Oral administration of dextran sodium sulphate induces a caecum-localized colitis in rabbits.

Trichuris suis ova (TSO) have shown promising results in the treatment of inflammatory bowel disease (IBD) but the mechanisms which underlies this therapeutic effect cannot be studied in mice and rats as T. suis fails to colonize the rodent intestine, whilst hatching in humans and rabbits. As a suitable rabbit IBD model is currently not available, we developed a rabbit colitis model by administration of dextran sodium sulphate (DSS). White Himalayan rabbits (n = 12) received 0.1% DSS in the daily water supply for five days. Clinical symptoms were monitored daily, and rabbits were sacrificed at different time points. A genomewide expression analysis was performed with RNA isolated from caecal lamina propria mononuclear cells (LPMC) and intestinal epithelial cells (IEC). The disease activity index of DSS rabbits increased up to 2.1 ± 0.4 (n = 6) at day 10 (controls <0.5). DSS induced a caecum-localized pathology with crypt architectural distortion, stunted villous surface and inflammatory infiltrate in the lamina propria. The histopathology score reached a peak of 14.2 ± 4.9 (n = 4) at day 10 (controls 7.7 ± 0.9, n = 5). Expression profiling revealed an enrichment of IBD-related genes in both LPMC and IEC. Innate inflammatory response, Th17 signalling and chemotaxis were among the pathways affected significantly. We describe a reproducible and reliable rabbit model of DSS colitis. Localization of the inflammation in the caecum and its similarities to IBD make this model particularly suitable to study TSO therapy in vivo.

Three-Dimensional Printed Hydroxyapatite Bone Substitutes Designed by a Novel Periodic Minimal Surface Algorithm Are Highly Osteoconductive.

Autologous bone remains the gold standard bone substitute in clinical practice. Therefore, the microarchitecture of newly developed synthetic bone substitutes, which reflects the spatial distribution of materials in the scaffold, aims to recapitulate the natural bone microarchitecture. However, the natural bone microarchitecture is optimized to obtain a mechanically stable, lightweight structure adapted to the biomechanical loading situation. In the context of synthetic bone substitutes, the application of a Triply Periodic Minimum Surface (TPMS) algorithm can yield stable lightweight microarchitectures that, despite their demanding architectural complexity, can be produced by additive manufacturing. In this study, we applied the TPMS derivative Adaptive Density Minimal Surfaces (ADMS) algorithm to produce scaffolds from hydroxyapatite (HA) using a lithography-based layer-by-layer methodology and compared them with an established highly osteoconductive lattice microarchitecture. We characterized them for compression strength, osteoconductivity, and bone regeneration. The in vivo results, based on a rabbit calvaria defect model, showed that bony ingrowth into ADMS constructs as a measure of osteoconduction depended on minimal constriction as it limited the maximum apparent pore diameter in these scaffolds to 1.53 mm. Osteoconduction decreased significantly at a diameter of 1.76 mm. The most suitable ADMS microarchitecture was as osteoconductive as a highly osteoconductive orthogonal lattice microarchitecture in noncritical- and critical-size calvarial defects. However, the compression strength and microarchitectural integrity in vivo were significantly higher for scaffolds with their microarchitecture based on the ADMS algorithm when compared with high-connectivity lattice microarchitectures. Therefore, bone substitutes with high osteoconductivity can be designed with the advantages of the ADMS-based microarchitectures. As TPMS and ADMS microarchitectures are true lightweight structures optimized for high mechanical stability with a minimal amount of material, such microarchitectures appear most suitable for bone substitutes used in clinical settings to treat bone defects in weight-bearing and non-weight-bearing sites.

Coronary optical frequency domain imaging (OFDI) for in vivo evaluation of stent healing: comparison with light and electron microscopy.

