Renal Glucose Release after Unilateral Renal Denervation during a Hypoglycemic Clamp in Pigs with an Altered Hypothalamic Pituitary Adrenal Axis after Late-Gestational Dexamethasone Injection.

Previously, we demonstrated in pigs that renal denervation halves glucose release during hypoglycaemia and that a prenatal dexamethasone injection caused increased ACTH and cortisol concentrations as markers of a heightened hypothalamic pituitary adrenal axis (HPAA) during hypoglycaemia. In this study, we investigated the influence of an altered HPAA on renal glucose release during hypoglycaemia. Pigs whose mothers had received two late-gestational dexamethasone injections were subjected to a 75 min hyperinsulinaemic-hypoglycaemic clamp (<3 mmol/L) after unilateral surgical denervation. Para-aminohippurate (PAH) clearance, inulin, sodium excretion and arterio-venous blood glucose difference were measured every fifteen minutes. The statistical analysis was performed with a Wilcoxon signed-rank test. PAH, inulin, the calculated glomerular filtration rate and plasma flow did not change through renal denervation. Urinary sodium excretion increased significantly (p = 0.019). Side-dependent renal net glucose release (SGN) decreased by 25 ± 23% (p = 0.004). At 25 percent, the SGN decrease was only half of that observed in non-HPAA-altered animals in our prior investigation. The current findings may suggest that specimens with an elevated HPAA undergo long-term adaptations to maintain glucose homeostasis. Nonetheless, the decrease in SGN warrants further investigations and potentially caution in performing renal denervation in certain patient groups, such as diabetics at risk of hypoglycaemia.

The old sheep: a convenient and suitable model for senile osteopenia.

INTRODUCTION: Existing osteoporosis models in sheep exhibit some disadvantages, e.g., challenging surgical procedures, serious ethical concerns, failure of reliable induction of substantial bone loss, or lack of comparability to the human condition. This study aimed to compare bone morphological and mechanical properties of old and young sheep, and to evaluate the suitability of the old sheep as a model for senile osteopenia. MATERIALS AND METHODS: The lumbar vertebral body L3 of female merino sheep with two age ranges, i.e., old animals (6-10 years; n = 41) and young animals (2-4 years; n = 40), was analyzed concerning its morphological and mechanical properties by bone densitometry, quantitative histomorphometry, and biomechanical testing of the corticalis and/or central spongious region. RESULTS: In comparison with young sheep, old animals showed only marginally diminished bone mineral density of the vertebral bodies, but significantly decreased structural (bone volume, - 15.1%; ventral cortical thickness, - 11.8%; lateral cortical thickness, - 12.2%) and bone formation parameters (osteoid volume, osteoid surface, osteoid thickness, osteoblast surface, all - 100.0%), as well as significantly increased bone erosion (eroded surface, osteoclast surface). This resulted in numerically decreased biomechanical properties (compressive strength; - 6.4%). CONCLUSION: Old sheep may represent a suitable model of senile osteopenia with markedly diminished bone structure and formation, and substantially augmented bone erosion. The underlying physiological aging concept reduces challenging surgical procedures and ethical concerns and, due to complex alteration of different facets of bone turnover, may be well representative of the human condition.

Altered Cerebral Blood Flow and Potential Neuroprotective Effect of Human Relaxin-2 (Serelaxin) During Hypoxia or Severe Hypovolemia in a Sheep Model.

