNS and may hold importance to CNS homeostasis. Thus, variations in CSF sEMMPRIN levels in different MS subtypes may indicate homeostatic alterations. Metabolic studies in the MS brain have revealed that lactate (a monocarboxylate) is elevated in the CSF of SPMS subjects compared to normal control subjects . FurtherTo the best of our knowledge, this is the first study highlighting the presence of sEMMPRIN in the CSF. Although EMMPRIN’s role in multiple inflammatory processes has been recognized , 38, itsS1 FigThese samples were stored at -80°C until analyzed. Error bars indicate differences from duplicate wells for each individual ELISA.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


A receptor α1(H101R)mutant mice. The α1(H101R)mutation renders α1-subunit containing GABAA receptors insensitive towards benzodiazepines. This GABAA receptor subtype is thought to mediate sedation. As midazolam is more potent than its metabolite 1-hydroxymidazolam, the major clinical effects are thus likely caused by midazolam itself. However, 1-hydroxymidazolam could add to the effects of midazolam, especially after the application of high doses of midazolam, and in case of impaired drug metabolism.The benzodiazepine midazolam is widely used in critical care medicine. Midazolam has a clinically active metabolite, 1-hydroxymidazolam. The contribution of 1-hydroxymidazolam to the effects of midazolam is controversial. The aim of the current study was to compare the actions of midazolam and 1-hydroxymidazolam on network activity of cortical neurons. Midazolam depressed neuronal activity at a low concentration of 5 nM. When midazolam concentration was increased, it depressed neuronal discharge rates in a biphasic manner. In comparison, 1-hydroxymidazolam did not depress the cortical network activity at low nanomolar concentrations. Higher concentrations of 1-hydroxymidazolam consistently inhibited neuronal activity. Moreover, midazolam shortened cortical up states at low, but not at high concentrations, while the opposite effect was observed with 1-hydroxymidazolam. The network depressant action of midazolam at low concentrations was absent in slices from GABA In addition, Crevoisier et al. have demonstrated that cognitive and motor impairments induced by midazolam are different after oral or intravenous application, an observation that was attributed to the relatively higher amount of 1-hydroxymidazolam after oral administration6.Midazolam is a commonly used benzodiazepine in anaesthesia and intensive care medicine. Midazolam has a clinical active metabolite: 1-hydroxymidazolam, which is, like the parent drug midazolam a neuronal depressant drug. However, 1-hydroxymidazolam’s contribution to the depression of neuronal activity and thus its clinical relevance still remains a matter of debate. There are numerous studies in which the statements concerning the contribution of 1-hydroxymidazolam to the clinical actions of midazolam range from “almost equipotent” to “no major contributing factor”The current study was therefore designed to directly compare the effects of midazolam and 1-hydroxymidazolam on the activity of neocortical neurons in organotypic slice cultures, a model system in which, to the best of our knowledge, there is no relevant metabolism from midazolam to 1-hydroxymidazolam.A receptors harbouring either an α1, α2, α3 or α5-subunit. About 40% of all GABAA receptors in the brain contain α1-subunits, making them the major GABAA receptor subtype in the brain7. Mice carrying a point mutation in the α1 subunit of the GABAA receptor at position 101 are resistant to the sedative action of diazepam, indicating that benzodiazepine-induced sedation is predominantly mediated via GABAA receptors containing the α1-subunit9. Moreover, it was suggested that sedation with diazepam depends on GABAA receptors located on glutamatergic neurons in the cerebral cortex10. On the cellular level, this molecular action of diazepam translates into a significant depression of cortical network action potential firing11. Therefore, in order to examine the contribution of α1-containing GABAA receptors to this phenomenon, in the current study clinically relevant concentrations of midazolam were also tested in neocortical slice cultures from GABAA receptor α1(H101R) knock-in mice.Basically, benzodiazepines like midazolam predominantly act via GABA12. The median of the spontaneous action potential firing rate (in C57/BL6J mice) was at 10.12 Hz; interquartile range (iqr): 12.92; (number of recordings n = 481).Spontaneous firing patterns from neocortical organotypic slice cultures were characterized by phases of high neuronal activity (up states) separated by periods of neuronal silence. A typical extracellular recording is displayed in Fig. Midazolam in a concentration range from 5 nM to 100 µM inhibited the neuronal firing rate in a biphasic manner. From 5 nM to 1 µM, midazolam significantly depressed action potential firing. However, the inhibitory action of midazolam did not rise continuously with increasing concentrations. Actually, 500 nM and 1 µM midazolam depressed the neuronal activity only slightly less than the administration of 100 nM midazolam. Though, raising the concentration of midazolam to 25 µM and beyond led to a stronger depression, which was significantly different from the concentration range of 5 nM to 1 µM .To further elucidate the actions of midazolam at 5 nM we calculated a peri-event time histogram (PETH) for the first 1000 ms of the average up state. The sham application of ACSF only led to negligible changes at the beginning of the up states Fig. . By compA receptor, we performed experiments with flumazenil, a selective antagonist at the benzodiazepine binding site of the GABAA receptor14. As shown in Fig. To test whether this action of midazolam is mediated via the classical benzodiazepine binding site of the GABAin vivo concentration range of 1-hydroxymidazolam is lower than that of midazolam itself 3.To compare the actions of midazolam with those of 1-hydroxymidazolam we tested a concentration range of 1-hydroxymidazolam from 5 nM to 1 µM, because the The concentration response relationship for 1-hydroxymidazolam is displayed in Fig. To test whether the effect of 1-hydroxymidazolam is also mediated by the classical benzodiazepine binding site, we measured the network activity after administering a combination of 1 µM 1-hydroxymidazolam with 1 µM flumazenil. The depression of neocortical activity triggered by 1-hydroxymidazolam was almost completely antagonized by flumazenil Fig. .50 values of both concentration response curves differed by over an order of magnitude : F = 11.98, p = 8.6 * 10−6).To further explain the quantitative differences of action between the two compounds in detail, we calculated PETHs for 1-hydroxymidazolam. As can be seen in Fig. −10, eta squared with 95% CI = 0.15 [0.09 0.25]).To further investigate the differences in action of midazolam and 1-hydroxymidazolam we analysed the length of up states. Under control conditions, the median length of up states was 664 ms (iqr: 2400). When midazolam was administered at low concentrations, it shortened the length of up states, while at 500 nM and 1 µM this effect was completely absent. On the other hand, 1-hydroxymidazolam only reduced the length of up states significantly at 500 nM and 1 µM Fig. . It is oTo further elucidate the actions of midazolam and 1-hydroxymidazolam at the synaptic level, we performed patch-clamp recordings in neocortical slice cultures from wild-type C57BL/6J mice. In a first set of experiments the effects on phasic inhibition were tested by recording miniature IPSCs from pyramidal neurons. To quantify drug actions on synaptical inhibition we used the total charge transfer, calculated as the area under the averaged IPSC multiplied by the frequency of IPSCs. Midazolam at a concentration of 50 nM increased total charge transfer by 71.6 ± 27.7% . At the same concentration, 1-hydroxymidazolam appeared to increase total charge transfer to a lesser extent, but this effect was not significant  knock-in mice, in which α1-containing GABAA receptors are insensitive to benzodiazepines, including midazolam16. The basal activity of cultures derived from α1(H101R)knock-in mice was 16.64 Hz; 14.87 . This value was slightly higher than the value obtained with wild-type mice. However, when comparing PETH of the averaged up states, no differences could be detected between wild-type and α1(H101R) mice knock-in mice for concentrations of 5 nM and 10 nM midazolam, neither during up states nor for the overall action potential firing rate. Only at a concentration of 100 nM midazolam did induce significant depression during phases of high neuronal activity  mice to those seen in wild-type mice, we observed that the inhibitory action of midazolam once again did not increase steadily with increasing concentrations. The concentration response relationship curves nearly run parallel to each other  is in line with previous studiesin vivo studies showing that sedation and changes of EEG frequencies could be induced by 1-hydroxymidazolam alone21 we found that 1-hydroxymidazolam inhibits neocortical network firing.In accordance with previous clinical investigations and 3, 1-hydroxymidazolam may, therefore, contribute to the effects of midazolam, above all, in cases of deep sedation or in patients with