Virtual Reality in Biomedical Education in the sense of the 3Rs.

Article 23(2) of EU Directive 2010/63 on the protection of animals used for scientific purposes requires staff involved in the care and use of animals to be adequately educated and trained before carrying out procedures. Therefore, the 3Rs (refinement, reduction, and replacement) and knowledge of alternative methods should be part of the education and training itself. For this purpose, the digital learning concept "Virtual Reality (VR) in Biomedical Education" evolved, which successfully combines VR components with classical learning content. Procedures, such as anesthesia induction, substance application, and blood sampling in rats, as well as aspects of the laboratory environment were recorded in 360° videos. The generated VR teaching/learning modules (VR modules) were used to better prepare participants for hands-on training (refinement) or as a complete replacement for a live demonstration; thus, reducing the number of animals used for hands-on skills training (reduction). The current study evaluated users' experience of the VR modules. Despite little previous VR experience, participants strongly appreciated the VR modules and indicated that they believed VR has the potential to enhance delivery of procedures and demonstrations. Interestingly, participants with previous experience of laboratory animal science were more convinced about VR's potential to support the 3Rs principle, and endorsed its use for further educational purposes. In conclusion, VR appeared to be highly accepted as a learning/teaching method, indicating its great potential to further replace and reduce the use of animals in experimental animal courses.

Characterization of blunt chest trauma in a long-term porcine model of severe multiple trauma.

Chest trauma has a significant relevance on outcome after severe trauma. Clinically, impaired lung function typically occurs within 72 hours after trauma. However, the underlying pathophysiological mechanisms are still not fully elucidated. Therefore, we aimed to establish an experimental long-term model to investigate physiological, morphologic and inflammatory changes, after severe trauma. Male pigs (sus scrofa) sustained severe trauma (including unilateral chest trauma, femur fracture, liver laceration and hemorrhagic shock). Additionally, non-injured animals served as sham controls. Chest trauma resulted in severe lung damage on both CT and histological analyses. Furthermore, severe inflammation with a systemic increase of IL-6 (p = 0.0305) and a local increase of IL-8 in BAL (p = 0.0009) was observed. The pO2/FiO2 ratio in trauma animals decreased over the observation period (p < 0.0001) but not in the sham group (p = 0.2967). Electrical Impedance Tomography (EIT) revealed differences between the traumatized and healthy lung (p < 0.0001). In conclusion, a clinically relevant, long-term model of blunt chest trauma with concomitant injuries has been developed. This reproducible model allows to examine local and systemic consequences of trauma and is valid for investigation of potential diagnostic or therapeutic options. In this context, EIT might represent a radiation-free method for bedside diagnostics.

Recommendation for severity assessment following liver resection and liver transplantation in rats: Part I.

Score sheets were first introduced 30 years ago to assess pain, distress and suffering in animals. To date, however, there is still no general agreement on their use in research practice, and only a few publications can be found on this topic. In the present work, we demonstrate the use of a special score sheet for severity assessment in the first three postoperative days in two showcased studies performed on Wistar and Lewis rats undergoing liver resection or orthotopic liver transplantation, respectively. Scoring of different criteria and the total score were evaluated within each intervention. Additionally, both procedures were compared regarding their degree of severity. Suitability of these score sheets was evaluated for assessing severity of the procedures and these showed a minor severity within each investigated study. A comparison of both studies showed slightly higher scores involving liver transplantation. In contradiction to the common classification of these procedures as a moderate severity grade the score sheets applied here indicates a minor severity grade within each investigated study. Also, limitations and possible improvements in the design of our score sheets for defined interventions are reconsidered.

Severity assessment in rabbits after partial hepatectomy: Part II.

