Several studies suggest that circulating bone marrow derived stem cells promote the regeneration of ischemic tissues. For hematopoietic stem cell transplantation combinatorial granulocyte-colony stimulating factor (G-CSF)/Plerixafor (AMD3100) administration was shown to enhance mobilization of bone marrow derived stem cells compared to G-CSF monotherapy. Here we tested the hypothesis whether combinatorial G-CSF/AMD3100 therapy has beneficial effects in cardiac recovery in a mouse model of myocardial infarction.We analyzed the effect of single G-CSF (250 µg/kg/day) and combinatorial G-CSF/AMD3100 (100 µg/kg/day) treatment on cardiac morphology, vascularization, and hemodynamics 28 days after permanent ligation of the left anterior descending artery (LAD). G-CSF treatment started directly after induction of myocardial infarction (MI) for 3 consecutive days followed by a single AMD3100 application on day three after MI in the G-CSF/AMD3100 group. Cell mobilization was assessed by flow cytometry of blood samples drawn from tail vein on day 0, 7, and 14.Peripheral blood analysis 7 days after MI showed enhanced mobilization of white blood cells (WBC) and endothelial progenitor cells (EPC) upon G-CSF and combinatorial G-CSF/AMD3100 treatment. However, single or combinatorial treatment showed no improvement in survival, left ventricular function, and infarction size compared to the saline treated control group 28 days after MI. Furthermore, no differences in histology and vascularization of infarcted hearts could be observed.Although the implemented treatment regimen caused no adverse effects, our data show that combinatorial G-CSF/AMD therapy does not promote myocardial regeneration after permanent LAD occlusion. Cytokine mediated mobilization of peripheral blood stem cells for autologous stem cell transplantation is a generally accepted therapeutic option for the hematopoietic reconstitution after myoablative chemotherapy. The clinically used cytokine granulocyte-colony stimulating factor (G-CSF) is known to mobilize various subsets of hematopoietic stem and progenitor cells (HSPC) into blood circulation that may contribute to tissue repair. Additionally G-CSF was shown to have anti-apoptotic, anti-inflammatory and antioxidant effects Especially in the field of ischemic heart disease numerous studies investigated the efficacy of G-CSF induced stem cell mobilization in myocardial regeneration. While early animal studies and small clinical trials indicated beneficial effects on cardiac regeneration, these results were later challenged by studies that could not confirm these positive effects or even reported deleterious effects of G-CSF therapy on cardiac recovery  was shown to rapidly mobilize stem cells by reversibly disrupting the interaction between CXCR4 and SDF-1α that tethers stem cells to the bone marrow (BM) environment On the basis of these results we explored possible beneficial effects of combinatorial G-CSF/AMD therapy in myocardial regeneration in a mouse model of MI. We applied a treatment regimen were G-CSF administration started directly after induction of MI for 3 consecutive days followed by a single dose of AMD in order to attain positive effects on stem cell mobilization while avoiding negative effects on stem cell homing.Eight to ten weeks old male FVB/NJ mice (Charles River) were anaesthetized with an intraperitoneal injection of midazolam (5.0 mg/kg), fentanyl (0.05 mg/kg), and medetomidin (0.5 mg/kg). The animals were intubated and ventilated using a rodent ventilator  with a stroke volume of 0.2 ml and respiration rate of 200 strokes/min. Inhalation anesthesia was maintained with 1.5% isoflurane through a vaporizer with 100% oxygen. After left lateral thoracotomy at the left third intercostal space, the left anterior descending coronary (LAD) was ligated with 7–0 prolene sutures (Ethicon) 1 mm below the tip of the left atrial auricle. The chest and skin were closed with 6–0 vicryl (Ethicon) sutures. The sham group underwent the same procedure except for the ligation of the LAD. After induction of MI animals were randomly divided into 3 groups and stem cell mobilization was induced by the following dosing regimen:1) G-CSF  subcutaneously (s.c.) starting 1 h post-MI and then daily on days 1 and 2; 2) Combination of G-CSF s.c. starting 1 h post-MI and then daily on days 1 and 2+ AMD3100  s.c. as single dose on day 3 post-MI; 3) the control MI group received at the same time points equal volumes of saline (0.9% NaCl). Data acquired from control MI and sham groups contributed in parallel to another study but were pooled with new animals 3) and incubated with Alexa Fluor 647-labeled anti-Flk-1/VEGFR2 (clone 89B3A5), Phycoerythrin (PE)-conjugated anti-Ly-6A/E (Sca-1) (clone D7), and allophycocyanin(APC)-labeled anti-CD117 (c-kit) (clone 2B8) . Appropriate isotype controls were always included. Cells were analyzed on FACSCanto II flow cytometer using FACSDiva software (BD Biosciences) and analyzed with FlowJo software (TreeStar).Whole blood samples were drawn from the tail vein 0, 7 and 14 days after MI and circulating white blood cells (WBC) were counted with an animal blood counter (Scil Vet abc). For fluorescence activated cell sorting (FACS) analysis, blood mononuclear cells were separated via gradient-density centrifugation using Histopaque-1083 (Sigma-Aldrich). Cells were blocked with normal rat serum and anti-CD16/32 monoclonal antibody (mAb) (clone 93) in FACS buffer  was inserted through the right carotid artery into the left ventricle to record baseline pressure-volume loops in the closed chest. Conversion of raw conductance data to calibrated volumes was performed by determination of parallel conductance (Vp) using hypertonic saline dilution method 2) in 10 sections per heart (n = 5–8 for each group) within the border zone, infarcted and remote area. Alpha-smooth muscle actin positive vascular structures were counted per area assessed with ImageJ software. Both parameters are expressed as positive stained vascular structures per mm2.At day 28, hearts were excised, fixed overnight with 4% formalin/PBS-buffered and embedded in paraffin. Transversal sections of a thickness of 3 µm were cut from apex to base and mounted on glass slides for histological and immunhistochemical staining. Masson trichrome (MT) staining was done according to standard protocols. Infarction size was determined using midline length measurement on MT stained sections from the surgical LAD occlusion to the base Data are reported as mean value ± standard error of the mean (SEM), and were analyzed by two-tailed unpaired Student's t-test. A p-value of less than 0.1 was considered as a trend a p-value less than 0.05 was considered significant. Group comparisons were performed using One-way ANOVA followed by the Tukey's test. Survival analysis was assessed by Kaplan-Meier method.Circulating white blood cells (WBC) and progenitor cells were determined before (0 days) and after MI in drug treated mice and untreated controls. Treatment with G-CSF (*p<0.05 vs. day 0) and G-CSF/AMD (p = 0.075 vs. day 0) enhanced the MI induced mobilization of circulating WBC, without reaching statistical significance compared to the untreated group 7 days after MI . AlthougA total of 106 mice  were included into cumulative Kaplan-Meier survival analysis. There were no statistical differences in overall mortality between saline and drug treated groups . In ordeSurgical induction of MI severely impaired cardiac function compared to sham-operated animals as assessed by hemodynamic measurements 28 days after surgery. In sham-operated mice the left ventricular ejection fraction (EF) was 66.2%±5.2 and declined to 23.8%±2.9 (p<0.001) in control MI mice. G-CSF and G-CSF/AMD treatment did not improve EF 28 days after MI . DetaileDetermination of infarction size 28 days post MI either by TTC staining or midline infarct length method on Masson trichrome stained sections revealed no significant difference between drug treated animals and control MI mice . QuantifTwenty-eight days after LAD ligation characteristic signs of late phase postinfarction remodelling of the heart were visible. Infarcted left ventricles showed typical loss of myocardium, left ventricular wall thinning and fibrous scar formation. Masson trichrome staining of infarcted regions revealed a viable subendocardial layer followed by collagen rich fibrous tissue reaching into border zone myocardium . No obviEPC as well as certain subsets of WBC are known to exert angiogenic properties in ischemic tissues. Using antibodies against endothelial (CD31/PECAM-1) and smooth muscle cells (α-smooth muscle actin), the abundance of capillaries and arterioles in remote area (RA), border zone (BZ) and infarct zone (IF) was analyzed . TreatmeIn the present study, the potential regenerative properties of a combinatorial G-CSF/AMD therapy were tested in a model of permanent LAD occlusion. During the last decade numerous studies investigated the hypothesis that cytokine mediated mobilization of stem cells contribute to cardiac regeneration after MI. Implementing different cytokines and mobilization protocols these studies yielded controversial results ranging from beneficial effects to even deleterious effects on cardiac regeneration G-CSF as well as the CXCR4 inhibitor AMD3100 has been shown to mobilize HSPC and potential angiogenic cells from BM + cells, but with huge animal-to-animal variations was also seen in a rat model of MI after G-CSF treatment by Werneck-de-Castro et al. In the present study we found that G-CSF and G-CSF/AMD treatment promoted mobilization of WBC and EPC into peripheral blood. Mobilization tended to be higher in treatment groups compared to control mice without reaching statistical significance. This was due to large variations within treatment groups suggesting that not all animals responded to the treatment in the same way. Inter-individual variations in G-CSF induced HSPC mobilization are also evident in humans + or CD133+ HSPC.Combination of G-CSF with AMD did not synergistically increase WBC counts, HPC or EPC numbers at day 7 after MI (4 days after AMD treatment). The rapid AMD mediated mobilization might be an explanation for this, however, significantly increased EPC mobilization could be detected even 7 days after single AMD injection in a mouse MI model Survival analysis showed no significant differences in mortality between saline and drug treated groups. On the basis of the applied drug regimen, beneficial effects on cardiac recovery resulting from either direct or paracrine actions of mobilized stem cells would not be conceivable until day 4 after MI. However, exclusion of animals that died in the first 4 days after MI did not uncover a reduced mortality of drug treated animals. Furthermore, pressure volume relationships of control and drug treated animals were recorded to evaluate heart function 28 days after MI. Drastically reduced heart function was evident in control MI mice compared to sham operated mice. Although G-CSF and G-CSF/AMD treatment led to a slight improvement of some hemodynamic parameters, no significant changes compared to the control MI group were observable. Moreover, there was no reduction in infarction size visible in drug-treated versus control MI animals. Since these basic parameters indicated no improvement of heart regeneration with respect to the applied therapy, histology and vascularization of infarcted hearts was evaluated. Histological analysis showed drastically reduced myocardium at the ischemic site of the left ventricle that was replaced by a thin collagen rich, fibrous tissue layer. In control MI as well as treatment groups similar histological pattern were visible showing no obvious signs of cardiac regeneration.Besides postulated direct actions of cytokines or stem cells on myocyte regeneration, numerous studies linked HSPC mobilization to favorable angiogenic effects promoting neovascularization of ischemic tissues In conclusion, although the applied drug regimen enhanced the mobilization of potentially regenerative cells, the combination of G-CSF and AMD did not significantly improve cardiac recovery after MI compared to control MI mice. On the other hand, no adverse effects of the applied drug treatment on cardiac function and remodeling could be observed. Further studies are needed to elucidate the complex mechanisms after cardiac injury to figure out treatment regimens that more specifically promote cardiac healing.Beneficial effects of stem cells or cytokines were already observed after permanent LAD occlusion Figure S1FACS analysis of peripheral white blood cells of naïve (day 0) mice and 7 days post MI. (A) Mononuclear cells were gated on forward scatter (FSC) and side scatter (SSC) plot to exclude blood cells, debris and dead cells. Percentages of (B) c-Kit/Sca-1 double positive and (C) Flk1/Sca-1 double positive sub-populations were recorded. Representative dots plots are shown.(TIF)Click here for additional data file.