severe renal dysfunction22. A summary of clinical and experimental findings concerning effects of midazolam and 1-hydroxymidazolam at different concentrations can be found in Table When comparing midazolam with 1-hydroxymidazolam it is evident that 1-hydroxymidazolam is far less potent. Inhibitory actions of 1-hydroxymidazolam could be observed starting at a concentration of 100 nM, corresponding to a plasma concentration of nearly 40 ng/ml. Considering the plasma concentration measured in a previous study1(H101R)knock-in mice. Therefore it seems reasonable to assume that midazolam at low nanomolar concentrations predominantly acts via α1-containing GABAA receptors. Thus, despite being a non-GABAA receptor subtype-selective benzodiazepine23, midazolam’s sedative actions are largely mediated by α1-containing GABAA receptors. In contrast to diazepam, at recombinant receptors midazaolam displays a preference for α1-containing GABAA receptors over α2-containing GABAA receptors: a recent study showed an approximately two-fold larger potentiation of GABA-induced currents by midazolam at GABAA receptors containing α1-subunits compared with GABAA receptors containing α2-subunits, whereas diazepam potentiated both receptor subtypes to a similar extent16.In addition, we detected that midazolam administered at the low concentration of 5 nM caused a significant inhibition of the cortical network activity in cultures from wild-type, but not from α1-containing GABAA receptors have been shown to be of major importance for mediating sedation, amnesia and anticonvulsant activity of benzodiazepines24. Due to midazolam’s high efficacy at this GABAA receptor subtype, changes in quantity or distribution of this receptor subtype might have some clinical implications. For example, there is growing evidence that inflammation changes GABAA receptor composition in the brain. Pribiag and Stellwagen demonstrated that TNFα decreases inhibitory synaptic strength by downregulation of cell-surface levels of GABAA receptors25. In addition, interleukin-1β increases surface expression of α5-containing GABAA receptors and a tonic current generated by these receptors, resulting in memory deficits26. Furthermore, it is known that assembling, trafficking, clustering and endocytosis of GABAA receptors are highly dynamic and, thus, can be influenced by other factors like repeated seizures or alcohol abuse27. These changes in GABAA receptor populations might well be contributing to the high variability of duration and the different qualities of sedation with midazolam. Considering that midazolam shows a preference especially for α1-subunit containing GABAA receptors, small changes in the cell surface concentration of this GABAA receptor subtype may have major implications for midazolam’s clinical actions.To date, α1-containing GABAA receptors29. Finally, α1-subunits containing GABAA receptors are involved in developmental plasticity. The present ex vivo results raise questions regarding the potential induction of undesired long-term changes by midazolam in immature neuronal networks30.Moreover, there is evidence indicating that the addictive properties of benzodiazepines are mediated via α1-containing GABAA receptors. This GABAA receptor subtype is the major molecular target of benzodiazepines for sedation. Small changes of this receptor population, e.g., due to inflammatory diseases, may therefore serve as an explanation for the large variability of midazolam’s clinical properties.In summary, midazolam depresses cortical networks at the low concentration of 5 nM, predominantly mediated via αA receptor subtype, possibly also at different GABAA receptor subtypes, as has recently been described for clobazam and its metabolite N-desmethyl-clobazam31.Midazolam’s main metabolite, 1-hydroxymidazolam, is far less potent than midazolam itself. The qualitatively different actions of 1-hydroxymidazolam compared with those of midazolam might argue for differential affinities of these two drugs at the α1 GABAin vivo studies could be due to other types of GABAA receptors in lower brain structures, i.e. δ-subunit containing GABAA receptors.We conclude that when considering the clinically relevant concentrations of midazolam, used for sedation in intensive care medicine, midazolam is clearly more potent than its metabolite, i.e. the major clinical effects are caused by the parent drug midazolam itself, presumably acting on cortical neurons. The higher potency of 1-hydroxymidazolam seen in some A receptor α1(H101R)mutant mice on C57BL/6J background15 of both sexes were used. Neocortical organotypic slice cultures were prepared from three to five day old mice as described before32. In brief, animals were deeply anaesthetized with isoflurane and then decapitated. Cortical hemispheres were removed aseptically and 300 µm thick coronal slices were cut. Slices were fixed on glass coverslips by means of a plasma clot, transferred into plastic tubes containing each 750 µL of nutrition medium, to be then incubated in a roller drum at 37 °C. After 1 day in culture, antimitotics  were added. The suspension was renewed twice a week.All procedures were approved by the Animal Care Committee  and were in accordance with the institutional and federal guidelines of the German Animal Welfare Act (TierSchG). Wild-type C57BL/6J mice and GABA2PO4 1.13, MgCl2 1.0, NaHCO3 26, CaCl2 1.2, and glucose 11; then bubbled with 95% oxygen and 5% carbon dioxide. All experiments were conducted at 34 °C. For this purpose, a heating wire was glued onto a metal frame of the recording chamber and then heated using a direct current. ACSF-filled glass electrodes with a resistance of 3 to 5 MΩ were advanced into the tissue until extracellular multi-unit spike activity exceeding 100 µV in amplitude was visible. Data were acquired with the digidata 1200 AD/DA interface and AxoScope 9 software.Extracellular multi-unit recordings were performed in a recording chamber mounted on an inverted microscope. Slices were perfused with artificial cerebrospinal fluid (ACSF) consisting of (in mM) NaCl 120, KCl 3.5, NaH2 and 4 ATP, adjusted to pH 7.2 with 1 N HCl. Cells were voltage-clamped at −70 mV. To suppress glutamatergic synaptic transmission, we added 25 µM D-L-2-amino-5-phosphonopentanoic acid, 10 µM 6-cyano-7-nitroquinoxaline-2.3-dione and 1 µM tetrodotoxin. For the detection of tonic currents 20 µM bicuculline was added to the ACSF. Analysis of tonic currents followed the method proposed by Glykys and Mody33 and is described in detail in reference34.Intracellular voltage clamp recordings were performed on visually identified pyramidal neurons at room temperature. Pipettes were filled with a solution containing (in mM) 121 CsCL, 24 CsOH, 10 HEPES, 5 EGTA, 1 MgCl36 because diffusion times in slice cultures are considerably shorter compared to acute slice preparations38.Midazolam and 1-hydroxymidazolam were dissolved in dimethyl sulfoxide to a 1 mM stock solution. Flumazenil was dissolved in 99% ethanol to a 5 mM stock solution. Drugs were diluted in ACSF to the tested concentration and filled into glass syringes immediately before the experiment was conducted. The drug containing ACSF was applied via bath perfusion using syringe pumps, connected to the experimental chamber via Teflon tubing. When switching from ACSF to drug-containing solutions, the medium was replaced by at least 95% within two minutes. To ensure steady-state conditions, recordings were conducted 12 minutes after commencing the change of the perfusate. Previous studies have demonstrated that this time interval is sufficient for steady-state conditionsth percentile and upper quartile = 75th percentile; whisker: 1.5*iqr (interquartile range); iqr: difference between the upper and the lower quartiles). For statistical analysis of extracellular detected neuronal spiking activity, we performed a combination of a non-parametric analysis of variance  and a multiple comparison post-hoc analysis (Bonferroni), followed by a Mann-Whitney U test to check for significant differences between the two groups.Extracellular recorded signals were filtered and counted offline using self-written Matlab (R2008b) routines. The activity pattern of neocortical slice cultures was characterized by phases of spontaneous action potential firing (up states) separated by periods of neuronal silence. Action potentials were detected and the average firing rate was computed, using an automated event detection algorithm with a threshold set approximately 2 times higher than baseline noise. An up state was defined by an initial inter-event interval of 75 ms and a preceding silent period of at least 500 ms. All automated up state detection was checked by visual inspection based on field potentials. Parameters are shown as relative change compared to ACSF sham application. Each data set was checked for normal distribution by using qq plots. However, the hypothesis that data sets were normally distributed had to be rejected for the majority of these data sets. Therefore, grouped data are expressed as boxplots  peri-event time histograms (PETH) were calculated. For this purpose, the up states of every experimental condition were collected, divided into bins of 10 ms, and averaged. Because the length of up states was variable, the PETH were constrained to the first 1000 ms. To test for statistically significant effects, the