Although the recognition of pain, distress and discomfort has already been described in 1985 by Morton and Griffiths there is still very little known about the establishment of score sheets especially, regarding post-surgical pain and severity assessment for laboratory animals such as rabbits. In this paper we describe the estimation of severity and recovery status of 36 female New Zealand White rabbits (NZW) in a standardized liver resection model using two different adhesive treatments and one control group. Welfare was assessed at 3-4 consecutive days after surgery using a scoring system which included the following criteria: body weight, general state, clinical results, spontaneous behavior and clinical examination. Values could range from 0 to 20 where increasing values indicated increasing severity with a predefined humane endpoint for a score ≥20 points. Documented score points were almost exclusively a result of body weight loss, whereas clinical signs and general health status had no influence on the overall sum of points scored. Behavioral variation was solely observed postoperatively, within the first 24 h, with an average score ≤1. In contrast to the classification of a laparotomy as a moderate procedure in the EU Directive 2010/63 (annex VIII) the assessment herein presented showed a mild burden in all groups according to the scoring system used. The partial hepatectomy itself, as well as the adhesive treatment using either synthetic glue VIVO-107 or fibrin glue, were well tolerated.

Potential in two types of collagen scaffolds for urological tissue engineering applications - Are there differences in growth behaviour of juvenile and adult vesical cells?

The aging society has a deep impact on patient care in urology. The number of patients in need of partial or whole bladder wall replacement is increasing simultaneously with the number of cancer incidents. Therefore, urological research requires a model of bladder wall replacement in adult and elderly people. Two types of porcine collagen I/III scaffolds were used in vitro for comparison of cell growth of two different pig breeds at different growth stages. Scaffolds were characterised with scanning electron and laser scanning microscopy. Urothelial and detrusor smooth muscle cells were isolated from 15 adult Göttingen minipigs and 15 juvenile German Landrace pigs. Growth behaviour was examined in cell culture and seeded onto the collagen scaffolds via immunohistochemistry, two-photon laser scanning microscopy and a viability assay. The collagen scaffolds showed different structured surfaces which are appropriate for seeding of the two different cell types. Moisturisation of the scaffolds resulted in a change of the structure. Cell growth of German Landrace urothelial cells and smooth muscle cells was significantly higher than cell growth of the Göttingen minipig cells. Seeding of scaffolds with both cell types from both pig races was possible which could be shown by immunohistochemistry and two-photon laser scanning microscopy. Growth behaviour on the scaffolds was significantly increased for the German Landrace compared to Göttingen minipig. Nevertheless, seeding with the adult Göttingen minipig cells resulted in a closed layer on the surface and urothelial cells and smooth muscle cells showed increasing growth until day 14. The results show that these collagen scaffolds are adequate for the seeding with vesical cells. Moreover, they seem appropriate for the use as an in vitro model for the adult or elderly as the cells of the adult Göttingen minipig too, show good growth behaviour.

Efficient bridging of 20 mm rat sciatic nerve lesions with a longitudinally micro-structured collagen scaffold.

An increasing number of biomaterial nerve guides has been developed that await direct comparative testing with the 'gold-standard' autologous nerve graft in functional repair of peripheral nerve defects. In the present study, 20 mm rat sciatic nerve defects were bridged with either a collagen-based micro-structured nerve guide (Perimaix) or an autologous nerve graft. Axons regenerated well into the Perimaix scaffold and, the majority of these axons grew across the 20 mm defect into the distal nerve segment. In fact, both the total axon number and the number of retrogradely traced somatosensory and motor neurons extending their axons across the implant was similar between Perimaix and autologous nerve graft groups. Implantation of Schwann cell-seeded Perimaix scaffolds provided only a beneficial effect on myelination within the scaffold. Functional recovery supported by the implanted, non-seeded Perimaix scaffold was as good as that observed after the autologous nerve graft, despite the presence of thinner myelin sheaths in the Perimaix implanted nerves. These findings support the potential of the Perimaix collagen scaffold as a future off-the-shelf device for clinical applications in selected cases of traumatic peripheral nerve injury.

Rogue waves lead to the instability in GaN semiconductors.

A new approach to understand the electron/hole interfaced plasma in GaN high electron mobility transistors (HEMTs). A quantum hydrodynamic model is constructed to include electrons/holes degenerate pressure, Bohm potential, and the exchange/correlation effect and then reduced to the nonlinear Schrödinger equation (NLSE). Numerical analysis of the latter predicts the rough (in)stability domains, which allow for the rogue waves to occur. Our results might give physical solution rather than the engineering one to the intrinsic problems in these high frequency/power transistors.

Diethylnitrosamine (DEN)-induced carcinogenic liver injury in mice.