Cerebrospinal fluid (CSF) amyloid-beta 38 (Aβ38), 40 (Aβ40), 42 (Aβ42) and total tau (T-tau) are finding increasing utility as biomarkers of Alzheimer’s disease (AD). The purpose of this study was to determine whether measured CSF biomarker concentrations were affected by transfer of CSF between tubes, and whether addition of a non-ionic surfactant mitigates any observed effects.AD and control CSF was transferred consecutively between polypropylene tubes. Aβ peptides and T-tau were measured with and without addition of Tween 20 (0.05%).Measured concentrations of Aβ42 decreased by approximately 25% with each consecutive transfer. Measured concentrations of Aβ38 and Aβ40 were also observed to decrease significantly with each consecutive transfer (approximately 16% loss per transfer). Measured concentrations of T-tau also decreased significantly, but at much smaller magnitude than the Aβ peptides (approximately 4% loss per transfer). The addition of Tween 20 mitigated this effect in all samples.Consecutive CSF transfer between tubes has a significant impact on the measured concentration of all Aβ peptides, and significant effect of lesser magnitude on T-tau. This would be sufficient to alter biomarker ratios enough to mislead diagnosis. The introduction of Tween 20 at the initial aliquoting stage was observed to significantly mitigate this effect. Cerebrospinal fluid (CSF) amyloid beta 38 (Aβ38), amyloid beta 40 (Aβ40), amyloid beta 42 (Aβ42) and total tau (T-tau) are protein biomarkers used in the clinical diagnosis and research analysis of, and drug development for, Alzheimer’s disease (AD). Inter-site variation in the measured concentration of these proteins, even in the same samples, is well-recognised. Known confounding factors in the measurement of Aβ and tau concentrations include delays in sample analysis , diurnalFollowing an anomalous decrease in assayed Aβ42 concentration in the same CSF between two laboratories, an investigation was made to ascertain the cause. As the CSF tested was the same this ruled out collection factors as a likely variable. Additionally, both laboratories had assayed the sample using the same protocol, suggesting that differences in technique and timing, though impossible to eliminate, should have been minimal. Based on the propensity for certain proteins to adsorb to container surfaces it was hypothesised that, as CSF is transferred between surfaces  the concentration of these proteins in solution would decrease. Given a standardised volume, it was also conjectured that this concentration decrease would be linear, with a relative proportion of the protein being lost at each step. It was predicted that the Aβ peptides would behave in this way, and that T-tau would not be significantly affected. Furthermore, this study explored whether such effects could be mitigated by pre-treating the sample with a non-ionic surfactant  known to reduce tube surface adsorption and/or aggregation of Aβ42 -9.We conducted an initial pilot study to demonstrate proof of concept, which was then followed by a larger experiment consisting of three replicate rounds. All assays were run on a Meso Scale Discovery 6000 platform. The pilot experiment used MSD Human Aβ42 and MSD Human Total Tau kits . Samples were added to Aβ42 and T-tau plates in triplicate.The replication experiment used MSD Human Aβ42 (V-plex), MSD Human Total Tau kits (V-plex) and MSD Aβ Peptide Panel 1  (V-plex). V-plex refers to the updated validation versions that superseded the same kits used in the pilot at the time of these experiments. Samples were added to T-tau and Aβ peptide plates in duplicate. Each assay was repeated at 24 hour intervals a total of three times. The kit manufacturer’s protocol was followed for all pilot and replicate assays.This study tested two pools of de-identified CSF. The first was from a cohort of subjects with CSF biomarker profiles consistent with AD pathology. The combination of Aβ42 <530 ng/L and T-tau >350 ng/L taken together have a sensitivity and specificity for diagnosing AD pathology of approximately 90% [The two pools were provided, pre-mixed and pre-spun, by the Clinical Neurochemistry Laboratory at the Sahlgrenska University Hospital, Sweden, in Greiner Bio-one  polypropylene (PP), sterile, 50 mL tubes (cat. 210261). Each pool was split into two 25 mL aliquots, 12.5 μL Tween 20 (0.05%) was introduced to one aliquot, and the other was kept neat. From these pools four 925 μL aliquots were derived – Neat AD (NAD), Tween AD (TAD), Neat control (NCT), and Tween control (TCT). These aliquots were stored in Sarstedt , PP, DNase/RNase free, 2 mL screw top tubes (cat. 72.694.406).Two pools of CSF were provided by the Clinical Neurochemistry Laboratory at the Sahlgrenska University Hospital, Sweden. These pools were not of the same CSF as those used in the pilot, but met the same criteria for AD and CTRL detailed above. Samples were received in eight Sarstedt 10 mL, screw cap, PP tubes (cat: 62.9924.284), four AD and four CTRL. Both AD and CTRL pools were treated by the same procedure which follows, and is illustrated in Figure At the start of the experiment, the four sample aliquots  were thawed together at room temperature for approximately one hour. A portion of CSF from each was then transferred consecutively to seven other tubes. Starting with Tube 0  with 925 μL, 710 μL was transferred to Tube 1; 510 μL was then transferred from Tube 1 to Tube 2; 310 μL from Tube 2 to Tube 3; and 110 μL from Tube 3 to Tube 4. Thus, the volumes in the experiment aliquots were as follows: Tube 0 = 215 μL, Tube 1 = 200 μL, Tube 2 = 200 μL, Tube 3 = 200 μL, and Tube 5 = 110 μL. Tube 5 had a lower volume due to total volume restrictions. Aβ42 and T-tau aliquots were dispensed into the same disposable PP plate for dilution at factors 1:8 and 1:2, respectively.It was recognised that the transfer volumes used in the pilot were inconsistent and that this was a flaw in its design. Therefore, these were adjusted in the replication study to be uniform. Additionally, three extra transfer steps were included in the transfer series of the replication. At the start of the experiment the four sample aliquots  were thawed together at room temperature for approximately one hour. A portion of CSF from each was then transferred consecutively to seven other tubes, leaving 100 μL in each  was incorporated as a covariate. All statistical analyses were conducted in Stata Version 12.1. Graphs were created using SPSS version 21.P = 0.002). The average percentage difference per transfer was 27.0% of the starting value. For neat control CSF this predicted change was −72.1 pg/mL . The average percentage difference per transfer was 28.6%. Tween 20 acted to mitigate the magnitude of this decrease in both pools. One transfer of AD CSF in the presence of Tween 20 predicted a change in measured Aβ42 concentration of −9.9 pg/mL . The average percentage difference per transfer was 5.1%. For control CSF with added Tween the trend was a non-significant −27.2 pg/mL . The average percentage difference per transfer was 4.9%.Figure P = <0.005). The average percentage difference per transfer was 21.1% of the starting value. For neat control CSF this predicted change was −52.2 pg/mL . The average percentage difference per transfer was 22.7%. Tween 20 acted to mitigate the magnitude of this decrease in both pools. One transfer of AD CSF with Tween 20 predicted a change in measured Aβ42 concentration of −25.2 pg/mL . The average percentage difference per transfer was 7.8%. For control CSF with added Tween one transfer predicted a change in measured Aβ42 concentration of −34.0 pg/mL . The average percentage difference per transfer was 8.0%.Figure P = <0.005). The average percentage difference per transfer was 22.2% of the starting value. One transfer of neat control CSF predicted a change in measured Aβ42 concentration of −41.4 pg/mL . The average percentage difference per transfer was 23.4%. Tween 20 acted to mitigate the magnitude of this decrease in both pools. In AD CSF with Tween 20 one transfer step predicted a concentration change of −17.3 pg/mL . The average percentage difference per transfer was 4.9%. In control CSF with Tween 20 one transfer step predicted a concentration change of −28.1 pg/mL . The average percentage difference per transfer was 5.5%.Figure P = <0.005). The average percentage difference per transfer was 16.0% of the starting value. One transfer of neat control CSF predicted a change in measured Aβ38 concentration of −224.0 pg/mL . The average percentage difference per transfer was 16.1%. Tween 20 acted to mitigate the magnitude of this decrease in both pools. In AD CSF with Tween 20 one transfer step predicted a concentration change of −53.1 pg/mL . The average percentage difference per transfer was 1.4%. In control CSF with Tween 20 one transfer step predicted a concentration change of −38.4 pg/mL . The average percentage difference per transfer was 1.1%.Figure P = <0.005). The average percentage difference per transfer was 15.6% of the starting value. One transfer of neat control CSF predicted a change in measured Aβ40 concentration of −513.3 pg/mL . The average percentage difference per transfer was 17.5%. Tween 20 acted to mitigate the magnitude of this decrease in both pools. In AD CSF with Tween 20 one transfer step predicted a concentration change of −121.5 pg/mL . The average percentage difference per transfer was 1.2%. In control CSF with Tween 20 one transfer step predicted a concentration change of −112.9 pg/mL . The average percentage difference per transfer was 1.6%.Figure P = 0.060, 5.1%; and −34.5 pg/mL, CI: −73.3 to +4.3 pg/mL, P = 0.066, 5.8% respectively), but no evidence for a decline with measured T-Tau concentration in AD and control CSF with Tween .Figure P = 0.003). The average percentage difference per transfer was 4.4% of the starting value. For neat control CSF this predicted change was −11.6 pg/mL . The average percentage difference per transfer was 3.5%. One transfer of AD CSF with Tween 20 did not demonstrate a significant change in