Name: coordinate mapping of hyolaryngeal mechanics in swallowing
Uploaded: 2014-05-06T15:30:00
Description: Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

Forfatterne erkender Kendrea Focht, CSCD, CCC-SLP og Evelyn Trammell Institut for Voice og synke på Medical University of South Carolina, for at dele MBS billeddannelse filer, der bruges til at demonstrere denne metode. Disse MBS data blev indsamlet via extramural støtte finansieret af Grant Number TL1TR000061 (PI: Focht) fra National Center for Fremme Translationelle Sciences og Grant Number 1K24DC12801 (PI: Martin-Harris) fra National Institute on Døvhed og andre kommunikations Disorders, og intramuralt støtte fra Mark og Evelyn Trammell Trust. Disse metoder blev oprindeligt udviklet af principal investigator mens støttet af Grant Number F31DC011705 fra National Institute on Døvhed og andre kommunikations Disorders. Indholdet er alene forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter National Institute on Døvhed og andre kommunikations Disorders eller National InstitutesSundhedsstyrelsen.

1.. Konfiguration af en computer <ol><li> For Macintosh hente følgende open source eller freeware software: ImageJ, MACX Video Converter Free Edition (Mac) og QuickTime (se tabel materiale / udstyr). </li><li> For en pc, kan du downloade følgende open source eller freeware software: ImageJ, MPEG Streamclip (PC) og QuickTime (se tabel materiale / udstyr). </li></ol> 2.. Forberedelse videoklip <ol><li> Fil konvertering. Konvertere rå videofiler i. Mov til indsamling af data i ImageJ. Bemærk...

<ol>
	<li>Leonard, R. J., Kendall, K. A., McKenzie, S., Gon&#231;alves, M. I., Walker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Displacements+in+Normal+Swallowing:+A+Videofluoroscopic+Study.">Structural Displacements in Normal Swallowing: A Videofluoroscopic Study.</a> Dysphagia. 15, 146-152 (2000).</li><li>Molfenter, S. M., Steele, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+Variability+in+the+Deglutition+Literature:+Hyoid+and+Laryngeal+Kinematics.">Physiological Variability in the Deglutition Literature: Hyoid and Laryngeal Kinematics.</a> Dysphagia. 26, 67-74 (2010).</li><li>Pearson, W. G., Hindson, D. F., Langmore, S. E., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Swallowing+Muscles+Essential+for+Hyolaryngeal+Elevation+by+Using+Muscle+Functional+Magnetic+Resonance+Imaging.">Evaluating Swallowing Muscles Essential for Hyolaryngeal Elevation by Using Muscle Functional Magnetic Resonance Imaging.</a> International Journal of Radiation Oncology* Biology* Physics. 85, 735-740 (2013).</li><li>Pearson, W. G., Langmore, S. E., Yu, L. B., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Analysis+of+Muscles+Elevating+the+Hyolaryngeal+Complex.">Structural Analysis of Muscles Elevating the Hyolaryngeal Complex.</a> Dysphagia. 27, 445-451 (2012).</li><li>Kim, Y., McCullough, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+hyoid+displacement+in+normal+swallowing.">Maximum hyoid displacement in normal swallowing.</a> Dysphagia. 23, 274-279 (2008).</li><li>Steele, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+hyoid+and+laryngeal+displacement+and+swallowing+impairment.">The relationship between hyoid and laryngeal displacement and swallowing impairment.</a> Clin. Otolaryngol. 36, 30-36 (2011).</li><li>Logemann, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+Biomechanical+Characteristics+of+Oropharyngeal+Swallow+in+Younger+and+Older+Men.">Temporal and Biomechanical Characteristics of Oropharyngeal Swallow in Younger and Older Men.</a> Journal of Speech, Language and Hearing Research. 43, 1264-1274 (2000).</li><li>Pearson, W., Langmore, S., Zumwalt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Structural+Properties+of+Suprahyoid+Muscles+and+their+Potential+for+Moving+the+Hyoid.">Evaluating the Structural Properties of Suprahyoid Muscles and their Potential for Moving the Hyoid.</a> Dysphagia. 26, 345-351 (2011).</li><li>Hopkins, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+reliability+in+sports+medicine+and+science.">Measures of reliability in sports medicine and science.</a> Sports Med. 30, 1-15 (2000).</li><li>Bingjie, L., Zhang, T., Sun, X., Xu, J., Jiang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+videofluoroscopic+analysis+of+penetration-aspiration+in+post-stroke+patients.">Quantitative videofluoroscopic analysis of penetration-aspiration in post-stroke patients.</a> Neurol. India. 58, 42-47 (2010).</li><li>Webster, M., Sheets, H. D., Alroy, J., Hunt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+introduction+to+landmark-based+geometric+morphometrics.">A practical introduction to landmark-based geometric morphometrics.</a> Quantitative Methods in Paleobiology. Paleontological Society Papers. 16, 163-188 (2010).</li><li>Inamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+swallowing+using+320-detector-row+multislice+CT.+Part+II:+Kinematic+analysis+of+laryngeal+closure+during+normal+swallowing.">Evaluation of swallowing using 320-detector-row multislice CT. Part II: Kinematic analysis of laryngeal closure during normal swallowing.</a> Dysphagia. 26, 209-217 (2011).</li></ol>