effect size  was calculated. The 95% confidence interval excluding zero indicates significance at p < 0.05. A general rule of thumb says that absolute Hedges’ g values >0.8 indicate a strong effect and values >0.5 indicate a medium effect.39 was performed to statistically compare IC50 values of the concentration-response relations of normalized firing rates for midazolam and 1-hydroxymidazolam. The underlying null hypothesis was that both data sets (midazolam and 1-hydroxymidazolam) stemmed from concentration-response relations with the same IC50. Briefly, Hill functions with three and two free parameters  were each fitted to each data set. In the constrained model, the IC50 (Kd) value was set to a fixed value, determined from a full Hill fit to the union of both data sets. An F value was computed as F = (SS1-SS2)/(DF1-DF2)/(SS2/DF2) where SS1 is the sum-of-squares of the constrained model (summed for both data sets), DF1 the degrees of freedom of the constrained model (again summed for both data sets), and SS2 and DF2 the equivalent values of the full model. From this F value  a p value was computed, which specified the probability of obtaining data sets at least as extreme as the measured samples, given the null hypothesis.An extra sums-of-squares F test40 as the distribution of normalized burst length often had long tails towards high values: for each concentration of both drugs, the transformation parameter lambda was estimated; from the resulting collection of lambda values an average weighted by the number of samples in each group was computed (lambda = 0.214) and all data transformed with this value.As midazolam and 1-hydroxymidazolam altered up state length in qualitatively different ways, a fitting approach for statistical comparison of both drugs’ effects was not deemed reasonable. Instead, we opted for a two-way analysis of variance (ANOVA) with drug type (midazolam and 1-hydroxymidazolam) and drug concentration as the independent factors, focusing on their interaction. We computed both the p-value and eta squared, a measure of effect size, for the interaction of drug type with drug concentration. 95% confidence intervals of eta squared were computed by bootstrapping. Prior to analysis via ANOVA, the data were Box-Cox-transformed41.All analyses were implemented in Matlab (R2016b) and partly made use of the measure of effect size toolbox


We present a case of post-traumatic headache complicated by intracranial hypotension resulting in an acquired Chiari malformation and myelopathy with syringomyelia. This constellation of findings suggest a possible series of events that started with a traumatic cerebral spinal fluid (CSF) leak, followed by descent of the cerebellar tonsils and disruption of CSF circulation that caused spinal cord swelling and syrinx. This unusual presentation of post-traumatic headache highlights the varying presentations and the potential sequelae of intracranial hypotension. In addition, the delayed onset of upper motor neuron symptoms along with initially normal head computerized tomography scan (CT) findings, beg the question of whether or not a post-traumatic headache warrants earlier magnetic resonance imaging (MRI). A 27 year-old man, previously healthy and without history of headache, presented for evaluation of persistent headache seven months after suffering blunt trauma to the back of the head. There was no associated loss of consciousness. He developed a headache the day after injury, which was later accompanied by nausea and vomiting. The headache was described as dull occipital pain with intermittent severe throbbing. Head CT performed one week after the initial injury was unremarkable. At the time of presentation, he described his headache as daily, lasting three to four hours per day with partial pain relief using combination aspirin-acetaminophen-caffeine. His headache was alleviated by lying down and sleeping; and was exacerbated by coughing, lack of sleep and alcohol consumption. Prior to presentation to our clinic, the patient had unsuccessful treatment with amitriptyline, propranolol, sumatriptan, and hydrocodone-acetaminophen.Past medical and surgical histories were unremarkable. There was no family history of migraine, neurologic disease, or collagen vascular disease. He was a former smoker and reported infrequent alcohol consumption. Caffeine intake included two to three sodas daily.General and neurologic physical exam were unremarkable. Initial diagnosis was of chronic post-traumatic headache with a chronic migraine phenotype and possible component of medication overuse headache due to frequent use of combination analgesics. He was started on preventive treatment with venlafaxine and instructed to reduce his abortive medication use and caffeine intake.At three month follow up the patient reported modest improvement in headache symptoms. However, he had subsequently developed bilateral hand weakness and upper thoracic back pain. Physical exam was notable for bilateral finger flexor weakness, intrinsic hand muscle wasting, tongue fasciculations and ankle clonus. MRI of the brain and cervical cord with and without contrast revealed diffuse pachymeningeal enhancement, sagging brainstem, low lying cerebellar tonsils (10 mm descent) with crowding at the foramen magnum, and venous engorgement of the cervical epidural space A,B. ThisUpon reviewing the MRI findings with the patient, he confirmed that the headache had always been alleviated by lying supine. A CT guided epidural blood patch was performed at L1/L2, and resulted in only modest improvement in headache pain. A second epidural blood patch was performed at T12/L1, again without significant clinical improvement. Repeat MRI of the brain and spine showed progression of spinal cord signal abnormality from T9 to T10. A CT myelogram showed an epidural fluid collection along the right lateral thecal sac extending from T5 to T12, most prominent at T10 A. CT-guiThe patient continues to have improvement of bilateral hand strength. His headaches, neck, and back pain all resolved over the following month. Repeat imaging performed approximately three months following the fifth and final therapeutic blood patch showed complete resolution of pachymeningeal enhancement, tonsillar herniation and cord edema with only a tiny residual syrinx .This case presents several unusual complications of post-traumatic headache. Curiously, the patient developed delayed symptoms that were found to be a result of upper thoracic syrinx and cord edema. The positional nature of the headache and exacerbation by coughing were consistent with that of intracranial hypotension . EtiologAlthough syrinx formation has somewhat of an elusive pathophysiology, several theories have been developed to explain its cause. One of such proposes that in accordance with the Bernoulli theorem, the narrowed flow created by sagging cerebellar tonsils at the foramen magnum, causes an increase in CSF velocity and a resultant low CSF pressure in the narrowed canal. This low CSF pressure creates a suction effect on the spinal cord that distends the cord during each systole, causing extracellular fluid to develop within the distended cord, enlarging the central canal to form a syrinx ,6. This We propose that trauma was the cause of CSF leak that led to intracranial hypotension and sagging of cerebellar tonsils, causing distention of the cord and culminating in syrinx formation. An alternative theory that may better explain the delayed onset of weakness is that the patient sustained a second traumatic event that enlarged an initially small dural leak into a larger tear. The larger tear may have resulted in more CSF leakage and caused occlusion of CSF flow at the foramen magnum with the descent of the cerebellar tonsils, ultimately resulting in cord edema and syrinx. A more rudimentary explanation may be that the events occurred semi-independently and not as a causal sequence which we have described. Although the initial head CT did not show evidence of pathology, it is possible the lack of sensitivity CT has for detecting these processes concealed a pre-existing chiari and/or syrinx. In this scenario, a traumatic CSF leaf would have disrupted CSF dynamics and resulted in intracranial hypotension.Known trauma was most likely to be the precipitating event in this presentation. However, in other cases of spontaneous CSF leak, connective tissue diseases leading to weakness of the dural sac may be considered a potential cause. Studies showing dural ectasia and meningeal diverticula to be common in connective tissue disorders may predispose this population to spontaneous CSF leaks ,14. A faThis case report highlights the importance of correctly identifying headaches associated with a CSF leak. Our young healthy man who presented with a seemingly straightforward case of post-traumatic headache developed delayed synrix formation that resulted in intrinsic hand weakness. CSF leakage is not a benign condition. Potential sequelae including syrinx formation can cause profound debilitation and prove to be quite challenging to treat. Given the risk of the afore-mentioned complications, it is vital a detailed history of the headache characteristics be obtained as well as a thorough neurologic examination performed. Should symptoms or signs consistent with intracranial hypotension be elicited, MR imaging may be warranted.