measured T-Tau concentration; T-Tau: −1.6 pg/mL . The average percentage difference per transfer was 0.2%. For control CSF with added Tween one transfer did not predict a significant change in measured T-Tau concentration: −0.4 pg/mL . The average percentage difference per transfer was 0.5%.Figure All results are summarised in Table This study demonstrates that the consecutive transfer of CSF samples between tubes has significant impact on the measured concentration of Aβ42. Figure The addition of the non-ionic surfactant Tween 20 to pooled samples prior to aliquoting had a mitigating effect on the reduction in measured Aβ42 over the transfer series, although reduction was still significant. The storage concentration of Tween 20 used in this study was 0.05% and after 1:8 sample dilution would have been approximately 0.006%. The generally accepted critical micelle concentration (CMC) for Tween 20 is 0.007%, but micelle formation has been shown to initiate at 0.002% . This suData show that the consecutive transfer of CSF samples between tubes has a significant impact on the measured concentration of Aβ38. Figure This study demonstrates that the consecutive transfer of CSF samples between tubes has a significant impact on the measured concentration of Aβ40. Figure P values for neat AD (P = 0.06) and control (P = 0.066) pools approached significance. Due to the difference in dilution factor between the pilot (1:2) and the replication study (1:4), and given the apparent tendency for protein loss to be concentration dependent, results may not be directly comparable between the two studies. The replication study showed significant reduction in T-tau concentration over seven transfer steps in both neat pools , it was not possible to maintain consistent 200 μL volume between all the transferral stage aliquots. Based on the previous data  the following discrepancies would be expected relative to an aliquot at 200 μL:Aβ42 Tube 0 (215 μL):Aβ42 Tube 4 (110 μL):Therefore, volume effects are not likely to have been significantly above noise and thus sufficient to bias the results of this study.In the replication study storage aliquots were 1000 μL and transfer aliquots were kept equal at 100 μL so as to exclude any effect on volume. It should be noted that the samples used in the pilot and replication studies were of different pools, and the comparisons of this study are based on proportional concentration loss not absolute values. Direct comparisons of Aβ42 between the pilot and replication study are not likely to be valid.et al. [2. pH change can affect the ‘tertiary and quaternary structure, enzymatic rate constants, solubility, tendency to aggregate, susceptibility to chemical degradation and propensity to adsorb to surfaces’ of constituent proteins [Murphy et al.  have ideproteins . In our in vivo reality. A number of these issues have been addressed in other studies [Standard procedure for CSF collection by lumbar puncture  (However studies ,14.Aβ42, Aβ42:T-tau ratio and Aβ38:40:42 ratio are now widely used to help diagnose Alzheimer’s disease pathology in individuals with cognitive impairment. Between lumbar puncture and laboratory analysis, CSF can be transferred to different containers a number of times, frequently unknown to scientists running the assays, and potentially different between individuals and between sites. We have shown that Aβ42 can be reduced by approximately 25% simply through tube transfer. Effort should be made to minimise multiple transfers of CSF between surfaces, and record how many such steps a sample has gone through. The addition of 0.05% Tween 20 to aliquots at initial sample storage may mitigate at least some of these effects and should be the subject of further study.AD: Alzheimer’s disease; Aβ42: amyloid beta 1–42 peptide; CI: confidence interval; CMC: critical micelle concentration; CSF: cerebrospinal fluid; CTRL: control sample; NAD: neat Alzheimer’s disease; NCT: neat control; PP: polypropylene; TAD: Tween Alzheimer’s disease; TCT: Tween control; T-tau: Total tau.The authors declare that they have no competing interests.JT conceived of the study, participated in its design, conducted the immunoassays and drafted the manuscript. RWP performed the statistical analysis and helped to draft the manuscript. JMS participated in coordination and helped to draft the manuscript. HZ participated in study design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Unblind. Original sample ID’s matched with their blinded aliases.Click here for filePilot Ab42. Assay raw data.Click here for filePilot Ttau. Assay raw data.Click here for fileReplication Ab42 #1. Assay raw data.Click here for fileReplication Ab42 #2. Assay raw data.Click here for fileReplication Ab42 #3. Assay raw data.Click here for fileReplication Ttau #1. Assay raw data.Click here for fileReplication Ttau #2. Assay raw data.Click here for fileReplication Ttau #3. Assay raw data.Click here for fileAb triplex #1. Assay raw data.Click here for fileAb triplex #2. Assay raw data.Click here for fileAb triplex #3. Assay raw data.Click here for file


The aim of this study is to present the five-year longitudinal magnetic resonance imaging (MRI) follow up of a patient with incidental finding of near-obstruction stenosis of the aqueduct of Sylvius due to a large pineal cyst. The patient was scanned 3 times on a 3T MR device using a set of standard structural sequences supplemented with high-resolution constructive interference of steady state (CISS) T2 sequence for precise delineation of the aqueduct of Sylvius and cardiac-gated phase-contrast sequences for the analysis of cerebrospinal fluid (CSF) movement. On all MR scans, the size of the pineal cyst and severity of near-obstruction aqueductal stenosis did not show any morphological changes. There was no significant ventricular enlargement although structural CISS sequence showed a near-obstruction stenosis and cardiac-gated phase-contrast sequences did not detect CSF movement through the aqueduct of Sylvius. Our findings are contradictory to the classic hypothesis of CSF physiology based on secretion, circulation, and absorption of CSF, which states that the impairment of CSF circulation through the aqueduct of Sylvius inevitably leads to a hypertensive hydrocephalus development involving the third and the lateral ventricle. Our research group previously proposed a new hypothesis of CSF physiology, which offers more suitable explanation for such clinical cases. Clinical practice has shown that an aqueduct of Sylvius obstruction or severe stenosis  could lead to a development of the triventricular hypertensive hydrocephalus . AccordiThe aqueduct of Sylvius is a narrow canal inside the mesencephalon that connects the third and the fourth lateral ventricle, thus, according to the classic hypothesis, its passability is of key importance for normal CSF physiology ,3. An aqThe patient presented in this case is a 40-year old woman with 25-year-long history of epilepsy, which is well controlled by 1 mg of clonazepam (the last seizure occurred before 15 years). She does not have a history of any other clinical symptoms . Structural brain magnetic resonance imaging (MRI) performed as a routine diagnostic procedure for epilepsy revealed a large pineal cyst that compressed the quadrigeminal plate. Control follow up MRI exams were performed four and five years after the first exam. The patient’s informed consent was obtained before every MR exam. All MR exams were performed at the Policlinic Neuron at the Croatian Institute for Brain Research in 2009-2014 period.All MRI exams were performed on a 3T MR scanner  using 12-channels head-coil. At the first MRI exam standard MR sequences for epileptic patients were used and the aqueduct of Sylvius was analyzed on sagittal T1 sequence . At the second MRI exam, standard protocol was supplemented with a high-resolution magnetization-prepared rapid acquisition with gradient echo (MPRAGE) sequence . At the third MRI exam we also applied a high-resolution T2 CISS sequence , which showed excellent contrast between the CSF and the brain parenchyma, indicating its suitability for precise morphological analysis and estimation of passability through the aqueduct of Sylvius. Also, sagittal and axial cardiac-gated phase-contrast sequences  were applied for qualitative assessment of CSF movement through the aqueduct of Sylvius. Volumetric analysis at all MRI exams was performed on T2 coronal slices  using Analyze 8.1 software .A series of three MRI examinations showed a large pineal cyst (17 × 10 × 15 mm), which compressed the quadrigeminal plate and caused severe stenosis of the aqueduct of Sylvius . During 3, at the second it increased to 47.15 cm3, and at the third to 48.73 cm3. High-resolution T2 CISS sequence performed at the third MR exam showed a severe stenosis of the aqueduct of Sylvius, which was reduced to almost a virtual diameter, smaller than the size of a single voxel (Volumetric analysis showed a slight increase in the size of the lateral and the third ventricles. At the first MRI exam, summated volume of the lateral and the third ventricles was 42.57 cmle voxel . At the le voxel . On otheDespite the presence of a large pineal gland cyst, a near-obstruction stenosis of the aqueduct of Sylvius, and a complete absence of CSF movement through the aqueduct between the third and the fourth ventricle in our patient, there were no signs of hypertensive triventricular hydrocephalus development, as it would be expected according to the classical CSF hypothesis ,3. We obA similar clinical picture has been described in patients with obstructive hydrocephalus following the treatment with Holter ventriculo-cardial shunt ,10. NameSimilar results were also obtained on experimental animals after a complete artificial obstruction of the aqueduct of Sylvius, which did not induce an enlargement of ventricular system or increase in ventricular CSF pressure . In addiAlthough these clinical observations cannot be explained by the classic CSF hypothesis, they can be explained by our new hypothesis of CSF physiology, which offers a different perspective on the mechanisms responsible for the hydrocephalus development -7. AccorIn conclusion, our previous experimental data  and the


