Akute Abstoßung in der Tunica muscularis bei der allogenen Dünndarmtransplantation im Rattenmodell

Das Patienten- und Organüberleben bei der klinischen Dünndarmtransplantation wird deutlich durch das Auftreten einer akuten Abstoßungsreaktion beeinflusst. Dies liegt im Wesentlichen in den, in hohem Ausmass in den Gewebskompartimenten des Dünndarmes vorhandenen, immunologisch aktiven Zellen begründet. In der vorliegenden Arbeit wurde speziell die Rolle der Tunica muscularis und der dort ablaufenden Inflammationsvorgänge während der akuten Abstoßung nach Dünndarmtransplantation untersucht. Syngene Dünndarmtransplantation (LEW -> LEW Ratten) verursacht durch den Ischämie/Reperfusionsschaden und die Darmmanipulation eine lokale Entzündungsreaktion in der Tunica muscularis welche mit Dyskontraktilität der Muskelstrips in vitro verbunden ist. Diese unspezifische Entzündungsreaktion ist reversibel. Allogene Dünndarmtransplantation in einem high-responder Modell (BN -> LEW) führt zunächst zu ähnlichen inflammatorischen Vorgängen in der Tunica muscularis. Mit Einsetzen der akuten Abstoßung jedoch kommt es zu einer immunologisch bedingten weiteren Schädigung der Tunica muscularis des Transplantats, die sich unter anderem durch eine massive inflammatorische Reaktion, spezifische Zellinfiltrate und eine noch stärkere Dyskontraktilität auszeichnet und im Verlauf (168h) zum Transplantatversagen führt. Während der unspezifischen Entzündungsphase fand sich in der Tunica muscularis eine starke Infiltration mit Neutrophilen, Monozyten und Makrophagen. Die Expressionslevel von IL6, ICAM1, MCP1, iNOS und TNF α wurden als erhöht in der SYBR Green RT PCR gemessen. Es zeigte sich eine Dyskontraktilität der Muskelstrips in vitro. Die Histologie zeigte unspezifische entzündliche Veränderungen. Bei der akuten Abstoßung, die ab 96h post transplantationem nachweisbar war, zeigte sich ein spezifisches Infiltrat aus lymphozytären Zellen in der Tunica muscularis (zumeist CD8+) sowie phagozytären Zellen. Die genannten Mediatoren zeigten ein zweites, absolutes Induktionsmaximum in der PCR. Zusätzlich wurden die abstoßungsassoziierten Mediatoren IFNγ, IL10, IL2, TNF α und FasL als erhöht detektiert. Die Dyskontraktilität (verursacht durch iNOS vermittelte NO Synthese und zelldestruktive Vorgänge in der Tunica muscularis) war stärker ausgeprägt als bei der syngenen Gruppe und verschlechterte sich im Verlauf der Abstoßung. Der selektive iNOS Antagonist L-NIL konnte die Dyskontraktilität in vitro um den Faktor 5 bessern, jedoch nicht auf Niveau der syngenen Grafts. Mittels TUNEL Test wurden erhöhte Apoptoseraten in der Tunica muscularis der allogenen Grafts festgestellt, gleichzeitig mit Induktionsmaxima für TNFα und Fas Ligand als apoptoseinduzierende Mediatoren. Die Histologie ergab Zeichen der milden (96h) bis moderaten (168h) akuten Abstoßung. Wir schlussfolgern, dass die sich während des Transplantationsvorganges manifestierende Entzündungsreaktion in der Tunica muscularis, möglicherweise schon zeitlich vor der Tunica mucosa, über die lokale Expression von chemotaktischen Substanzen und Adhäsionsmolekülen, zu einer schnellen Rekrutierung alloreaktiver Leukozyten in das Transplantat führt. Abstoßungsmechanismen werden hierdurch verstärkt, bzw. möglicherweise erst in Gang gesetzt. Die unspezifische posttraumatische Entzündung und die spezifische Abstoßungsreaktion bewirken dann kofaktoriell und wechselseitig den Funktionsverlust bis zum Untergang des Transplantates. Dementsprechend sollten die Vorgänge in der Tunica muscularis sowohl in der Diagnostik als auch in der Therapie, zur Prävention und Behandlung der akuten Abstoßung nach intestinaler Transplantation mitberücksichtigt werden. Aus dem Gesagten folgern wir außerdem, dass durch eine gezielte und frühzeitige Blockade der Entzündungskaskade, möglicherweise schon während der Transplantation, das Outcome der Dünndarmtransplantation verbessert werden kann