Specific neuroprotective strategies to minimize cerebral damage caused by severe hypoxia or hypovolemia are lacking. Based on previous studies showing that relaxin-2/serelaxin increases cortical cerebral blood flow, we postulated that serelaxin might provide a neuroprotective effect. Therefore, we tested serelaxin in two emergency models: hypoxia was induced via inhalation of 5% oxygen and 95% nitrogen for 12 min; thereafter, the animals were reoxygenated. Hypovolemia was induced and maintained for 20 min by removal of 50% of the total blood volume; thereafter, the animals were retransfused. In each damage model, the serelaxin group received an intravenous injection of 30 µg/kg of serelaxin in saline, while control animals received saline only. Blood gases, shock index values, heart frequency, blood pressure, and renal blood flow showed almost no significant differences between control and treatment groups in both settings. However, serelaxin significantly blunted the increase of lactate during hypovolemia. Serelaxin treatment resulted in significantly elevated cortical cerebral blood flow (CBF) in both damage models, compared with the respective control groups. Measurements of the neuroproteins S100B and neuron-specific enolase in cerebrospinal fluid revealed a neuroprotective effect of serelaxin treatment in both hypoxic and hypovolemic animals, whereas in control animals, neuroproteins increased during the experiment. Western blotting showed the expression of relaxin receptors and indicated region-specific differences in relaxin receptor-mediated signaling in cortical and subcortical brain arterioles, respectively. Our findings support the hypothesis that serelaxin is a potential neuroprotectant during hypoxia and hypovolemia. Due to its preferential improvement of cortical CBF, serelaxin might reduce cognitive impairments associated with these emergencies.

Assessment of MR imaging during one-lung flooding in a large animal model.

OBJECTIVE: Magnetic resonance imaging (MRI) of the lung remains challenging due to the low tissue density, susceptibility artefacts, unfavourable relaxation times and motion. Previously, we demonstrated in vivo that one-lung flooding (OLF) with saline is a viable and safe approach. This study investigates the feasibility of OLF in an MRI environment and evaluates the flooding process on MR images. METHODS: OLF of the left lung was performed on five animals using a porcine model. Before, during and after OLF, standard T2w and T1w spin-echo (SE) and gradient-echo (GRE) sequences were applied at 3 T. RESULTS: The procedure was successfully performed in all animals. On T1w MRI, the flooded lung appeared homogenous and isointense with muscle tissue. On T2w images, vascular structures were highly hypointense, while the bronchi were clearly demarcated with hypointense wall and hyperintense lumen. The anatomical demarcation of the flooded lung from the surrounding organs was superior on T2w images. No outflow effects were seen, and no respiration triggering was required. DISCUSSION: OLF can be safely performed in an MR scanner with highly detailed visualization of the pulmonary structures on T2w images. The method provides new approaches to MRI-based image-guided pulmonary interventions using the presented experimental model.

Impact of chronic maternal stress during early gestation on maternal-fetal stress transfer and fetal stress sensitivity in sheep.

Acute stress-induced reduction of uterine blood flow (UBF) is an indirect mechanism of maternal-fetal stress transfer during late gestation. Effects of chronic psychosocial maternal stress (CMS) during early gestation, as may be experienced by many working women, on this stress signaling mechanism are unclear. We hypothesized that CMS in sheep during early gestation augments later acute stress-induced decreases of UBF, and aggravates the fetal hormonal, cardiovascular, and metabolic stress responses during later development. Six pregnant ewes underwent repeated isolation stress (CMS) between 30 and 100 days of gestation (dGA, term: 150 dGA) and seven pregnant ewes served as controls. At 110 dGA, ewes were chronically instrumented and underwent acute isolation stress. The acute stress decreased UBF by 19% in both the CMS and control groups (p < .05), but this was prolonged in CMS versus control ewes (74 vs. 30 min, p < .05). CMS increased fetal circulating baseline and stress-induced cortisol and norepinephrine concentrations indicating a hyperactive hypothalamus-pituitary-adrenal (HPA)-axis and sympathetic-adrenal-medullary system. Increased fetal norepinephrine is endogenous as maternal catecholamines do not cross the placenta. Cortisol in the control but not in the CMS fetuses was correlated with maternal cortisol blood concentrations; these findings indicate: (1) no increased maternal-fetal cortisol transfer with CMS, (2) cortisol production in CMS fetuses when the HPA-axis is normally inactive, due to early maturation of the fetal HPA-axis. CMS fetuses were better oxygenated, without shift towards acidosis compared to the controls, potentially reflecting adaptation to repeated stress. Hence, CMS enhances maternal-fetal stress transfer by prolonged reduction in UBF and increased fetal HPA responsiveness.

