Time-lapse imaging as a tool to investigate contractility of the epididymal duct--effects of cGMP signaling.

The well orchestrated function of epididymal smooth muscle cells ensures transit of spermatozoa through the epididymal duct during which spermatozoa acquire motility and fertilizing capacity. Relaxation of smooth muscle cells is mediated by cGMP signaling and components of this pathway are found within the male reproductive tract. Whereas contractile function of caudal parts of the rat epididymal duct can be examined in organ bath studies, caput and corpus regions are fragile and make it difficult to mount them in an organ bath. We developed an ex vivo time-lapse imaging-based approach to investigate the contractile pattern in these parts of the epididymal duct. Collagen-embedding allowed immobilization without impeding contractility or diffusion of drugs towards the duct and therefore facilitated subsequent movie analyses. The contractile pattern was made visible by placing virtual sections through the acquired image stack to track wall movements over time. By this, simultaneous evaluation of contractile activity at different positions of the observed duct segment was possible. With each contraction translating into a spike, drug-induced alterations in contraction frequency could be assessed easily. Peristaltic contractions were also detectable and throughout all regions in the proximal epididymis we found regular spontaneous contractile activity that elicited movement of intraluminal contents. Stimulating cGMP production by natriuretic peptide ANP or inhibiting degradation of cGMP by the phosphodiesterase 5 inhibitor sildenafil significantly reduced contractile frequency in isolated duct segments from caput and corpus. RT-PCR analysis after laser-capture microdissection localized the corresponding molecules to the smooth muscle layer of the duct. Our time-lapse imaging approach proved to be feasible to assess contractile function in all regions of the epididymal duct under near physiological conditions and provides a tool to evaluate acute (side) effects of drugs and to investigate various signaling pathways

Barrieren der Leitlinienumsetzung und Fortbildungsbedarf von Hausärzten zur Herzinsuffizienz: eine qualitative Studie [Barriers to guideline implementation and educational needs of general practitioners regarding heart failure: a qualitative study]

[english] Objectives: A clinical practice guideline (CPG) contains specifically developed recommendations that can serve physicians as a decision aid in evidence-based practice. The implementation of heart failure (HF) CPGs represents a challenge in general practice. As part of the development of a tailored curriculum, aim of this study was to identify barriers of guideline adherence and needs for medical education (CME) in HF care.Methods: We conducted a modified focus group with elements of a workshop of three hours duration. Thirteen GPs collected and discussed together and parallel in smaller groups barriers of guideline implementation. Afterwards they performed a needs assessment for a tailored CME curriculum for chronic HF. The content of the discussions was analysed qualitatively according to Mayring and categorised thematically.Results: Barriers of guideline adherence were found in the following areas: doctor: procedural knowledge (knowledge gaps), communicative and organisational skills (e.g. time management) and attitude (dissatisfaction with time-money-relation). Patients: individual case-related problems (multimorbidity, psychiatric comorbidity, expectations and beliefs). Doctor and patient: Adherence and barriers of communication. Main measures for improvement of care concerned the areas of the identified barriers of guideline adherence with the focus on application-oriented training of the abovementioned procedural knowledge and skills, but also the supply of tools (like patient information leaflets) and patient education. Conclusion: For a CME-curriculum for HF tailored to the needs of GPs, a comprehensive educational approach seems necessary. It should be broad-based and include elements of knowledge and skills to be addressed and trained case-related. Additional elements should include support in the implementation of organisational processes in the practice and patient education.<br>[german] Zielsetzung: Leitlinien zur Herzinsuffizienz (HI) enthalten systematisch entwickelte Empfehlungen, deren Umsetzung speziell in der hausärztlichen Praxis eine bekannte Herausforderung darstellt. Ziel der vorliegenden Studie war es, Barrieren bei der Umsetzung der Leitlinienempfehlungen zu identifizieren, Vorschläge zu Verbesserungsmaßnahmen der hausärztlichen Versorgung und für die Entwicklung einer bedarfsgerechten Fortbildung zu gewinnen.Methodik: Es wurde eine modifizierte Fokusgruppe mit Workshopcharakter durchgeführt. In drei parallelen Kleingruppen erarbeiteten 13 Hausärzte Barrieren bei der Leitlinienumsetzung zur HI. Darauf aufbauend wurde eine Bedarfsanalyse bzgl. der Lernziele und der spezifischen Verbesserungsmaßnahmen für eine hausärztliche Fortbildung zur HI durchgeführt. Die protokollierten Aussagen der Ärzte wurden mittels Inhaltsanalyse nach Mayring ausgewertet und anschließend thematisch kategorisiert.Ergebnisse: Als Barrieren der Umsetzung vorhandener Leitlinien nannten die teilnehmenden Ärzte arztseitige Defizite und Unterstützungsbedarf in den Dimensionen Wissen, kommunikativen und organisatorischen Fähigkeiten (z. B. Zeitmangel/-management) und der Haltung gegenüber Leitlinien (z.B. Faulheit). Den Patienten wurden individuelle, patientenbezogene Probleme (z.B. Komplexität des Einzelfalles bei Multimorbidität, psychische Komorbidität) sowie ablehnende Einstellungen (z.B. gegenüber „Schulmedizin“) zugeschrieben. Im Bereich der Arzt-Patient-Interaktion wurden u.a. mangelnde medikamentöse Adhärenz und Kommunikationsprobleme als Barrieren genannt. Vorschläge zu Verbesserungs- bzw. Schulungsmaßnahmen umfassten breit angelegte Schulungskonzepte (z.B. in interdisziplinären Qualitätszirkeln mit Fall-Audit), die neben der Vermittlung von Wissensaspekten aus einer (idealerweise einheitlichen) Leitlinie auch praktische Fertigkeiten hinsichtlich Arzt-Patienten-Kommunikation und Praxis-Organisation einschlossen. Die Bereitstellung praktikabler Arbeitsmaterialien und Patientenschulungen sollten aus Sicht der Teilnehmer die Arztschulungen ergänzen.Schlussfolgerung: Ein Fortbildungs-Curriculum für Hausärzte zur HI scheint einer umfassenden Leitlinienschulung zu bedürfen, in welchen insbesondere Handlungskompetenz und kommunikative Fertigkeiten anwendungsorientiert geübt werden sollten. Weitere Bestandteile sollten Hilfestellungen zur Implementierung von Organisationsabläufen und Patientenschulungen sein