AIMS: Coronary late stent thrombosis, a rare but devastating complication, remains an important concern in particular with the increasing use of drug-eluting stents. Notably, pathological studies have indicated that the proportion of uncovered coronary stent struts represents the best morphometric predictor of late stent thrombosis. Intracoronary optical frequency domain imaging (OFDI), a novel second-generation optical coherence tomography (OCT)-derived imaging method, may allow rapid imaging for the detection of coronary stent strut coverage with a markedly higher precision when compared with intravascular ultrasound, due to a microscopic resolution (axial approximately 10-20 microm), and at a substantially increased speed of image acquisition when compared with first-generation time-domain OCT. However, a histological validation of coronary OFDI for the evaluation of stent strut coverage in vivo is urgently needed. Hence, the present study was designed to evaluate the capacity of coronary OFDI by electron (SEM) and light microscopy (LM) analysis to detect and evaluate stent strut coverage in a porcine model. METHODS AND RESULTS: Twenty stents were implanted into 10 pigs and coronary OFDI was performed after 1, 3, 10, 14, and 28 days. Neointimal thickness as detected by OFDI correlated closely with neointimal thickness as measured by LM (r = 0.90, P < 0.01). The comparison of stent strut coverage as detected by OFDI and SEM analysis revealed an excellent agreement (r = 0.96, P < 0.01). In particular, stents completely covered by OFDI analysis were also completely covered by SEM analysis. All incompletely covered stents by OFDI were also incompletely covered by SEM. Analyses of fibrin-covered stent struts suggested that these may rarely be detected as uncovered stent struts by OFDI. Importantly, optical density measurements revealed a significant difference between fibrin- and neointima-covered coronary stent struts [0.395 (0.35-0.43) vs. 0.53 (0.47-0.57); P < 0.001], suggesting that differences in optical density provide information on the type of stent strut coverage. The sensitivity and specificity for detection of fibrin vs. neointimal coverage was evaluated using receiver-operating characteristic analysis. CONCLUSION: The present study demonstrates that OFDI is a highly promising tool for accurate evaluation of coronary stent strut coverage, as supported by a high agreement between OFDI and light and electron microscopic analysis. Furthermore, our data indicate that optical density measurements can provide additional information with respect to the type of stent strut coverage, i.e. fibrin vs. neointimal coverage. Therefore, coronary OFDI analysis will provide important information on the biocompatibility of coronary stents.

Transplantation of Autologous Dermo-Epidermal Skin Substitutes in a Pig Model.

Due to its similarity of skin anatomy and physiology, the pig appears to be a well-suited animal model for preclinical studies of skin analog transplantations. The choice of the location of the skin defect and appropriate postoperative measures are essential for the protection of the transplanted graft. This protocol describes in detail a porcine skin transplantation model including peri- and postoperative measures taken to improve and refine the study outcome.

Preventive Trichuris suis ova (TSO) treatment protects immunocompetent rabbits from DSS colitis but may be detrimental under conditions of immunosuppression.

Trichuris suis ova (TSO) have been tested for therapeutic application in inflammatory bowel diseases (IBD) yet understanding of the underlying mechanisms and safety in an immunocompromised host is limited due to lack of a suitable animal model. We used a recently established rabbit model of dextran sodium sulphate (DSS) induced colitis to study the efficacy, mechanisms and safety of TSO therapy in immunocompetent and immunosuppressed animals. TSO treatment prevented the DSS induced weight loss, delayed the onset of DSS induced symptoms by 2 days and significantly reduced the disease activity (DAI). TSO treatment protected caecal histology and prevented the colitis-associated loss in faecal microbiota diversity. Mainly the transcriptome of lamina propria mononuclear cells (LPMC) was affected by TSO treatment, showing dampened innate and adaptive inflammatory responses. The protective effect of TSO was lost in immunosuppressed rabbits, where TSO exacerbated colitis. Our data show that preventive TSO treatment ameliorates colitis severity in immunocompetent rabbits, modulates LPMC immune responses and reduces faecal dysbiosis. In contrast, the same TSO treatment exacerbates colitis in immunosuppressed animals. Our data provide further evidence for a therapeutic effect of TSO in IBD, yet caution is required with regard to TSO treatment in immunosuppressed patients.

Echinomycin did not affect the safety of fracture healing: an experimental pilot study on a murine femur fracture model.

BACKGROUND: There is a need for effective drugs in the prevention and treatment of heterotopic ossifications (HO) after fractures. Echinomycin has been shown to prevent formation of HO in an animal model. However, before it may be considered as an option against HO, it needs to be studied whether it prevents fracture healing similar to non-steroidal anti-inflammatory drugs (NSAIDS). Therefore, the hypothesis was that echinomycin prevents fracture healing and callus formation. METHODS: In an experimental murine pilot study, standard blunt femur fractures were induced and retrograde intramedullary compression fixation of the femur was performed. The treatment group (n = 8) received echinomycin (0.3 mg/kg body weight) and the control group (n = 8) did not receive echinomycin. The fractures and implant positions were verified by conventional X-rays immediately postoperatively. As the primary outcome variable, fracture healing (osseous consolidation) was evaluated by conventional X-rays and micro-computed tomography (CT) scans after ten weeks and graded as healed, partial or complete pseudarthrosis. The secondary outcome, callus formation, was graded semi-quantitatively from 0 (mostly absent) to 3 (maximum). RESULTS: Fracture healing was present in all living cases after ten weeks concerning the treatment group. Partial pseudarthrosis was seen in two cases, one in the treatment and another one in the control group. Complete pseudarthrosis was seen in one case of the control group after an open fracture. Callus formation was similar in both groups with a mean grade of 1.5 within each group. Two cases of the treatment group died. CONCLUSION: As a novel finding, echinomycin did not inhibit fracture healing or callus formation in this in vivo murine standard femur fracture model pilot study. Further studies involving a larger number of cases, quantitative assessment with CT scans and histopathological analysis are needed before generalizing the results of this pilot study.

