In agreement with the modeling, our recordings demonstrate the dependence of magnetic stimulation-triggered action potentials on the type and state of the neuron and its orientation within the magnetic field. Our results suggest that the observed effects of TMS are deeply rooted in the biophysical properties of single neurons in the central nervous system and provide a framework both for interpreting existing TMS data and developing new simulation-based tools and therapies.Although transcranial magnetic stimulation (TMS) is a popular tool for both basic research and clinical applications, its actions on nerve cells are only partially understood. We have previously predicted, using compartmental modeling, that magnetic stimulation of central nervous system neurons depolarized the soma followed by initiation of an action potential in the initial segment of the axon. The simulations also predict that neurons with low current threshold are more susceptible to magnetic stimulation. Here we tested these theoretical predictions by combining  TMS is a popular tool for human brain stimulation and for modulating cognitive tasks . The electric field induced in the plane of the brain slice was calculated using MATLAB  for a magnetic coil with a mean radius of 1 cm Figure , using t0 is the permeability constant, N is the number of loops, I is the current, r is the radius, z is the distance of the point from the coil plane, x is the distance of the point from the center of the coil, K(m) and E(m) are elliptic integrals of the first and second order and where μThe changes to the membrane potential induced by the magnetic field were calculated using the activating function:E is the induced electric field, and a is a unit vector parallel to the axial direction of the segment. This function, used to calculate membrane polarization due to changes in the external electric field, is known as the activating function  was connected to an oscilloscope. The pick-up coil was centered on top of our coil while the potential across the capacitor bank was increased Figure . A bi-moThirteen to fifteen day old Wistar rats of either sex were killed by rapid decapitation after anesthesia with isoflurane, according to the guidelines of the Bar-Ilan University animal welfare committee. This procedure was approved by the national committee for experiments on laboratory animals at the Israeli Ministry of Health. Slices  were prepared from the somatosensory cortex using previously described techniques  or artificial cerebrospinal fluid 2 (ACSF2) containing: (mM) 125 NaCl, 25 NaHCO3, 4.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 1.2 CaCl2, 0.499 Na-ascorbate, and 25 glucose (pH 7.4 with 5% CO2). ACSF2 had a higher KCl concentration (2.5–4.5) and lower CaCl2 concentration (2–1.2) than ACSF in order to excite neurons in the slice  containing: (mM) 125 NaCl, 25 NaHCORecordings from neuron somata used a BVC-700A amplifier (Dagan Corp.). Voltage was filtered at 5 kHz and sampled at 10 or 40 kHz using a National Instruments analog-to-digital interface operated by procedures custom written in IgorPro 6  and stored on the hard disk of a personal computer. Patch pipettes were pulled (5–10 MΩ) from thick-walled borosilicate glass capillaries . The electrophysiological recordings were first performed in the whole-cell patch-clamp configuration followed by the magnetic threshold measurement in the loose-patch configuration.t-test either paired or unpaired depending on the experiment. The type of test is indicated in the text. The squared correlation coefficient and the statistical significance of the correlation are reported for linear correlations.Data were analyzed off-line with IgorPro 6.0  on a personal computer. Experimental results were observed in cells from two or more animals. Therefore, all the results for a particular experiment were pooled and displayed as means ±SD. Groups were compared by Student's We investigated the response of a single neuron to magnetic stimulation by combining a patch-clamp setup with a magnetic coil Figures , 1A. At n = 21) in the whole-cell mode than in the loose-patch configuration  pointing to possible interaction of the stimulus with the whole-cell pipette. We therefore performed all the recordings in this study in the loose-patch configuration. To gain access to intracellular parameters we briefly recorded the membrane potential in a current-clamp recording from each neuron in the whole-cell configuration. From these recordings we calculated the input resistance of the neuron and the current threshold of the action potential. Following this brief whole-cell recording, the patch electrode was retracted from the cell and then brought back in contact with the membrane to form a loose-patch recording configuration. In the whole-cell configuration the cytoplasm is replaced by the pipette solution. This may lead to unwanted changes in the cellular function. To rule out this possibility we recorded the magnetic threshold from several neurons in the loose-patch configuration without prior whole-cell recording. The magnetic threshold recorded under these conditions  was not significantly different than that recorded following a brief whole-cell recording .Ideally, the best configuration of the patch-clamp technique for investigating intracellular mechanisms is the whole-cell configuration . We have previously shown that this modified ACSF increases synaptic input to cortical neurons leading to somatic depolarization . Taken together, these experiments agree with our simulations and suggest that magnetic stimulation activates cortical neurons primarily by somatic depolarization.A third prediction from our modeling was that an increase in synaptic input would reduce magnetic threshold  for L5 pyramidal neurons and 0.6 ± 0.2 T (n = 9) for low threshold interneurons. The mean latency for L5 pyramidal neurons was 0.48 ± 0.24 ms  and for interneurons 0.28 ± 0.13 ms  for pyramidal neurons. Given the average magnetic threshold recorded for L5 pyramidal neurons in our recording setup, this simulation predicted that the experimental magnetic threshold in the Y direction should be ~4 T. This was above the upper intensity limit of our magnetic stimulator. Therefore, we repeated the same simulations using morphological reconstructions of low threshold interneurons. The somata of these neurons are less elongated than those of L5 pyramidal neurons. Thus, based on our biophysical model  for low threshold interneurons  agreeing with our simulations. It was possible to hypothesize, based on our theoretical predictions, that somatic depolarization will lower the magnetic threshold ratio since the membrane potential will be closer to threshold and its orientation compared to the induced electric field will be less relevant. We again induced somatic depolarization by increasing synaptic activity in the brain slice by replacing ACSF with ACSF2. Under these conditions the magnetic threshold ratio was 1.5 ± 0.3 (n = 9) for low threshold interneurons, significantly smaller than the ratio recorded in ACSF . In all four experimental conditions the latencies between the magnetic stimulation and the action potential were short, supporting the hypothesis that the action potential was generated at the axon's initial segment regardless of the orientation of the magnetic coil . Obviously, this pause may be the result of a combination of cellular refractory period and network activity that could not be told apart while recording in the loose-patch configuration. This experiment could not be performed with L5 pyramidal neurons; the residual mechanical vibrations at high magnetic pulse intensities did not allow collecting enough stimulation sweeps to generate a raster plot and PSTH. However, a similar pattern of activity was qualitatively observed in three L5 pyramidal neurons.Assuming that magnetic stimulation induces action potential firing in the axon's initial segment, then suprathreshold magnetic stimulation should cause the neuron to enter a refractory period phase-locked with the stimulus. To test this hypothesis the standard ACSF was replaced with ACSFin vitro by combining magnetic stimulation with patch-clamp recordings in rat brain slices (Figures Here we investigated the basic mechanisms of magnetic stimulation of cortical neurons  Figures , 2. Usin Figures , 4. We p Figures  and supp Figures , 6, 7.We modified a standard patch-clamp setup adding a custom made coil between the slice chamber and the condenser Figure . It was Similar to inducing an axial current in the solution contained within the patch pipette the induced electric field also generates an axial current when it interacts with the cytoplasmic resistor in dendrites, axons, and somata Rattay, . Thus, fWe tested these predictions using our experimental setup. We were able to show that, as predicted, the magnetic threshold was a function of current threshold Figure  and of tin vitro have shown short latency initiation of action potentials by magnetic stimulation suggesting action potential generation at the axon's initial segment (Bonmassar et al., This suggestion is also supported by several recent experiments. For example, imaging in primary cultures of hippocampus neurons has provided some support for the relationship between magnetic threshold and intrinsic neuronal excitability; a small group of neurons responded with higher sensitivity to magnetic stimulation, promoting the concept of initiating cells in the network (Rotem and Moses, in vitro results have clear correlates with TMS studies. For example, stimulation of the motor cortex with TMS generates activity that can be monitored as pyramidal tract volleys (Day et al., in vitro recordings. However, our instrument generated relatively low magnetic fields and we did not observe large network activation in the brain slice. Therefore, it is probably safe to assume that our recordings were performed below what would have been the motor threshold in vivo.What is the relation between our cellular findings and the numerous results obtained when applying TMS to human subjects? Obviously, the effects of TMS on humans are complex, including a large contribution from local and distal networks (Walsh and Rushworth, Using this assumption, it may be possible to hypothesize that at low TMS intensities, the somata of low threshold excitatory cortical neurons are depolarized enough to trigger action potentials in the axon's initial segment. This initial activation of local, low threshold, cortical networks may then drive deep pyramidal neurons to fire that may culminate in I-waves. The pause in the firing we observe following magnetic stimulation Figure , occurriIn conclusion, the convergence of our cellular study with behavioral data in humans strongly suggests that the effect of TMS is correlated with the cell type and network state. This may explain, in part, the considerable variability observed between and within many brain stimulation studies. Moreover, our work demonstrates impressive correlation between the biophysical properties of single cortical neurons and results obtained when applying TMS to humans and lab animals. Thus, it is possible to suggest a conceptual model in which a single pulse of TMS activates a large population of somata in the cortex depending on their biophysical properties and their level of synaptic input at the moment of the pulse. The almost immediate firing of these neurons followed by a refractory period perturbs the cortical network, perhaps initiating the process termed “virtual lesion” (Pascual-Leone et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Post-traumatic stress disorder patients experience chronic systemic inflammation. However, the molecular pathways involved and mechanisms regulating the expression of genes involved in inflammatory pathways in PTSD are reported inadequately. Through RNA sequencing and miRNA microarray, we identified 326 genes and 190 miRNAs that were significantly different in their expression levels in the PBMCs of PTSD patients. Expression pairing of the differentially expressed genes and miRNAs indicated an inverse relationship in their expression. Functional analysis of the differentially expressed genes indicated their involvement in the canonical pathways specific to immune system biology. DNA methylation analysis of differentially expressed genes also showed a gradual trend towards differences between control and PTSD patients, again indicating a possible role of this epigenetic mechanism in PTSD