This method and the equation for Vf calculation are based on the assumption that the dilution of the indicator substance is a consequence of the newly formed CSF, ie, that a higher CSF formation rate will result in a higher degree of dilution. However, it has been experimentally shown that the indicator substance dilution inside the CSF system does not occur because of a “newly formed” CSF, but as consequence of a number of other factors . This technique allows “calculation” of the CSF formation even in dead animals, in an in vitro model, and in any other part of the CSF system outside the ventricles that is being perfused. Therefore, this method is indirect and any dilution of the indicator substance in the perfusate caused by other reasons would result in questionable and often contradictory conclusions regarding CSF formation rates.The generally accepted hypothesis on cerebrospinal fluid (CSF) hydrodynamics suggests that CSF is actively formed mainly by choroid plexuses, circulates unidirectionally along the brain ventricles and subarachnoid space, and is passively absorbed mainly into the dural venous sinuses. CSF formation rate (V This hypothesis has so far been presented as a proven fact in all textbooks and review articles.According to the classic hypothesis of cerebrospinal fluid (CSF) physiology, CSF is actively formed inside the brain ventricles, after which it flows unidirectionally along the subarachnoid spaces (SAS) to be absorbed into the venous sinuses across the arachnoid villi and/or via the paraneural sheaths into the lymphatics -8. Thus,According to the classic hypothesis, it is unquestionable that in physiological conditions CSF secretion and absorption inside the CSF space are balanced:f = Va [1]VCSF). Namely, CSF secretion and absorption take place at different CSF system sites, and therefore, the flow rate (QCSF) between these two sites has to be of the same magnitude to keep Vf equal to Va.In other words, the secreted CSF volume (inside brain ventricles) has to be the same as the passively absorbed CSF volume (into the venous sinuses and/or lymphatics). If this is not so, a pathological state may occur . The same is true for the CSF flow  to flow into the lateral ventricle at a constant perfusion rate (µL/min). The outflow cannula is connected to a plastic tube, through which the outflow perfusate is collected from the brain ventricles and the CM  via polyethylene tubing and on the other to a syringe containing artificial fluid with the diluted marker. The syringe is fixed on the perfusion pump that allows the artificial fluid containing the marker  in the outflow perfusate. Considering the importance of Vey et al  for CSF fRate of Formation of CSF in the ventricle, V“Evidence will be presented that diffusion of inulin from the ventricular system is negligible; almost all inulin lost from CSF can be accounted for by bulk absorption distal to the fourth ventricle. It follows that any dilution of inulin during passage through the ventricles results from newly formed inulin-free fluid. Stated mathematicallyf = Vi (ci – co)/co (I)VAn alternative form of equation (I) isf = CIn + Vo – Vi (II)VaRate of Bulk Absorption of Fluid, VIf net loss of inulin can only occur by bulk absorption distal to the fourth ventricle, thena = (Vi ci – Vo co)/co = clearance of inulin, CIn (III)VClearance of inulin from the CSF system may be compared with glomerular elimination of inulin in the kidney. Renal clearance of inulin is a measure of bulk filtration at the glomerulus; CSF clearance of inulin is a measure of bulk absorption distal to the fourth ventricle,”V = rate of flow, mL/min; i and o = subscript referring to inflow and outflow; c = concentration, quantity per milliliter; f and a = subscript referring to formation and bulk absorption of fluid.where Since the equations were elaborated on the basis of a well-known principle for glomerular filtration quantity calculation using inulin clearance in renal physiology, it is very important to analyze and compare the physiological conditions inside the kidney and CSF system during the use of both methods. The conditions of ventriculo-cisternal perfusion are thoroughly described above. Let us now briefly describe the method for inulin clearance calculation in renal physiology. Inulin is applied intravenously to the patient, who afterwards urinates into a special container. After some time, a certain volume of venous blood is taken as a sample, and urine collection is stopped. Inulin concentration in both blood and urine is calculated, and the volume of collected urine is measured. Glomerular filtration volume is equal to inulin clearance, since all of the glomerular filtrate is cleared from inulin. Therefore, based on the data regarding inulin concentration in the blood, inulin concentration in the urine, volume of collected urine, and the period required for its collection, one can calculate inulin clearance, ie, glomerular filtration (GF). The mathematical equation is the following:K = V × U/P [3]P = inulin concentration in urine (mg/mL).where K = plasma clearance; V = volume of urine excreted in minutes (mL/min); U = inulin concentration in plasma (mg/mL); If we compare these two methods, it is crucial to emphasize the following: while calculating inulin clearance , inulin is being administered intravenously, and after some time the blood sample is collected. In the meantime, the patient spontaneously urinates and urine is collected. Hence, inulin from the blood is transported (filtered) to the kidneys, where it is spontaneously eliminated via urine into the bladder, and afterwards collected into a special kind of canister.f will be “falsely” smaller; 2) all CSF must be absorbed outside the ventricles; if any CSF is absorbed within the brain ventricles, the amount of the formed CSF would be reduced by the amount of the CSF absorbed within the ventricles; 3) marker substance cannot be absorbed from the CSF system into the brain tissue and the blood circulation between the inflow and outflow cannula; if it is absorbed, this will lead to marker “dilution,” which will result in a “falsely” higher Vf; 4) CSF formation must not depend on the perfusion rate and hydrostatic pressure under which perfusion is performed; namely, CSF formation is an active process that should not be affected by these parameters; 5) only a substance that is not present inside the CSF can be used as a marker substance.During ventriculo-cisternal perfusion, after animals have been anaesthetized the inflow and outflow cannulas are introduced into the CSF system . Inulin It should be stressed that these conditions are based mainly on the classic hypothesis according to which CSF is secreted exclusively inside the brain ventricles, absorbed exclusively on the brain surface outside the ventricles, and circulates between those two spaces. It should also be pointed out that these conditions are taken as indisputable scientific facts, and not as unverified presumptions. Therefore, this represents a scientific discrepancy: parameters which have yet to be confirmed as scientific facts, already exist as ultimate requirements necessary for this method to be used.f) calculation, although each evaluation of CSF formation is simultaneously an evaluation of CSF absorption, which is derived from equation [1] we have already discussed:Although these considerations put into question the ventriculo-cisternal method, further effort should be invested into the evaluation of the conditions for the CSF formation and absorption calculation. Our attention will primarily be focused on the evaluation of CSF formation  can also be applied to the original equations (equation 2 and 3) for CSF absorption calculation by Heisey et al , but the amount of absorbed CSF (Va) should be zero. Namely, as it has been previously mentioned, ventriculo-cisternal perfusion method could be used only if the entire CSF was produced inside the ventricles and absorbed outside of them. However, it was found that, at physiological CSF pressure, only about one third of the total CSF was formed inside the brain ventricles (Vf = 16.00 µL/min in dogs and 4.50 µL/min in cats) and that CSF absorption took place within the ventricles as well as in the subarachnoid space. In other words, it is obvious that CSF is also formed outside the ventricles, and that it is also absorbed inside of them. Furthermore, at physiological CSF pressure, the amount of CSF formed inside the ventricles is equal to the CSF amount absorbed inside of them , especially because a force weaker than 30 cm H2O (the limit within which the experiments were performed) is not sufficient to affect an active process (3H-water and blue dextran (m.w. 2 × 106), a hydrostatic pressure increase in the perfusate from negative (-10 cm H2O) to positive (+20 cm H2O) values had opposite effects on these two substances: increase in blue dextran and decrease in 3H-water concentration  (f) (f) rate is calculated. In other words, a change in the perfusion rate should not cause a change in calculated Vf. However, ventriculo-cisternal perfusion in cats showed that an increase in the perfusion rate  significantly decreased the calculated CSF formation rate   should rter) (f) ,19,33-36ter) (f) ,37, it sf discredits the ventriculo-cisternal perfusion model as a method Vf calculation using the equation derived from Heisey et al (f) are not satisfied.ey et al . This maf for 4% each hour during the final five hours of perfusion, although the variables known to affect Vf remained stable . If this occurred in dead animals and in a plastic cylinder, the question arises as to what exactly the perfusion process does in live animals.The results obtained in cat model showed persisting CSF formation (between 3.0 and 5.0 µL/min) even 80 minutes after the animal sacrifice . As CSF f is simultaneously a criticism of Va, and all that has been said for CSF formation can be