Comparison of medetomidine, thiopental and ketamine/midazolam anesthesia in chick embryos for in ovo Magnetic Resonance Imaging free of motion artifacts.

Non-invasive assessment of the perfusion capacity of tissue engineered constructs grown on the chorioallantoic membrane by MRI is often hampered by motion artifacts. Therefore, we examined the suitability of three anesthetic regimes for sufficient sedation of the chick embryo. Medetomidine at a dosage of 0.3 mg/kg, was compared to thiopental at 100 mg/kg and ketamine/midazolam at 50 mg/kg and 1 mg/kg, respectively. These soluble anesthetics were applied by dropping a total volume of 0.3 mL onto the surface of the CAM. Motion was videotaped through the window of the eggshell and scored semi-quantitatively. Medetomidine performed best in terms of reduced motion; onset of anesthesia occurred within 10 minutes and for the following 30 minutes, allowing proper in vivo MRI measurements. The other regimen were not sedating deep enough (ketamine/midazolam) and not long enough (thiopental). In sum, medetomidine allows proper sedation for MRI assessment of the perfusion capacity in a tissue engineered construct placed on the CAM.

HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats.

OBJECTIVES: HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272-specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders. METHODS: The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry. RESULTS: HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM). HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules. CONCLUSION: HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.

2D motion analysis of rabbits after Achilles tendon rupture repair and histological analysis of extracted tendons: can the number of animals be reduced by operating both hind legs simultaneously?

BACKGROUND: Considering the 3Rs principle in animal experiments, there is a demand to perform research experiments with the fewest number of animals possible while warranting the welfare of the animals. Orthopaedic experimental studies involving operations on the hind legs of rabbits are either performed on one hind leg with the second hind leg serving as control or on both hind legs simultaneously (control: rabbits with no operations at all). METHODS: The Achilles tendon of rabbits was transected and sutured, and the two-dimensional motion pattern of animals having only one leg operated was compared to rabbits having both hind legs operated (control: non-treated animals). Step length, maximum ankle angle, minimum ankle angle and the resulting range of motion of both hind legs were determined weekly over a time span from 3 weeks to 12 weeks post-operation. The results were fitted by a linear mixed effects model including time dependency. Moreover, all tendon specimen were analysed histologically. Tenocyte and tenoblast density, tenocyte and tenoblast nuclei width, inflammation level and collagen fibre alignment were determined. RESULTS: Statistically significant differences in the motion pattern were found when one-leg treated and two-leg treated animals were compared. However, the absolute differences were on average less than 20%. Histologically, 1-leg treated animals had tendon tissue with higher cell density, but lower inflammation and less ondulated collagen fibres compared to 2-leg treated animals; the nuclei width was the same for both groups. With regard to welfare, all animals were fine during the experiments. CONCLUSIONS: While comparative studies should be performed with one-leg treated animals due to interaction effects, for proof-of-principle studies, operating two legs per animal may be justified as the welfare of the animals is warranted. This is a great benefit in the sense of the 3Rs because up to 50% of animals can be spared.

Human miR223 promoter as a novel myelo-specific promoter for chronic granulomatous disease gene therapy.

Targeting transgene expression to specific hematopoietic cell lineages could contribute to the safety of retroviral vectors in gene therapeutic applications. Chronic granulomatous disease (CGD), a defect of phagocytic cells, can be managed by gene therapy, using retroviral vectors with targeted expression to myeloid cells. In this context, we analyzed the myelospecificity of the human miR223 promoter, which is known to be strongly upregulated during myeloid differentiation, to drive myeloid-restricted expression of p47(phox) and gp91(phox) in mouse models of CGD and in primary patient-derived cells. The miR223 promoter restricted the expression of p47(phox), gp91(phox), and green fluorescent protein (GFP) within self-inactivating (SIN) gamma- and lentiviral vectors to granulocytes and macrophages, with only marginal expression in lymphocytes or hematopoietic stem and progenitor cells. Furthermore, gene transfer into primary CD34+ cells derived from a p47(phox) patient followed by ex vivo differentiation to neutrophils resulted in restoration of Escherichia coli killing activity by miR223 promoter-mediated p47(phox) expression. These results indicate that the miR223 promoter as an internal promoter within SIN gene therapy vectors is able to efficiently correct the CGD phenotype with negligible activity in hematopoietic progenitors, thereby limiting the risk of insertional oncogenesis and development of clonal dominance.