The toxic properties of various nitrosamines in animals and humans are well established. The parenteral or oral administration of the smallest quantities of diethylnitrosamine (DEN) or dimethylnitrosamine (DMN) results in severe liver damage. Most prominent are intense neutrophilic infiltration, extensive centrilobular haemorrhagic necrosis, bile duct proliferation, fibrosis, and bridging necrosis that ends in hepatocarcinogenesis. Due to the robustness of the induced hepatic alterations, the application of DEN in rodents has become an attractive experimental model for studies aimed at understanding the pathogenetic alterations underlying the formation of liver cancer, which represents one of the most common malignancies in humans worldwide. However, several studies have shown that the hepatocarcinogenic effects of nitrosamines might vary with the genetic background of the animals, their sex, their age, and other factors that might impact the outcome of experimentation. We present general guidelines for working with DEN, and a detailed protocol that allows the establishment of highly reproducible liver cancer in mice. The outcome of liver injury after the application of DEN in mice, as estimated by the formation of cirrhosis and cancer, appears to be a suitable animal model for the analysis of some aspects and processes that promote the pathogenesis of hepatocellular carcinoma in humans.

Induction of experimental obstructive cholestasis in mice.

The induction of experimental obstructive cholestasis is a reliable model for cholestatic liver diseases in rodents. Bile duct ligation (BDL) in mice provokes typical time-dependent morphological and structural changes in the liver, ranging from liver cell injury and elevated serum enzyme levels after several days, to a severe inflammatory response in the liver after 5-7 days, up to an advanced hepatic fibrosis as soon as three to four weeks after surgical ligation of the common biliary duct. Upon BDL induction, hepatic stellate cells become activated and transdifferentiate into myofibroblasts that produce extracellular matrix proteins such as collagen. In principle, the periportal fibrosis induced by BDL in rat livers is reversible. After the relief of a biliary obstruction, the liver has the capacity to revert to a nearly normal histological architecture and a fully normal biochemical function. When BDL surgery is performed by an experienced scientist, this model has very high reproducibility among all fibrotic models. All these factors corroborate the outstanding value of this model for basic and translational research in biomedicine and hepatology. Nevertheless, this model can result in significant variations when surgery is carried out by untrained personnel or when unconscious modifications are implemented that affect the quality of the intervention. A detailed protocol is provided here for the provision of reliable and reproducible BDL in mice.

Partial hepatectomy in mice.

The surgical procedure of two-thirds partial hepatectomy (PH) in rodents was first described more than 80 years ago by Higgins and Anderson. Nevertheless, this technique is still a state-of-the-art method for the community of liver researchers as it allows the in-depth analysis of signalling pathways involved in liver regeneration and hepatocarcinogenesis. The importance of PH as a key method in experimental hepatology has even increased in the last decade due to the increasing availability of genetically-modified mouse strains. Here, we propose a standard operating procedure (SOP) for the implementation of PH in mice, which is based on our experience of more than 10 years. In particular, the SOP offers all relevant background information on the PH model and provides comprehensive guidelines for planning and performing PH experiments. We provide established recommendations regarding optimal age and gender of animals, use of appropriate anaesthesia and biometric calculation of the experiments. We finally present an easy-to-follow step-by-step description of the complete surgical procedure including required materials, critical steps and postoperative management. This SOP especially takes into account the latest changes in animal welfare rules in the European Union but is still in agreement with current international regulations. In summary, this article provides comprehensive information for the legal application, design and implementation of PH experiments.

Micturition in Göttingen minipigs: first reference in vivo data for urological research and review of literature.

One possible symptom of overactive bladder (OAB) is urinary incontinence, which is generally considered to be an age-associated disease and which is rapidly increasing with demographic changes. Rodent models are commonly used for the investigation of lower urinary tract functions, although the use of these species has limitations in several translational aspects. In biomedical research and preclinical toxicology, Göttingen minipigs are used increasingly. But in urological research, only few data are available for Göttingen minipigs. To the best of our knowledge, this study is one of the first to provide reference data of micturition in female Göttingen minipigs. Micturition frequency and volumes were monitored and analyzed in five female Göttingen minipigs. Voided volume was 520 ± 383 mL (mean ± standard deviation of mean [SD]) and micturition frequency 6.17 ± 3.68 (mean ± SD). We also performed a review of the literature to compare our data with data from different species (humans, pigs, rats and mice). Our findings revealed that micturition volume and frequency of Göttingen minipigs are more comparable with that of humans, leading to the conclusion that Göttingen minipigs may be the better choice for translational research in different research fields, such as urology, neurology and nephrology, etc. The provision of in vivo reference values meets with the 3R concept of 'reduction, refinement and replacement' of laboratory animals, because they allow comprehensive statistical power calculations (reduction), planning of telemetric approaches (refinement), and generation of computer-based modulation for the development of intravesical drug delivery systems (replacement).