also said for CSF absorption. Nevertheless, it is necessary to comment on illogical and hardly explicable experimental Va results obtained using ventriculo-cisternal perfusion and the equation derived from Heisey et al . In other words, any criticism of Vey et al .f and Va calculation during ventriculo-cisternal perfusion with dextran blue in cats at negative (-10 cm H2O) and positive hydrostatic pressure (+20 cm H2O) are shown in 2O resulted in a substantial Vf decrease and Va increase. Vf changes related to the hydrostatic pressure are described above, but it is interesting to notice that at negative pressure (-10 cm H2O), negative CSF absorption was obtained . The exact meaning of this result from a pathophysiological point of view is not clear, except that it is a possible artifact of the method. It should be emphasized that the same results  were obtained in other studies  by using the equation of Heisey et al (a) and a significant CSF absorption rate in conditions where CSF absorption should not exist, seriously discredits ventriculo-cisternal perfusion and the equations for Vf and Va calculation as a method that can reliably represent CSF formation and absorption.To summarize, if we are to obtain a correct CSF formation rate . If we take into account that CSF is secreted predominantly inside the brain ventricles and absorbed predominantly in the subarachnoid space, it is reasonable to assume that under normal CSF pressure it should circulate through the aqueduct of Sylvius, and in our model, through the plastic cannula positioned in the aqueduct. This means that CSF outflow through the external cannula adjusted to the normal CSF pressure values should visually have the direction that corresponds to the CSF flow direction from the ventricles to the subarachnoid space. CSF formation rate is calculated by dividing the collected CSF volume with the time of collection. In this way artificial circulation  has been achieved. However, during the collection time of 1, 2, or 3 hours no CSF was observed running out from the system. Only small pulsatile movements of the CSF at the end of the cannula were detected . This model was tested by using a pump that infused artificial fluid into the LV (imitation of “CSF secretion”), which showed that the volume of infused fluid can be accurately measured. In other words, the exact fluid volume that was infused into the LV was collected into the test tube through the outflow cannula . ,15. 9,15detected . In othe cannula  (15).f is studied in dead and live animals in in vitro experiments, during perfusion of any part of the CSF system, etc). If we also take into consideration the results obtained by the direct method, which showed that there was no net CSF formation and circulation, it can be concluded that ventriculo-cisternal perfusion method does not represent the CSF formation and absorption rate. In other words, ventriculo-cisternal perfusion is not a method that will give reliable answers about the existence of CSF formation and absorption.Finally, we can conclude that ventriculo-cisternal perfusion is neither a precise nor physiological method for studying cerebrospinal fluid formation and absorption rate. Primarily, the postulates necessary for calculation using the equation of Heisy et al  are not All of this is in accordance with our new hypothesis -12 on CS


Biological augmentation of rotator cuff repair is of growing interest to improve biomechanical properties and prevent re-tearing. But intraoperative single shot growth factor application appears not sufficient to provide healing support in the physiologic growth factor expression peaks. The purpose of this study was to establish a sustained release of granulocyte-colony stimulating factor (G-CSF) from injectable vesicular phospholipid gels (VPGs) in vitro and to examine biocompatibility and influence on histology and biomechanical behavior of G-CSF loaded VPGs in a chronic supraspinatus tear rat model.G-CSF loaded VPGs were produced by dual asymmetric centrifugation. In vitro the integrity, stability and release rate were analyzed. In vivo supraspinatus tendons of 60 rats were detached and after 3 weeks a transosseous refixation with G-CSF loaded VPGs augmentation  was performed. 6 weeks postoperatively the healing site was analyzed histologically  and biomechanically (n = 6).In vitro testing revealed stable proteins after centrifugation and a continuous G-CSF release of up to 4 weeks. Placebo VPGs showed histologically no negative side effects on the healing process. Histologically in vivo testing demonstrated significant advantages for G-CSF 1 μg/d but not for G-CSF 10 μg/d in Collagen III content (p = 0.035) and a higher Collagen I/III ratio compared to the other groups. Biomechanically G-CSF 1 μg/d revealed a significant higher load to failure ratio (p = 0.020) compared to control but no significant differences in stiffness.By use of VPGs a continuous growth factor release could be obtained in vitro. The in vivo results demonstrate an improvement of immunohistology and biomechanical properties with a low dose G-CSF application via VPG. The VPG itself was well tolerated and had no negative influence on the healing behavior. Due to the favorable properties  VPGs are a very interesting option for biologic augmentation. The study may serve as basis for further research in growth factor application models. Rotator cuff tears are a common cause of shoulder pain, reduced function and weakness of the arm in the elderly patient . The retA previous study has shown that continuous release of G-CSF via an osmotic pump positively affected the supraspinatus tendon (SSP) remodeling in a chronic SSP tear rat model . NeverthThe purpose of the present study was to evaluate the in vitro release kinematics of G-CSF by phospholipid gels and in the following to examine the effect of continuous G-CSF release via phospholipid gels on tendon remodeling in a chronic supraspinatus tear rat model.According to the purpose of the study we evaluated the hypothesis that a continuous G-CSF release by phospholipid gels could be achieved and that the application of G-CSF loaded VPGs as biological augmentation in a chronic supraspinatus rat model results in a significant improvement of histological and biomechanical healing properties.In a first step, the aim was to evaluate in vitro the best combination of the gel’s release properties and viscosity for optimized intraoperative handling, therefore different variation of pH, G-CSF and lipid content were analyzed. Second the in-vivo effects of this developed gel were evaluated in a chronic tendon rat model. The systemic effects were analyzed direct postoperatively by G-CSF ELISA and 6 weeks postoperatively by histological and biomechanical testing Figure .Figure 1The preparation of VPGs by dual asymmetric centrifugation (DAC) was based on the method described by Massing et al. . In brieThe integrity and stability of G-CSF was analyzed by non-reducing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) with subsequent silver staining. In vitro release tests were conducted within flow through cells based on the design described by Tardi et al.  at 37°C.The influence of the lipid content on the specific release behavior of G-CSF VPGs was first investigated. VPGs were prepared with various lipid  contents ranging from 400 mg/g to 490 mg/g. The concentration of G-CSF was 4 mg/g in all these formulations. In a second step, the influence of the G-CSF content on the release behavior of the system was investigated. VPGs were prepared with various concentrations of G-CSF (4 mg/ml and 8.4 mg/ml). The lipid content in these VPG-formulations was 450 mg/g  Table .Table 1For the sterile production of the VPGs the protein solutions were sterile filtered and added to the previously sterilized lipid (gamma radiated) in the sterile sample containers  under aseptic conditions. From that point on, aseptic production was achieved using the hermetically sealed sample containers for the asymmetric centrifugation processes. The prepared gels were then transferred into 1 ml syringes  under aseptic conditions and were implanted within 3 days after production.Sixty healthy  Sprague–Dawley rats were obtained by Charles Rivers  and included into the study. All animals received care in compliance with the guidelines of the local animal care committee following National of Institute of Health guidelines. The supraspinatus tendon (SSP) was completely detached and a transosseous refixation with additional VPG augmentation was performed after 3 weeks as described before  , designed to measure G-CSF in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human G-CSF and antibodies raised against the protein. It has been shown to accurately quantitate recombinant human G-CSF without obvious cross reactivity with rabbit serums .Blood samples (2 μl) of the G-CSF high group (Release rate: 10 μg/24 hr) and placebo group were taken from the contralateral subclavian vein following short anesthesia with Isoflurane on day 0, 2, 5 and 8. The detailed steps of the assay followed the manual by the supplier.All rats were euthanized 6 weeks after the SSP reconstruction. For n = 9 in each group the musculotendinous unit of the supraspinatus was exposed and removed proximally from the supraspinatus fossa and distally from the bony insertion of the humeral head for histological analysis and stored in 4% buffered formalin solution . For anaAfter scarification, the complete supraspinatus muscle was resected from the scapula in toto in 6 rats of each group and the humero-ulnary joint was disarticulated. Each specimen was frozen at −18°C separately and slowly thawed under room temperature for testing. During testing the thawed supraspinatus tendon was constantly moistened with sprayed isotone solution of sodium chlorid to anticipate drying out. Subsequently the proximal end (musculotendinous junction) was press-fixed in a cryoclamp while the humerus was placed in a mounting grid with bony humeral fixation Figure A. TestinThe study-protocol was approved by the local government .Between group differences (MOVIN) were compared using Wilcoxon Test . Statistical significance was set at p ≤ 0.05. A pretesting power-analysis (power > 0.80) was performed for the sum score (MOVIN) with a predicted score difference of 3 points and a SD of 2.25 points. The biomechanical properties of the tendons were analyzed across treatments with use of analysis of variance with the level of alpha set at 0.05. Pairwise comparisons were performed with use of two-tailed paired t tests. Statistical analysis was performed with use of SPSS software .No detrimental effect of the preparation process in a dual asymmetric centrifuge on the quality of G-CSF was found when analyzed by SDS-PAGE. The in-vitro release studies clearly demonstrated the potential of VPGs for sustained delivery of G-CSF. It was observed that G-CSF was released from all formulations in a linear manner following zero-order kinetics. Increasing the lipid content from 400 mg/g to 450 mg/g resulted in a slower release rate. However, the VPG-system appeared rather robust when the lipid content was changed from 450 mg/g to 490 mg/g, i.e. the release rate was not much affected. For VPGs based on 400 mg/g lipids and 4.0 mg/g G-CSF 63% of the total entrapped protein was delivered over 408 hrs whereas VPGs based on 450 mg/g lipids delivered 43% and VPGs with 490 mg/g lipids delivered 35% within the same period  for the preclinical batches are provided in Tables The final product combined the required properties of the daily release rate (1 μg/24 hours respectively 10 μg/24) and the viscosity to allow to inject the gel or also to use it arthroscopically.The Quantikine G-CSF Immunoassay was validated for the quantification of G-CSF in the serum samples. No cross-reactivity of the assay was observed with the rat serum. The results revealed no significant differences within the first 8 postoperative days  between Placebo VPGs and G-CSF high (10 μg/24 h) . The Placebo group also showed a significant higher grade of hyalinization compared to control (p = .021). Detailed data is shown in Figure For collagen I there was no significant intergroup difference. Collagen III staining revealed a significant lower level for Placebo (p = .003) and G-CSF low (p = .035) compared to the control group. G-CSF high showed a significant increased Collagen III content compared to Placebo (p = .004) and a tendency of a higher content compared to G-CSF low (p = .053). The Collagen I/III ratio was increased for G-CSF low 0.6) compared to Control (0.5), Placebo (0.42) and G-CSF High (0.29) . The load to failure ratio  revealed a significant better restoration of contralateral values for G-CSF low compared to Placebo (p = .020). For G-CSF high the difference was not significant (p = .062) . The stiffness failure ratio  revealed a higher but not significant restoration of contralateral values for G-CSF high compared to Placebo (p = .078). For G-CSF low there was no difference . Accordingly significant differences in immunohistology (absolute staining as well as the ratio of Collagen I/III staining) as a marker of tendon organization and maturation confirmed the positive effect of G-CSF low (1 μg/24 hr).Additionally the previously described excellent biocompatibility of the VPGs was proven in the animal study as the placebo group showed no consistent significant difference in histology and biomechanics compared to the control group ,12. AlsoThe in-vitro release studies provided clear evidence, that VPGs are a feasible scaffold for sustained delivery of G-CSF. G-CSF can be encapsulated by dual asymmetric centrifugation in vesicular phospholipid matrices without inducing protein destabilization. Furthermore, the phospholipid gel (VPG) enables sustained G-CSF release. In a wide range of formulation variables the release occurs in a quasi-linear manner without burst effect which is of particular advantage for indications that require constant drug levels over prolonged time periods. The desired release kinetics can be achieved by adjusting the lipid composition or the G-CSF loading amount. Both parameters were shown to affect the matrix erosion which in turn is the dominant mechanism controlling protein release .In current literature, a vast number of growth factors were used in animal research on tendon repair ,15,21-23The application method should have a significant effect on the results of studies for biological tendon augmentation, since recent data estimate the time of the peak for physiological GF expression between week 1 and 2 after SSP refixation in animal models (rat/rabbit) ,9. RespeIn the previous study G-CSF was used in a dose of 5 μg/day . To ensuThe accuracy of the release rate transferred from in vitro to in vivo highly depends on the local erosion situation, in this case the subacromial space, which is unpredictable and hard to simulate in vitro . A standIn the current study, we performed a continuous subacromial GF application, i.e. we achieved a high growth factor dose also during the expression peaks. The manufacturing process of these phospholipid gels includes as well the disadvantage that for each protein the optimal composition (protein and lipid content) has to be tested separately. But that also allows adaptable release rates for different implementations. Due to the technical limitations of practicable slow release gel formulations, a physiological growth factor release profile could not be simulated and we do not know how this influences the tendon healing. The experiments were performed in a small animal model, which has been used in previous studies and shown to create a chronic supraspinatus tear, which is comparable to the tissue found in chronic human tendon tears. According to current literature, the rat model has been found superior to other animal models for rotator cuff pathologies but still some restrictions remain  ,31,32. ABy use of VPGs a continuous growth factor release could be obtained in vitro. The in vivo results demonstrate an improvement of immunohistology and biomechanical properties with a low dose G-CSF application via VPG. The VPG itself was well tolerated and had no negative influence on the healing behavior.


The aim of this article is to provide a different perspective to choroid plexus (CP) physiology and pathophysiology from that presented in the recently published review by Spector et al . This reIn this article we would like to present a different perspective from the mainstream view described by Spector et al . Throughex vivo and in vitro conditions, which could significantly differ from those in living organisms  were constant and present everywhere in the CSF system and partially in the CP, mainly as a consequence of water filtration between the capillaries and interstitial fluid ,6-8. BecAlthough examination of substances passing through CP is important, one must not forget that those substances account to (less than) 1% of total CSF volume, whereas the remaining 99% accounts to water . For thi2O appearance) could be detected before and after choroid plexectomy. The curves of D2O could almost be superimposed on each other . A series of highly sophisticated experiments were performed in control and AQP-1 or AQP-4 loss of function mice . It was ch other . The autricles.” . It is i3H2O curves within the lateral brain ventricles and cisterna magna suggested the same mechanism of water entry from the bloodstream into these compartments in terms of dynamics and volume  cannot be a consequence of CSF flow from the cerebral ventricles. According to all this, it should be concluded that CPs in humans only partially contribute to the CSF/water volume.Very similar results were also obtained in dogs, where overlapping d volume . But if d volume . These rd volume , accordiFurthermore, it is well known that CSF communicates freely with the brain extracellular fluid . There iNo one has done so much for the promotion of the idea that the CP is the site of CSF formation as professor Dandy , connectWith the development of endoscopic methods in the mid 1990s, new attempts were made to cure hydrocephalus with different surgical procedures on the CP ,15,16. A”The common concept that CSF flows slowly but steadily from the cerebral ventricles into the subarachnoid space up over the cerebral hemispheres to the arachnoid villi is not the case, and it leads to misinterpretation of experimental data and clinical interpretation.” Furthermore, our findings on the absence of CSF circulation are anything but unexpected, since our group has continuously been publishing articles on CSF “circulation” since 1991 (For some reason, Spector et al  describence 1991 . Howevernce 1991  is less nce 1991 -8,19-21.nce 1991 . Thus, rnce 1991 . ResultsIt is unquestionable that CSF is formed within the ventricular cavities of some lower vertebrates without CPs ,23,24. IFurthermore, in spite of the presence of the CPs in isolated brain ventricles in some sharks, there was no open communication between internal and external CSF and no tendency for ventricular dilatation in physiological conditions under stable and constant presence of CSF in subarachnoid space ,23,24. ASpector et al have misinterpreted the article of Lorenzo and Sondgrass  by conclAlthough the above-mentioned results and controversies do not fit into the classic hypothesis, they can be explained by the new hypothesis of CSF physiology, which offers a different perspective on CSF physiology and pathophysiology ,11,31,32The fact that after plexectomy the patient continues living with normal CSF turnover ,15,33,34The controversy regarding the role of brain plexuses is not yet at the point to be resolved. The cumulating evidence and application of new technologies, such as MRI, have already made their contributions, and we should further strive to improve our concepts and create the right therapies for patients. Our contribution to this discussion is rather straightforward – the role of CP in the physiology and pathophysiology of CSF shown in the textbooks, atlases, and review articles is highly overemphasized and needs to be revised.