inflammation. Overall, combining data from the three techniques provided a holistic view of several pathways in which the differentially expressed genes were impacted through epigenetic mechanisms, in PTSD. Thus, analysis combining data from RNA-Seq, miRNA array and DNA methylation, can provide key evidence about dysregulated pathways and the controlling mechanism in PTSD. Most importantly, the present study provides further evidence that inflammation in PTSD could be epigenetically regulated. PTSD develops after exposure to a traumatic event, such as military combat, violence, natural disaster, and the like. While in the US general population alone, the prevalence rate of PTSD is estimated to be ~3.5%, the rate increases significantly to ~20% in U.S. military service personnel, following combat167A recent review analyzed published literature on immune status and concluded that PTSD patients exhibit excessive inflammatory state10121315Recent use of powerful techniques like Next Generation Sequencing (NGS) and microarrays has led to the identification of differentially expressed mRNAs and miRNAs at the global level. Moreover, combining data from these techniques with various bioinformatics tools for data analysis has made it possible to predict and discern biological pathways that are affected in a diseased state, thereby making it possible to overcome the limitations of single gene-based studies. Furthermore, data from miRNA expression arrays and DNA methylation studies can be used to predict and study the regulation of expression of differentially expressed genes at the epigenetic level. Epigenetic regulation of gene expression includes the influence from processes like DNA methylation, histone modifications and miRNA expression171821672425272830In the present study, we could identify and predict many differentially expressed genes involved in canonical pathways related to the immune system biology, in the PBMCs of PTSD patients. We further provide preliminary evidence at the global level that the differential expression of the genes is possibly an outcome of differential expression of miRNAs and change in DNA methylation level.We performed RNA-Seq on RNA samples from PBMCs of PTSD patients  and 4851p value less than or equal to 0.05 (significant) and linear fold change of at least ±1.5 or more. Thus, we obtained a total of 190 miRNAs  ontology was ‘immune system process’ with 40 genes from our dataset  and genes (326) by using the IPA Expression pairing tool. From the total 190 differentially expressed miRNAs in the dataset, 44 (23.16%) miRNAs were identified to have targets in the RNA-Seq dataset during the time of analysis in IPA. Of the 326 differentially expressed genes, >70 (21.48%) were identified to be targets of the 44 paired miRNAs . A list www.targetscan.org website, we could identify many more miRNAs  that target the genes  from our dataset. www.targetscan.org.As mentioned earlier, because all miRNAs or genes are not covered by one single database or tool, we wanted to see whether there are genes and miRNAs in our dataset that were not picked during Expression pairing in IPA, but are predicted to be target(s) of miRNAs in our dataset. Based on the miRNA target gene information available on t- test or Wilcoxon test. However, in all the genes listed in this study, there was a gradual trend of DNA methylation values supporting the expression levels of the genes  present in our dataset. Based on the Illumina CpG site designations, only 138 genes from our dataset had true CpG sites. As per our set criteria used for this analysis, in the PTSD group, 40 (12.27%) genes had higher DNA methylation average β-values at their corresponding CpG sites when compared to that of controls  and thesMTRNR2L1, MMP25, CXCL8, G0S2, GZMB, CXCL3 and STAT4) as representative for qRT-PCR analysis. MTRNR2L1, MMP25, CXCL8 and G0S2 were shown to be significantly downregulated and GZMB, CXCL3 and STAT4 significantly up-regulated by RNA-Seq analysis. The entire above mentioned gene expression matched with that of RNA-Seq results as per qRT-PCR data  present from the significant set of genes. STAT4 and TBX21 have crucial roles in regulating T cell functions. For example, TBX21 is the main transcription factor for the expression of interferon gamma, a pro-inflammatory gene and already reported by our group (Bam IFNG)IL12B)43TBX-21 and STAT4, the key genes in the Th cell differentiation pathway, were upregulated in PTSD. TBX-21 is one of the main transcription factors involved in the differentiation and functioning of Th1 cells and also plays an important role in the functioning of other cells of the immune system45STAT4 is induced in response to signaling via the IL-12 pathway, leading to induction of IFN in Th1 type CD4+ cells47TBX-21 and STAT4. These observations suggested that several of the up-regulated genes in the PBMCs of PTSD patients could be resulting from the decreased presence of miRNAs, as a result of their downregulation, that target the genes post transcription. Further analysis of the miRNA dataset indicated that, up-regulated chemokines CXCL2, CXCL3, CCL4 and CCL5 are targets of numerous downregulated miRNAs from our dataset. Altogether, these observations suggest that there is a breakdown in the miRNA-mediated gene regulation in the PBMCs during PTSD and strikingly, it includes several of the up-regulated genes having pro-inflammatory properties. In contrast to the above observations, we found only 7 upregulated miRNAs and very few genes from our dataset were shown to be their targets. Moreover, these genes were not present in the top canonical pathways from our analysis.Micro-RNAs are critical regulators of gene expression and their interaction with target mRNAs can lead to destabilization of the mRNA in most (>90%) of the cases in mammalsIL-12 transcription is increased in PTSD which correlated with lower DNA methylation at its promoter regionCSRNP1 (aka AXUD1), a tumor suppressor, is one of the significantly downregulated genes in PTSD and it has higher level of DNA methylation in PTSD (82.08%) than control (79.09%). Downregulation of CSRNP1 correlates with progression of cancerSTAT4 is among the significantly up-regulated genes and has correspondingly lower DNA methylation trend at the CpG island. STAT4, induced by IL-12, leads to activation and proliferation of Th1 type CD4+ cells, which correlates well with our observation of increased T cells and alteration in the Th cell differentiation pathway in PTSDWe observed that a higher percentage 80.36%) of the differentially expressed genes in PTSD patients were downregulated. Correlating with the downregulated gene expression, there was a gradual but clear trend of higher DNA methylation level at CpG sites. Higher DNA methylation in the promoter region of a gene correlates with lower transcription of the gene and vice versa2728% of the In summary, the present work has identified differentially expressed genes and miRNAs and the related canonical immune system pathways in the PBMCs of PTSD inflicted war veterans. Furthermore, we provide evidence that many genes have altered DNA methylation at their CpG islands and the expression of the associated genes inversely correlate in PTSD patients. Taken together, the present and previous reports from our lab, and from other research groups, clearly indicate that miRNAs and DNA methylation play a critical role in the modulation of the immune system, with a special emphasis on chronic inflammation seen in PTSD. Most importantly, our findings, although preliminary, open future directions for studies in a pathway specific manner and targeting specific gene regulators to develop novel management strategies and therapies to control the inflammatory response seen during PTSD in war return veterans and the general population.All procedures performed in studies involving human participants were approved by the University of South Carolina Institutional Review Board and experimental methods and protocols were carried out in accordance with the approved guidelines. Samples were collected after obtaining proper informed and written consent from every participant. PTSD patients were Veterans of either the 1991 Persian Gulf war, or of the recent Iraq or Afghanistan wars, recruited from William Jennings Bryan Dorn Veterans Medical Center, as described earlierPeripheral blood samples (10–20 ml) were collected in EDTA coated collection tubes and PBMCs were isolated using Ficoll-Paque  within 1 h from sample collection. PBMC viability was determined by trypan-blue exclusion. Using a universal kit  recommended for simultaneous isolation of high quality DNA and total RNA including miRNAs, all the three entities were isolated from the same ~ten million PBMCs and immediately frozen at −80 °C until use. For the miRNA microarray analysis, we used 4 controls and 8 PTSD samples. In the RNA-Seq analysis we used 5 controls and 5 PTSD samples. Two PTSD and 4 control samples were common for microarray and RNA-Seq analysis. For the qRT-PCR validation experiments, we included all the controls and PTSD samples used for the microarray and RNA-Seq analysis, in addition to more samples collected later, thereby making the total number of samples for each group to 24. For the DNA methylation data analysis, we included all the individuals’ (76 control and 23 PTSD) result submitted in the public database by the authorsFor RNA-Seq, five controls and five PTSD patients were analyzed. RNA-Seq libraries were constructed using Illumina TruSeq RNA Sample Preparation kit. Briefly, total RNA was purified from PBMCs using the Qiagen RNA easy kit. The oligo-dT beads were added to 1 μg of total RNA to isolate mRNA. The purified mRNA was fragmented to 200–400 bases. The RNA fragments were then reverse transcribed into double stranded cDNA fragments. The DNA fragments were repaired to generate blunt ends using T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. After DNA fragments were purified using Qiagen PCR purification kit , an “A” base was added to the 3′ end of the blunt DNA fragment by Klenow fragment. Sequencing adapters were ligated to the ends of DNA fragments using DNA ligase. The libraries were then amplified by limited PCR (15 cycles) using primers provided by the kit. The PCR products were then separated by 2% agarose gel electrophoresis and fragments with sizes ranging from 250 bp to 400 bp were excised and purified using the QIAquick Gel Extraction Kit . The concentration and distribution of the library were determined by a NanoDrop spectrophotometer . The library was sequenced by Illumina HiSeq 2000 at Tufts University Genomic core facility. During analysis, we trimmed three nucleotides from the 5′ end. Raw sequencing reads (50 bp single-end) were mapped to human genome build hg19 using Tophat 2et al.p value  calculated using student’s t test, were included for the analysis. We call these miRNAs differentially expressed. We used two tailed Student’s t test to get the p values. The miRNA array data is now available in ArrayExpress (Accession# E-MTAB-4880).For the miRNA microarray, four controls and eight PTSD patients were included in the study. Microarray for the miRNAs was performed by Johns Hopkins Memorial Institute (Deep Sequencing and Microarray Core Facility), Baltimore. Total RNA, including mRNA, miRNA and other small RNA molecules, were isolated from PBMC samples as described above. Next, total RNA samples were used in the analysis of miRNA differential expression by miRNA array hybridization assay using the Affymetrix miRNA-v1 gene chip. Array data normalization and quality control was performed as described previously by Zhou http://www.ingenuity.com/, QIAGEN, CA), Panther58We employed Ingenuity Pathway Analysis . The three bioinformatics tools are similar in that they search for evidence of enrichment of genes in particular list of genes. However, not all databases have complete information for all the genes and their interactions. Thus, a more complete analysis is obtained if the same dataset is analyzed using different tools.Functional enrichment of a set of genes can help to identify the biological pathways in which the genes in a dataset are involved and also other genes with which a certain gene interacts, thereby providing a more meaningful understanding of the data. Consequently, we performed functional enrichment analysis in IPA, Panther pathways and DAVID with the RNA-Seq data which included all the genes with a fold-change of 2 or more and In this part of the analysis, the