Retrieval of migrated coils with stent retrievers: an animal study.

BACKGROUND AND PURPOSE: Coil migration is a potentially serious complication of endovascular aneurysm treatment. The aim of the study was to systematically investigate the effectiveness of coil retrieval with a stent retriever in an animal model. MATERIALS AND METHODS: A total of 148 coils of various types and sizes were placed into arteries of varying diameters in a porcine in vivo model. Coil retrieval was performed by placing a Trevo ProVue stent retriever over the coil and trying to trap a part of the platinum coil within the stent mesh by advancing the microcatheter over the stent or simply by retrieving the stent without trying to trap the coil by advancing the microcatheter. RESULTS: Coil retrieval was successful in 101 of 102 cases (99%), in which trapping of the coil within the stent retriever by advancing the microcathter was applied. When we only pulled back the stent without trapping the coil, retrieval was successful in only 5 of 46 cases (11%). Coil type, coil structure (2D versus 3D), actual coil shape in the affected vessel, investigator experience, aspiration, coil localization, and vessel diameter had no significant influence on retrieval outcome. There was no case of vessel perforation. CONCLUSIONS: Retrieval of migrated platinum coils with a stent retriever is an effective treatment option for migrated coils when the correct technique is applied.

Insular infarct size but not levosimendan influenced myocardial injury triggered by cerebral ischemia in rats.

Cerebral injuries can trigger stress-related cardiomyopathy. The extent of cerebral injury and the involvement of the insular cortex influence the incidence and extent of myocardial injury (MI), and drugs with proven neuroprotective and cardioprotective properties such as levosimendan might be beneficial. This hypothesis was addressed in a rat model of transient middle cerebral artery occlusion. Transient brain ischemia was induced for 60 min by intraluminal occlusion of the middle cerebral artery in 40 male Wistar rats. Treatment with levosimendan (24 µg/kg) was started briefly before reperfusion. Hemodynamic parameters were recorded and cerebral and MI quantified after 24 h. Levosimendan treatment significantly reduced cerebral infarct size in the cortex, but not in the striatal and insular regions. However, its effects on survival (28 vs. 45%), incidence of MI (8 vs. 33%) as indicated by a troponin I (sTnI) threshold of 4.8 µg/L and large insular infarcts of ≥10 mm(3) (23 vs. 50%) failed to reach statistical significance. Blood pressure demonstrated significant differences related to insular infarct size during reperfusion. Levosimendan demonstrated no relevant effects on markers of MI (sTnI = 1.5 ± 2.8 vs. 5.3 ± 7.2 µg/L, P = 0.121). Insular infarct size could be identified as the only predictor of MI (odds ratio = 1.86, P = 0.037). In conclusion, the current investigation confirmed insular infarct size as a predictor of MI and source of hemodynamic compromise, but failed to demonstrate an effect of levosimendan on MI trigged by brain ischemia. A hardly protectable insular region might explain this.

Promising future for the transgenic rat in transplantation research.

The rat is the most widely used animal species in surgical research and offers distinct advantages over the mouse in transplantation models due to its size and close genetic similarity to humans. Sequencing of the rat genome and successful application of transgenic technologies which had only been available for mice have since led to a resurgence of the use of rat models. Transplantation provides the possibility to deliver transgenes through a variety of routes which can potentially offer treatment modalities for post-transplant dysfunction and rejection. Moreover, the use of genetically encoded fluorescent light probes has enabled in vivo visualization of organs and tissue in living animals. In recent years, generation of gene knockout rats via the zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies has offered alternatives to the sophisticated embryonic stem cell based gene-targeting. In this review, we aim to provide an overview of transplantation studies involving transgenic techniques using rat models and recent advances in methods to modify the rat genome. Through novel gene modification techniques, precise, complete and conditional knockout and knockin rat models have become available which can provide promising new treatment options and opportunities for studying human transplant-related pathophysiology.

