Experimental Animal Research Research Articles

The Effect of Ellagic Acid on Paraoxanase - 1 Activity and DNA Damage in Acute Exercise

The purpose of this study was to evaluate the correlation between exhaustive and intensive exercise with changes in paraoxonase-1 enzyme activity, oxidative DNA damage and the role of ellagic acid against possible damage.
 The study was carried out on 32 male and adult Spraque - Dawley rats at the Experimental Animal Research and Research Center of Afyon Kocatepe University. The experimental animals were equally divided into four groups. Swimming exercises were performed as acute exercises for once and experimental animals are made to swim in groups including two rats following the completion of the study and before the decapitation.
 At the end of the experiment, obtained blood samples; Paraoxonase-1 (PON-1), Malondialdehyde (MDA) and 8-HydroxyGuanine (8-OhdG) levels were measured to determine DNA damage and DNA damage was assessed by Comet Assay method.
 As a result, PON-1 levels in rats with intense swimming training were found to be significantly lower (p <0.05) than the control group. MDA and (8-OhdG) levels were significantly higher in the swimming group than in the control group (p <0.05). As to the DNA damage determination by COMET analysis, DNA damage was observed in the swimming groups according to the control groups. When the ellagic acid groups were compared with the swimming groups, there was a significant increase in PON-1 levels, and the levels of MDA and (8-OhdG) were significantly lower than the swimming groups. The DNA damage was also found to be low in these groups.

A simulation model for the degradation of magnesium-based bone implants

The development of degradable bone implants, in particular made of metal materials, is an emerging field. The advantage of degradable implants is that they do not have to be removed later. In order to be able to develop and scale appropriate implants for different applications, it is necessary to know the change in mechanical properties of the implant during the degradation process in general and at different locations. One area of bone implants are bone substitute materials. They are deployed when there is a defect in the bone which cannot be filled autonomously by the body. In this study, a numerical degradation model of magnesium-based bone substitute materials is developed using the finite element method. Computational models are being developed to reduce experimental animal research in future. Magnesium is a naturally occurring material which is needed to build enzymes in the body. Additionally, magnesium has a Young's modulus close to native bone, wherefore it is attractive for medical applications with bone contact. The simulation model is based on the assumption that the degradation is a diffusion-controlled process driven by the dissolution of magnesium. The model is adapted to a 3D open-pored structure made of the magnesium alloy LAE442. Previous studies showed that implants made of LAE442 lose stiffness without a volume reduction. To simulate the change in mechanical properties, a concentration-dependent Young's modulus is assumed. With this model the formation of the degradation layer is computable as well as the change in mechanical properties, as measured by the effective Young's modulus of the structure. The movement of the interface between the not-degraded and degraded material is modelled using the level set method.

Assessment of knowledge and attitude towards alternative to animal experimentation in research and education among interns and post graduate medical students in a teaching hospital, Tamilnadu, India

Background: Use of animals for various purposes like food, transportation, pets, sports, recreation and companionship is as old as the human beings itself. Animals also serve as a tool for education, research, medical procedures, toxicological screening, for several decades. Since pain, distress and death of animals occur commonly during scientific experiments, various guidelines have been proposed and posed many restrictions over the experimental use of animals.To assess the knowledge and attitude towards alternative to animal experimentation in research and education among interns and postgraduate medical students in a teaching hospital.Methods: A cross sectional study was carried out by self-administered questionnaire among interns (92) and postgraduate medical students (53) in a teaching hospital in July 2018. The data was analysed by descriptive statistics and expressed in percentage.Results: Among 145 participants, interns 92 (63.4%), postgraduates 53 (36.6%). 70.2% interns and 68.8% postgraduates had adequate knowledge about alternative animal experimentation and 67% and 67.1% of interns and postgraduates had knowledge about animal experiments. 69.4% interns and 68.8% postgraduates had positive attitude towards alternative animal experimentation.83.8% interns and 70.9% postgraduates were agreed to have various barriers to alternative animal experimentation.Conclusions: Majority of interns and postgraduates have appreciable knowledge of alternative to animal experimentation, but their attitude is scarce. It is imperative to incorporate continuous training through workshops for budding medical professionals to provide innovative scientific knowledge in research and education towards alternative to animal experimentation.