Denne undersøgelse viser nytten af ​​en metode, ved hjælp af koordinere data fra anatomiske kendetegn at beregne flere kinematiske målinger af hyolaryngeal bevægelse i synke. Inter-Rater pålideligheden af ​​seks raters, herunder to nybegyndere raters, for koordinater og beregnede variable var stærk (ICC&gt; 0,90). Repræsentative resultater fra en stikprøveundersøgelse af sunde, ikke-dysphagic voksne viste forskelle i flere kinematiske variable i svar på to bolus typer. Vi fandt også, at bruge forskell...

Svælg fase synke er en kompleks proces, der involverer mere end tyve muskler og flere skeletelementer at overføre en bolus fra mundhulen i spiserøret og samtidig beskytte luftvejene. Foregående svælg konstriktion, elementer af hyolaryngeal kompleks (tungebenet, strubehoved og tilknyttede strukturer, herunder den øverste esophageal sphincter) er forskudt til at konvertere en respiratorisk ledning ind i en fordøjelseskanalen. Strubehovedet er re-placeret anteriort væk fra bane af en modkørende bolus, og den øverste esophageal sphincter strækkes åben. Kinematiske målinger taget fra video fluoroskopiske synke undersøgelser (også kendt som en MBS eller Modified Barium Swallow Studies) er den primære forskning metode til at kvantificere de mange bevægelser hyolaryngeal kompleks 1.. Mens kvantitativ video fluoroskopiske målinger er nyttige til måling synke funktion, de forskellige akser henvisningog skalarer af måleresultatet i fund, der er uforenelige blandt de forskellige metoder til kinematiske målinger 2. Bevægelsen af ​​patienten og fluoroscope under manuel kliniker kontrol forvirrer også nøjagtigheden af ​​måling af dette kompleks fysiologisk proces. Endnu vigtigere, behøver kinematiske målinger ikke nødvendigvis afspejler struktur-til-funktion relationer vigtige for at vurdere, uorganiseret synke. Kinematik hyoid især er designet til at spore bevægelse i en anterior eller bedre retning i forhold til en anatomisk plan på linie med hvirvlerne. Men denne konfiguration ikke repræsenterer aktionslinje af muskler, der suspenderer hyoid. En to-slynge mekanisme hyolaryngeal elevation i svælg fase synke er blevet identificeret (figur 1) 3,4. De suprahyoid muskler omfatter forreste muskuløs slynge, og de lange svælg muskler omfatter posterior muskuløs slynge. Disse muskler ophøje forskellige elementer af hyolaryngeal kompleks herunder hyoid, strubehoved, og strukturer, der danner den øvre esophageal sphincter. Koordinere kortlægning af hyolaryngeal mekanik udnytter ni let identificerbare anatomiske kendetegn at kortlægge tre skelet håndtag og funktioner i hyolaryngeal komplekse repræsenterer fastgørelsespunkter i den forreste og bageste muskuløse slynger (figur 2). Under synke, hver skelet håndtag og træk den hyolaryngeal kompleks er i bevægelse. Ved at samle koordinater, kan systemet blive fanget i en tidsramme. Trigonometrisk konvertering af koordinater kan bruges til at generere flere kinematiske målinger af hyolaryngeal bevægelse under synke. Variable kan beregnes til sammenligning med resultater rapporteret i litteraturen, eller bruges til at generere nye målinger repræsenterer struktur-til-funktion