Slice Protocol Research Articles

Genetically and pharmacologically limiting RyR2 open time prevents neuronal hyperactivity of hippocampal CA1 neurons in brain slices of 5xFAD mice

Neuronal hyperactivity is an early, common manifestation of Alzheimer’s disease (AD), and is believed to drive AD progression. Neuronal hyperactivity in the form of baseline activity (or spontaneous Ca2+ transients) has consistently been demonstrated in mouse models of AD using two-photon in vivo Ca2+ imaging of cortical or hippocampal neurons in anesthetized animals. Notably, these AD-related spontaneous Ca2+ transients were hardly detected in acute hippocampal slices, probably due to neuronal damage during brain slicing. To better preserve neuronal activity, we employed the N-methyl-D-glucamine (NMDG) protective brain slicing protocol. We performed confocal in vitro Ca2+ imaging of hippocampal CA1 neurons in optimized hippocampal slices. Consistent with previous in vivo studies, our in vitro studies using optimized brain slices also showed that limiting the open duration of the ryanodine receptor 2 (RyR2) by the RyR2 mutation E4872Q or by the R-carvedilol enantiomer prevented and rescued neuronal hyperactivity of hippocampal CA1 neurons from 5xFAD mice. Thus, genetically and pharmacologically limiting RyR2 open time prevented and rescued AD-related neuronal hyperactivity in vitro in optimized brain slices in the absence of anesthetics’ influence. Our data also suggest that the NMDG protective brain slicing preparation offers an alternative means to study neuronal hyperactivity of various cell types in different brain regions, especially in regions that are not readily accessible to two-photon in vivo Ca2+ imaging.

A Systematic Approach to Dissection of the Equine Brain-Evaluation of a Species-Adapted Protocol for Beginners and Experts.

Introduction of new imaging modalities for the equine brain have refocused attention on the horse as a natural model for ethological, neuroanatomical, and neuroscientific investigations. As opposed to imaging studies, strategies for equine neurodissection still lack a structured approach, standardization and reproducibility. In contrast to other species, where adapted protocols for sampling have been published, no comparable guideline is currently available for equids. Hence, we developed a species-specific slice protocol for whole brain vs. hemispheric dissection and tested its applicability and practicability in the field, as well as its neuroanatomical accuracy and reproducibility. Dissection steps are concisely described and depicted by schematic illustrations, photographs and instructional videos. Care was taken to show the brain in relation to the raters' hands, cutting devices and bench surface. Guidance is based on a minimum of external anatomical landmarks followed by geometric instructions that led to procurement of 14 targeted slabs. The protocol was performed on 55 formalin-fixed brains by three groups of investigators with different neuroanatomical skills. Validation of brain dissection outcomes addressed the aptitude of slabs for neuroanatomical studies as opposed to simplified routine diagnostic purposes. Across all raters, as much as 95.2% of slabs were appropriate for neuroanatomical studies, and 100% of slabs qualified for a routine diagnostic setting. Neither autolysis nor subfixation significantly affected neuroanatomical accuracy score, while a significant negative effect was observed with brain extraction artifacts. Procedure times ranged from 14 to 66 min and reached a mean duration of 23.25 ± 7.93 min in the last of five trials in inexperienced raters vs. 16 ± 2.83 min in experts, while acceleration of the dissection did not negatively impact neuroanatomical accuracy. This protocol, derived analogously to the consensus report of the International Veterinary Epilepsy Task Force in dogs and cats, allows for systematic, quick and easy dissection of the equine brain, even for inexperienced investigators. Obtained slabs feature virtually all functional subcompartments at suitable planes for both diagnostic and neuroscientific investigations and complement the data obtained from imaging studies. The instructive protocol and brain dissection videos are available in Supplementary Material.

Diffusion kurtosis imaging in the characterisation of rectal cancer: utilizing the most repeatable region-of-interest strategy for diffusion parameters on a 3T scanner.