Visualization of transport of intraluminal contents.

<p>Snapshot of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092603#pone.0092603.s004" target="_blank">Movie S4</a> resulting from time-lapse imaging (1 frame/s) of a piece of the epididymal duct in the caput region (scale bar: 100 μm) showing net transport of intraluminal contents. In the cross section of the time stack, this net transport is indirectly visible when observing the pattern between the epididymis walls. The darker areas in the cross section do not remain constant over time indicating that intraluminal contents have moved out of the cross section.</p

Visualization of sildenafil effects on spontaneous contractile activity.

<p>Effects of sildenafil and the NO donor SNP on spontaneous contractility of caput segments of the epididymal duct (A–C). A: Visualization of contractility derived from virtual sections through the corresponding time stacks (scale bar: 100 μm) in examples of SNP and subsequent sildenafil treatment of a duct segment from the caput region. Enlarging the regions that surround the time of drug addition, indicated by colored frames, illustrate transient effects of the substances. B: Statistical analyses compared the contractile frequency during 2 minutes preceding and following the addition of the substances. A non-parametric one-way ANOVA for repeated measurements (Friedman's test for paired samples) was used followed by Dunn's test for multiple comparisons. Adjusted p-values for each comparison are given in the graphs with “*” indicating p<0.05 and “**” indicating p<0.01. Statistical analyses of SNP and sildenafil treatments show that sildenafil significantly reduced contractile activity whether given alone or after SNP. In contrast, SNP effects remained non significant. “Spont” indicates spontaneous contractile frequency. C: Visualization of sildenafil and SNP effects in another duct segment originating from caput (scale bar: 100 μm). In this example sildenafil results in a complete loss of contractility. When the NO donor SNP was added in this situation, it was without additional effect as expected. The addition of noradrenaline at the end of the experiment lead to a resumption of contractile activity indicating that the duct segment was still viable. Movies were captured at 1 frame/s.</p

Simultaneous demonstration of contractile activity in different regions of one epididymal duct segment.

<p>Left side: Snapshot of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092603#pone.0092603.s001" target="_blank">Movie S1</a> resulting from time-lapse digital photography of a piece of the epididymal duct in the caput region (scale bar: 100 μm). The different positions at which this time stack was virtually dissected are indicated by colored bars (A: green, B: yellow, C: red, D: black). Right side: Virtual sections through the time stack at indicated positions. Each contraction elicits a small movement of the epididymal duct with changes of its diameter resulting in a series of spikes (marked by vertical arrows). A–C: Virtual sections at different positions (1 frame/s). With the contractions spreading over the observed duct segment, the detection of contractions at different places yields equivalent results. D: Demonstration of contraction-derived pattern of spikes resulting from a movie captured at an accelerated rate of 7 frames/s providing more details. Contractile frequency in A–D is 7.5 beats/min.</p

Spontaneous contractions in caput and corpus epididymidis.

<p>Visualization of contractility derived from virtual sections through the corresponding time stacks (as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092603#pone-0092603-g001" target="_blank">Fig. 1</a>) in examples from duct segments originating from caput (A) and corpus (B) epididymidis of different individuals (samples 1, 2 and 3). All movies were captured at 1 frame/s. Regular spontaneous contractility is visible in all samples of the caput and corpus region.</p

Demonstration of GC-A, GC-B and sGC expression in smooth muscle cells of the rat epididymal duct by LCM+RT-PCR.

<p>Laser-assisted microdissection (A,B) combined with RT-PCR (C) was used to localize GC-A, GC-B and sGC mRNA within the epididymis. A,B: Examples of excised segments of the epididymal smooth muscle layer. Scale bar corresponds to 150 μm. C: Microdissected epididymal smooth muscle cells (M) were analyzed by RT-PCR. Preparation from whole epididymis tissue (ET) served as positive control. The ribosomal protein RPS18 served as loading control. “+”, “−” and “0” indicate lanes with reverse transcriptase, without reverse transcriptase and water control, respectively. GC-A (172 bp), GC-B (234 bp) and sGC (261 bp) transcripts could be detected in microdissected smooth muscle layer. The quality of microdissection was assessed using additional primers for SMA (148 bp) as marker for smooth muscle cells and TRPV6 (259 bp) as marker for epithelial cells, respectively, to exclude the contamination of dissected parts of the smooth muscle layer with epithelial cells.</p