Chronic Otitis Media (COM) is characterized by middle ear effusion (MEE) and conductive hearing loss. MEE reflect mucus hypersecretion, but global proteomic profiling of the mucosal components are limited.This study aimed at characterizing the proteome of MEEs from children with COM with the goal of elucidating important innate immune responses.MEEs were collected from children (n = 49) with COM undergoing myringotomy. Mass spectrometry was employed for proteomic profiling in nine samples. Independent samples were further analyzed by cytokine multiplex assay, immunoblotting, neutrophil elastase activity, next generation DNA sequencing, and/or immunofluorescence analysis.109 unique and common proteins were identified by MS. A majority were innate immune molecules, along with typically intracellular proteins such as histones and actin. 19.5% percent of all mapped peptide counts were from proteins known to be released by neutrophils. Immunofluorescence and immunoblotting demonstrated the presence of neutrophil extracellular traps (NETs) in every MEE, along with MUC5B colocalization. DNA found in effusions revealed unfragmented DNA of human origin.Proteomic analysis of MEEs revealed a predominantly neutrophilic innate mucosal response in which MUC5B is associated with NET DNA. NETs are a primary macromolecular constituent of human COM middle ear effusions. Otitis Media (OM) is one of the most common conditions of early childhood accounting for a very high proportion of all pediatric office visits and surgeries annually,2 at a nLittle is known about the biological mechanisms in OM that fully explain the progression in OM from acute OM (AOM) to COM. It has been well-demonstrated that during this process, the healthy single layered middle ear epithelium remodels into a pseudo stratified epithelium in COM able to potently produce mucins,16,17. TRecently, separate studies of MEE from children with either recurrent AOM or COMdemonstrated the presence of neutrophil extracellular traps (NETs) in the middle ear fluid and showed that the extensive DNA stranding evident within the MEE is largely NET derived ,22 NETs For this study we posited that, an unbiased proteomics approach would reveal neutrophilic markers and NETs would be abundant in COM fluid. We aimed to demonstrate that NETs were present in the MEE of children with COM and characterize the COM MEE NET and proteome focusing the analysis on mediators associated with innate immune responses.The Institutional Review Board Committee of Children’s National Health System approved this study. After obtaining written informed consent from the legal guardian, effusions from children aged 0–35 months with COM, undergoing myringotomy with tube placement irrespective of race/ethnicity/gender were collected. For sample analysis, effusions from each ear were pooled into one sample per patient. Exclusion criteria included: cleft palate or other craniofacial dysmorphic syndromes, immunosuppressive states or conditions, cystic fibrosis, immotile cilia syndrome, or prior history of skull base radiation therapy or skull base malignancy. A total of 49 samples of MEE from x pediatric patients with COM were collected fresh, aliqouted, and if not immediately used for experiments, frozen at -80°C as previously described. ImportaProtein concentration was determined from MEE aliquots using Bicinchoninic Acid Microtiter Plate Assays . As some of the effusion samples had blood contamination, a serum sample was also collected as a control for protein presence for the proteomic analysis.Proteins were separated by SDS-PAGE electrophoresis and analyzed by proteomics as previously described. BrieflyMS was carried out as previously described. BrieflyBecause proteins less than 20kDa in size are typically too small to be detected by one-dimensional gel mass spectrometry proteomics approaches, we also analyzed MEE samples (n = 49) for quantitative levels of IL10, macrophage derived chemokine (MDC), IL13, IL17A, IL1β, IL6, IL8, RANTES, TNFα, and VEGF using a commercially available multiplex magnetic bead immunoassay  according to the manufacturers’ instructions using provided standards and quality controls as previously described. For thiFreshly obtained MEE samples were gently spread directly over a glass slide and allowed to dry for 2 to 4 hours on ice, before fixation in paraformaldehyde 3% for 20 min and storage at 4°C in PBS 1X until immunostaining was performed. Cells were permeabilized with PBS/Tween 20 0.05% for 5 min; non-specific sites were saturated using 0.01% PBS/Tween-20/3% Bovine Serum Albumin for 30 min. This solution was also used to incubate primary anti-human antibodies for -neutrophil elastase , Citrullinated H3 , MUC5B , Lysozyme , Lactoferrin , Myeloperoxidase , S100A8/9 , S100A9 , SPLUNC  at 1/500 dilution and the secondary antibodies anti-goat Alexa 488 A11055 oranti-rabbit Alexa A10042 at 1/500 . Three washes of 30 min were done in order to reduce the non-specific signal of the antibodies. 4',6-diamidino-2-phenylindole(DAPI) at 1:8000 in PBS was finally used to stain DNA. The chambers were removed and the slide was mounted using Permount mounting medium  with a coverslip. Slides were stored at 4°C in dark before immunofluorescent analysis using an Olympus FV1000 confocal microscope .® West Dura Extended Duration Substrate kit  according to the manufacturer’s instructions.A previously described protocol was used for the western blotting of non-m® West Dura Extended Duration Substrate kit  according to the manufacturer’s instructions.For mucins, western blot analysis was performed as previously described. BrieflyNeutrophil elastase protein activity was assayed with the Elastase Substrate I, Colorimetric . The substrate was reconstituted in 0.1M Hepes, 0.5M NaCl and 10% dimethylsulfoxide at 1 mg/ml as performed previously. 200μl oDNA from 200 uL of middle ear effusions was purified using the QiaAmp mini kit  prior to electrophoresis in 1% Agarose Gels run at 100 V in 1X TAE buffer containing ethidium bromide for visualization under ultraviolet lighting. Quantification of relative DNA concentrations in each effusion sample was performed by the Qubit double stranded DNA kit .Quality control was performed on 49 DNA samples from middle ear effusion using the Qubit 2.0 fluorometer system  and the nanodrop. These samples were further processed for shotgun sequencing using a Truseq Nano prep kit . Genomic libraries were validated (concentration and size) with the Qubit and 2100 Bioanalyzer. Forty-four samples passed the validation and were sequenced on the Nextseq 500 using the 2x151 basepair paired end protocol, after an initial loading titration trial sequencing run. Raw reads (FASTQ format) were preprocessed (QC) using PRINSEQ-lite 0.20.4 . FiltereThe entire middle ear proteome from 6 middle ear effusions was previously reported by out group. This prDue to their very high abundance in COM samples, immunoglobulins as well as complement proteins are summarized in Surprisingly, five actin and three histones were detected in the high abundance list, but only in the MEE samples and not in serum. Additionally, the three neutrophil/leukocyte proteins in the highly abundant list were present only in MEE and not in serum.We hypothesized the presence of NETs as an explanation for the high abundance of actin, histones and neutrophil markers in MEE. Given the above mentioned results and findings we postulated that visualization of fresh, independent MEE samples would reveal a copious amount of NETs. Through DAPI staining of DNA and antibody staining of specific proteins, IF unequivocally revealed abundant extravasation of DNA from cellular structures resulting in variable stranding of DNA in MEE specimens. Validation with NE, LYZ, and LTF confirmed these cells to be neutrophils. Colocalization of extracellular DNA strands with citrullinated histone-3 (CitH3) confirmed these networks of extravasated DNA and extracellular proteins in MEE fluid are indeed NETs, as the citrullination of histones is characteristic of NET DNA release,33 Fig  Notably,Immunoblotting was subsequently used to further evaluate the presence or absence of NET markers in independent samples. Western blot analyses demonstrated the presence of NET markers lactotransferrin, CitH3, Histone H4, lysozyme, and neutrophil elastase, as well as the innate immune mediators LPLUNC, α-antitrypsin, and proteins S100A9 and A8 in a majority of the samples evaluated. . As expeIn order to determine whether the DNA present in MEE from COME patients  was more-18 and p = 4.29x10-14 respectively)   was then used to analyze the relationships between the proteins of the MEE proteomic dataset. Notably, abundant MEE proteins were predicted to be directly linked to the action of IL-8, which given these relationships, was determined to most likely be involved in MEE for the process of neutrophil chemotaxis as well as for the movement of phagocytes/myeloid cells (p = 8.33x10ctively) . Almost Because proteomic techniques are limited in their ability to detect small molecules such as cytokines, we performed a multiplex assay to quantify the levels of a group of representative Th1, Th2 and other cytokines that have previously been associated with COM,36 Fig . As predThis study comprehensively validates the fact that NETs are present in the MEE of pediatric patients and reports for that proteins derived from NETS are the predominant innate immune response mediators in COM, providing a detailed proteomic analysis of COM MEEs.ex vivo and airway epithelial cell secretions in vitro. Joo et al. compared their proteomic analysis of normal human airway gland secretions with proteomic studies of of bronchial alveolar lavage fluid, apical fluid of primary human tracheobronchial epithelial cell cultures, and nasal lavage fluid and demonstrated an overall similarity in the dataset profiles from each of the sample sources[Analysis of the MEEs proteome revealed a majority of proteins implicated in mucosal innate immune responses as observed in proteomic analysis of airway secretions e sources. Our MEEe sources,38,39,40e sources and thate sources,17,42.Our MEE dataset was further characterized by the abundance of proteins produced by neutrophils, such as neutrophil elastase , myeloperoxidase (MPO), Cathepsin G (CTSG), Lactotransferrin (LTF) but also intracellular proteins typically not expected in such abundance in secretions: actin and histone H4. This finding led us to investigate the presence of NETs in the MEE of patients with COME, as recently reported ,22. In 2in vitro NETosis in neutrophils from healthy subjects [S. Aureus (MRSA) challenge,  with a parallel reduced ability for neutrophils from aged mice to form NETs kill MRSA[Of particular note, IL-8 was found to be by far the most abundant cytokine detected by multiplex assay in out MEE samples. This is of major relevance to the finding of NETosis in MEE, because not only is IL-8 a well known chemoattractant for neutrophils, its presence has been shown to positively correlate with the abundance of NETs in bronchial fluids. Furthersubjects . Aged mikill MRSA. Finallykill MRSA, suggestIt thus appears that rapid clearance of NETs from mucosal surfaces the airways is critical in preventing their deleterious effects. In the lung, the effective clearance of NET structures is a critical process in the maintenance of healthy airways. The lack of appropriate NET homeostasis, with accumulation of bacteria, extracellular DNA and NET-associated enzymes such as MPO and elastases, worsening of lung inflammation and tissue damage has been demonstrated in diseases such as CF and asthOur work confirms and establishes the presence of abundant NETs in COM fluids using immunofluorescence analysis of fresh MEEs. This is important in order to avoid sample degradation and spontaneous neutrophil activation due to temperature among other factors. All the mucoid MEEs were characterized by the presence of cells releasing high amounts of DNA in the extracellular space, which co-localized with CitH3 and other antibacterial proteins as lysozyme, lactotransferrin, neutrophil elastase which are proteins known to be characteristic of NETs along wiBased on our results we hypothesize a model for OM progression whereby acute bacterial infection leads to middle ear epithelial secretion of mediators , which sClearly our report is limited though by the fact that our characterization of mediators in middle ear fluid only represent a “snapshot” in time, and as such the proposed model is only an extrapolation of the potential role of the identified mediators in OM progression over time, and cannot necessarily be directly surmised from the data on hand. Another limitation of our work includes the fact that not every single middle ear fluid specimen could be profiled by each experimental technique. However, we believe the clear uniformity of NET presence in all specimens (stained fresh and immediately to avoid spontaneous production of NETs) by each technique employed demonstrates the importance of NETs in COM. Notably, detection by WB in every single specimen proved to be difficult given the fact that proteins might be degraded by the high abundance of proteases likely coming from neutrophils. As such mass spectrometry techniques, which recognize peptides and not specific epitopes, are more sensitive for protein identification than western blotting and less affected by natural protein degradation during sample procurement and processing. A final limitation of our work is the fact that we did not measure viscosity of the effusions, as such although we surmise that the noted association between NETs and MUC5B is likely to significantly contribute to increased MEE viscosity, we cannot demonstrably conclude as much.In conclusion, an unbiased proteomic analysis of MEE revealed that innate immune responses in COME are typically neutrophilic in nature, with NETs being a primary macromolecular constituent of human mucoid middle ear effusions.S1 TableThis table shows the peptide counts of the serum sample and the average peptide count of 9 MEEs for each protein. The cutoff peptide count was set at 2 per MEE.(DOC)Click here for additional data file.