However, it remains elusive why a limited overall effect was observed. We aim to investigate the limiting factors underlying functional recovery promoted by epoB. The same SCI model treated by epoB was established as discussed previously. We used a cerebrospinal fluid (CSF) sample to assess the changes in cytokines in milieu of the SCI lesion site after epoB treatment. We then analyzed the source of cytokines, the state of microglia/macrophages/monocytes (M/Ms), and the recruitment of neutrophil in the lesion site by using the results of antibody array. Following these findings, we further evaluated the motor functional recovery caused by the reshaped microenvironment. Systemic administration of epoB significantly increased levels of several cytokines in the CSF of the rat SCI model; macrophage colony-stimulating factor (M-CSF) secreted by intact central nervous system (CNS) cells was one of the cytokines with increased levels. Along with epoB and other cytokines, M-CSF reshapes the SCI milieu by activating the microglias, killing bone marrow-derived macrophages, polarizing the M/M to M1 phenotype, and activating downstream cytokines to exacerbate the SCI injury, but it also increases the expression of neurotrophic factors. Anti-inflammatory therapy using a neutralizing antibody mix shows encouraging results. Using  First, epoB is a blood–brain barrier-permeable microtubule-stabilizing drug that is absorbed in the central nervous system (CNS) after being administered.1 Second, through neuron-specific protein Tau expression, epoB promotes axon elongation and reduces fibrotic scarring simultaneously.1 Third, epoB reduces axonal dieback and promotes serotonergic axon regrowth. Thus, the rat SCI model exhibits functional recovery after epoB administration.1 It seems epoB matches every step of SCI treatment perfectly.1Epothilone B (epoB) is an ideal drug for the treatment of spinal cord injury (SCI), which can reduce scar formation in the lesion site, reactivate the axons’ regeneration potential, and is convenient for operation.1 Furthermore, epoB does not appear superior to any other monotherapies.4 To further evaluate the therapeutic limitation of epoB, we can refer to the similar microtubule-stabilizing drug taxol, which was tested as a treatment for SCI for many years.5 Hellal et al.6 showed that taxol also possessed multitargeted abilities in treatment of SCI similar to epoB. Nevertheless, recently an independent evaluation reported that taxol is able to reduce the scar formation in the SCI lesion site; however, it does not possess the ability to promote serotonergic axon growth and protect neurons from damage.7 It is still unclear why these contradictions occur.However, the overall efficiency of epoB is not quite so satisfactory. As a matter of fact, the number of footfall errors was reduced ~50% on the horizontal ladder compared with the control. These effects could be counteracted by ablation of 5-hydroxytryptamine.A fundamental question is whether epoB is beneficial for the SCI functional recovery overall and it also consists of some detrimental factors. Here, we found the changes of cytokines in the cerebrospinal fluid (CSF) instead of lesion site milieu. We investigated the source and function of elevated macrophage colony-stimulating factor (M-CSF) focus on the microglia/macrophages/monocytes (M/Ms) in the SCI lesion site after epoB treatment. We further evaluated the downstream cytokine LIX (CXCL5) focused on the effects of neutrophil recruitment. These results indicate that combined with elevated cytokines, epoB suppresses SCI functional recovery by killing bone marrow-derived macrophages (BMDMs), activating microglia, polarizing M/M to M1 phenotype, and recruiting neutrophils to increased lesion inflammation burden. Unlike previous reports, these effects retarded the functional recovery after SCI.n=3) or solvent control (n=3). Interestingly, epoB-treated SCI rats were only M-CSF elevated compared with the control at 1 day post epoB treatment (DPE) (α (IL-1α), IL-4, M-CSF, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β) were elevated in the CSF of epoB-treated SCI models at 3 DPE  and LIX were elevated 7 days after epoB treatment  ; howeverat 3 DPE . In addireatment  and onlyreatment . Accordin=3) or intact model (n=3). Interestingly, the elevated six cytokines 3 DPE of epoB-treated SCI model shows no obvious difference compared with the epoB-treated intact model (n=6) using M-CSF and specific antigen of CNS cells. It is shown that astrocytes, T cells, M/Ms, ependymal cells, and neurons were the main sources of elevated M-CSF at 3 DPE (n=3); the concentration of M-CSF peaked at ~1.6 ng/ml at 4 DPE  followed by 2 ng/ml M-CSF plus PBS and 1 nM, or 5 nM epoB treatment in vitro. Either 3 or 5 days after stimulating, the NR8383 cells were detected by flow cytometry (FC) for cell proliferation. As a matter of fact, even at the highest level and longest stimulation used, M-CSF did not significantly stimulate M/M division compared with controls; on the contrary, in epoB-treated NR8383 cells, division reduced  (n=6) further shows a decrease in BMDMs but not microglias at 3 and 7 DPE (n=3)  . Next, wFC (n=6) . UnexpecFC (n=6) . Immunofnd 7 DPE . FurtherPE (n=3) . The nex9 MCP-1, also known as CCL2, is a chemotactic factor that attracts M1 to the SCI lesion site through spinal cord (SC) leptomeninges.9 The corresponding cytokine is M-CSF, which recruits M2 to the SCI lesion site through choroid plexus. In our research, both M1 and M2 chemotactic factors elevated at 3 DPE. We therefore tested whether the elevated two factors promote the homing process of M/M to the SCI lesion. Immunofluorescent staining of the third ventricle choroid plexus (n=6) shows an increase of homing M/M in SCI-epoB compared with SCI-vehicle. On the contrary, these recruitment effects could be reduced by M-CSF-neutralizing antibody treatment (n=6) of the SCI-epoB group; these effects could be weakened by corresponding neutralizing antibody treatment. Quantification analysis shows that the elevated two factors M-CSF and MCP-1 contribute to ~2- and 2.5-fold increase in homing M/M, respectively. These findings suggested that both increased M1 and M2 cells infiltrated in the SCI microenvironment after epoB administration, and the homing M/Ms were killed by epoB mainly in the CNS.M-CSF and MCP-1 recruit two different types of M/M that go through two different paths to the SCI site.reatment . Similarn=6) shows that phagocytic markers of M/M CD68 in SCI lesions were not changed significantly at 3 or 7 DPE between the SCI-epoB- and vehicle-treated groups (n=6) for CD68 and Iba-1. Interestingly, in the SCI-vehicle group, CNS M/M phagocytic phenotypes were altered to turgid and smooth. By contrast, phagocytic M/M experience shrinkage and size reduction in the SCI-epoB group (n=6)  . These rα as M1 markers and CD206, CD163, and TGF-β as M2 markers.11 Cells isolated from SCI lesions were triple-labeled M/M pan-marker CD11b and M/M phenotype markers for FC analysis (n=6). α expressed an M/M increase and was associated with decreasing M/M with CD206 expression 3 DPE. However, CD163- and TGF-β-expressed M/M was not detected. It is worth noting that using all six neutralizing antibodies, M/Ms tend to possess less M1 phenotype compared with SCI-epoB group 3 DPE. The findings indicate that beyond our antibody-array-detected cytokines, the rest of the cytokines tend to be in the anti-inflammatory state. The polarization is weakened; meanwhile, the neutralizing antibody mix tends to be unable to counteract the inflammatory milieu 7 DPE  illustrated that myeloperoxidase activity obtained from SCI lesions of the SCI-epoB group increased 1.6-fold compared