Dual endothelin-converting enzyme/neutral endopeptidase blockade in rats with D-galactosamine-induced liver failure

Secondary activation of the endothelin system is thought to be involved in toxic liver injury. This study tested the hypothesis that dual endothelin-converting enzyme / neutral endopeptidase blockade might be able to attenuate acute toxic liver injury

Effects of DPP-4 Inhibitors on the Heart in a Rat Model of Uremic Cardiomyopathy

BACKGROUND: Uremic cardiomyopathy contributes substantially to mortality in chronic kidney disease (CKD) patients. Glucagon-like peptide-1 (GLP-1) may improve cardiac function, but is mainly degraded by dipeptidyl peptidase-4 (DPP-4). METHODOLOGY/PRINCIPAL FINDINGS: In a rat model of chronic renal failure, 5/6-nephrectomized [5/6N] rats were treated orally with DPP-4 inhibitors (linagliptin, sitagliptin, alogliptin) or placebo once daily for 4 days from 8 weeks after surgery, to identify the most appropriate treatment for cardiac dysfunction associated with CKD. Linagliptin showed no significant change in blood level AUC(0-∞) in 5/6N rats, but sitagliptin and alogliptin had significantly higher AUC(0-∞) values; 41% and 28% (p = 0.0001 and p = 0.0324), respectively. No correlation of markers of renal tubular and glomerular function with AUC was observed for linagliptin, which required no dose adjustment in uremic rats. Linagliptin 7 µmol/kg caused a 2-fold increase in GLP-1 (AUC 201.0 ng/l*h) in 5/6N rats compared with sham-treated rats (AUC 108.6 ng/l*h) (p = 0.01). The mRNA levels of heart tissue fibrosis markers were all significantly increased in 5/6N vs control rats and reduced/normalized by linagliptin. CONCLUSIONS/SIGNIFICANCE: DPP-4 inhibition increases plasma GLP-1 levels, particularly in uremia, and reduces expression of cardiac mRNA levels of matrix proteins and B-type natriuretic peptides (BNP). Linagliptin may offer a unique approach for treating uremic cardiomyopathy in CKD patients, with no need for dose-adjustment

Effects of High Salt-Exposure on the Development of Retina and Lens in 5.5-Day Chick Embryo

Background/Aims: Excess maternal salt intake during pregnancy may alter fetal development. However, our knowledge on how an increased salt intake during pregnancy influences fetal eye development is limited. In this study, we investigated the effects of high-salt treatment on the developing eyes in chick embryos, especially focusing on the development of the retina and the lens. Methods: 5.5-day chick embryos were exposed to 280mosm/l (n=17), or 300mosm/l (n=16) NaCl. The treated embryos were then incubated for 96 hours before they were fixed with 4% paraformaldehyde for H&E staining, whole-mount embryo immunostaining and TUNEL staining. BrdU and PH3 incorporation experiments were performed on the chick embryos after high-salt treatment. RT-PCR analyses were conducted from chick retina tissues. Results: We demonstrated that high-salt treatment altered the size of eyes in chick embryos, induced malformation of the eyes and impaired the development of the lens and the retina. We found an impaired expression of Paired box 6 (PAX6) and neuronal cells in the developing retina as revealed by neurofilament immunofluorescent staining. There was a reduction in the number of BrdU-positive cells and PH3-positive cells in the retina, indicating an impaired cell proliferation with high-salt treatment. High-salt treatment also resulted in an increased number of TUNEL-positive cells in the retina, indicating a higher amount of cell death. RT-PCR data displayed that the expression of the pro-apoptotic molecule nerve growth factor (NGF) in chick retina was increased and CyclinD1 was reduced with high-salt treatment. The size of the lens was reduced and Pax6 expression in the lens was significantly inhibited. High salt-treatment was detrimental to the migration of neural crest cells. Conclusion: Taken together, our study demonstrated that high-salt exposure of 5.5-day chick embryos led to an impairment of retina and lens development, possibly through interfering with Pax6 expression

Chronic kidney disease induces a systemic microangiopathy, tissue hypoxia and dysfunctional angiogenesis