Combining sevoflurane anesthesia with fentanyl-midazolam or s-ketamine in laboratory mice.

Laboratory mice typically are anesthetized by either inhalation of volatile anesthetics or injection of drugs. Here we compared the acute and postanesthetic effects of combining both methods with standard inhalant monoanesthesia using sevoflurane in mice. After injection of fentanyl-midazolam or S-ketamine as premedication, a standard 50-min anesthesia was conducted by using sevoflurane. Addition of fentanyl-midazolam (0.04 mg/kg-4 mg/kg) induced sedation, attenuation of aversive behaviors at induction, shortening of the induction phase, and reduced the sevoflurane concentration required by one third (3.3% compared with 5%), compared with S-ketamine (30 mg/kg) premedication or sevoflurane alone. During anesthesia, heart rate and core body temperature were depressed significantly by both premedications but in general remained within normal ranges. In contrast, with or without premedication, substantial respiratory depression was evident, with a marked decline in respiratory rate accompanied by hypoxia, hypercapnia, and acidosis. Arrhythmia, apnea, and occasionally death occurred under S-ketamine-sevoflurane. Postanesthetic telemetric measurements showed unchanged locomotor activity but elevated heart rate and core body temperature at 12 h; these changes were most prominent during sevoflurane monoanesthesia and least pronounced or absent during fentanyl-midazolam-sevoflurane. In conclusion, combining injectable and inhalant anesthetics in mice can be advantageous compared with inhalation monoanesthesia at induction and postanesthetically. However, adverse physiologic side effects during anesthesia can be exacerbated by premedications, requiring careful selection of drugs and dosages.

Burrowing behavior as an indicator of post-laparotomy pain in mice.

Detection of persistent pain of a mild-to-moderate degree in laboratory mice is difficult because mice do not show unambiguous symptoms of pain or suffering using standard methods of short-term observational or clinical monitoring. This study investigated the potential use of burrowing performance - a spontaneous and highly motivated behavior - as a measure of post-operative pain in laboratory mice. The influence of minor surgery on burrowing was investigated in adult C57BL/6J mice of both genders in a modified rodent burrowing test (displacement of food pellets from a pellet-filled tube) within the animal's home cage. Almost all (98%) healthy mice burrowed (mean latency 1.3 h, SEM 0.5 h). After surgery without pain treatment, latency of burrowing was significantly prolonged (mean Δ latency 10 h). Analgesic treatment using the anti-inflammatory drug carprofen (5 mg/kg bodyweight) decreased latency of burrowing after surgery (mean Δ latency 5.5 h) to the level found in mice that had been anesthetized (mean Δ latency 5.4 h) or had received anesthesia and analgesia (mean Δ latency 4.6 h). Analgesia during surgery was associated with a significantly earlier onset of burrowing compared to surgery without pain treatment. A distinct gradation in burrowing performance was found ranging from the undisturbed pre-operative status to the intermediate level following anesthesia/analgesia and surgery with analgesia, to the pronounced prolongation of latency to burrow after surgery without pain relief. In conclusion, post-surgical impairment of general condition, probably mainly attributable to pain, can be conveniently assessed in laboratory mice on the basis of the burrowing test.

Isoflurane and sevoflurane provide equally effective anaesthesia in laboratory mice.

Isoflurane is currently the most common volatile anaesthetic used in laboratory mice, whereas in human medicine the more modern sevoflurane is often used for inhalation anaesthesia. This study aimed to characterize and compare the clinical properties of both anaesthetics for inhalation anaesthesia in mice. In an approach mirroring routine laboratory conditions (spontaneous breathing, gas supply via nose mask, preventing hypothermia by a warming mat) a 50 min anaesthesia was performed. Anaesthetics were administered in oxygen as carrier gas at standardized dosages of 1.5 minimum alveolar concentrations, which was 2.8% for isoflurane and 4.9% for sevoflurane. Both induction and recovery from anaesthesia proceeded quickly, within 1-2 min. During anaesthesia, all reflex testing was negative and no serious impairment of vital functions was found; all animals survived. The most prominent side-effect during anaesthesia was respiratory depression with hypercapnia, acidosis and a marked decrease in respiration rate. Under anaesthesia, heart rate and core body temperature remained within the normal range, but were significantly increased for 12 h after anaesthesia. Locomotor activity, daily food and water consumption and body weight progression showed no abnormalities after anaesthesia. No significant difference was found between the two anaesthetics. In conclusion, isoflurane and sevoflurane provided an equally reliable anaesthesia in laboratory mice.