with the SCI-vehicle 10 DPE. Similarly, the infiltrating neutrophils rose in the SCI-epoB group compared with the vehicle control, this effect could be neutralized on different levels by LIX-neutralizing antibody administration 10 DPE (n=6) (n=9) show that compared with SCI-epoB, the SCI-epoB-NAM group recovers the best among the three groups from 7 DPE. This score was followed by the SCI-epoB-M-CSF Ab group; it recovers better than SCI-epoB from 21 DPE. At last, SCI-epoB-LIX Ab group presents superior to SCI-epoB at the beginning of 42 DPE  . BBB scof 42 DPE .1 On the contrary, in the current study we found that epoB is detrimental for SCI recovery via killing BMDMs, activating microglias, skewing M/M to M1 phenotype in CNS, and recruiting neutrophils to aggravating SCI lesion inflammation in the acute phase, as well as therapeutic benefits for SCI.Recent data indicate that epoB is a promising strategy for SCI treatment.15 However, it is very hard to perform the experiments technically by using the intercellular washing fluid of the lesion site. CSF-brain and SC barrier are the weakest in the three parts of blood–brain barriers and the majority of CSF cytokines produced by CNS cells.18 As a result, we evaluated the cytokines changing by CSF sample to instead of intercellular washing fluid of lesion site.These findings reveal that epoB could be a double-edged sword for SCI recovery. These adverse effects are mainly due to epoB reshaping the SCI microenvironment by increasing the cytokine secretion in CSF. Actually, cytokines existing in intercellular washing fluid of lesion site cells modulate the reshaping of the microenvironment.9 After that, initial cytokines stimulate other cells to secrete downstream cytokines in the second or third cascade reactions in CNS.19 However, we found that only M-CSF shows 1.5-fold increase in CSF of SCI-epoB 24 h later, while six kinds of cytokines rose ~3-fold in CSF 72 h after epoB treatment. This phenomenon suggests that the six elevated cytokines may be the second or third cascade reaction after epoB administration. On the other hand, previous research reported that M-CSF could increase expression level of many cytokines, which include TNF-α, IL-1, TGF-β, and MCP-1.20 These results strongly suggest that except for IL-4, M-CSF may be the central and initial cytokine in the SCI superacute stage after epoB administration.Generally speaking, in inflammatory cascade reactions, initially elevated cytokines reach peak within 12–48 h after SCI.21 In fact, M-CSF is a milieu-dependent cytokine that exhibits both neuroprotective and/or neurotoxic effects in CNS diseases.20 Our study revealed that treating SCI models with epoB generates M-CSF increases in CSF; M/Ms of CNS exposed to both elevated M-CSF and epoB showed contradictory responses. Results showed that the number of CD45+ M/Ms in SCI lesion sites decreased as microglias (CD45− M/Ms) of milieu exhibited active states. In other words, the SCI milieu presents cytotoxic effects to homing M/Ms, which are sensitive to microtubule-stabilizing drugs, while resident microglias show activated states, which are sensitive to M-CSF even when they are both exposed to the same microenvironment. How could this dichotomy happen? First of all, one of the most common side effects of epoB is myelosuppression; meanwhile, epoB shows condensed forms in CNS after administration.23 Second, recruited M/Ms via ventricle choroid plexus and adjacent SC leptomeninges were still increased because of the elevated M-CSF and MCP-1. Third, microglias are more stable than homing M/Ms in CNS.25 It is reasonable to infer that the main reason for this contradiction is that the absorbed epoB exhibit cytotoxicity for homing M/Ms and nonkilling effects for microglias. On the other hand, elevated M-CSF activates microglia rather than dying BMDMs. Then, activated microglia exhibits a variety of neurotoxic effects, including secreting reactive oxygen species, nitric oxide, TNF-α, excitatory amino acids, and so on, and also promotes neuroprotection by secreting IGF-1 and increasing macrophage phagocytosis, which is crucial for debris removal.25 Unexpectedly, there was no significant change in the M/M number of phagocytic markers of CD68 expression in the SCI-epoB and SCI-vehicle groups. Nevertheless, CD68+ M/M shrink and become crenulated morphologically after epoB treatment, which reminds us that epoB shows quite a bit of cytotoxic effects to phagocytic M/Ms and elevated M-CSF stimulated the M/M phagocytosis insufficiently.In the CNS, M-CSF modulates the survival, proliferation, maturation, differentiation, recruitment, and activation of M/Ms.α, MCP-1, and TNF-α) and two anti-inflammatory cytokines (IL-4 and TGF-β).26 Many studies reported that M-CSF-treated M/Ms changed to M2 phenotypes.27 Interestingly, which phenotypes are lesion site M/Ms tend to when exposing well-balanced anti- and proinflammatory cytokines in CSF? These questions guide us to evaluate the M/M phenotype conversion after epoB treatment.Beyond M-CSF, there are five other cytokines elevated in CSF at the same time, which contains three proinflammatory cytokines  were used. All rats were housed in stainless-steel cages (six per cage) in a room kept at 22±1 °C on a 12–12 h light–dark cycle. All rats were acclimatized to their environment for 1 week and free access to food and water. A total of 198 animals were used for all the experiments . The stu41 Briefly, the animals were anesthetized with an intraperitoneal injection of chloral hydrate (0.33 ml/kg), and the intact dorsal cord surface was exposed by laminectomy at T9. Subsequently, moderate contusive injury was conducted by impounder striking  at the surface of T9 SC. The SCI models were maintained on twice-daily bladder evacuation manually until the bladder function of the rat was re-established. After injury, the rats were randomly assigned to various groups. First, in accordance with previous literature, SCI models were treated with epoB  or vehicle  and 64.3% saline) via intraperitoneal 1  DPI systemically, which is referred to as SCI-epoB or SCI-vehicle. Second, SCI-epoB were randomly administered corresponding neutralizing antibodies according to the experiment design. Rat did not undergo SCI surgery, which was treated with epoB  is referred to as intact-epoB. Rats used for histological analysis were killed by intracardiac perfusion with 0.9% NaCl, followed by 4% paraformaldehyde after anesthesia. Rats used for others assays were killed with an overdose of anesthetic.SCI models were prepared as described previously.