Redistribution of Cerebral Blood Flow during Severe Hypovolemia and Reperfusion in a Sheep Model: Critical Role of α1-Adrenergic Signaling.

BACKGROUND: Maintenance of brain circulation during shock is sufficient to prevent subcortical injury but the cerebral cortex is not spared. This suggests area-specific regulation of cerebral blood flow (CBF) during hemorrhage. METHODS: Cortical and subcortical CBF were continuously measured during blood loss (≤50%) and subsequent reperfusion using laser Doppler flowmetry. Blood gases, mean arterial blood pressure (MABP), heart rate and renal blood flow were also monitored. Urapidil was used for α1A-adrenergic receptor blockade in dosages, which did not modify the MABP-response to blood loss. Western blot and quantitative reverse transcription polymerase chain reactions were used to determine adrenergic receptor expression in brain arterioles. RESULTS: During hypovolemia subcortical CBF was maintained at 81 ± 6% of baseline, whereas cortical CBF decreased to 40 ± 4% (p < 0.001). Reperfusion led to peak CBFs of about 70% above baseline in both brain regions. α1A-Adrenergic blockade massively reduced subcortical CBF during hemorrhage and reperfusion, and prevented hyperperfusion during reperfusion in the cortex. α1A-mRNA expression was significantly higher in the cortex, whereas α1D-mRNA expression was higher in the subcortex (p < 0.001). CONCLUSIONS: α1-Adrenergic receptors are critical for perfusion redistribution: activity of the α1A-receptor subtype is a prerequisite for redistribution of CBF, whereas the α1D-receptor subtype may determine the magnitude of redistribution responses.

Increase of cortical cerebral blood flow and further cerebral microcirculatory effects of Serelaxin in a sheep model.

Serelaxin, recombinant human relaxin-2, modulates endothelial vasodilatory functionality and is under evaluation for treatment of acute heart failure. Little is known about acute effects on cerebral perfusion. We tested the hypothesis that Serelaxin might also have effects on the cerebral microcirculation in a sheep model, which resembles human brain structure quite well. We used laser Doppler flowmetry and sidestream dark-field (SDF) imaging techniques, which are reliable tools to continuously assess dynamic changes in cerebral perfusion. Laser Doppler flowmetry shows that bolus injection of 30 µg Serelaxin/kg body wt induces an increase (P = 0.006) to roughly 150% of cortical cerebral blood flow (CBF), whereas subcortical CBF remains unchanged (P = 0.688). The effects on area-dependent CBF were significantly different after the bolus injection (P = 0.042). Effects on cortical CBF were further confirmed by SDF imaging. The bolus injection of Serelaxin increased total vessel density to 127% (P = 0.00046), perfused vessel density to 145% (P = 0.024), and perfused capillary density to 153% (P = 0.024). Western blotting confirmed the expression of relaxin receptors RXFP1 and truncated RXFP2-variants in the respective brain regions, suggesting a possible contribution of RXFP1 on the effects of Serelaxin. In conclusion, the injection of a high dose of Serelaxin exerts quick effects on the cerebral microcirculation. Therefore, Serelaxin might be suitable to improve cortical microcirculation and exert neuroprotective effects in clinically relevant scenarios that involve cortical hypoperfusion. These findings need to be confirmed in relevant experimental settings involving cerebral cortical hypoperfusion and can possibly be translated into clinical practice.

Stress-induced decrease of uterine blood flow in sheep is mediated by alpha 1-adrenergic receptors.