differentially expressed genes and the miRNAs were analyzed together to see whether there are gene(s) that are targets of the miRNAs from our dataset obtained from similar samples in the present study. This type of analysis can help to simultaneously identify the differentially expressed genes and their regulators  in the same cells. To do this, we used a tool called Expression pairing, available in the Ingenuity’s microRNA target filter analysis section. This tool is useful to determine the miRNA(s) and gene(s) that interact with each other from a given set of miRNAs and genes when provided simultaneously as input datasets. To perform expression pairing, both the miRNA array followed by the RNA-Seq dataset was analyzed in IPA simultaneously. The tool then paired miRNAs with the genes from the dataset based on Ingenuity’s knowledge base for miRNAs and their targets. The genes obtained after expression pairing are either known or predicted to be a target of miRNA(s) from the list. Consequently, we got a list of genes from the input dataset and also miRNAs that could be their possible regulator for the observed differential expression. Subsequently, the genes obtained after expression pairing were used for another round of functional enrichment analysis for which, only IPA was used. This functional enrichment was performed to obtain the pathways in which the genes targeted by the miRNAs are involved.www.targetscan.org for performing this analysis. One at a time, the genes from our dataset were analyzed in www.targetscan.org. After obtaining the list of miRNAs that target the genes, we manually searched for the miRNAs that are present in our dataset. As we had a very long list of differentially expressed genes, we listed miRNAs for only the top up- or downregulated genes as per the RNA-Seq analysis.In our miRNA dataset, most of the miRNAs from PTSD patients were downregulated. Based on our experience, IPA did not cover all of the miRNAs and their targets. So, we performed miRNA-target search to identify additional differentially expressed miRNAs and genes in our dataset, which possibly could be targeted by the miRNAs. We used the publicly available websitewww.ncbi.nlm.nih.gov/). This dataset contains DNA methylation analysis results from 76 controls and 23 PTSD individuals . In this dataset, the DNA methylation of CpG sites was obtained from whole blood cells. The observations in the above mentioned dataset are different from ours in that our observations are collected from PBMCs rather than whole blood cells. However, using this dataset and our own earlier Methylated DNA immunoprecipitation (MeDIP) data (not provided), we could previously identify and report differential expression of IL12 as a result of alteration in DNA methylation and other epigenetic mechanisms in PBMCs from PTSD patientsFor identifying differences in the DNA methylation level in specific genes, we used the publicly available Gene Expression Omnibus (GEO) datasets (GSE21282)TM 7 Real-Time PCR system  real time PCR instrument. As an internal control, 18S rRNA and GAPDH message was quantified along with the genes. The expression level of the genes was expressed as the relative abundance value to the internal control. For the validation of gene expression by employing qRT-PCR, we included 24 controls and 24 PTSD samples.For validation purpose of the RNA-Seq data, we selected seven genes for analysis. Complementary DNA (cDNA) was prepared from 0.5 μg of total RNA using miScript RT II kit  in a 20 μl system and used 7 ng of the original amount for qRT-PCR. Quantitative RT-PCR in triplicate wells was performed in an Applied Biosystem ViiAHow to cite this article: Bam, M. et al. Dysregulated immune system networks in war veterans with PTSD is an outcome of altered miRNA expression and DNA methylation. Sci. Rep.6, 31209; doi: 10.1038/srep31209 (2016).


Type 2 diabetes (T2D) is very prevalent among the elderly. Insulin therapy is often required for glycemic control. The association of starting this therapy with depressive symptoms as well the health-related quality of life (HRQoL) is unknown among the elderly patients.Evaluate the association of starting insulin therapy depressive symptoms as well with HRQoL of elderly people with T2D.36 T2D participants  were recruited, 26 of whom completed the follow-up. Generic  and specific (Problem Areas in Diabetes - PAID) HRQoL questionnaires, Beck Depression Inventory (BDI), clinical, laboratorial and socio-demographic data were recorded on baseline and 6 months after the beginning of insulin treatment.p = 0008). There were no statistically significant differences in HRQoL scores between the two time periods There was also a reduction in HbA1c (Before/After: median – 8.7/7.9). Otherwise, there were no statistically significant differences in: BMI (28.1/28.3); Abdominal circumference:(100.5/99.5) and chronic complications status.There was a reduction in the BDI score after the use of insulin, which means an improvement in depressive symptoms (Before/After: median – 10.5 / 7; Insulin therapy in elderly people with type 2 diabetes can lead to an improvement of depressive symptoms and does not seem to affect negatively HRQoL of the participants. Type 2 diabetes mellitus (T2D) is prevalent among adults, especially among elderly people. New data from International Federation of Diabetes (IDF) indicates that there are currently more than 285 million people with T2D around world. It is estimated that by 2030 this figure will have increased to more than 552 million people with T2D. Population growth and improvements in public health leading to an increase on life expectancy have contributed to a steady increase in these numbers and consequently to an increase in the number of older people with diabetes. Aged subjects with diabetes have an increased rate of diabetes-related complications and are more likely to have comorbid conditions, such as physical disability, cognitive dysfunction and depression, all of which could impair health-related-quality-of-life (HRQoL) .Geriatric diabetes care guidelines recognize the importance of improving HRQoL for older subjects with diabetes. Almost all studies have compared HRQoL in older adults with T2D with HRQoL in those without T2D. Studies evaluating the impact of different types of T2D therapies on HRQoL and depressive symptoms in this population are scarce. Three central goals in the treatment of diabetes mellitus are the avoidance of hyperglycaemia (prevention of the development or progression of diabetes complications over time), the avoidance of hypoglycaemia and the maintenance or achievement of good HRQoL. Having multiple diabetic complications is clearly associated with decreased HRQoL. Data from large studies, such as the Diabetes Control and Complications Trial (DCCT) and United Kingdom Prospective Diabetes Study (UKPDS), suggest that intensive treatment itself (using insulin or not) does not impair HRQoL .The role of insulin therapy over HRQoL of patients with diabetes is controversial. Data from A1chieve study, which evaluated people with T2D with unsatisfactory glycemic control, has showed benefit of starting insulin on HRQoL of the sample studied. In this study, people with T2D starting insulin significantly improved overall HRQoL across all component health dimensions. However, the average age of the population in the study was 54.9 (±14.4) years. So far, we could not extrapolate these data to elderly population . OtherwiThe link between depressive symptoms and T2D remains unknown. If depression was a causal factor for the development of T2D, depressed patients would be at an increased risk of developing pre-diabetes, which is not true. A suggestion of a link between depression and diabetes comes from reports of a positive association between continuous measures of depression  and serum glucose levels . However, it had also been showed that both hypoglycaemia as well hyperglycaemia were associated with higher levels of depressive symptoms, contradicting the idea of a direct link between depression and dysglycaemia . The rolFinally, the role of insulin therapy over depressive symptoms and HRQoL in adults with T2D remains controversial. Indeed, it has not been studying specifically in elderly population so far. The aim of our study was to evaluate the role of insulin therapy on depressive symptoms as well HRQoL of elderly people with T2D. Our study is especially of value because of its prospective design.The Brazilian National Institute of Health defines as an elderly a person who is 60 years or older. Patients with 60 years or older and diabetes from the outpatient unit of nutrology and diabetes from Hospital Universitário Clementino Fraga Filho with indication of starting insulin therapy between January 2012 and June 2013 were invited to be included in the research. We adopted a convenience sample, one of the main types of non-probability sampling methods. Patients who had been included gave written informed consent to participate. Data was collected using paper and pen in the waiting room. The Research and Ethics Committee of Federal University of Rio de Janeiro, Brazil, approved the study protocol, participant information sheet, and consent inform. We have recruited thirty-six patients were recruited, 10 of which did not complete follow up. We excluded one of them from the research due to severe episode of hypoglycemia and subsequent interruption of insulin therapy. We evaluated all the participants at monthly intervals in the 6 months of the study and the assistant physician adjusted the dosage schedule of insulin therapy when necessary.Short-Form 36 Health Survey (SF-36), Problem Areas in Diabetes (PAID) and Beck Depression Inventory (BDI) questionnaires, body mass index (BMI), waist circumference and glycosylated hemoglobin (HbA1c) have been recorded at baseline and 6 months after the beginning of insulin therapy. Socio-demographics characteristics , presence of complications related to T2D and comorbidities were also registered. The ophthalmologist examined all patients and evaluated the presence/degree of diabetic retinopathy by fundoscopy in mydriasis. Neuropathy was clinically diagnosed. Sensitivity was tested by the Semmes-Weinstein pressure asthesiometer. The 24-hour urinary excretion of albumin (UAER) was measured. UAER was considered abnormal when it was > 30 mg/24 h. Microalbuminuria was defined as 30–300 mg/24 h and macroalbuminuria as > 300 mg/24 h. All patients with micro or macroalbuminuria were defined as having nephropathy. Hypoglycemia was defined as blood glucose < 50 mg/dL in the presence or not of clinical symptoms.HRQoL was measured using a generic  and a disease-specific questionnaire (Problem Areas in Diabetes: PAID). SF-36 provides scores related to both the physical and mental dimensions of HRQoL. This scale consists of 36 items, which constitutes 8 domains: physical function (PF), role physical (RP), bodily pain (BP), general health perceptions (GH), vitality (VT), social functioning (SF), role emotional (RE), and mental health (MH). Scores ranged from 0 to 100, with higher scores indicating better quality of life in those dimensions . The BraData was analyzed using SAS 6.11 . Numerical variables were expressed as mean, standard deviation (SD), median, minimum and maximum. Categorical data was expressed in percentage (%). The distribution of the sample was analyzed by Kolmogorov-Smirnoff test. Comparison between the two time periods (before/after insulin) has been performed by Wilcoxon test for the numerical variables and by McNemar’s test for categorical data. Non-parametric tests were used because the sample did not present a Gaussian distribution. To verify the existence of a significant association between range of scores  and baseline categorical variables the Mann-Whitney’s test was used, and to verify the association with baseline numerical variables the Spearman’ s rank correlation coefficient (rs) was used. A p-value smaller than 0.05 on the two-tail was considered to indicate statistical significance.Thirty-six T2D patients  were recruited, 26 of whom completed the follow-up. Generic  and specific (Problem Areas in Diabetes - PAID) HRQoL questionnaires, Beck Depression Inventory (BDI), clinical, laboratorial and socio-demographic data were recorded at baseline and after 6 months of insulin therapy. Baseline characteristics of the sample are shown in Table p = 0.07). So we could infer that our sample was relatively representative of our population [p = 0.019). There