Chronic kidney disease (CKD) is associated with excessive mortality from cardiovascular disease (CVD). Endothelial dysfunction, an early manifestation of CVD, is consistently observed in CKD patients and might be linked to structural defects of the microcirculation including microvascular rarefaction. However, patterns of microvascular rarefaction in CKD and their relation to functional deficits in perfusion and oxygen delivery are currently unknown. In this in-vivo microscopy study of the cremaster muscle microcirculation in BALB/c mice with moderate to severe uremia, we show in two experimental models (adenine feeding or subtotal nephrectomy), that serum urea levels associate incrementally with a distinct microangiopathy. Structural changes were characterized by a heterogeneous pattern of focal microvascular rarefaction with loss of coherent microvascular networks resulting in large avascular areas. Corresponding microvascular dysfunction was evident by significantly diminished blood flow velocity, vascular tone, and oxygen uptake. Microvascular rarefaction in the cremaster muscle paralleled rarefaction in the myocardium, which was accompanied by a decrease in transcription levels not only of the transcriptional regulator HIF-1 alpha, but also of its target genes Angpt-2, TIE-1 and TIE-2, Flkt-1 and MMP-9, indicating an impaired hypoxia-driven angiogenesis. Thus, experimental uremia in mice associates with systemic microvascular disease with rarefaction, tissue hypoxia and dysfunctional angiogenesis

Access to a Simulator is Not Enough: The Benefits of Virtual Reality Training Based on Peer-Group-Derived Benchmarks—A Randomized Controlled Trial

Background: Virtual reality (VR) simulators are widely used to familiarize surgical novices with laparoscopy, but VR training methods differ in efficacy. In the present trial, self-controlled basic VR training (SC-training) was tested against training based on peer-group-derived benchmarks (PGD-training). Methods: First, novice laparoscopic residents were randomized into a SC group (n=34), and a group using PGD-benchmarks (n=34) for basic laparoscopic training. After completing basic training, both groups performed 60 VR laparoscopic cholecystectomies for performance analysis. Primary endpoints were simulator metrics; secondary endpoints were program adherence, trainee motivation, and training efficacy. Results: Altogether, 66 residents completed basic training, and 3,837 of 3,960 (96.8%) cholecystectomies were available for analysis. Course adherence was good, with only two dropouts, both in the SC-group. The PGD-group spent more time and repetitions in basic training until the benchmarks were reached and subsequently showed better performance in the readout cholecystectomies: Median time (gallbladder extraction) showed significant differences of 520s (IQR 354-738s) in SC-training versus 390s (IQR 278-536s) in the PGD-group (p<0.001) and 215s (IQR 175-276s) in experts, respectively. Path length of the right instrument also showed significant differences, again with the PGD-training group being more efficient. Conclusions: Basic VR laparoscopic training based on PGD benchmarks with external assessment is superior to SC training, resulting in higher trainee motivation and better performance in simulated laparoscopic cholecystectomies. We recommend such a basic course based on PGD benchmarks before advancing to more elaborate VR trainin

Renal Effects of the Novel Selective Adenosine A1 Receptor Blocker SLV329 in Experimental Liver Cirrhosis in Rats

Liver cirrhosis is often complicated by an impaired renal excretion of water and sodium. Diuretics tend to further deteriorate renal function. It is unknown whether chronic selective adenosine A1 receptor blockade, via inhibition of the hepatorenal reflex and the tubuloglomerular feedback, might exert diuretic and natriuretic effects without a reduction of the glomerular filtration rate. In healthy animals intravenous treatment with the novel A1 receptor antagonist SLV329 resulted in a strong dose-dependent diuretic (up to 3.4-fold) and natriuretic (up to 13.5-fold) effect without affecting creatinine clearance. Male Wistar rats with thioacetamide-induced liver cirrhosis received SLV329, vehicle or furosemide for 12 weeks. The creatinine clearance of cirrhotic animals decreased significantly (−36.5%, p<0.05), especially in those receiving furosemide (−41.9%, p<0.01). SLV329 was able to prevent this decline of creatinine clearance. Mortality was significantly lower in cirrhotic animals treated with SLV329 in comparison to animals treated with furosemide (17% vs. 54%, p<0.05). SLV329 did not relevantly influence the degree of liver fibrosis, kidney histology or expression of hepatic or renal adenosine receptors. In conclusion, chronic treatment with SLV329 prevented the decrease of creatinine clearance in a rat model of liver cirrhosis. Further studies will have to establish whether adenosine A1 receptor antagonists are clinically beneficial at different stages of liver cirrhosis