Prenatal maternal stress can be transferred to the fetus via a catecholamine-dependent decrease of uterine blood flow (UBF). However, it is unclear which group of adrenergic receptors mediates this mechanism of maternal-fetal stress transfer. We hypothesized that in sheep, alpha 1-adrenergic receptors may play a key role in catecholamine mediated UBF decrease, as these receptors are mainly involved in peripheral vasoconstriction and are present in significant number in the uterine vasculature. After chronic instrumentation at 125 ± 1 days of gestation (dGA; term 150 dGA), nine pregnant sheep were exposed at 130 ± 1 dGA to acute isolation stress for one hour without visual, tactile, or auditory contact with their flockmates. UBF, blood pressure (BP), heart rate (HR), stress hormones, and blood gases were determined before and during this isolation challenge. Twenty-four hours later, experiments were repeated during alpha 1-adrenergic receptor blockage induced by a continuous intravenous infusion of urapidil. In both experiments, ewes reacted to isolation with an increase in serum norepinephrine, cortisol, BP, and HR as typical signs of activation of sympatho-adrenal and the hypothalamic-pituitary-adrenal axis. Stress-induced UBF decrease was prevented by alpha 1-adrenergic receptor blockage. We conclude that UBF decrease induced by maternal stress in sheep is mediated by alpha 1-adrenergic receptors. Future studies investigating prevention strategies of impact of prenatal maternal stress on fetal health should consider selective blockage of alpha 1-receptors to interrupt maternal-fetal stress transfer mediated by utero-placental malperfusion.

Pre-clinical evaluation of a minimally invasive laryngeal pacemaker system in mini-pig.

Microlaryngoscopic enlargement techniques have been the standard treatment for bilateral vocal fold paralysis (BVFP) for decades. Laryngeal pacing is a promising alternative treatment based on the electrostimulation of the posterior cricoarytenoid (PCA) muscle. This paper reports on the results of a pre-clinical study aiming to evaluate this method. Eight Göttingen mini-pigs were implanted with a laryngeal pacemaker (LP) implant prototype and with two LP electrodes, one in each PCA muscle. The 6-week follow-up included endoscopic stimulation controls in general anaesthesia and radiographic controls of electrode integrity and position stability. Stimulation parameters for optimal glottal opening were evaluated via videolaryngoscopy. Histopathology was performed upon conclusion of the study. 7/8 (87.5 %) animals were successfully implanted with the LP implant prototype and two LP electrodes. In general, stimulation was effectively delivered and correlated with the expected PCA muscle activation. 2/14 (14.3 %) electrodes dislocated and 1/14 (7.1 %) electrode tip broke. The LP system used in this experiment to induce vocal fold abduction by means of selective functional electrical stimulation of the PCA showed promising results. It may be a valid alternative to the current golden standard for BVFP treatment. Clinical studies are needed to confirm the medical relevance of the LP.

Transcatheter treatment of tricuspid regurgitation by caval valve implantation--experimental evaluation of decellularized tissue valves in central venous position.

BACKGROUND: Caval valve implantation has been suggested for transcatheter treatment of severe tricuspid regurgitation (TR). Combining the interventional technique with the promising surgical experience with decellularized valves, we sought to evaluate the functional and structural outcome of decellularized pericardial tissue valves (dTVs) in the low-pressure venous circulation in a chronic model of TR. METHODS AND RESULTS: Sixteen pericardial tissue valves were heterotopically implanted in the inferior and superior vena cava in a sheep model (54-98 kg; median 74.5 kg, n = 8) of severe TR. The devices were assembled using self-expanding nitinol stents and bovine pericardia decellularized by a detergent-based protocol (group dTV; n = 8). Glutaraldehyde-fixed pericardial tissue valves served as control (GaTV, n = 8). After 6 months, device function and structural maturation were analyzed using echocardiographic, histologic, immunohistologic, and electron microscopic approaches. After implantation, cardiac output increased significantly from 3.7 ± 1.1 l/min to 4.8 ± 1.1 l/min (P < 0.05) and competent valve function was verified by angiography. At 6 months, angiographic and echocardiographic evaluation revealed moderate to severe regurgitation in all GaTV. In contrast, five of the eight dTVs functioned well with only minor regurgitation. In these animals, autopsy revealed preserved valve structure with tender leaflets without signs of thrombosis or calcification. Conversely, GaTV showed severe degeneration with large calcification areas. Microscopic and histologic analysis confirmed endothelial repopulation in both valve types. However, additional interstitial reseeding was observed in decellularized valves. CONCLUSIONS: In the venous circulation in severe TR, decellularized valves show superior functional performance compared to Ga-fixed tissue valves. Macroscopic and microscopic analyses suggest preserved structural integrity and advanced endothelial and interstitial repopulation with evidence of less degradation in dTV. © 2014 Wiley Periodicals, Inc.