were no significant changes in BMI  and waist circumference . There was a significant reduction in BDI score when compared to baseline values , indicating an improvement in depressive symptoms. Despite the robust drop in BDI score, HRQoL did not change significantly in any of domains when the total sample was analyzed as a whole, as shown in Table The mean in BDI score was 10.5, similar to other report in a Brazilian sample, in which the mean was 10.0 , physical role  and pain ranges  as well between baseline BDI score and BDI range . Otherwise, there was a positive correlation between the BDI range and PAID range .However, when correlation between the range of scores , baseline variables and their respective ranges has been assessed, it was shown that there was an negative correlation between BDI range and physical functioning  as well those with higher educational level did in mental health domain when compared with those with lower levels .When gender and educational level have been taken into account, It was observed that men had a greater improvement in physical functioning domain than women  and HRQoL results . However, participants with nephropathy had a lower range in physical functioning than those without nephropathy . Data is shown in Table As far as chronic complications related to T2D are concerned, participants with or without retinopathy or neuropathy did not present any statistical difference in the BDI , it is of value that studies evaluating this specific population are conducted. Indeed, this population is more vulnerable to lose their independence and the introduction of insulin therapy increases treatment complexity, requiring some cognitive and visual skills [We observed that starting insulin therapy in elderly participants with T2D was associated with significant improvement in depressive symptoms, as indicated by an important reduction in BDI score. We also found that the higher the improvement in depressive symptoms, the higher the improvement in physical functioning and role physical, as well as in pain domain. The influence of depressive symptoms in HRQoL of people with T2D is known and has been published elsewhere . Recent We found a direct correlation between the BDI and PAID ranges, which signifies the higher the improvement on depressive symptoms, the bigger the improvement on HRQoL complaints registered in PAID score. We also found an inverse correlation between baseline BDI score and BDI range, which means the more severely depressed the individual at baseline, the smaller the improvement on depressive symptoms.We did not find a clear change in HRQoL scores. However, when gender and educational level have been taken into account, we also observed that men had a greater improvement in physical functioning domain than women. This gender interaction with HRQoL has also been showed previously by Eljedi et al. His group have studied a sample of patients with T2D and evaluated HRQoL in this population in a cross-sectional design. Their results indicated that HRQoL of patients with T2D was strongly reduced and indeed identified that women were specially affected . In ZhanEducational level seems also to have a different meaning on our population. Participants with higher educational levels had a greater improvement in mental health when compared with those with lower educational levels, as previously shown . Our patFinally, we observed a trend of direct correlation between the range of BDI score and HbA1c levels, which means that the higher the reduction in HbA1c levels, the higher the improvement in depressive symptoms. However, this did not achieve statistical significance, possibly because of relatively small sample.Indeed our data showed reduction in HbA1c levels, although of smaller magnitude than in non-elderly adult population studies, presumably because of the lower doses used in our sample (related to less strict glycemic targets) and the relatively short follow-up –7. We dip < 0.05). The results in SF-36 and PAID scores were very similar, when whole sample was analyzed, which makes improbable that if the sample was greater we would find significant differences. However, it is important to note that the exclusion of the case of serious hypoglycemia may have influenced the results. The participants lost in the follow up maybe could have been those in whom insulin therapy had a different meaning. It could be also considered a bias. Furthermore, the design of the study (lack of a control-group and the open-label format) does not guarantee that the increase in BDI score was related to insulin therapy or to the improvement in glycemic control. A possible improvement in depressive symptoms related to the starting of insulin therapy in elderly diabetic subjects could be relevant in clinical setting in which starting insulin faces some barriers, especially in elderly subjects. However, these data must be investigated in a randomized controlled study and further research is warranted.The major limitation of our study is its relatively small sample size that could have influenced the statistical power to show a significant change in HRQoL scores. However, the number of individuals included is similar to the studies published previously , 26. We In our study, the beginning of insulin therapy in elderly participants with T2D was associated with a significant improvement in depressive symptoms and did not seem to affect adversely the HRQoL of this population, both fundamental aspects when deciding for any therapy directed to people with T2D.


While uncommon, post-traumatic pelvic malunions present reconstructive challenges and are associated with significant disability and financial burden. A transiliac osteotomy is a surgical technique useful to correct certain types of pelvic fracture malunions, and is only used when the correction of a limb-length discrepancy is the primary goal. This study aims to present our experience with this technique in the treatment of post-traumatic pelvic malunions.Eight patients who underwent transiliac osteotomies for post-traumatic pelvic malunions at our department from 2006 to 2011 were included in this study. We reviewed the clinical and radiographic results of these patients.By the time of their last follow-up, all osteotomy sites and iliac bone graft had healed with no evidence of internal fixation failure. Of the 3 patients who complained of preoperative posterior pain, 2 reported an improvement. All 8 patients noted the resolution of their lower back pain. At the time of their final follow-up, 4 patients could walk normally, 2 had a slight limp without a cane, 1 patient used a cane to help with standing and walking, and the final felt limited during ambulation with a cane. Limb-lengthening relative to preoperative measurements was 2.86 cm (2.2–3.0 cm) at the time of the last follow-up. Two patients were able to return to their previous jobs, 4 patients changed their jobs or engaged in light manual labor while the final 2 were able to perform activities of daily living but were unable to participate in work or labor. Three patients reported being “extremely satisfied” with their outcomes, 3 were “satisfied,” and 2 were “unsatisfied.”A transiliac osteotomy can be used to manage selected cases of post-traumatic pelvic malunions that are unable to be corrected with a traditional release and osteotomy. However, in these cases the correction of limb-length discrepancies should be the primary reconstructive goal. The surgical treatment of pelvic fractures has improved tremendously over the past few decades.8 In some cases, restorations are impossible.Although the ideal way to treat a fracture malunion is to reproduce the original fracture and then re-reduce and fix it, this is not consistently possible with pelvic fracture malunions. Fractures of the posterior pelvic ring, especially when accompanied by the vertical displacement of the lateral sacrum or the sacroiliac joint, often heal with a complex deformity. The loss of bony landmarks and the displacement of neurovascular structures makes a local osteotomy difficult and dangerous.The transiliac osteotomy is a surgical technique that has been found to be useful in certain types of pelvic fracture malunions. While it is commonly not the surgeon's first choice in the treatment of malunions, transiliac osteotomies have been used in select cases to correct limb-length discrepancies. Here, we present our experience with this technique.Permission for this retrospective study was obtained from the Medical Ethics Committee of our Institution. Eight patients who had post-traumatic pelvic malunions managed with a transiliac osteotomy by our department between 2006 and 2011 were included in this study  and the average Majeed score 9 was 44.88 points (38−54 points). Of the 8 patients, 3 complained of mild posterior pelvic pain that required daily analgesic use. All patients had lower back complaints such as lumbar fatigue. Lower back pain was most common during weight-bearing activities and was alleviated with rest.In all cases, our initial preferred method was to correct the pelvic malunion with a traditional technique like a 1-, 2-, or 3-stage procedure. A transiliac osteotomy was chosen as the operative technique when restoration with traditional methods was deemed impossible.10 The height of the bone graft should therefore be greater than the intended limb-length correction.When conducting a transiliac osteotomy, a well-measured operative lengthening is crucial. Limb-length discrepancies were measured radiographically as the vertical distance between the inferior border of the L5 (or the superior border of S1) vertebrae to a line connecting the center of the femoral heads. Based upon prior findings, the actual lengthening distance should be less than that of the initial osteotomy gap.In our cases, a Smith–Peterson incision was used to expose the iliac crest and the osteotomy, therefore permitting the iliac bone removal and the transiliac osteotomy creation through 1 incision. Taking care to avoid injuring the lateral femoral cutaneous nerve, the iliac bone was excised as determined by our preoperative plan. The osteotomy line ran from the area between the anterior inferior iliac spine and the anterior superior iliac spine to the greater sciatic notch. A pelvic distraction device was then placed, allowing the surgeon to gradually distract the osteotomy site with 1 assistant applying traction to the lower limbs while another fixed the pelvis while protecting the ipsilateral posterior ring. Iliac crest bone graft was placed in contact with both sides of the osteotomy gap. X-ray imaging was used to verify the correct placement of the graft  with a mean blood loss of 2225 mL (1200–4200 mL). The average length of hospitalization was 23.13 days (16–38 days). After a mean follow-up of 23.25 months (12–36 months), all iliac grafts were healed, no osteotomy gap was noted and no internal fixation failure was observed  is minimal when limb-lengthening is limited to 4 cm.19 However, the higher quantity and increased complexity of the nerves of the pelvic area make the fixation of pelvic fractures more challenging than THAs. We therefore recommend limiting limb-lengthening to 3 cm. We had no sciatic nerve stretch injuries or postoperative nerve damage in our case series.The incidence rate of neurovascular injuries associated with pelvic fractures is approximately 10%,A transiliac osteotomy corrects the limb-length discrepancies of pelvic malunions and places the ischial tuberosities in or near the same horizon. Leg-length discrepancies and sitting imbalance problems are corrected by surgery, and this method can correct the pelvic vertical deformity that is difficult to do with a general release and osteotomy. However, as the surgery does not involve the posterior pelvis and cannot correct a posterior rotational deformity, patients with intractable posterior pain or posterior ring instability should not undergo this procedure. Postoperatively, patients need time to adapt to the changed spatial relationship of their pelvic ring and acetabulum and the impact of the procedure on the soft tissues of the hip, femoral head, and spine. We have successfully corrected pelvic fracture malunions using the transiliac osteotomy. However, because of the typically unstable condition of these patients and the small sample size available in our study, additional high quality trials with larger samples and longer follow-up periods are needed to permit conclusive recommendations.