μl CSF from each rat without blood contamination. Finally, we sutured the skin and muscle, sterilized the incision, and recuperated the rats at 37 °C. CSF was sampled one time a rat for array (n=3) and once every other day in experiment groups  for ELISA (n=3)  . Specifin=3) and total 30 CSF samples were analyzed. We centrifuged the CSF samples at 1000 × g for 5 min after thawing to remove any particulates, aspirated 100 μl CSF samples, and diluted them to 1 ml for all later array procedures. The manufacturer’s instructions were followed for all steps involving the cytokine array. We briefly placed each membrane into a well of the incubation tray and incubated in blocking buffer for 30 min at room temperature. After washing, we added diluted CSF into each well and incubated it overnight at 4 °C. After consecutive washes, we pipetted 1 ml of the prepared detection antibody cocktail into each well and incubated it overnight at 4 °C. With the same washes, 2 ml of HRP-streptavidin was added into each well and incubated overnight at 4 °C. After consecutive washes, we then added 500 μl of the detection buffer mixture onto each membrane and incubated them for 2 min at room temperature. Afterwards, we transferred the membranes to the CCD camera and exposed them. The intensity of the positive control signal was used to normalize the cytokine signal between the two arrays.RayBiotech Neuro Discovery Array C Series  was used for detecting changes of CSF cytokine levels after epoB or vehicle treatment (n=3)  according to the manufacturer's instructions.The rats’ CSF of the SCI-vehicle and SCI-epoB groups were collected as described above in consecutive time. Level and duration of the increasing M-CSF was measured using ELISA , goat anti-IL-4 , mouse anti-M-CSF , rabbit anti-MCP-1 , rabbit anti-TNF-α , mouse anti-TGF-β .46 A mix of isotype antibody served as the control. The number and start time of neutralizing antibodies injection was based on results of cytokines antibody array. SCI-epoB injected with M-CSF antibody or MCP-1 antibody from 0 DPE until 7 DPE or killing are referred to as SCI-epoB-M-CSF Ab or SCI-epoB-MCP-1 Ab, respectively. SCI-epoB injected with neutralizing antibody mix  from 0 DPE until 7 DPE or killing is referred to as SCI-epoB-NAM, and SCI-epoB injected with LIX (C–X–C motif chemokine 5) antibody from 3 DPE until 14 DPE or killing is referred to as SCI-epoB-LIX Ab.All neutralizing antibodies were intravenously injected into the tail vein every other day after epoB or vehicle administration. The injection doses of neutralizing antibodies are specified according to the published studies, as follows: goat anti-IL-1in vivo experiments, SCI models were anesthetized and killed via intracardiac perfusion with 0.9% NaCl, followed by 4% paraformaldehyde 3, 7 or 10 DPE. Then SC and brain samples were removed and fixed in 4% paraformaldehyde. After 3 days fixation, tissues were equilibrated in paraformaldehyde supplemented with 30% sucrose. After that, the samples were cut into 10-μm sections horizontally, transversely or sagittally, and the slides were kept in a cryoprotective solution at −80 °C.For μg/ml; Abcam), mouse anti-M-CSF , rabbit anti-M-CSF  and mouse anti-O4 . After overnight incubation, primary antibodies were discarded and slides were rinsed gently in PBS three times. Afterwards, the fluorescent secondary antibodies were added to sections for single or double staining: FITC-conjugated goat anti-rabbit , PE-conjugated goat anti-mouse , FITC-conjugated goat anti-mouse , PE-conjugated goat anti-rabbit , and CY3-conjugated goat anti-chicken  for 2 h at room temperature. Finally, nuclei were stained with Prolong Gold Antifade reagent with DAPI . Sections were captured with a fluorescent microscope  at equal exposure time.At the beginning, we warmed the frozen slides to room temperature and washed them once gently in phosphate-buffered solution (PBS), and then we blocked the slides in blocking buffer (PBS supplementary with 1% BSA+5% goat serum). After washing two times gently, the following primary anti-rat antibodies were incubated with the sections in the blocking buffer at 4 °C overnight: APC-conjugated anti-CD11b , chicken anti-GFAP , rabbit anti-NeuN , rabbit anti-CD45 , mouse anti-CD31 , chicken anti-vimentin , mouse anti-CD8 , rabbit anti-Iba-1 , rabbit anti-myeloperoxidase (MPO) , mouse anti-CD68  assay and trypan blue staining. In brief, NR8383 cells were planted at a density of 1x104 per well in 96-well plates. After incubation for 24 h, the cells were treated by medium or 2 ng/ml M-CSF, and meanwhile they were also exposed to epoB at the final concentration of 1 or 5 nM. After 3 or 5 days of stimulation, 20 μl MTT (5 mg/ml) was added to each well, and 4 h later, cells were lysed by 100 μl of DMSO. The absorbance was determined at 570 nm on a scanning multiwell spectrophotometer . For further assessing the cytotoxic effects of epoB on M/Ms, trypan blue dye technique was used for distinguishing the dead cells. Briefly, NR8383 was plated in 8-well chamber slides  and treated by M-CSF plus epoB as above. Three days after stimulation, slides were centrifuged to sedimentate suspension cells and stained with trypan blue at the final concentration of 0.04% for 3 min at 37 °C. Data were collected as described above.NR8383 cells  were cultured in DMEM/F12K medium  supplement with 20% heat-inactivated fetal bovine serum , 50 U/ml penicillin, and 50 n=6) of rats were killed for FC 3 or 7 DPE. For the groups, SCI-epoB-M-CSF group serves as a positive control because only 24 cytokines were detected by our antibody array, and six of these cytokines were elevated in the CSF. Hundreds of cytokines changing were still unknown, so SCI-epoB-NAM was used for detecting the combined effect of all unknown cytokines in the SCI milieu. Sample collection was conducted by dissecting out the pieces of 0.5 cm length SC using the lesion site as a center. We transferred the samples into cold Dulbecco's phosphate-buffered saline (D-PBS) immediately after resection, and then washed the samples two times with cold D-PBS gently to remove blood cells from the surface of the SC. After that, we cut the sample into multiple small segments, and they were digested by trypsin for 30 min at 37 °C. After digestion, trypsin was removed by centrifugation and cells were resuspended in staining buffer (PBS with 2%FBS). Single-cell suspension was obtained from filtering through the 48μm mesh. Samples were stained according to the manufacturer’s instructions in the subsequent steps. Briefly, 1 × 106 isolated cells were stained in 100 μl staining buffer with the following primary antibodies: APC-conjugated anti-CD11b , rabbit anti-CD16/32 , mouse anti-CD206 , PE-conjugated anti-CD163 , FITC-conjugated anti-CD86 , rabbit anti-TNF-α , mouse anti-TGF-β , mouse anti-CD68 , and rabbit anti-CD45. After incubation for 30 min at 4 °C with the first antibodies, and washing twice by staining buffer, the following fluorescent-labeled secondary antibodies were added to bind corresponding primary non-fluorescent antibodies: FITC-conjugated goat anti-rabbit  and PE-conjugated goat anti-mouse . After incubating and washing the same as the last step, cells were fixed with 1% paraformaldehyde before analysis by FC. A total of 50 000 events were collected and analyzed on FACSCalibur Flow Cytometer  using the FlowJo Software . Isotype-matched antibody-stained cells, positive-stained cells, and unstained cells were used as controls to gate the cell populations of interest.For assessing the quantitative, phenotypic, polarized, and functional status of M/Ms, the SCI-vehicle, SCI-epoB-M-CSF, SCI-epoB, and SCI-epoB-NAM group . In brief, cultured NR8383 was digested and resuspended in CFSE staining buffer without FBS as 2 × 106 cells/ml for 10 min at 37 °C. After incubation, adding FBS, washing, and labeling, cells were seeded in six-well plates as 5 × 105 cells/well. Subsequently, the cells were treated with medium or 2 ng/ml rat recombinant M-CSF while being exposed to 1 nM/5 nM epoB or not. Either 3 or 5 days after labeling, cells were analyzed on FACSCalibur flow cytometer as described above.To detect the elevated M-CSF’s proliferative effect on M/Ms, the NR8383 cell line and CFSE cell proliferation assay were used 47 In short, 1 ml saline containing 50 μl APC-conjugated anti-CD11b (Miltenyi) was injected via the coccygeal vein in SCI model 1 DPE. SC tissues were taken for visualization of recruited M/Ms with immunofluorescence staining (n=3) 3 DPE.For assessing the cytotoxic effect of epoB in SCI milieu M/Ms, fluorophore-conjugated primary antibody was used for labeling the M/Ms as described previously.n=6) . The experiment was conducted according to the instructions. Briefly, SC tissue was washed by cold PBS to remove blood cells from the surface. Then tissues were homogenized in 