Role of catecholamines in maternal-fetal stress transfer in sheep.

OBJECTIVE: We sought to evaluate whether in addition to cortisol, catecholamines also transfer psychosocial stress indirectly to the fetus by decreasing uterine blood flow (UBF) and increasing fetal anaerobic metabolism and stress hormones. STUDY DESIGN: Seven pregnant sheep chronically instrumented with uterine ultrasound flow probes and catheters at 0.77 gestation underwent 2 hours of psychosocial stress by isolation. We used adrenergic blockade with labetalol to examine whether decreased UBF is catecholamine mediated and to determine to what extent stress transfer from mother to fetus is catecholamine dependent. RESULTS: Stress induced transient increases in maternal cortisol and norepinephrine (NE). Maximum fetal plasma cortisol concentrations were 8.1 ± 2.1% of those in the mother suggesting its maternal origin. In parallel to the maternal NE increase, UBF decreased by maximum 22% for 30 minutes (P < .05). Fetal NE remained elevated for >2 hours accompanied by a prolonged blood pressure increase (P < .05). Fetuses developed a delayed and prolonged shift toward anaerobic metabolism in the presence of an unaltered oxygen supply. Adrenergic blockade prevented the stress-induced UBF decrease and, consequently, the fetal NE and blood pressure increase and the shift toward anaerobic metabolism. CONCLUSION: We conclude that catecholamine-induced decrease of UBF is a mechanism of maternal-fetal stress transfer. It may explain the influence of maternal stress on fetal development and on programming of adverse health outcomes in later life especially during early pregnancy when fetal glucocorticoid receptor expression is limited.

Effects of early- and late-gestational maternal stress and synthetic glucocorticoid on development of the fetal hypothalamus-pituitary-adrenal axis in sheep.

Prenatal maternal stress (PMS) programs dysregulation of the hypothalamus-pituitary-adrenal axis (HPAA) in postnatal life, though time periods vulnerable to PMS, are still unclear. We evaluated in pregnant sheep the effect of PMS during early gestation [30-100 days of gestation (dGA); term is 150 dGA] or late gestation (100-120 dGA) on development of fetal HPAA function. We compared the effects of endogenous cortisol with synthetic glucocorticoid (GC) exposure, as used clinically to enhance fetal lung maturation. Pregnant sheep were exposed to repeated isolation stress twice per week for 3 h in a separate box with no visual, tactile, or auditory contact with their flock-mates either during early (n = 7) or late (n = 7) gestation. Additional groups received two courses of betamethasone (BM; n = 7; 2 × 110 µg kg(- 1) body weight, 24 h apart) during late gestation (106/107 and 112/113 dGA, n = 7) or acted as controls (n = 7). Fetal cortisol responses to hypotensive challenge, a physiological fetal stressor, were measured at 112 and 129 dGA, i.e. before and during maturation of the HPAA. Hypotension was induced by fetal infusion of sodium nitroprusside, a potent vasodilator. At 112 dGA, neither PMS nor BM altered fetal cortisol responses. PMS, during early or late gestation, and BM treatment increased fetal cortisol responses at 129 dGA with the greatest increase achieved in stressed early pregnant sheep. Thus, development of the HPAA is vulnerable to inappropriate levels of GCs during long periods of fetal life, whereas early gestation is most vulnerable to PMS.

Laryngeal pacing in minipigs: in vivo test of a new minimal invasive transcricoidal electrode insertion method for functional electrical stimulation of the PCA.