Despite their abundance, unspliced EST data has received little attention as a source of information on non-coding RNAs. Very little is known, therefore, about the genomic distribution of unspliced non-coding transcripts and their relationship with the much better studied regularly spliced products. In particular, their evolution has remained virtually unstudied. A subclass of the unspliced EST cluster consists of so called 3'UTR-derived RNAs (uaRNAs). They can have functions in cis and trans, independent from the harboring gene. uaRNAs can be detected by combining EST data with predicted transcription start sites.We systematically study the evidence on unspliced transcripts available in EST annotation tracks for human and mouse, comprising 104,980 and 66,109 unspliced EST clusters, respectively. 15-20\% of the unspliced EST cluster are conserved between human and mouse. More than 7,000 human and 6,000 mouse unspliced EST cluster overlap the 3'UTR of a RefSeq gene or are located within 5kb downstream of the 3' end. Using TSS predicted by chromatin data we identify a total of 1,547 bona fide uaRNA candidates in human. Integrating only the public available CAGE data by the FANTOM5 consortium we predict a total of 1,891 uaRNA candidates in human and 2,477 candidates in mouse.We also give a first glimpse on the sequence and structure conservation of these uaRNA candidates.Expressed sequence tag data combined with experimentally predicted promoter data, e.g. CAGE, is a powerful tool to identify candidate uaRNAs. This combination of data sets could also be applied to non-model organisms without a sequenced genome. uaRNAs are a quite new class of non-coding RNAs and have not been extensively analysed yet. We present a catalog of candidates which are excellent targets for experimental verification. Increasing evidence hints to additional regulatory functions of 3'UTRs independent from the processing of the corresponding gene. It is very likely that this mechanism is not only present in humans and mice but also other eukaryotes. Using public data is a great way to get a glimpse of the uaRNAome of the respective species.


It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established invitro/exvivo methods.The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to  To date, the rabbit Draize eye irritation test According to an expert group In the presented study we aimed to establish an in vitro test method which reliably predicts the eye-irritation potential of chemicals for all three GHS categories within one test. For this purpose the previously established and well characterized reconstructed cornea model, consisting of a collagenous stroma covered with a multilayered corneal epithelium Therefore it was our aim to increase the predictive capacity of the 3D cornea-based test system in order to eliminate false negative responses on the one hand and to discriminate between all 3 GHS categories in one stand-alone test system on the other hand. This method refinement was performed by integrating an additional physiologically relevant endpoint, namely the initial depth of injury in both epithelium and stroma, into the eye irritation assessment. We developed a new technique to quantify the initial DOI in the reconstructed cornea tissues by combining the MTT viability assay with cryosectioning procedures (MTT-DOI method). The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. A panel of 25 chemicals which covered all GHS categories in a balanced manner was tested on the cornea models in order to assess performance and predictivity of the MTT-DOI method. The test results were then compared with high-quality in vivo reference data.Although corneal damage is the most influential driver of eye irritation for all GHS categories, also conjunctival damage was found to be of importance, particularly as driver of irritation for GHS Cat. 2 classification 2+ and Mg2+ (PBS−), isopropanol, Nunc cell culture inserts  were from Thermo Scientific . Rat tail collagen solution was purchased from CellSystems . Media 199 and Ham’s F12, as well as TrypLE Express were from Invitrogen . For construction of cornea equivalents, Keratinocyte Basal Medium (KBM) with a Bullet-kit and the chemically defined Keratinocyte Growth Medium (KGM-CD) were obtained from Lonza . To build up the conjunctiva equivalents two different media were used: Keratinocyte serum-free medium was from Invitrogen , and supplemented with 25 g/mL bovine pituitary extract, 0.2 ng/ml epidermal growth factor , and 0.4 mM calcium chloride . DMEM/F12  supplemented with 10% newborn calf serum  and 10 ng/mL EGF was used for stratification (stratification medium). Penicillin/streptomycin, PBS- and PBS with Ca2+, Mg2+ (PBS+) were purchased from Biochrom . Bola Teflon O-rings (with 3 inner and 6 outer diameter) were from Bohlender GmbH .Thiazolyl blue tetrazolium bromide (MTT reagent), sodium hydroxide, sodium bicarbonate, HEPES and sucrose were obtained from Sigma-Aldrich . Calcium chloride, PBS without CaMost of the test materials used in this study were chosen from the database published by the European Center for Ecotoxicology and Toxicology of Chemicals The cornea model consists of a 3D stromal biomatrix with embedded human corneal keratocytes covered with a multilayer of human corneal epithelial cells. The corneal keratocytes (HCK), immortalized with SV-40 adenovirus, were established by Zorn-Kruppa The cornea models were prepared according to Engelke The human conjunctival epithelial cells , were kindly provided by Ilene Gipson from Schepens Eye Research Institute at Harvard Medical School in Boston, Massachusetts, USA. The cells were taken from a human bulbar conjunctiva, immortalized by expression of hTERT and characterized by Gipson and coworkers When conjunctiva epithelial models lacking the collagen matrix were used, HCjE cells were directly plated into membrane inserts with 0.4 µm pore size and cultivated as described above for the full thickness models.Barrier function testing by transepithelial electrical resistance (TER) measurements of conjunctiva epithelial models was carried out using the Endohm chamber and the EVOM resistance meter .Immunohistochemistry was conducted as described in 2. Then sucrose solution was added to a final concentration of 20% and incubated for another 60 min. Thereafter the tissues were removed from the inserts, transferred into cryomolds and embedded in cryomatrix. The tissues were placed at 4°C for 30 minutes and then frozen in liquid nitrogen. The frozen tissues were stored at −20°C prior to cryosectioning.The cornea models were treated topically with 50 µl of the liquid test substances described in The cornea and conjunctiva models were sectioned with a Leica CM3050 cryostat at a chamber temperature of −23 to −25°C and object temperature of −19 to −21°C. The thickness of the cryosections was 30 µm. 3 sections per sample from the center of the models were taken for further analysis. For long time preservation, the cryosections were mounted in Fluoromount G. For evaluation, a Leica DMLS binocular transmitted light microscope  equipped with a Leica EC3 digital imaging camera and LAS EZ software were used.For the quantitative analysis of cell damage and for the determination of the DOI in both tissue models, the images of the sections were processed in ImageJ open source software We developed a method to analyze the DOI by combining the MTT viability assay with cryosectioning techniques and computer-aided analysis with the aim to establish a prediction model to discriminate all three GHS classes for eye irritation within one model.When applying common cryosectioning procedures to the bioengineered 3D-cornea models, insufficient tissue preservation was a general issue, in particular induced by shattering due to the high water content of the artificial collagenous stroma. To avoid these freezing artifacts, the unfixed MTT-stained tissues were saturated with 20% sucrose before embedding in cryomatrix When testing the reference chemicals we observed that the overall tissue thickness of cornea models was influenced by the test substances itself during 60 min exposure . This waRegarding the epithelial portion of the tissue model, multilayer growth was ensured in all cases.To develop an adequate prediction model, 25 chemicals with different physicochemical properties were selected . The cheUsing the 60 min exposition protocol, all rMTT-DOIs found for the Cat. 1 chemicals were higher than 90%, indicating that these chemicals severely damage the cells of the whole tissue models . A seconHowever, when using the 10 min exposure protocol , we obseWhen comparing the rMTT-DOI values obtained after both exposure times, most of the substances led to higher values after 60 min than after 10 min. This was most noticeable for the Cat 2B substances.According to the results of the 60 min exposures a preliminary prediction model was defined using two cut-off values for the in vitro classification of the test chemicals into the