Our goal was to investigate the correlation between histopathology and diffusion parameters by utilising the most repeatable region-of-interest (ROI) strategy for diffusion parameters in rectal cancer on a 3T scanner. 113 patients underwent DKI-MR and 66 of these patients received surgery without neoadjuvant chemoradiotherapy. Two readers independently measured the parameters using three slice protocols including single slice, three slices and whole-tumour slice (WTS), combined with one of two ROIs, including outline and round ROI. ANOVA, Kruskal-Wallis, a paired sample t-test, interclass correlation coefficient (ICC), Bland-Altman, Student's t-tests, receiver operating characteristic curves and z statistic were used for statistical analysis. There were no significant differences among the three slice protocols in ADC values (p = 0.822, 0.987), K values (p = 0.842, 0.859) and D values (p = 0.917, 0.988) using round and outline ROI, respectively. The ADC and D values derived from outline ROIs were higher than those from round ROIs (all p < 0.001 for ADC, all p < 0.001 for D), while K values derived from outline ROIs were lower than those from round ROIs (p < 0.001, p = 0.001, p < 0.001) using three slice protocols, respectively. The WTS-outline ROI resulted in the best intra- and inter-observer ICC. Utilising the WTS-outline ROI method, the AUC for assessment of well-differentiated tumours was 0.871 by K and 0.809 by ADC; and the AUC for T2 was 0.768 by K. The most repeatable strategy was the WTS-outline ROI method. In addition to DWI, DKI also have diagnostic value for rectal cancer histopathological characteristics utilising the WTS-outline ROI on a 3T scanner. • DKI using a 3T scanner is feasible for assessing rectal cancer. • ROI and slice protocol show considerable influence on DKI parameters. • DKI parameters exhibit excellent repeatability using whole-tumour slice-outline ROI on 3T scanner. • DKI has considerable diagnostic value for the estimation of rectal cancer characteristics.

Computing voxelised representations of macromolecular surfaces

Voxel-based representations of surfaces have received a lot of interest in bioinformatics and computational biology as a simple and effective way of representing geometrical and physicochemical properties of proteins and other biomolecules. Processing such surfaces for large molecules can be challenging, as space-demanding data structures with associated high computational costs are required. In this paper, we present a methodology for the fast computation of voxelised macromolecular surface representations (namely the van der Waals, solvent-accessible and solvent-excluded surfaces). The proposed method implements a spatial slicing procedure on top of compact data structures to efficiently calculate the three molecular surface representations at high-resolutions, in parallel. The spatial slicing protocol ensures a balanced workload distribution and allows the computation of the solvent-excluded surface with minimal synchronisation and communication between processes. This is achieved by adapting a multi-step region-growing EDT algorithm. At each step, distance values are first calculated independently for every slice, then, a small portion of the borders’ information is exchanged between adjacent slices. Very little process communication is also required in the pocket detection procedure, where the algorithm distinguishes surface portions belonging to solvent-accessible pockets from cavities buried inside the molecule. Experimental results are presented to validate the proposed approach.

Protein synthesis and consolidation of memory-related synaptic changes

Although sometimes disputed, it has been assumed for several decades that new proteins synthesized following a learning event are required for consolidation of subsequent memory. Published findings and new results described here challenge this idea. Protein synthesis inhibitors did not prevent Theta Bust Stimulation (TBS) from producing extremely stable long-term potentiation (LTP) in experiments using standard hippocampal slice protocols. However, the inhibitors were effective under conditions that likely depleted protein levels prior to attempts to induce the potentiation effect. Experiments showed that induction of LTP at one input, and thus a prior episode of protein synthesis, eliminated the effects of inhibitors on potentiation of a second input even in depleted slices. These observations suggest that a primary role of translation and transcription processes initiated by learning events is to prepare neurons to support future learning. Other work has provided support for an alternative theory of consolidation. Specifically, if the synaptic changes that support memory are to endure, learning events/TBS must engage a complex set of signaling processes that reorganize and re-stabilize the spine actin cytoskeleton. This is accomplished in fast (10min) and slow (50min) stages with the first requiring integrin activation and the second a recovery of integrin functioning. These results align with, and provide mechanisms for, the long-held view that memories are established and consolidated over a set of temporally distinct phases.This article is part of a Special Issue entitled SI: Brain and Memory.