Functional electrical stimulation (FES) of the posterior cricoarytenoid muscle (PCA) to restore respiratory function of the larynx may become an option for the treatment of bilateral recurrent laryngeal nerve paralysis (RLNP) in the near future. The feasibility of this has been shown in several animal trials and in a human pilot study. The common open surgical inferolateral approach for electrode insertion into the PCA for FES has a risk of damaging the recurrent laryngeal nerve (RLN) and may result in postoperative swelling and scaring of the larynx. Therefore, a minimal invasive electrode insertion technique is needed. A new miniaturized bipolar spiral tip electrode and a new electrical stimulatable insertion needle were tested in a short-term trial for an endoscopically guided and functionally controlled transcricoidal electrode insertion in eight Göttingen minipigs with bilateral normal RLN function. The feasibility of this technique was evaluated and the achieved positions of the electrodes in the PCA were analyzed using intraoperative stimulation threshold data and 3D-CT reconstructions. In seven cases it was possible to place two well-performing electrodes into the PCA. They were positioned one on either side. In one animal no functioning electrode position could be achieved because the PCA was missed. Thresholds of the electrode tips varied between 0.2 and 2.5 mA (mean 0.71 mA). In any case maximal glottal opening could be reached before adductors were co-activated. The majority of electrodes were placed into the central lower part of the PCA with no apparent correlation between threshold and electrode position. Surgical trauma might be further reduced by using endoscopy via a laryngeal mask avoiding the temporary tracheostomy used in this trial. If the implanted electrodes remain stable in long-term tests, we suggest that this method could soon be transferred into human application.

CIRS-LAS - a novel approach to increase transparency in laboratory animal science for improving animal welfare by reducing laboratory animal distress.

The 3Rs principle is highly topical in animal-based research. These include, above all, new scientific methods for conducting experiments without an animal model, by using non-animal models (Replace), reducing the number of laboratory animals (Reduction) or taking measures to keep the stress on the laboratory animal as low as possible (Refinement). Despite numerous modern alternative approaches, the complete replacement of animal experiments is not yet possible. The exchange in the team about the daily work with laboratory animals, about open questions and problems, contributes to a reflection of one's own work and to a better understanding of the work of the others. CIRS-LAS (Critical Incident Reporting System in Laboratory Animal Science) represents a reporting system for incidents in laboratory animal science. It is urgently needed because the lack of transparency about incidents leads to the repetition of failed experiments. Negative experiences from animal-based experiments are often not mentioned in publications, and the fear of hostility is still very high. Therefore, a constructive approach to errors is not a matter of course. To overcome this barrier, CIRS-LAS was created as a web-based database. It addresses the areas of reduction and refinement of the 3Rs principle by providing a platform to collect and analyze incidents. CIRS-LAS is open to all individuals working with laboratory animals worldwide and currently exists with 303 registered members, 52 reports, and an average of 71 visitors per month. The development of CIRS-LAS shows, that an open and constructive error culture is difficult to establish. Nevertheless, the upload of a case report or the search in the database leads to an active reflection of critical occurrences. Thus, it is an important step towards more transparency in laboratory animal science. As expected, the collected events in the database concern different categories and animal species and are primarily reported by persons involved in an experiment. However, reliable conclusions about observed effects require further analysis and continuous collection of case reports. Looking at the development of CIRS-LAS, its high potential is shown in considering the 3Rs principle in daily scientific work.

Femtosecond laser treatment of the crystalline lens: a 1-year study of possible cataractogenesis in minipigs.

BACKGROUND: To investigate the long-term stability and possible cataractogenesis after femtosecond laser treatment of the crystalline lens. METHODS: The crystalline lenses of ten Göttingen minipigs® underwent femtosecond laser treatment. During a subsequent 1-year follow-up, the pigs were monitored by means of slit-lamp examination of the anterior segment and Scheimpflug imaging of the lens. RESULTS: No laser-induced cataractogenesis was observed during the 1-year follow-up. The laser pattern showed a stable appearance under all examination devices. CONCLUSION: Femtosecond laser treatment seems to be no trigger for cataract formation. Moreover, the long-term stability of the laser pattern makes it suitable for applications such as presbyopia treatment.

Percutaneous caval stent valve implantation: investigation of an interventional approach for treatment of tricuspid regurgitation.