three GHS categories as shown in The outcome of the evaluation of all test substances and their predicted categorization according to the above defined prediction model is summarized in From the 25 substances tested, 19 were categorized correctly. Of the other six substances, toluene, 2-heptanone and n-bromohexane were classified false positive as Cat. 2, and 1% sodium hydroxide false positive as Cat. 1.For two chemicals no reliable MTT-DOIs could be determined: Glycolic acid led to a complete decomposition of the collagen matrix. Quinacrine resulted in a patchy MTT-formazan stain even in the epithelium, indicating some remaining viability, although Quinacrine seemed to affect the whole tissue.To assess the relevance of the test system, reliability and predictive capacity were determined from a contingency table .Accordingly, using the 60 min exposure times, all Cat. 1 chemicals and 89% of the Cat. 2 chemicals were predicted correctly. Thus the accuracy of prediction for the severe and the mild and moderate irritant substances is very good. Furthermore, no compound was false negative predicted in the MTT-DOI test, but 43% of the non-irritants were overestimated.In consequence, the overall concordance rate of 83% implies a substantial agreement between the in vitro and in vivo estimates. However, the test system is over-predictive with regard to non-irritant chemicals.Regarding the physicochemical properties, all alcohols and surfactants and 2 of the 3 solid compounds were correctly predicted. However the pH extreme compounds were over-predicted or led to no result, especially strongly acidic compounds destroy the collagen matrix, as it was found for 10% glycolic acid.With the aim to test its ability to improve predictivity of the 3D cornea model, we established human 3D conjunctiva equivalent models  constructed from human immortalized conjunctiva cells . When testing these models we generally took a 10 min exposure to minimize the time of usage of the Teflon ring which might affect the measurements.Twelve reference substances with different physicochemical properties were tested on conjunctiva models. Boxplot analysis of the resulting rMTT-DOI values are depicted in In order to provide a straightforward and reliable method to predict the eye irritation potential of chemicals, we developed a cryosectioning procedure for MTT-stained unfixed 3D cornea equivalents to measure the initial depth of injury in relation to the total tissue thickness. By using this method supplemented by ImageJ-based quantitative analysis tools, we can clearly distinguish reference substances of different GHS categories by means of their rMTT-DOIs.In contrast to the ex vivo DOI measurements on isolated rabbit eyes (IRE-DOI) described by Jester In the presented study, 23 of the selected 25 test chemicals caused MTT-DOIs which were detectable with our MTT-DOI/cornea model test system. 10% glycolic acid did not result in any measurable MTT-DOI, because it dissolved the collagenous stromal matrix leading to total destruction of the reconstructed tissue. Since also 1% sodium hydroxide was over-predicted as a severe irritant compound, we conclude that pH extreme compounds (pH<2.0 or >11.5) in general might be overestimated or rather incompatible with the MTT-DOI test in reconstructed cornea models as it has already been described for other in vitro methods The second substance which could not be evaluated with our method was quinacrine. In contrast to all the other test chemicals, quinacrine induces cell death in the stroma while parts of the epithelium are still viable. This observation indicates that the impact on the epithelium is not preceding the impact on the stromal keratocytes. Our observation corresponds to the European Commission/British Home Office (EC/HO) validation study which showed that also the BCOP test cannot predict the actual degree or depth of injury for quinacrine The investigation of our test substances showed, that all severe irritant substances were categorized correctly. Further this was true for 89% of the mild and moderate irritant substances and 57% of non-irritants. Hence, the MTT-DOI test method promises to be suitable to discriminate between severe irritants (Cat. 1), and mild to moderately irritant Cat. 2 substances, which cannot be covered by any other test system published so far.According to the ECETOC database, toluene, 2-heptanone and n-bromohexane, belong to the non-irritant GHS category and are therefore over-predicted as mild and moderate irritants using the MTT-DOI method. However, toluene has also been misclassified in other studies: For example, the European Union Risk Assessment Report Also for 2-heptanone and n-bromohexane contradicting results to the Draize test exist in literature. According to NIOSH Pocket Guide to Chemical Hazards, 2-heptanone is indeed irritating to the eye and for n-bromohexane it was found by Kojima that this substance could only be correctly predicted with modification of the rinsing protocol within their in vitro test system Taken into account these fundamental limitations in reliability of predictions due to inconsistent in vivo data, the MTT-DOI test method promises to be suitable to discriminate not only between severe irritants (Cat. 1), and mild to moderately irritant Cat. 2 substances but also identify the non-irritating substances with high accuracy.It is worth mentioning that no compound was false negative predicted in the MTT-DOI test and no surfactant and no alcohol resulted in a false prediction. Thus, compared to BCOP and ICE tests, the MTT-DOI test promises to cause less false results of alcohols, surfactants as well as ketones Further, the predictive capacity of the MTT-DOI method for volatile chemicals seems to be higher than in other in vitro systems Compared to the prediction models from our previous studies where spectrophotometric determination of MTT reduction of the whole tissue was used as endpoint, the prediction model based on rMTT-DOI values thus represents a significant and promising improvement.In our hands, 60 min of incubation showed a more reliable result to categorize the test substances compared to 10 min of incubation. However, in view of quickness of the method, it might be worthwhile testing more substances for this purpose. In addition, the increase of rMTT-DOI values from 10 min to 60 min exposure was most noticeable for the Cat 2B substances, indicating that the exposure time might be a crucial factor for separation of Cat. 2A and 2B.Although damage to the cornea is the crucial driver of eye irritation for all GHS categories, conjunctiva damage gains more importance particularly as driver of irritation for GHS Cat. 2 classification For this purpose the human hTERT-immortalized human conjunctiva epithelial cell line HCjE In order to compare the effects of chemicals with conjunctival damage as the main driver of irritation A few comparative studies have been published so far in terms of cytotoxicity testing in corneal and conjunctival cultures, dealing mainly with pharmacological relevant compounds like benzalkonium chloride or thiomersal Nonetheless, the conjunctiva full thickness model might be used for other studies, e.g. for basic biological questions using knock-out strategies of specific proteins. In addition, the epithelial conjunctiva model, which was used here mainly as an intermediate step for the generation of full thickness conjunctiva models may be useful, e.g. for the investigation of the influence of substances on barrier function.In order to present an adequate, stand-alone strategy for the assessment of all 3 GHS categories of eye irritation we developed a MTT-based method to determine the initial depth of injury in 3D tissue models of the human eye. The test was applied to the previously developed cornea equivalent system and 25 reference chemicals of different GHS categories were tested. Our results reveal that the MTT-DOI test allows us to distinguish between the cytotoxic effects of all GHS categories in one test system. The overall concordance rate of 83% implies a substantial agreement between the in vitro and in vivo estimates. The system is slightly over-predictive with respect to the non-irritant chemicals. The method is currently being tested in a ring trial with a small set of carefully selected chemicals. In order to meet the requirements for additional test systems assessing more specifically the conjunctiva involvement within an eye irritating response, we established a human cell based in vitro full thickness conjunctiva equivalent. The conjunctiva model reveals quite similar to in vivo conjunctiva regarding the initial depth of injury. However, it could not provide additional information to the cornea-based test method even when testing those chemicals which display conjunctiva damage prior to corneal effects, and thus seems to be dispensable for in vitro safety assessment testing. However, it may be beneficial for barrier function analysis and further biological questions.