Sensor monitoring of organotypic human tumor short term cultures may reveal profiles of metabolism and chemosensitivity

Event Abstract Back to Event Sensor monitoring of organotypic human tumor short term cultures may reveal profiles of metabolism and chemosensitivity Martin Brischwein1*, Regina Kleinhans1, Marina Haas1 and Bernhard Wolf1 1 Technische Universitaet Muenchen, Germany Background and aim. There is an urgent clinical demand for improved stratification of cancer patients and allocation into distinct therapeutic regimens. In this proof-of-principle study we analyse the feasibilty of a sensor-based profiling of metabolism and chemosensitivity on an organotypic human breast cancer model preserving the native, heterogeneous character of the tumor tissue. Methods. The approach is based on surgically explanted human breast cancer tissue samples which were subjected to careful slicing preparation (Kleinhans, 2012). These organotypic slices in a size of ≈ 4 µl were subsequently cultured for 3-5 days in an automated assay platform (Wolf, 2013). This platform analyzes the pH and dissolved oxygen values with optochemical sensors in the close vicinity of the tumor slices. Acid extrusion and oxygen consumption rates are determined before, during and after treatments with pharmaceutical compounds. Results . A number of 23 surgical samples was analysed. After the introduction of a careful vibratome slicing protocol the success rate of generating viable short term tumor cultures was generally good. The optimum slice thickness was found to be at 200-250 µm. Both a recording of pre-treatment metabolic profiles (i.e. ratios of extracellular acidification rates and oxygen consumption rates) and assessments of chemoresponses to drugs such as chloracetaldehyde and doxorubicine is feasible. Additionally, a computational reaction-diffusion modelling (Grundl, 2013) allows an estimation of pH and dissolved oxygen values in temporal and spatial resolution for an improved control about physico-chemical conditions of the cellular microenvironment. Conclusions / Discussion. A non-interventional, prospective clinical study should be initiated on the basis of this proof-of-principle research. In such a study, the microphysiometric, sensor-based approach should be complemented by data from histopathological, metabolomic and genomic profiling.

The necessity for full ventricular coverage with noncontrast T1 mapping in hypertrophic cardiomyopathy

Background Hypertrophic cardiomyopathy (HCM) is a genetic heart disease characterized by diverse phenotypic expression. T1 mapping is a novel CMR imaging sequence aimed at characterizing abnormal myocardial substrate. Current protocols obtain a mean T1 value derived from a single mid-LV short axis slice (single slice protocol) or three short-axis locations: apical, mid-LV and basal (three slice protocol). However, given the heterogeneity of disease expression within HCM, it is unclear if T1 values obtained from these limited portions of the LV chamber are representative of values derived from the entire LV myocardium (global).

Accelerating global left-ventricular function assessment in mice using reduced slice acquisition and three-dimensional guide-point modelling.

BackgroundTo investigate the utility of three-dimensional guide-point modeling (GPM) to reduce the time required for CMR evaluation of global cardiac function in mice, by reducing the number of image slices required for accurate quantification of left-ventricular (LV) mass and volumes.MethodsFive female C57Bl/6 mice 8 weeks post myocardial infarction induced by permanent occlusion of the left coronary artery, and six male control (un-operated) C57Bl/6 mice, were subject to CMR examination under isoflurane anaesthesia. Contiguous short axis (SAX) slices (1 mm thick 7-9 slices) were obtained together with two long axis (LAX) slices in two chamber and four chamber orientations. Using a mathematical model of the heart to interpolate information between the available slices, GPM LV mass and volumes were determined using full slice (all SAX and two LAX), six slice (four SAX and two LAX) and four slice (two SAX and two LAX) analysis protocols. All results were compared with standard manual volumetric analysis using all SAX slices.ResultsInfarct size was 39.1 ± 5.1% of LV myocardium. No significant differences were found in left ventricular mass and volumes between the standard and GPM full and six slice protocols in infarcted mice (113 ± 10, 116 ± 11, and 117 ± 11 mg respectively for mass), or between the standard and GPM full, six and four slice protocols in control mice, (105 ± 14, 106 ± 10, 104 ± 12, and 105 ± 7 mg respectively for mass). Significant differences were found in LV mass (135 ± 18 mg) and EF using the GPM four slice protocol in infarcted mice (p < 0.05).ConclusionGPM enables accurate analysis of LV function in mice with relatively large infarcts using a reduced six slice acquisition protocol, and in mice with normal/symmetrical left-ventricular topology using a four slice protocol.