Contribution of Experimental Animal Research Studies to the Emergency Medicine Literature.

The aim of this study is to provide a detailed analysis of emergency medicine (EM) research literature to unveil the trends while underlining the importance of experimental research for all territories of science. To this end, the experimental animal research articles published in EM journals indexed to the Science Citation Index Expanded database with a date of publication between January 1, 2008, and December 31, 2017, were reviewed retrospectively. All data regarding the journal name, publication year, country, department and institution of the first author, subject species, type of the experimental model, target organ/system/functions, evaluation method, outcome measures, and citation counts were noted. Resultantly, a total of 736 articles were found to be published in 18 journals. Resuscitation (n=285, 38.7%) had the highest number of articles followed by Injury (n=143, 19.4%), Turkish Journal of Trauma and Emergency Surgery (n=128, 17.4%), and American Journal of Emergency Medicine (n=63, 8.6%). The USA was the largest contributor with 199 studies (27%). The department of the first author was EM in 190 (28.8%) of the reports. Various versions of cardiac arrest models were applied in 257 (34.9%) studies while brain (n=101, 13.7%) was the most commonly explored area. The main outcome measures were clinical outcomes/survival rates (n=408, 55.43%). The molecular mechanisms of the injury were evaluated in 37 (5%) of the studies. In conclusion, experimental animal studies are essential in the progress of contemporary scientific knowledge. EM journals should encourage and consider giving more place to experimental research given their undisputed worth and potential future contributions to science, including the field of EM.

A Neuroscience Perspective of Physical Treatment of Headache and Neck Pain.

The most prevalent primary headaches tension-type headache and migraine are frequently associated with neck pain. A wide variety of treatment options is available for people with headache and neck pain. Some of these interventions are recommended in guidelines on headache: self-management strategies, pharmacological and non-pharmacological interventions. Physical treatment is a frequently applied treatment for headache. Although this treatment for headache is predominantly targeted on the cervical spine, the neurophysiological background of this intervention remains unclear. Recent knowledge from neuroscience will enhance clinical reasoning in physical treatment of headache. Therefore, we summarize the neuro- anatomical and—physiological findings on headache and neck pain from experimental research in both animals and humans. Several neurophysiological models (referred pain, central sensitization) are proposed to understand the co-occurrence of headache and neck pain. This information can be of added value in understanding the use of physical treatment as a treatment option for patients with headache and neck pain.

Paying the Toll for Inflammation

Paying the Toll for Inflammation

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier.

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p < 0.01) in the experimental group and 35% vs. 85% (p < 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 ± 0.75 vs. 2.90 ± 0.31, p < 0.05; 1.15 ± 0.37 vs. 1.55 ± 0.76, p < 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.

Silkworm as an experimental animal for research on fungal infections.

Silkworm, Bombyx mori, has various advantages as an experimental animal, such as the low cost for rearing and fewer ethical problems. Models utilizing silkworms of infection with pathogenic bacteria have been established for identification of genes encoding virulence factors by large‐scale in vivo screening. In this review, we describe recent progress in the study of silkworm infection models for elucidating the mechanisms of fungi infection. Silkworm infection models have been established for Candida albicans, Candida tropicalis, Candida glabrata and Cryptococcus neoformans, which are yeast type fungi, and Aspergillus fumigatus, Arthroderma vanbreuseghemii, Arthroderma benhamiae, Microsporum canis, Trichophyton rubrum, and Rhizopus oryzae, which are filamentous fungi. Novel genes encoding virulence factors in C. albicans and C. glabrata have been identified by using the silkworm infection models. We here outline the benefits of using silkworm infection models and a strategy for identifying the genes responsible for pathogenicity of microorganisms such as fungi. © 2019 The Authors. Microbiology and Immunology Published by The Societies and John Wiley & Sons Australia, Ltd.