Procedural sedation in children continues to be a problem in the emergency department (ED). Midazolam is the first water-soluble benzodiazepine and it has been widely used for procedural sedation in pediatric patients.The aim of this study was evaluation of clinical safety and effectiveness of intramuscular Midazolam for pediatric sedation in the ED setting.We performed a self-controlled clinical trial on 30 children who referred to the Baqiyatallah Hospital ED between 2009 and 2010. They received intramuscular Midazolam 0.3 mg/kg for procedural sedation and then they were followed for sedative effectiveness and safety. Vital signs and O2 saturation were also observed. The findings were compared using SPSS ver. 16 software.The mean age was 5.50 ± 2.70 years, the mean weight was 19.50 ± 6.63 kilograms and 16 patients (53.3%) were females. The most common adverse effect was euphoria (66.66%) and vertigo (6.7%); 27.7% did not show any side effects. There was an overall complication rate of 72.3%. The vital signs including heart rate, respiratory rate, systolic and diastolic blood pressure and O2 saturation decreased significantly during sedation .Midazolam is an effective and relatively safe sedative for pediatric patients in the ED. The patient should be observed closely and monitored for psychological and hemodynamic side effects. Procedural sedation in children continues to be a problem in the emergency department (ED). Midazolam is a benzodiazepine that has been widely used for procedural sedation in adults . VariousThe aim of this study was evaluation of clinical safety and effectiveness of intramuscular midazolam for pediatric sedation.We conducted a before-after clinical trial on a highly selective group of 30 children between 2 and 12 years-old. The children who presented to the ED of the Baqiyatallah Hospital between 2009 and 2010 were enrolled. The patients that met the inclusion criteria received intramuscular midazolam 0.3 mg/kg, before imaging (CT-Scan or magnetic resonance imaging). Midazolam was administrated at least 30 minutes before beginning the procedure. Sedation, irritability and cooperation scores were followed every 15 minutes during the first hour after receiving the drug. Five stages for sedation were assessed ..2 saturation were observed during the sedation. The findings were analyzed by using t-test, Chi-square and repeated measure ANOVA SPSS ver. 16; and P value < 0.05 was considered statistically significant. This study was approved by ethics committee of our university and the parents filled an informed consent before enrollment.Also, the vital signs and OThe mean age was 5.50 ± 2.70 years, the mean weight was 19.50 ± 6.63 Kg and 16 patients (53.3%) were female. All of the patients were sedated completely after the first dose. The trend of sedation staging progressed to deep sleep; irritability progressed to complete calmness . These t2 saturation at first was 97.50 ± 1.30 that at the last check changed to 96.33 ± 1.68. The trend of O2 saturation changes during sedation had significant decreases . None of the children suffered hypoxemia (O2 saturation under 90%). The mean RR at the onset was 22.23 ± 6.54 ; at the last visit it changed to 18.80 ± 4.81. Also the mean of HR at the onset was 112.46 ± 14.82 and at the last visit it changed to 103.90 ± 14.57. The trends of RR and HR changes had significant decreases . Moreover, the trends of systolic and diastolic BP changes also had significant decreases .The mean OThere was an overall side effect rate of 72.3%. The most common was euphoria (66.66%) followed by vertigo (6.7%); 27.7% did not present any side effects. All of the adverse effects resolved by observation only.Effectiveness of midazolam compared closely to other routine sedative agents such as propofol, fentanyl and ketamine , 6. MidaPsychological side effects such as hallucination and agitation have been commonly reported for benzodiazepines but euphoria with this high incidence has been reported rarely. One reason for this high incidence might be race -9. Previ


Heart transplantation is limited by severe donor organ shortage. Regardless of the changes made in the acceptance of marginal donors, any such mechanism cannot be considered successful unless recipient graft survival rates remain acceptable. A stress echo-driven selection of donors has proven successful in older donors with normal left ventricular resting function and in standard donors with reversible resting left ventricular dysfunction acutely improving during stress, or slowly improving (over hours) during intensive hormonal treatment. Aim of this study is to assess the medium-term outcome of recipients of marginal donor hearts selected with new echocardiographic techniques over standard criteria.We enrolled 43 recipients of marginal donor hearts: age > 55 years, or < 55 years but with concomitant risk factors, n = 32; acutely improving during stress, n = 3; or slowly improving during hormonal treatment, n = 8. At follow-up , 37 of the recipients were still alive. One-year survival was 93%.The strict use of new stress-echocardiographic techniques over standard criteria of marginal donor management, together with comprehensive monitoring of the donor, has the potential to substantially increase the number of donor hearts without adverse effects on recipient medium-term outcome. Heart transplantation is an established procedure in end-stage heart failure patients, albeit limited by severe and incremental donor organ shortage. In Europe every year a pool of ≈ 4500 unused hearts  with permission granted for heart donation is estimated, from which additional transplants could be generated, with more confidence in their post-transplantation performance  ,4; 2) reAccording to a methodology previously described in detail, in this analysis we enrolled three different categories of potential marginal donors: 1) 97 patients enrolled in the Adonhers project ,4,12, coThe pts in the Ht protocol were considered eligible only after coronary angiography ruled out the presence of coronary stenosis.The ethics committee of the Emilia-Romagna and Tuscany regions of Italy approved the Aged Donor Heart Rescue by Stress Echo Project in 2004 . Partial funding for the Stress Echo project was provided by the Italian Health Ministry . On May 11, 2011 the Italian National Transplant Center/Italian National Institute of Health (CNT/ISS 2011) approved the Guidelines: Increase available organs for heart transplant with heart assessed by stress echocardiography in older donors or in donors with several risk factors, with a second-opinion telemedicine system from the core echo lab, IFC-Pisa.Donors enrolled in the stress echo protocols were enrolled in neurological intensive care units, without direct access to coronary angiography facilities. Standard coronary angiography with IVUS was performed 1 month after heart TX . Donors SPSS version 21 for Windows  was used for statistical analyses. The statistical analyses included descriptive statistics . Patient’s survival curves were estimated using the Kaplan–Meier method and compared between groups using the log-rank test. The survival was censored at the time of death. For patients who were still alive, survival was censored at date of last known follow-up. Two-tailed p < 0.05 was considered statistically significant.43 eligible transplanted hearts showed significant (70%) stenosis of a major coronary vessel  on 1-month post-HT coronary angiography, and underwent PCI with stenting. At follow-up , 37 of the recipients were still alive , epicardial coronary calcium at surgical inspection (N = 2), malignancy (N = 1), HCV positivity (N = 1), lack of a matching recipient (N = 3). Seven eligible donors enrolled in the HT protocol were not transplanted due to: opposition (N = 3), tubercolosis (N = 1), malignancy (N = 1), coronary artery stenosis at pre-harvesting angiography (N = 2); 43 marginal donor hearts were eventually transplanted Table This study shows that the surgical mortality rates of adults who underwent heart transplantation of marginal donors selected by means of new echocardiographic techniques are excellent, and that medium-term survival is acceptable. The most common cause of death within 1 year is graft failure, followed by infection. Notably, only one of our patients died due to cardiac allograft vasculopathy, in recurrent multiple myeloma. Additional studies are needed to assess the impact of cardiac allograft vasculopathy in marginal donors.Supply of donor hearts is a critical rate-limiting step in heart transplantation. An effective way to solve the current shortage would be to accept an upward shift of the age cut-off limit and to accept for transplant normal but temporarily stunned donor hearts ,5,13,22.The suggested first choice was dipyridamole, with dobutamine as the alternative, acceptable second choice. There were two reasons for the choice: dipyridamole is equally as accurate but technically simpler than dobutamine because of a lesser increase in heart rate, and the image quality is therefore less degraded during stress ,4  for all Italian regions ,26. The In theory, it may seem more convenient to perform stress echocardiography at the bedside rather than transport the donor to the cardiac catheterization laboratory -29. The The medium-term outcome of recipients of marginal donor hearts selected with new echocardiographic techniques over standard criteria demonstrated survival rates similar to that of recipients of “standard” donor hearts. As waiting lists for heart transplantation continue to grow, continuous changes in practice patterns of donor heart usage are most urgent. It is believed that about 15,000 patients would potentially benefit from a heart transplant, if the acceptance criteria included ‘marginal’ donors up to 55 years of age, and about 40,000-70,000 patients would benefit, if the acceptance age was extended to 65 years . In “youADONHERS: Aged donor heart rescue by stress echo project; CNT: Italian National Transplant Center; HCV: Hepatitis c virus; HT: Hormonal treatment; ISS: Italian National Institute of Health; LAD: Left anterior descending artery; LV: Left ventricular; PCI: Percutaneous coronary intervention; UNOS: United network for organ sharing; RCA: Right coronary artery; TX: Transplant; VAD: Ventricular assist device; WMSI: Wall motion score index.The authors of this manuscript have no conflicts of interest to disclose.TB, GA and EP conceived this study, performed the data analysis, and drafted the manuscript; MM, FP and OL gave a contribution to preparation of study design, data discussion, and critical revision of the manuscript; LP and SB were responsible for data collection and revised the manuscript. All authors read and approved the final manuscript.