Vascular closure devices after endovascular procedures in swine: a reliable method?

PURPOSE: To investigate the safety and feasibility of the use of a vascular closure device (VCD) after endovascular procedures in swine. MATERIAL AND METHODS: In a study on endovascular therapy, VCD (StarClose, Abbott Vascular, Il, USA) was used in 20 female swines to achieve immediate hemostasis after percutaneous right femoral artery (FA) access. 10 animals were sacrificed immediately after the study and 10 animals were sacrificed 28 days after the initial study. To ensure complete hemostasis and patency of the femoral artery, a CT-angiography of the puncture site was performed on day 1 (acute and chronic group) and day 28 (chronic group). After the sacrifice, the femoral artery was explanted and examined macroscopically for signs of VCD dysfunction. RESULTS: Technical success rate was 100% with immediate hemostasis being achieved in all animals. No animals showed evidence of hematoma. During explantation, only small traces of coagulated blood were found in the acute group, while there were no signs of hematoma in the chronic group. CT-angiography immediately after VCD application as well as before sacrifice (chronic group) showed patency of the FA in all cases. CONCLUSION: The use of VCD to achieve hemostasis after endovascular studies in swine is feasible and safe.

In vivo characterisation of the inflammatory reaction following mesh implantation in transgenic mice models.

INTRODUCTION: Hernia repair with prosthetic meshes represents one of the most common surgical procedures in the field of surgery. This intervention is always associated with an ensuing inflammatory response, angiogenesis and fibrotic encapsulation forming a foreign body granuloma (FBG) around the mesh fibres. Several studies have described this inflammatory reaction by characterising inflammatory cell infiltrate around the FBG after mesh explantation. However, very little is known about the real-time progression of such an inflammatory response. The aim of this study was to investigate the feasibility of monitoring the ongoing inflammatory response to mesh implantation using bioluminescence in vivo. MATERIALS AND METHODS: Three luciferase transgenic mice strains (FVB/N-Tg(Vegfr2-luc)-Xen, BALB/C-Tg(NFκB-RE-luc)-Xen and Tg(INS/EpRE-Luc)T20Rbl) were used. Mice were anaesthetized with 2 % isoflurane, and two incisions were made on the left and right sides of the abdomen of the mice. A 1-cm(2) propylene mesh was implanted subcutaneously in the right incision wound of each mouse, and the left wound served as control. Two hundred microliters of D-luciferin was injected into the mice, and bioluminescence measurements were done prior to the surgical intervention and subsequently every 3 days. After mesh explantation, histological analysis was done. Statistical analysis was done using prism GraphPad software. RESULTS: Bioluminescence results revealed different time points of maximum signal for the different mice strains. VEGFR2 gene expression peaked on day 6, NFkB on day 12 and ARE on day 3 post mesh implantation. We also observed much higher bioluminescent signal around the FBG surrounding the mesh as compared to the control wound, with p < 0.05 for all the different mice strains. CONCLUSION: Our results prove the possibility of monitoring the inflammatory reaction after mesh implantation in vivo using bioluminescence signal release. This provides a novel method of accessing and accurately describing the ongoing inflammatory response over a given period of time.

Pumpless extracorporeal CO(2) removal restores normocapnia and is associated with less regional perfusion in experimental acute lung injury.