0.5% hexadecyltrimethylammonium hydroxide PBS solution and centrifuged. After centrifugation, the supernatant was transferred into PBS (pH 6.0) containing 0.17 mg/ml 3,3′-dimethoxybenzidine and 0.0005% H2O2. Afterwards, supernatant MPO catalyzed H2O2-dependent oxidation of 3,3′-dimethoxybenzidine to produce yellow compound, which could be measured by 460 nm.MPO activity in the SC of the SCI-vehicle, SCI-epoB, and SCI-epoB-LIX Ab group 10 DPE were determined by MPO Detection Kit (12 Every group contained six or three (M/M labeling in vivo) animals, and the SCs from rats were cut into five 10-μm-thick sections spaced 20 μm apart serially through the entire injury site. The target area was a range of ±1000 μm surrounding the lesion epicenter, and the counting frame size was 550 μm × 550 μm. The injury size was detected by Luxol/Nissel staining, and the lesion epicenter was identified as the unlabeled region by Luxol. Quantifications of the M/M and neutrophils numbers, density per counting frame, and density per cell were performed using specific tools of the Image-Pro Plus Software .Quantification of immunolabeling was estimated by unbiased observers after removing the background threshold level using the Image-Pro Plus Software 5.1  as described previously.n=9) as described by Basso et al.48 Briefly, two investigators, who were not aware of the experiment design, operated the behavioral tests. BBB scores were observed and recorded at 1, 3, 5, and 7 DPE and then weekly until 7 weeks.Locomotion recovery was evaluated by the Basso, Beattie, and Bresnahan (BBB) open-field locomotor rating scale  and expressed as the mean±S.D. The differences among groups were compared and analyzed by one-way ANOVA, followed by the Fisher's LSD procedure. The rate of the group was analyzed by


In AD, degeneration of neurons is preceded by the formation of Aβ plaques, which show different morphological forms. Most of them are birefringent owing to the parallel arrangement of amyloid fibrils. Here, we present polarization sensitive optical coherence microscopy (PS-OCM) for imaging mature neuritic Aβ plaques based on their birefringent properties. Formalin-fixed, post-mortem brain samples of advanced stage AD patients were investigated. In several cortical brain regions, neuritic Aβ plaques were successfully visualized in tomographic and three-dimensional (3D) images. Cortical grey matter appeared polarization preserving, whereas neuritic plaques caused increased phase retardation. Consistent with the results from PS-OCM imaging, the 3D structure of senile Aβ plaques was computationally modelled for different illumination settings and plaque sizes. Furthermore, the birefringent properties of cortical and meningeal vessel walls in CAA were investigated in selected samples. Significantly increased birefringence was found in smaller vessels. Overall, these results provide evidence that PS-OCM is able to assess amyloidosis based on intrinsic birefringent properties.One major hallmark of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA) is the deposition of extracellular senile plaques and vessel wall deposits composed of amyloid-beta (A More than 46 million patients suffered from Alzheimer’s disease (AD) in 2015, making it the most common form of dementia in the world34β) protein and intracellular neurofibrillary tangles composed of hyperphosphorylated tau proteinβ, the concept of preclinical AD has been developedβ aggregates, respectively, magnetic resonance imaging (MRI) and positron emission tomography (PET) are promising imaging techniques111213β42 in their cerebrospinal fluidThe diagnosis of AD is challenging. Clinically, AD manifests with progressive cognitive impairment with increasingly accompanying neurocognitive and neurological disturbancesβ and tau protein deposits using immunohistochemical methods. Extracellular Aβ deposits show different morphologies from perineuronal and diffuse deposits to so-called focal deposits, which may be associated or not to a neuritic coronaβ has a fibrillary substructure and exhibits birefringence  appear reddish in bright field micrographs after staining with Congo red and have a positive signal in polarization contrast micrographs18The gold standard for the diagnosis of AD is the neuropathological examination of post-mortem brain including screening for Aringence 171819. Tex vivo human brain tissue252627in vivo and in vitro29313234Optical coherence tomography (OCT) is a noninvasive modality for three-dimensional (3D) imaging of transparent and translucent samples and tissuesex vivo PS-OCT imaging of neuritic Aβ plaques in the brains of AD patients. Complementary to the 3D image data, we used electron microscopy data to develop a computational model for polarization propagation in cortical Aβ plaques. Furthermore, we investigated the birefringent properties of cerebral vasculature in cerebral amyloid angiopathy (CAA) and controls.In this article, we present cerebral imaging of AD-related pathology using polarization-sensitive (PS) OCT. Other than standard OCT, which acquires images solely based on the intensity of light backscattered from tissue, PS-OCT can simultaneously measure its polarization state37384143Brain samples of patients diagnosed with end-stage AD  as well as with confirmed extensive CAA  were retrieved from the Neurobiobank of the Medical University of Vienna. Post-mortem specimens were obtained from patients who underwent autopsy at Medical University of Vienna. Informed consent for the use of their biological materials for research purposes was obtained by all patients. All methods were carried out in accordance with relevant guidelines and regulations. This study was approved by the institutional review board at Medical University of Vienna .μm for immunostaining. In addition, unstained sections were cut at a thickness of 50 μm and deparaffinized for examination by PS-OCM.From each post-mortem brain, several representative regions  were sampled. Formalin-fixed brain tissues were directly imaged using PS-OCT prior to paraffin-embedding for histopathologic work-up. Paraffin blocks were cut at a thickness of 3 Histologic assessment was based on conventional Hematoxylin Eosin and Congo red stainings as well as immunohistochemistry for beta A4-amyloid . Slides were assessed using a Nikon Eclipse 80i microscope (camera: Jenoptik ProgRes C5) comprising bright field and polarization contrast microscopy. For electron microscopy small areas of temporal cortex were postfixed in 1% osmium tetroxide for 1–2 hours, dehydrated through a series of graded ethanols and propylene oxide, and then embedded in Embed 812 resin. Ultrathin sections were stained with lead citrate and uranyl acetate. Specimens were examined using a transmission electron microscope (Carl Zeiss EM 109).