Super resolution imaging of brain tissue harbors difficulties due to light scattering and the density of signals. In order to reduce out of focus signal, we take advantage of the genetic tools available in the Drosophila and have fluorescently tagged synaptic proteins expressed in only a small number of neurons. These neurons form synapses within the calyx of the mushroom body, a distinct brain region involved in associative memory formation. Our results show that super resolution microscopy, in combination with genetically labeled synaptic proteins, is a powerful tool to investigate synapses in a quantitative fashion providing an entry point for studies on synaptic plasticity during learning and memory formation.Understanding synaptic connectivity and plasticity within brain circuits and their relationship to learning and behavior is a fundamental quest in neuroscience. Visualizing the fine details of synapses using optical microscopy remains however a major technical challenge. Super resolution microscopy opens the possibility to reveal molecular features of synapses beyond the diffraction limit. With direct stochastic optical reconstruction microscopy,  In recent years, an increasing effort has been put toward the characterization of neuronal connectivity on large scales , and UAS-Drep-2 mStrawberry .Fly strains are reared on standard Drosophila medium at 25 or 18°C with a 14/10 h light/dark cycle. To visualize synaptic proteins in specific neurons we cross the GAL4-driver lines Mz19-Gal4/+ or 201y-Gal4 with UAS-syt::GFP, UAS-brp-shortAdult female flies are cooled to immobility and groups of five or six flies are threaded by their necks into collar holders. Flies are fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) for 1 h at room temperature or for 4 h at 4°C. After washing three times 10 min with PBS the flies are immersed overnight with 20% sucrose in 0.1 M PBS for cryoprotection. Eight micrometers thin cryosections are cut on a Leica 2020 vibratome and mounted on Poly-L-Lysine coated coverslips .−1 Glucose oxidase, Catalase . The coverslips are sealed with nail polish or Secure Seal imaging spacer (Sigma-Aldrich).Cryosections are first dried on coverslips for 30 min. The sections are then blocked for 30 min with 5% Normal Goat Serum in PBT (PBS containing 0.1% Triton X-100) before they are incubated for 2 h with primary antibody in blocking solution. Primary antibodies used in this study include mouse antibody to GFP (Invitrogen) and rabbit antibody to DsRed (Clontech). Sections are washed three times for 10 min with PBS and then incubated for 1 h with the secondary antibody. Secondary antibodies applied are anti-mouse or anti-rabbit Alexa Fluor 647 antibodies (Invitrogen). After final washing of three times for 10 min with PBS, coverslips are mounted on slides with a freshly prepared imaging medium. The medium contains 100 mM cysteamine, 10% (m/v) glucose, 1 mg mlSuper resolution and confocal imaging is performed on a Nikon Ti Eclipse inverted microscope, equipped with an A1 Confocal Scan Head, an oil immersion objective , and a TIRF-arm for highly inclined illumination. For super resolution imaging specifically a powerful red laser  and a weaker violet one  are coupled into a single mode fiber through an AOTF , which also allows for intensity control. The fiber directs the light to the TIRF-arm through which collimation and angle of illumination can be controlled. The emitted fluorescence is directed through an adaptive optics system (MicAO 3D-SR), which corrects unwanted aberrations and adds astigmatism for 3D imaging. The light is finally captured on an Andor iXon Ultra 897 EMCCD camera. The pixel size is 104 nm, achieved with extra magnification.We first take a stack of confocal images of the region of interest with a step size of 0.2 μm. We then switch to weak wide-field illumination with the red laser and, once the plane of interest is identified, the laser is set to full power. Initially the angle of illumination is kept at 0° in order to render dark the whole thickness of the sample. After a few seconds the angle is increased to near TIRF  conditions, thereby reducing out of focus fluorescence and increasing local intensity. Once stable blinking conditions are reached, we adjust the camera integration time between 6 and 30 ms and acquire 60–100 thousand frames. During acquisition, the violet laser is sometimes used to adjust the density of blinking events.ThunderSTORM , which are imaged at different axial positions. All z-coordinates are rescaled by a factor of 0.72 due to the refractive index mismatch between glass and the sample in the buffer solution . With the To study protein organization of chemical synapses in the brain we investigated synapses formed between olfactory projection neurons (OPN) and Kenyon cells (KC) within the calyx of the fruit fly. KCs receive olfactory information through OPN from the antennal lobe Figure  and it hdSTORM imaging in tissue, fluorescently tagged synaptic proteins are expressed in a few defined neurons. By using the MZ19-Gal4 driver, tagged presynaptic proteins are expressed only in 10–13 OPNs . A subset of the MZ19 positive boutons is first imaged by confocal and subsequently with dSTORM. Figures cherry) under the control of MZ19-Gal4. BRP is localized at T-bar formations within the active zone and is widely used as a presynaptic marker. In earlier studies, it has been tested that the expression of UAS-brp-shortcherry does not affect the number of active zones . On the confocal images the red presynaptic and green postsynaptic signal overlap and result in a diffuse, yellow signal  is a transmembrane protein of synaptic vesicles and plays an important role in the fusion of vesicles at presynaptic sites. Figure e Figure , we can e Figure , arrow. cherry) expressed in a MZ19-Gal4 fly line. Similar to Figure Figure mStrawberry, we can see in the confocal image an expression restricted to the membrane of the claw-like structured dendrites, however the single synaptic sites cannot be distinguished , which corresponds to circular structures with a radius of 31 nm. This average cluster size is very close to the size of synaptic vesicles reported previously . For the two postsynaptic proteins even smaller object areas of 0.13 μm2 for Drep-2  and 0.12 μm2 for Dα7  were measured , while the cluster size for Drep-2 and Dα7 are the same (p = 0.607). On the underlying super resolution image for the two postsynaptic proteins Drep-2 and Dα7 we observe signal along the dendritic membrane with sites of higher density. By thresholding for clusters, we would get these sites of higher protein density within the postsynaptic density. In the case of BRP thresholding for the object gives an area of BRP expression close to the presynaptic site and, when we consider the size, clusters would represent the confined and dense expression of BRP and with it a T-bar in the active zone. In this way, thresholding based on local density reveals different sizes and shapes indicative for relevant structures within the bouton, dendrite and synapse.While the same threshold criteria for Syt results in cross section areas of boutons, we obtain for BRP, Drep-2 and Dα7 sizes indicative of synaptic membrane areas. When we apply subsequently the same threshold for clusters, we find an average cluster size of 0.006 μmdSTORM in two dimensions. In order to obtain the signal localization of all three dimensions, samples should exhibit little noise, robust blinking, and the regions of interest need to be sufficiently close to the surface as we image under TIRF or highly inclined illumination conditions. 3D super resolution images are obtained by introducing astigmatism in the imaging pathway . Within this depth, we can image entire synaptic sites but rarely complete bouton terminals. For this reason, we get an average surface area of 13 μmTo define the localization of proteins within the synapse or bouton in three dimensions adds important information especially when we want to study the relationship of different proteins expressed within a synapse. In this study, we determine the localization of specific synaptic proteins first alone and within two dimensions, as the accessible depth for SMLM is very limited. Three dimensional imaging can however reveal the relation of areas and clusters within the volume, resolve overlapping fine structures and add information about variation of signal density within a certain depth  as well as for the postsynaptic proteins Drep-2mStrawberry and Dα7-GFP(0.13 μm2). Butcher et al. also describe that almost all synapse were divergent polyads, meaning that there is more than one postsynaptic partner for each presynaptic site. They write that the presynaptic density is often of the same size as the width of all the opposing postsynaptic elements combined. From the values we obtain from super resolution imaging we can thus confirm that there is no significant difference in the local density of signals related to BRP-shortcherry and the Drep-2mStrawberry and Dα7-GFP, resulting in similar object sizes. However, the polyade synapse would also explain why the clusters found within the Drep-2 and Dα7 sites are smaller compared to the BRP. Having more than one postsynaptic partner implies that there is one active zone opposed to several postsynaptic partners. Clusters of higher signal within the postsynaptic site could thus represent local postsynaptic densities across the different postsynaptic partners.The accuracy of our values can be confirmed by another anatomical study of synapses in the calyx. Butcher et al. imaged boutons of different types of OPN under the EM. They reconstructed the boutons with serial ultrathin sections and quantified the surface area, volume, and the synapses density per bouton terminal  and obtain thus similar results. Taking the results from EM studies as the golden standard, we can conclude that imaging with dSTORM gives similarly accurate results as the anatomical study based on EM images.Butcher et al. have further measured the surface area of bouton terminals to be on average 14–25 μmDrosophila brain tissue before, however, a recent study has imaged BRP in larval neuromuscular junctions (NMJ) with dSTORM and quantified the number of proteins per active zone cytomatrix (CAZ; Ehmann et al., SMLM has not been applied to synapses in dSTORM for the imaging of tissue sections of the fly brain. With the genetic possibilities in the fly system and the specific labeling of proteins, we can examine the localization and expression density of specific proteins in few defined neurons. The fly brain thus offers important advantages to minimize noise or out of focus signals. To further improve the signal to noise, we apply dSTORM on 8 μm thin cryosections and image with highly inclined or TIRF illumination. Due to this illumination mode, we can access only structures within the first 1–3 μm. Within this depth, entire synapses can be imaged, though some may be incomplete due to sectioning or illumination angle. Further, to reconstruct several synapses along one axon or of an anatomical region such as the entire calyx is not feasible, unless one cuts serial and very thin sections (Nanguneri et al., In the current study, we demonstrate the potential of SR-Tesseler allows us to perform quantitative image analysis based on local density of signals so that we can identify object of interest and clusters within the objects (Levet et al., dSTORM originates from the blinking of AF647 and only relates indirectly, through immunostaining, to the protein of interest. The quality of immunostaining is therefore crucial for imaging with dSTORM and for the interpretation of the super resolution images obtained.Quantification of super resolution images and reliable analysis of the reconstructed signals is crucial to answer biological questions. Though, SMLM gives more accurate measurements than confocal microscopy and similar values to those obtained with EM, one needs to consider the systematically larger sizes due to antibody staining, especially for small structures. It has been estimated that the antibodies related to the staining add ~15 nm to the actual size of the observed protein (Ehmann et al., dSTORM we obtain very similar results for bouton areas as well as sizes of synapses compared to measurements based on serial reconstructions of images taken under the EM. With SMLM, the density and distribution of proteins can further be revealed in their subcellular structures and analyzed for clusters. We can thus confirm that imaging with dSTORM resolves the localization of proteins and can provide quantitative measures of synapse composition. With the aim to image pre- and post-synaptic proteins simultaneously, we recently started to apply dSTORM using two different fluorophore tags.Summarized, we are able to describe and map the protein organization at synapses formed between OPNs and KCs in very fine detail, beyond the diffraction limit. When we put our quantitative results in relation to what has been measured in the microglomeruli with confocal and electron microscopy, we see that with Concluding, we believe that super resolution images will help to examine in fine detail plasticity of synapses related to density and distribution of synaptic proteins and eventually investigate its role in memory formation.FS and SS directed the project. IS performed experiments and analyzed the data with the help of GC. All authors contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mechanical