Characterization of an Ovarian Slice Model In Vitro to View Ovulation.

Most studies of follicular maturation and ovulation in vitro utilize isolated follicles or whole ovaries through perfusion systems. Frequently, ovulation is established in vivo by the presence of an empty follicle. This study presents a method for real-time visualization of ovulation using an in vitro slice protocol that preserves the 3 dimensional structure of potentially key ovarian components. Ovulation is viewed from intact follicles within ovarian slices by either capturing daily images to examine progression at chosen time points or by using video microscopy to capture the continuity of events. Ovaries were collected from prepubertal (<5 wks) mice (C57 background), embedded in 8% agarose, cut at 200 µm and plated 3 slices per dish. Several different treatment combinations were investigated qualitatively, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), pregnant mare serum gonadotropin (PMSG), and PG600 (PMSG and hCG). For quantitative analysis, ovaries were treated with either no hormone, 5IU PG600, 5IU hCG, or 5IU PG600 followed 48 hours later by 5IU hCG. Results showed that dishes treated with both PG600 and hCG released approximately 22% of an average of 4.2 readily releasable oocytes per slice, while no oocytes were released without hCG. Ovulation was observed within 12 hours after the addition of hCG following PG600 pretreatment based on video microscopy or end point image comparisons. For other treatment groups, there was no evidence of stimulated oocyte release. Health and viability of the released oocytes was assessed by examining DNA labeled with 1mM Hoechst stain. Video microscopy indicated that one of the first steps in stimulated release is mobilization of the oocytes within the follicles within minutes. In summary, this study characterizes a novel method for studying the mechanisms of ovulation, the ability to examine and alter the progression of ovulation in a defined serum free media in vitro, and for the analysis of oocyte behavior within follicles. (platform)