AIMS: Severe tricuspid regurgitation (TR) reduces cardiac output (CO) and increases central venous pressure leading to secondary organ dysfunction. To date, the open surgical approach is the only option to treat TR. Herein, we report our experience of treatment by percutaneous implantation of valved stents in the inferior vena cava (IVC) and superior vena cava (SVC) to substitute tricuspid valve function in a model of acute insufficiency. METHODS AND RESULTS: Acute TR grades III-IV was created in 13 sheep (54-75 kg) via papillary muscle and chordae avulsion using a 0.07 inch wire blade. Successful creation of TR was confirmed using angiography and by a prominent ventricular wave in central venous pressure recording. Two self-expanding nitinol stents containing a porcine pulmonary valve were then implanted in the IVC and SVC in a transcatheter approach. Implantation was performed through the right jugular vein by means of a 21 F catheter and guided by fluoroscopy. Haemodynamics were continuously monitored and valve function was verified by angiography and epicardial echocardiography. After successful implantation and proof of concept in the acute study (acute group, n = 9), chronic studies were (n = 4, 4 weeks follow-up) performed. Tricuspid regurgitation grades III-IV was successfully created in all animals and resulted in a significant reduction of CO. A ventricular wave in the IVC of 16.2 +/- 2.33 mmHg (acute group) and 14.9 +/- 1.71 mmHg (chronic group) confirmed the presence of severe TR. After deployment of the IVC and the SVC valve, the ventricular wave in the IVC significantly decreased to 13.9 +/- 2.97 mmHg (acute group) and 12.7 +/- 1.15 (chronic group), whereas CO significantly increased to 4.20 +/- 0.84 L/min (acute group) and 5.4 +/- 0.67 L/min (chronic group). At autopsy, correct device position was verified in all successfully implanted animals, no macroscopic damage resulting from the implantation procedure was observed. CONCLUSION: In high-grade tricuspid insufficiency, percutaneous implantation of valved stents in the central venous position reduces venous regurgitation and improves haemodynamics in the animal experiment. Implantation of one or two valves in central venous position is technically feasible. Functional replacement of the insufficient tricuspid valve leads to an increase in CO. This technique expands the potential therapeutic options for patients with relevant tricuspid valve regurgitation having a high risk for open heart surgery.

Systematic Postoperative Assessment of a Minimally-Invasive Sheep Model for the Treatment of Osteochondral Defects.

To assess the clinical course of a sheep stifle joint model for osteochondral (OC) defects, medial femoral condyles (MFC) were exposed without patella luxation using medial parapatellar skin (3-4 cm) and deep incisions (2-3 cm). Two defects (7 mm diameter; 10 mm depth; OC punch) were left empty or refilled with osteochondral autologous transplantation cylinders (OATS) and explanted after six weeks. Incision-to-suture time, anesthesia time, and postoperative wound or impairment scores were compared to those in sham-operated animals. Implant performance was assessed by X-ray, micro-computed tomography, histology, and immunohistology (collagens 1, 2; aggrecan). There were no surgery-related infections or patellar luxations. Operation, anesthesia, and time to complete stand were short (0.5, 1.4, and 1.5 h, respectively). The wound trauma score was low (0.4 of maximally 4; day 7). Empty-defect and OATS animals reached an impairment score of 0 significantly later than sham animals (7.4 and 4.0 days, respectively, versus 1.5 days). Empty defects showed incomplete healing and dedifferentiation/heterotopic differentiation; OATS-filled defects displayed advanced bone healing with remaining cartilage gaps and orthotopic expression of bone and cartilage markers. Minimally-invasive, medial parapatellar surgery of OC defects on the sheep MFC allows rapid and low-trauma recovery and appears well-suited for implant testing.

Clinical application of dendritic cells and interleukin-2 and tools to study activated T cells in horses--first results and implications for quality control.