BACKGROUND: Lung protective ventilation may lead to hypoventilation with subsequent hypercapnic acidosis (HA). If HA cannot be tolerated or occurs despite increasing respiratory rate or buffering, extracorporeal CO2-removal using a percutaneous extracorporeal lung assist (pECLA) is an option. We hypothesised that compensation of HA using pECLA impairs regional perfusion. To test this hypothesis we determined organ blood flows in a lung-injury model with combined hypercapnic and metabolic acidosis. METHODS: After induction of lung injury using hydrochloric acid (HCl) aspiration and metabolic acidosis by intravenous HCl infusion in nine pigs, an arterial-venous pECLA device was inserted. In randomised order, four treatments were tested: pECLA shunt (1) with and (2) without HA, and clamped pECLA shunt (3) with and (4) without HA. Regional blood flows were measured with the coloured microsphere technique. RESULTS: HA resulted in higher perfusion in adrenal glands, spleen and parts of splanchnic area (P < 0.05) compared with normocapnia. During CO2-removal with pECLA, regional perfusion decreased to levels comparable with those without pECLA and normocapnia. Cardiac output (CO) increased during HA without a pECLA shunt and was highest during HA with a pECLA shunt compared with normocapnia. During CO2-removal with pECLA, this variable decreased but stayed higher than during normocapnia with clamped pECLA shunt (P < 0.05). CONCLUSION: In our lung-injury model, HA was associated with increased systemic and regional blood flow in several organs. pECLA provides effective CO2 removal, requiring a higher CO for perfusion of the pECLA device without improvement of regional organ perfusion.

A lipid anchor improves the protective effect of ectoine in inflammation.

Others and we have shown in several studies that the natural tetrahydropyrimidine ectoine protects mammalian cells and tissues against various stress factors including ischemia/reperfusion injury, UV-irradiation, and inflammation. Since little is known about the molecular mechanism of this protective effect, which was ascribed exclusively to an extracellular action of this small water-soluble molecule, we asked whether and how a hydrophobic anchor modulates the inflammation protective properties of ectoine. We therefore investigated the influence of ectoine and of its semi-synthetic derivative lauryl-ectoine on inflammation in RAW 264.7 macrophages and primary cultured rat intestinal smooth muscle (RISM) cells. Both, ectoine and lauryl-ectoine considerably decreased lipopolysaccharide (LPS)-induced interleukin (IL)- 1, IL-6, tumor necrosis factor (TNF)- α, and cyclooxygenase (COX)-2 gene expression in macrophages as well as TNF-α- induced IL-1, IL-6 and COX-2 expression in RISM cells. This reduction of inflammatory agents was accompanied on the one hand by a significant decrease of nuclear translocation of nuclear factor (NF)-κB and on the other hand by a reduction of cellular ceramide content. Interestingly, lauryl- ectoine was much more active exerting its effect at about 10-fold lower concentrations than its natural counterpart. Note that ectoine was almost completely recovered in the medium whereas lauryl-ectoine was found to be cell-associated. Together our data indicate that a lipid anchor considerably improves a possible preventive and/or therapeutic implementation of ectoine in inflammatory processes.

Effect of anesthesia and cerebral blood flow on neuronal injury in a rat middle cerebral artery occlusion (MCAO) model.

Middle cerebral artery occlusion (MCAO) models have become well established as the most suitable way to simulate stroke in experimental studies. The high variability in the size of the resulting infarct due to filament composition, rodent strain and vessel anatomy makes the setup of such models very complex. Beside controllable variables of homeostasis, the choice of anesthetics and the grade of ischemia and reperfusion played a major role for extent of neurological injury. Transient MCAO was induced during either isoflurane or ketamine/xylazine (ket/xyl) anesthesia with simultaneously measurement of cerebral blood flow (CBF) in 60 male Wistar rats (380-420 g). Neurological injury was quantified after 24 h. Isoflurane compared with ket/xyl improved mortality 24 h after MCAO (10 vs. 50 %, p = 0.037) and predominantly led to striatal infarcts (78 vs. 18 %, p = 0.009) without involvement of the neocortex and medial caudoputamen. Independent of anesthesia type, cortical infarcts could be predicted with a sensitivity of 67 % and a specificity of 100 % if CBF did not exceed 35 % of the baseline value during ischemia. In all other cases, cortical infarcts developed if the reperfusion values remained below 50 %. Hyperemia during reperfusion significantly increased infarct and edema volumes. The cause of frequent striatal infarcts after isoflurane anesthesia might be attributed to an improved CBF during ischemia (46 ± 15 % vs. 35 ± 19 %, p = 0.04). S-100ß release, edema volume and upregulation of IL-6 and IL-1ß expression were impeded by isoflurane. Thus, anesthetic management as well as the grade of ischemia and reperfusion after transient MCAO demonstrated important effects on neurological injury.