Guest Editors' Introduction: Special Section on Autonomic Network Computing

IN the paper “Slicing Distributed Systems,” Gramoli et al. consider the problem of slicing—the partitioning of nodes into subsets using an one-dimensional attribute—in distributed systems. This problem arises in peer-to-peer networks and other popular distributed systems used in today’s large data centers. Slicing can help autonomic systems shift work dynamically among nodes and react dynamically to nodes dropping from the system or new nodes joining. The paper reviews existing approaches such as sorting and ranking and shows their problems in convergence, resulting in slow adaptation to changed conditions or inaccurate slice assignments in nonstatic networks. The authors introduce Sliver, a simple distributed slicing protocol that samples attribute values from the network and estimates the slice index from the sample. The theoretical results show that Sliver has provably rapid conversion, is robust under stress, and is simple to implement which they also show with simulation results of the new protocol. The paper “Cholla: A Framework for Composing and Coordinating Adaptations in Networked Systems” by Bridges et al. describes Cholla, a software architecture that separates the policy decisions of how and when adaptive components in networked systems react to their environment into separate centralized controllers that are constructed from composable rule sets. A major goal of this work is to develop a controller architecture that can be used for any collection of adaptive networking components, not a specific architecture that has to be implemented for each application-specific configuration or reimplemented each time that configuration changes. To achieve this, controllers are designed to be composable, with each adaptive component in the system contributing logic that together implements the overall functionality of the controller. This logic is implemented as rule sets that describe particular adaptation policies, and allow rule sets that implement linear, heuristic, and fuzzy control policies. Using composable control logic allows the controller to be specialized based not only on the set of adaptive components in the system, but also on the target network architecture and application requirements. This architecture consists of adaptable components, composable adaptation controllers, and a runtime system. In addition to describing the architecture of Cholla, this paper also presents a Linux-based prototype implementation of this architecture that controls and coordinates adaptation policy decisions inside network protocols and multimedia applications. An experimental evaluation of this prototype demonstrates that Cholla’s controller architecture enables component-based construction and customization of adaptation policies in networked systems, and that these policies can effectively control and coordinate adaptation. The paper “An Online Mechanism for BGP Instability Detection and Analysis” by Deshpande et al. proposes an online instability detection architecture for the border gateway protocol (BGP). BGP is one of the core building blocks of the Internet and is used to direct traffic between different subnetworks of the Internet, known as Autonomous Systems (ASs). The stability of BGP is critical to the stability of the Internet, and under stressful conditions such as worm attacks or router failures, BGP’s basic update protocol can result in severe route fluctuations and loss of connectivity for impacted networks for extended periods of time. The proposed architecture can be implemented by individual routers autonomously without the need for central control or centralized data. This allows widespread deployment without the need for modification of standards and also allows partial deployments without the need of large-scale cooperation among networks. The proposed system uses statistical techniques to detect instabilities using the time domain characteristics of BGP update message data and does not need any additional probing. The experimental evaluation using data collected from routers in Europe including data from several anomaly periods show that the system performs well various kinds of instabilities and across different topologies and policies. The paper “An Integrated Planning and Adaptive Resource Management Architecture for Distributed RealTime Embedded Systems” by Shankaran et al. looks at autonomicity issues in an open distributed embedded realtime environment. Such systems which may arise in context such as space control missions have fundamentally different characteristics than traditional embedded systems with controllable inputs. Autonomic operations require a close coordination between the resource planning needs of the IEEE TRANSACTIONS ON COMPUTERS, VOL. 58, NO. 11, NOVEMBER 2009 1441

An In Vitro Protocol for Recording From Spinal Motoneurons of Adult Rats

In vitro slice preparations of CNS tissue are invaluable for studying neuronal function. However, up to now, slice protocols for adult mammal spinal motoneurons--the final common pathway for motor behaviors--have been available for only limited portions of the spinal cord. In most cases, these preparations have not been productive due to the poor viability of motoneurons in vitro. This report describes and validates a new slice protocol that for the first time provides reliable intracellular recordings from lumbar motoneurons of adult rats. The key features of this protocol are: preexposure to 100% oxygen; laminectomy prior to perfusion; anesthesia with ketamine/xylazine; embedding the spinal cord in agar prior to slicing; and, most important, brief incubation of spinal cord slices in a 30% solution of polyethylene glycol to promote resealing of the many motoneuron dendrites cut during sectioning. Together, these new features produce successful recordings in 76% of the experiments and an average action potential amplitude of 76 mV. Motoneuron properties measured in this new slice preparation (i.e., voltage and current thresholds for action potential initiation, input resistance, afterhyperpolarization size and duration, and onset and offset firing rates during current ramps) are comparable to those recorded in vivo. Given the mechanical stability and precise control over the extracellular environment afforded by an in vitro preparation, this new protocol can greatly facilitate electrophysiological and pharmacological study of these uniquely important neurons and other delicate neuronal populations in adult mammals.