Dendritic cells (DCs) are antigen-presenting cells, which are well known for their capacity to stimulate immunity. The ex vivo generation of myeloid DC from monocytes has facilitated the development of DC-vaccination protocols which have been extensively evaluated in tumour immunology and are regarded by some as a gold mine for clinical research. However, there is a considerable amount of work required to overcome the potential risks associated with such therapy. It is therefore mandatory to characterize the system to be applied and to study the reactions, particularly at the level of T cell responses. The first objective of the current study was to test if tumour lysates loaded autologous DC or recombinant human IL-2 are well tolerated in horses and performed an exploratory phase I study on equine sarcoids and squamous cell carcinomas. We consequently intended to establish a robust protocol for the magnetic separation of monocytes such as in use in human clinical studies. Finally we intended to address the limits in the reagents to study equine T cell based immune reactions, and analysed markers for CD25 and FoxP3. The data showed that local application of DC or IL-2 did not cause side effects. Additionally our data show that a polyclonal approach to detect antigens such as CD25 might be successful, where mAbs are not available. Our data also demonstrate that the mAb FJK16s, which has been used successfully in rodents, humans, and dogs, can also be applied in horses. We finally wish to share our concerns regarding quality control for clinical studies and encourage multi-central studies such as in human medicine to ensure that progress along established standards is made for the benefit of veterinary medicine.

Biomimetic multilayer coatings deliver gentamicin and reduce implant-related osteomyelitis in rats.

Implant-related infections like periprosthetic joint infections (PJI) are still a challenging issue in orthopedic surgery. In this study, we present a prophylactic anti-infective approach based on a local delivery of the antibiotic gentamicin. The local delivery is achieved via a nanoscale polyelectrolyte multilayer (PEM) coating that leaves the bulk material properties of the implant unaffected while tuning the surface properties. The main components of the coating, i.e. polypeptides and sulfated glycosaminoglycans (sGAG) render this coating both biomimetic (matrix mimetic) and biodegradable. We show how adaptions in the conditions of the multilayer assembly process and the antibiotic loading process affect the amount of delivered gentamicin. The highest concentration of gentamicin could be loaded into films composed of polypeptide poly-glutamic acid when the pH of the loading solution was acidic. The concentration of gentamicin on the surface could be tailored with the number of deposited PEM layers. The resulting coatings reveal a bacteriotoxic effect on Staphylococcus cells but show no signs of cytotoxic effects on MC3T3-E1 osteoblasts. Moreover, when multilayer-coated titanium rods were implanted into contaminated medullae of rat tibiae, a reduction in the development of implant-related osteomyelitis was observed. This reduction was more pronounced for the multifunctional, matrix-mimetic heparin-based coatings that only deliver lower amounts of gentamicin.

Comparison of Near-Infrared Spectroscopy with Needle Indentation and Histology for the Determination of Cartilage Thickness in the Large Animal Model Sheep.

The suitability of near-infrared spectroscopy (NIRS) for non-destructive measurement of cartilage thickness was compared with the gold standard needle indentation. A combination of NIRS and biomechanical indentation (NIRS-B) was used to address the influence of varying loads routinely applied for hand-guided NIRS during real-life surgery on the accuracy of NIRS-based thickness prediction. NIRS-B was performed under three different loading conditions in 40 osteochondral cylinders from the load-bearing area of the medial and lateral femur condyle of 20 cadaver joints (left stifle joints; female Merino sheep; 6.1 ± 0.6 years, mean ± standard error of the mean). The cartilage thickness measured by needle indentation within the region analyzed by NIRS-B was then compared with cartilage thickness prediction based on NIRS spectral data using partial least squares regression. NIRS-B repeat measurements yielded highly reproducible values concerning force and absorbance. Separate or combined models for the three loading conditions (the latter simulating load-independent measurements) resulted in models with optimized quality parameters (e.g., coefficients of determination R2 between 92.3 and 94.7) and a prediction accuracy of < 0.1 mm. NIRS appears well suited to determine cartilage thickness (possibly in a hand-guided, load-independent fashion), as shown by high reproducibility in repeat measurements and excellent reliability compared with tissue-destructive needle indentation. This may provide the basis for non-destructive, intra-operative assessment of cartilage status quo and fine-tuning of repair procedures.