λ = 837 nm and covering a FWHM bandwidth of 52 nm served as a light source. The measured axial resolution was 7.6 μm in air, corresponding to 5.6 μm in brain tissue . A 10× water immersion objective was used for some samples in order to reduce the strong reflection at the air-tissue interface. Formol or phosphate buffered saline was used for immersion and for preventing the samples from drying. The illuminating beam diameter was chosen to intentionally underfill the objectives’ back apertures such that vignetting effects during x-y scanning were minimal. The effective numerical apertures were between 0.072 for the 10× objective (0.096 with water immersion) and ~0.31 for the 40× objective. Samples were placed in a petri dish located on a three axis manual translation stage , which enabled precise alignment of the sample with respect to the beam. By raster scanning the beam over the tissue, PS-OCT data cubes covering a region of up to 1.8 mm (x) × 1.8 mm (y) × 2.7 mm  were recorded. Each data set comprised 700 (x) × 700 (y) × 1024 (z) voxels and was acquired in approximately 7.5 seconds.In the sample arm, a magnifying telescope was used in order to relay the pivot point of a pair of galvanometer scanners onto the back focal plane of a microscope objective. Objectives of different magnifications between 10× and 40× were used to provide high lateral resolution . Hence, the majority of the image areas in θ = 0°, the second set of simulations was performed. Again, plaques sized 6–80 μm were simulated. The results are shown in θ = ±45° cause an increase (red) or decrease (blue) of the signal in the respective areas after passing the analyzer. As a result, the intensity pattern is subject to an azimuthal variation. To validate these simulation results, we performed polarized light microscopy of potato starch samples, which have a radial, birefringent substructure similar to that of neuritic plaques shown in In order to study the effect of circularly polarized light on the observed polarization component for an analyzer orientation of β plaques were computed in the third set of simulations. A matrix of cumulative phase retardation images for simulated plaque diameters of 6–80 μm and for NA’s of 0.1–1.0 is shown in PS-OCT signals arising from birefringent Aψ contribute to a lateral spread of these retardation shadows (lower rows). This effect is very pronounced in the x-y projections shown for a 20-μm plaque at the right-hand side.No phase retardation change was observed in most of the grey matter tissue surrounding the plaques. Within the plaques, which are demarked by a dotted white circle in the top row, a cumulative increase of retardation visible as a color change from blue to red from top to bottom was caused by the birefringent fibers. Beneath the plaques, the retardation values stay at the plaques’ maximum value. Note that all images are scaled from zero to maximum retardation of the respective image in order to enable a direct comparison between the different scenarios. The focal plane was set to the central z-plane of the volume which reflects in the tightest focus at high NA. For low NA values, the plaques cast straight retardation shadows in z-direction. For higher NA values, ray components impinging at greater angles Cortical tissue of AD patients was imaged with the PS-OCM system. Exemplary 3D phase retardation images are shown in β plaques with increased phase retardation were observed in cortical tissue of the hippocampal formation, the frontal lobe and the occipital lobe of the AD patient. Inserts below the renderings show details of some plaque-laden regions.Larger phase retardation renderings of three representative regions in an AD brain are shown in β plaque located at an adjacent location in the same sample is shown in PS-OCM was also performed in thin sections of cerebral tissue. In order to investigate whether PS-OCM can detect increased birefringence in the vessel walls related to amyloidosis, selected cortical and meningeal vessels were imaged in 3 brains diagnosed with CAA and 2 control brains. For this purpose, larger vessels were identified in temporal, frontal and occipital tissue samples, respectively. PS-OCM imaging was performed with the beam oriented roughly perpendicular to the vessel cross-section, i.e., parallel to the vessel direction. Twenty-one 3D data sets in CAA brains and seven control data sets were acquired, each covering a field of view of 1.8 mm (x) × 1.8 mm (y) and containing at least one vessel.dmax) and minimum vessel diameters (dmin) were measured. The average vessel diameter was computed as μm . Average vessel wall birefringence was 0.21 ± 0.04 °/μm (range: 0.16 °/μm–0.32 °/μm). In μm, nCAA = 11), birefringence was significantly higher than in the control vessels . In contrast, in larger vessels, , no significant difference was observed. When comparing vessel wall birefringence in all CAA versus non-CAA brains, a trend  of slightly higher values (0.22 ± 0.04 °/μm) in CAA than in controls (0.19 ± 0.03 °/μm) was observed, PS-OCM projection images were computed . Using tβ plaques are a major hallmark of AD. A variety of imaging methods have been developed for the detection of cortical Aβ, including PET1350525455575836β plaques used in histopathological practice to PS-OCT imaging. We demonstrate that individual plaques can be identified and portrayed three-dimensionally with micrometer-scale resolution  is required to detect small Aβ plaques inducing only modest retardation. An analysis of the dependence of retardation measurement precision on the SNR of the PS-OCM imaging setting used here is provided in β plaques, this precision translates to a minimum detectable plaque size of ~20 μm for the PS-OCM imaging setting employed here. The detection capabilities of PS-OCM for small Aβ plaques may be further improved by image averaging45Cerebral Asolution , 4 and 5croscopy  and, at β plaque load. Still, OCT is a rapid imaging modality and datasets of several gigabytes of data can be acquired in just a few seconds. Second, it is known that formalin fixation is accompanied by both structural changes such as tissue shrinkage and changes of optical properties68β plaque load in PS-OCM volumesβ plaque. Finally, the image data only included patients with advanced AD. In future studies, it will be interesting to investigate tissue samples of patients at earlier stages in order to get an impression of the diagnostic sensitivity of PS-OCT during disease progression. An exciting application of the technique presented here would be diagnostic imaging of the retina in AD patients where disease-related neuropathologic features – including Aβ plaque deposition – were also reported7071β plaque load in a mouse model of AD, in vivo imaging of retinal plaques was demonstrated based on fluorescent labeling by systematic curcumin administration71β plaques were detectable even earlier than in the brain and accumulated with disease progression in the transgenic mice, retinal scans revealing Aβ load may serve as an easily accessible target for tracking disease progression also in human patientsβ plaques provide intrinsic polarization contrast.There are some limitations to the current imaging approach. First, in order to increase the currently rather limited 3D coverage of our PS-OCM, the technique could be implemented at longer wavelengths, which would enable deeper penetration into cerebral tissue6544β deposition than veinsin vivo in animal models of CAA would be an interesting continuation of the experimental results presented here.The vasculature of the CNS is affected by amyloidosis in CAA7574β plaques was computationally modelled for different illumination settings and plaque sizes. Furthermore, the birefringent properties of cortical and meningeal vessel walls in CAA were investigated. Significant differences were found in smaller vessels; however, the sample size for this experiment was small. In summary, PS-OCT may be a useful tool for three-dimensional imaging of amyloidosis based on intrinsic polarization properties.In this paper, we have demonstrated PS-OCM imaging in unstained, formalin-fixed brain samples of human AD and CAA patients. Based on their intrinsic birefringence properties, neuritic plaques were successfully visualized in representative cortical regions. In line with the results from PS-OCM imaging, the three-dimensional structure of senile AHow to cite this article: Baumann, B. et al. Visualization of neuritic plaques in Alzheimer’s disease by polarization-sensitive optical coherence microscopy. Sci. Rep.7, 43477; doi: 10.1038/srep43477 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.