chest compressions have been proposed to provide high-quality cardiopulmonary resuscitation (CPR), but despite the growing use of mechanical chest compression devices, only few studies have addressed their impact on CPR quality. This study aims to evaluate mechanical chest compressions provided by LUCAS-2 (Lund University Cardiac Assist System) compared with manual chest compression in a cohort of out-of-hospital cardiac arrest (OHCA) cases.In this prospective study conducted in the Central Denmark Region, Denmark, the emergency medical service attempted resuscitation and reported data on 696 non-traumatic OHCA patients between April 2011 and February 2013. Of these, 155 were treated with LUCAS CPR after an episode with manual CPR. The CPR quality was evaluated using transthoracic impedance measurements collected from the LIFEPAK 12 defibrillator, and the effect was assessed in terms of chest compression rate, no-flow time and no-flow fraction; the fraction of time during resuscitation in which the patient is without spontaneous circulation receiving no chest compression.The median total episode duration was 21 minutes, and the episode with LUCAS CPR was significantly longer than the manual CPR episode, 13 minutes vs. 5 minutes, p < 0.001. The no-flow fraction was significantly lower during LUCAS CPR (16%) than during manual CPR (35%); difference 19% . No differences were found in pre- and post-shock no-flow time throughout manual CPR and LUCAS CPR.Contrary to the manual CPR, the average compression rate during LUCAS CPR was in conformity with the current Guidelines for Resuscitation, 102/minute vs. 124/minute, p < 0.001.2 and chest compression depth surrogate parameters of CPR quality could not be reported.Mechanical chest compressions provided by the LUCAS device improve CPR quality by significantly reducing the NFF and by improving the quality of chest compression compared with manual CPR during OHCA resuscitation. However, data on end-tidal Co High-quality chest compression and early defibrillation are particularly essential for survival outcome after out-of-hospital cardiac arrest (OHCA) . Recent To address these shortcomings, a mechanical chest compression device, LUCAS-2 (Lund University Cardiopulmonary Assist System), has been developed Figure A. The LUDespite the growing use of these devices, randomised studies in humans have not been able to show better outcome for OHCA patients resuscitated with mechanical chest compressions -9. Resea2 inhabited by a total of 1.3 million people. The first tier consists of 75 conventional ambulances manned with two EMS providers. The second tier consists of nine physician-manned prehospital critical care teams and one HEMS available 24/7. The prehospital critical care teams and the HEMS have the competency to provide advanced life support (ALS). ALS includes the potential use of the LUCAS device. Conventional ambulances provide basic life support (BLS) and defibrillation only.The emergency medical service (EMS) in the Central Denmark Region is organised as a two-tier system; it operates a double-dispatch service that covers an area of 13,142 kmThe EMS in the Central Region Denmark has a standardised pre-hospital-resuscitation protocol, which was strictly adhered to in this study. A conventional ambulance is dispatched to all emergencies including OHCA. It normally arrives at the scene as a first responder. The prehospital critical care team or the HEMS is dispatched to patients with presumed OHCA as determined by the dispatcher triage and according to availability. All cardiac arrest patients are treated according to the 2010 Guidelines for Resuscitation. During the entire study period, the physicians of the prehospital critical care team and the HEMS serve jointly as the decision-maker who decides whether or not the LUCAS device is to be used in the attempt of resuscitation. In patients receiving LUCAS CPR, the EMS providers initiate the BLS until the arrival of the physician-manned mobile prehospital critical care team bringing the LUCAS device. Manual CPR is continued when the LUCAS device is being deployed; and in order to minimise the interruptions in CPR, it is only paused briefly when the back plate is inserted and the arms positioned. When the prehospital critical care team or the HEMS are involved, the patient is transported directly to the tertiary university hospital’s Heart Centre, which is staffed with dedicated anaesthesiologists, cardiologists and surgeons. Provided that active treatment is indicated, acute coronary angiography (CAG) and/or percutaneous coronary intervention (PCI) is performed, and therapeutic hypothermia is applied according to international guidelines. The patients are subsequently treated in the Cardiac Intensive Care Unit which is staffed by anaesthesiologists and cardiologists. However, if the prehospital critical care team or the HEMS are unable to assist the EMS providers, the operating procedure for the EMS providers state that after three defibrillation attempts or in the case of a non-shockable rhythm after three 2-minute cycles of CPR and persisting cardiac arrest, the patient is transported to the nearest hospital.A total of nine LUCAS devices were introduced in the prehospital critical care team and the HEMS. Prior to this introduction, the physicians and the EMS providers were informed about the study protocol and the procedures, and they were instructed how to handle the LUCAS device. Furthermore, they accomplished a manikin-scenario-training-session, which enabled them to use the device correctly, i.e. to deploy the device within 20 seconds while minimising interruptions in CPR. The physicians and the EMS providers who did not have the opportunity to attend the training session were trained and accredited by one of the physicians who took part in the session.The inclusion criteria for the present study were non-traumatic OHCA, age above 18 years and OHCA occurring between 1 October 2011 and 31 January 2013. Exclusion criteria were pregnancy, trauma, intoxication, inability to attach the LUCAS device to the patient, patients with missing data and total resuscitation episode < 2 minutes.Data were collected prospectively and registered according to the Utstein templates for resuscitation registries  Figure . The Dan2 (ETCO2), which served as a surrogate marker of CPR quality.Standard LIFEPAK 12 defibrillators  were used. De defibrillators continuously measure the transthoracic impedance (TTI) by applying a near constant current across the defibrillations pads. After a CPR effort, the ECGs and the TTI data of the OHCA case were transferred to a central server at the tertiary hospital; Aarhus University Hospital, Denmark. Furthermore, the prehospital critical care teams and the HEMS filled out a separate study form regarding the end-tidal COThe software programme CODE-STAT™ -8  was used to process the TTI data Figure B, C, D. We defined the CPR-pause interval as the time from the trailing edge of the last chest compression to the leading edge of the next chest compression. Mechanical compressions were distinguished from the manual compressions by their highly regular morphology Figure C. UsableAdditionally, nationally adapted Utstein style forms filled out by the EMS personnel were used to obtain data on bystander CPR, OHCA location and the use of an automated external defibrillator (AED) prior to arrival of the EMS and whether OHCA was witnessed or unwitnessed. Data on the patients who achieved ROSC, survived to hospital discharge and survived after 30 days were collected from the OHCA registration form, ambulance records and hospital records.LUCAS is a chest compression system that provides both active compression and decompression of the chest wall back to neutral position Figure A. The fiData management and statistical calculations were performed using STATA/SE 12.1. Normally distributed continuous variables are presented as mean ± standard deviation and non-normally distributed variables as median and quartiles. Differences were analysed with Student’s paired t-test or Wilcoxon’s sign rank test as appropriate. Categorical variables are presented as number (n) and percent (%) as appropriate, and differences are analysed with McNemar’s test. The a priori α-level was 0.05.Data were reported on 696 OHCA patients who were attempted resuscitated between 1 October 2011 and 31 January 2013. Of these, 196 were resuscitated with both manual CPR and LUCAS CPR. Due to incomplete TTI data, 31 (4%) patients were excluded. Another 41 (21%) patients were excluded due to the fact that only data with LUCAS CPR were available. The remaining 155 OHCA patients comprised the study population  from emergency call to first defibrillation by the EMS was 9 (8–19) minutes.CPR variables are shown in Tables There was no significant difference between the results of either NFT during rhythm analysis with and without defibrillation or pre- and post-shock NFT with manual or LUCAS CPR Table . LUCAS NTable While recent years have seen studies reporting on the outcome associated with mechanical CPR ,14, onlyOur results show that during the manual episode, NFF lasted 34% of the time in patients without ROSC. However, once the LUCAS device was deployed, the NFF was reduced significantly to 16% of the time. Although an NFF of 34% during manual CPR is lower than what has been reported in previous studies where CPR was performed according to the former resuscitation guidelines of 2005 [The low NFF with the LUCAS device may also have been achieved owing to fewer interruptions while loading the patient into the ambulance and during transport with on-going LUCAS.Another advantage of the LUCAS device is that it affords the possibility of delivering shocks during compression and enables users to shorten or eliminate pauses for defibrillation. Nevertheless, we found no difference in pre-shock and post-shock NFT in relation to automated rhythm analysis throughout the time with manual CPR or LUCAS CPR. The median pre-shock pause was 17 sec. (IQR 15–20) and the median post-shock pause was 7 sec. (IQR 6–8). While these pauses are reasonable for defibrillation in automatic mode during manual CPR and similar to pauses reported in previous research , they arAlthough they fell short of being optimal, the manual chest compression rate and the number of chest compressions per minute still appear superior to the manual compression rates (138 compressions per minute) reported in the study by Krarup et al. .The episode duration with LUCAS CPR was nearly three times longer than the manual CPR episode Table . This agIn a study of changes in bystander resuscitation attempts and survival during a 10-year period in which national initiatives were taken to improve rates of bystander CPR, Wissenberg et al.  concludeThere are some limitations to this study. First, it is a small observational, prospective study with potentially unknown confounders; however, the relative advantage of this study design is that being their own controls, the patients studied are the same during manual CPR and LUCAS CPR. Second, the physician of the prehospital critical care team decides if the LUCAS device is to be used in the resuscitation, which undoubtedly introduces selection bias. Third, even though TTI and ECG data transfer to the central server was mandatory, a large part of the OHCA data was never transferred to the server.2 (ETCO2), which is considered a surrogate measure of cardiac output during CPR [2 values in this study due to the lack of registered values/too many missing values. Data on adverse device events or injuries have likewise not been reported in those patients not surviving OHCA due to the fact that these events were not described in the medical reports. The 45 patients admitted to hospital alive did not have any device-related events or injuries.Ventilation and compression depth measurements during resuscitation are recommended for the evaluation of CPR quality; however, these measurements cannot be assessed from TTI data alone, and little data support the clinical importance of these variables in this setting . The prering CPR ; howeverMechanical chest compressions provided by the LUCAS device improve CPR quality by significantly reducing the NFF and by improving the quality of chest compression compared with manual CPR during OHCA resuscitation.Randomised clinical studies not have been able to demonstrate improved survival for patients resuscitated with mechanical chest compression devices, and the devices have to some extent been discredited following the ASPIRE trial, the LINC trial and the PARAMEDIC trial. However, these trials are impeded by methodological deficiencies such as different resuscitation protocols in the compared groups and, in particular, prolonged time to deployment of the device. Maintenance of high-quality CPR during OHCA is not easy because of the small number of crew present, fatigue, patient access, and the impossibility of performing resuscitation in a moving vehicle. These considerations must be balanced against the current evidence from previous studies when deciding on the future role of mechanical CPR in healthcare systems, but it seems reasonably that such devices will continue to play a role when manual CPR is impractical. Nevertheless, it is essential that resources are available to support regular training of healthcare personnel ensuring that resuscitation guidelines are followed and to monitor CPR quality continuously.