forhold af interesse. Det primære formål med dette oplæg er at demonstrere en metode til generering af flere kinematiske målinger beregnet ud fra et enkelt sæt anatomiske skelsættende koordinater indsamlet fra Modified Barium Swallow (MBS) undersøgelser. Vi dokumenterer pålideligheden af ​​denne metode ved hjælp Intraclass korrelationskoefficienter at bestemme den inter-rater pålidelighed 6 forskellige efterforskere herunder en ekspert, tre testere med erfaring, og to novicer. Fra de kinematiske resultater, er forskelle i synke mekanik ved bolus konsistens evalueret. Endelig foreslås af Molefenter og Steele spørgsmål om betydningen af ​​referenceaksen til måling hyoid bevægelse behandles. At nærme sig dette spørgsmål, vi sammenligne målinger af hyoid udflugt i forhold til ryghvirvlerne og i forhold til underkæben, beregnet ud fra den samme koordinater for begge bolus typer. Hvis disse to metoder til måling af hyoid bevægelse repræsenterer den samme struktur til at fungere relationerhofte, så resultaterne bør stærkt korreleret. Til denne undersøgelse blev 40 laterale view MBS undersøgelser tilfældigt udvalgt fra en samling af 139 normale undersøgelser under et forsknings-protokol godkendt af Georgien Regents University Institutional Review Board og i forskningssamarbejde med Evelyn Trammell Institut for Voice og synke på det medicinske universitet i South Carolina. For at demonstrere anvendeligheden af denne metode blev ti variable kendetegner hyolaryngeal kinematik beregnet ud fra det samme sæt af koordinere data (tabel 1). Syv af disse beregnede målinger har tidligere været anvendt i litteraturen, herunder: forreste og overlegen målinger hyoid distance i forhold til ryghvirvlerne 5; forreste og overlegen hyoid forskydning i forhold til C2-4 længde, også i forhold til ryghvirvlerne 6; overlegen bevægelse af strubehovedet i forhold til hvirvlerne 7; hyolaryngeal approximation 1; og maksimal hyoid udflugt i forhold til ryghvirvlerne 1.. Desuden blev tre nye målinger beregnes: svælg afkortning tilnærme de vedhæftede filer i palatopharyngeus muskel, larynx elevation efter en linje af handling repræsenterer stylopharyngeus og hyoid udflugt tilnærmelse af vedhæftede filer i de suprahyoid muskler 4,8. En ekspert hoved og hals anatom (WP), tre efterforskere med begrænset erfaring tager målinger (CJ, SR, TT), og to nybegyndere efterforskere (RS, JT) opnået koordinere kortdata anvendelse af protokollen beskrevet nedenfor. Eksperten (WP) er uddannet de tre bedømmere med erfaring, og disse til gengæld trænet de to nybegyndere raters. Inter-Rater pålidelighed koordinere data og resultater beregnet ud fra koordinater efter emne blev bestemt ved Intraclass korrelationskoefficienter 9. To tailed t-tests blev udført på hver variabel for at bestemme statistically signifikante forskelle i bolus typer. En Pearson korrelationskoefficient og en koefficient på beslutsomhed blev brugt til at vurdere overensstemmelse mellem resultaterne af hyoid udflugt beregnet med ryghvirvler som referenceaksen versus hyoid udflugt med underkæben som en akse referencepunkt for 5 ml tynd væske svale og 5 ml budding svale.