Computer-Aided Detection of Pulmonary Nodules in Computed Tomography

To evaluate diagnostic sensitivity of the pulmonary nodules computer-aided detection (CAD) in computed tomography. To analyze parameters that modify CAD performance. We made a critical analysis of the literature, and we described CAD sensitivity. Moreover, we compared CAD and CAD plus radiologist sensitivity in detection of pulmonary nodules, and we compared different acquisition techniques (thin slice vs thick slice and low dose vs normal dose). We used as major data sources the medical literature database of PubMed and MEDLINE, where we searched for articles in English language published from January 2001 to November 2006. We included studies that used spiral or multidetector row CT for CAD. Twenty studies met the inclusion criteria containing a total of more than 827 patients and 2717 pulmonary nodules detected by CAD. We observed an overall sensitivity of 79% for the CAD and of 92% for CAD plus radiologist; CAD sensitivity was 80% and 74% for thin slice and thick slice protocols, respectively. Results of our study suggest that CAD technique is an accurate tool in detection of pulmonary nodules, by working as useful second look for the physician. Sensitivity becomes higher by using it together with radiologist. Actually, the main limitation about the use of CAD to be solved is represented by the persistent high false-positive rate.

Prediction of Visceral Adipose Tissue Using Air Displacement Plethysmography in Children

To determine the ability of air displacement plethysmography (ADP) to predict visceral adipose tissue (VAT) volume in children. Fifty-five (33 boys/22 girls) white children 13 to 14 years old were studied. Anthropometric measures were collected for body mass, stature, BMI, and waist-to-hip ratio (WHR), and body fat percentage was estimated from triceps and subscapular skinfolds, bioelectrical impedance analysis, and ADP. VAT volume was determined using magnetic resonance imaging, using a multiple slice protocol at levels L1 to L5. Boys had significantly (p < or = 0.05) less VAT volume than girls [645.1 (360.5) cm(3) vs. 1035.8 (717.3) cm(3)]. ADP explained the greatest proportion of the variance in VAT volume compared with the other anthropometric measures. Multiple regression analysis indicated that VAT volume was best predicted by ADP body fat percentage in boys [r(2) = 0.81, SE of the estimate (SEE) = 160.1, SEE coefficient of variation = 25%] and by WHR and BMI in girls (r(2) = 0.80, SEE = 337.71, SEE coefficient of variation = 33%). Compared with the other anthropometric measures, ADP explains the greatest proportion of the variance in VAT volume in children 13 to 14 years old. For boys, ADP is the tool of choice to predict VAT volume, yet using the more simply collected measures of BMI and WHR is recommended for girls. However, large SE of the estimates remained, suggesting that if precision is needed, there is no surrogate for direct imaging of VAT.

Radiation dose in Spiral CT: The relative effects of collimation and pitch

The proliferation of helical CT and thin slice protocols has motivated a reevaluation of methods for determining radiation dose. Traditional measurements may be insufficient in that computed tomography dose index (CTDI) is undefined for the case of continuous table motion which characterizes helical CT. We have measured the radiation dose using thermoluminescent dosimeters (TLDs) under contiguous axial, contiguous helical, and noncontiguous helical scans. A device that holds tightly spaced TLDs was inserted into a 32‐cm‐diam Plexiglas™ (CTDI) phantom so that (1) TLDs were exposed to contiguous axial scans; (2) TLDs were exposed to a contiguous (pitch 1) helical scans, and (3) TLDs were exposed to noncontiguous (pitch &gt;1) helical scans. The TLDs integrated exposure from both primary and scattered radiation resulting from scanning a volume of the phantom. The TLD measurements were repeated at several slice thicknesses and for pitches of 1, 1.5, and 2. For a direct comparison, conventional pencil ionization chamber measurements were made at the same slice thicknesses. Our results show that contiguous helical CT scans (pitch of 1) give approximately the same radiation dose as contiguous axial scans acquired with the same technical factors. For noncontiguous scans (pitch &gt;1) at a given collimation, radiation dose decreases as pitch increases; specifically as 1/pitch. However, the dose remained relatively constant across collimations (±20% of the 10 mm slice dose) for both axial and helical scans. At smaller slice thicknesses, the radiation profile width (full width at half‐maximum) is greater than the nominal slice thickness, which results in extended radiation overlap between slices and no net change in radiation dose compared to thicker slices. Finally, for a given table speed, radiation dose decreases when a higher pitch is used with a thinner collimation.