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coordinate mapping of hyolaryngeal mechanics in swallowing

Karakterisering af hyolaryngeal bevægelse er vigtig for dysfagi forskning. Tidligere metoder kræver flere målinger for at opnå en kinematisk måling mens koordinere kortlægning af hyolaryngeal mekanik brug af modificeret Barium Swallow (MBS) bruger et sæt koordinater til at beregne flere variabler af interesse. Til demonstration blev ti kinematiske målinger genereres fra ét sæt koordinater til at bestemme forskelle i synke to forskellige bolus typer. Beregninger af hyoid udflugt mod ryghvirvler og underkæben er korreleret til at bestemme betydningen af ​​akser reference. For at demonstrere koordinere kortlægning metode blev 40 MBS undersøgelser tilfældigt udvalgt fra et datasæt af sunde normale forsøgspersoner uden kendt synke værdiforringelse. En 5 ml tynd væske bolus og et 5 ml budding svaler blev målt fra hvert emne. Ni koordinater kortlægning kranie base, underkæbe, ryghvirvler og elementer af Hyolarynx-kompleks, blev registreret ved rammerne for minimum og maksimum hyolaryngeal udflugt. Koordinater blev matematisk konverteret til ti variabler af hyolaryngeal mekanik. Inter-rater pålidelighed blev evalueret ved Intraclass korrelationskoefficienter (ICC). To-tailed t-test blev anvendt til at evaluere forskelle i kinematik efter bolus viskositet. Hyoid udflugt målinger mod forskellige akser referencerammer var korreleret. Inter-rater pålidelighed blandt seks testere for de 18 koordinater varierede fra ICC = 0,90 til 0,97. En skifer af ti kinematiske målinger blev sammenlignet efter emne mellem de seks raters. En outlier blev afvist, og gennemsnittet af de resterende reliabilitetsscores var ICC = 0,91, 0,84 til 0,96, 95% CI. Tohalede t-test med Bonferroni korrektioner sammenligner ti kinematiske variable (5 ml tynd flydende vs 5 ml budding svaler) viste statistisk signifikante forskelle i hyoid udflugt, overlegen larynx bevægelse og svælg shortening (p &lt;0,005). Pearson korrelationer af hyoid udflugt målinger fra to forskellige akser referencerammer var: r = 0,62, r 2 = 0,38, (tynd væske); r = 0,52, r2 = 0,27 (budding). Indhentning skelsættende koordinater er en pålidelig metode til at generere flere kinematiske variable fra video fluoroskopiske billeder er nyttige i dysfagi forskning.

Intraclass korrelationskoefficienter (ICC) koordinater indsamlet af seks efterforskere, der selvstændigt analyseret 80 video fluoroskopiske filer (to bolus forsøg fra 40 forsøgspersoner) varierede fra ICC = 0,90-0,97. En opdeling af ICC s koordinater efter gruppe er som følger: koordinerer nr. 1 - 5 (skelet elementer på minimum hyolaryngeal udflugt) betyder, = 0,93, 0,91 til 0,95, 95% CI; koordinerer nr. 6 - 9 (hyolaryngeal kompleks på minimum hyolaryngeal udflugt) betyder = 0,94, 092 til 0,96, 95% CI; koordinerer...

Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Koordinere mapping er en metode til at dokumentere iøjnefaldende træk ved hyolaryngeal biomekanik i svælg fase synke. Denne metode benytter billede analyse software til at registrere koordinaterne for anatomiske vartegn. Disse koordinater importeres til en Excel-makro og oversat til kinematiske variable af interesse nyttig i dysfagi forskning.

Koordinere Kortlægning af Hyolaryngeal Mechanics synke

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

coordinate-mapping-of-hyolaryngeal-mechanics-in-swallowing

Research

JoVE Journal

Medicine

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Heterogenitet Kortlægning af protein udtryk i tumorer ved hjælp af kvantitative Immunofluorescens

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

Label-fri Isolering og berigelse af celler gennem Contactless dielektroforese

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

MRI Kortlægning af cerebrovaskulær Reaktivitet via Gas Indånding Udfordringer

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Kirurgiske modeller af gastroøsofageal refluks med Mus

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Elektrofysiologiske vurdering af Murine forkamre med højopløselig optisk kortlægning

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Kvantitativ kortlægning af specifik ventilation i den menneskelige lunge ved hjælp af Proton magnetisk resonans imaging og ilt som kontrastmiddel

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Høj gennemløbs analyse af optiske kortlægnings data ved hjælp af Elektromap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

Sarcomere Afkortning af pluripotente stamcelle-afledte kardiomyocytter ved hjælp af fluorescerende-tagged Sarcomere proteiner.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...