Costs of Implementing Quality in Research Practice.

Using standardized guidelines in preclinical research has received increased interest in light of recent concerns about transparency in data reporting and apparent variation in data quality, as evidenced by irreproducibility of results. Although the costs associated with supporting quality through a quality management system are often obvious line items in laboratory budgets, the treatment of the costs associated with quality failure is often overlooked and difficult to quantify. Thus, general estimations of quality costs can be misleading and inaccurate, effectively undervaluing costs recovered by reducing quality defects. Here, we provide examples of quality costs in preclinical research and describe how we have addressed misconceptions of quality management implementation as only marginally beneficial and/or unduly burdensome. We provide two examples of implementing a quality management system (QMS) in preclinical experimental (animal) research environments - one in Europe, the German Mouse Clinic, having established ISO 9001 and the other in the United States, the University of Kentucky (UK), having established Good Laboratory Practice-compliant infrastructure. We present a summary of benefits to having an effective QMS, as may be useful in guiding discussions with funders or administrators to promote interest and investment in a QMS, which ultimately supports shared, mutually beneficial outcomes.

Experimental Rat Model for Brain Death Induction and Kidney Transplantation

Background: Experimental animal research has been pivotal in developing clinical kidney transplantation (KTx). One donor-associated risk factor with negative affect of transplantation outcome is brain death (BD). Many rat models for BD and KTx have been developed in the last decade, but no surgical guidelines have been developed for these models. Here, we describe a surgical technique for BD induction and the cuff technique for experimental KTx in rats.Methods: After intubation and mechanically ventilation of sixteen healthy adult male Sprague–Dawley rats were induction of BD performed. Animals were kept hemodynamically stable for eight hours. Then, the kidney was prepared and perfused with standard histidine–tryptophan–ketoglutarate solution. After explantation, grafts were immediately implanted in recipients using the cuff technique and reperfused. After 2 h of observation, animals were sacrificed by intravenous administration of potassium chloride.Results: In the early phase of BD, heart rate increased and mean arterial pressure decreased. Partial variations were observed in O2 partial pressure, O2 saturation, and HCO3. During the 2-h observation phase, all transplanted kidneys were sufficiently perfused macroscopically. There was no hyperacute rejection.Conclusions: It is feasible to observe BD for 8 h with maintained circulation in small experimental settings. The cuff technique for KTx is simple, the complication rate is low, and the warm ischemia time is short, therefore, this could be a suitable technique for KTx in the rat model.

PO-301 Study on the effects of bone marrow-derived stem cells were used in chronic hyperactivity rats to cardiac injury treatment

Objective overload and long-term overtraining can cause hypoxic and hypoxic damage to the myocardial structure of the body. In recent years, studies have shown that the stem cells promote angiogenesis in vivo, resistance to apoptosis, myocardial stem cell mobilization, and promote its proliferation in paracrine effect, such as vascular distribution. By animal experiments, this study explore MSCMs role in the improvement of heart function and its molecular mechanism to sports injury prevention and postoperative rehabilitation is of great significance of the heart, heart research provides the basis for the motion at the same time support.&#x0D; Methods Wistar rat model of excessive swimming training. Grouping: rats were randomly divided into 4 groups (n=10), quiet feeding group (Q), general training group (ET), over-training group (OT), and MSCMs transplant-over-training group (MOT). Source and preparation of stem cells: the rat autologous bone marrow was extracted 1 day before surgery, and the bone marrow mononuclear cells were isolated by Ficoll density gradient centrifugation. Methods of stem cell transplantation: perfusion via coronary artery in MOT group rats; Test indicators and methods: cardiac tissue was taken after the end of 1d training (group Q, ET and OT), MEF2A factor was tested by rcal-time, gata-4 expression was tested by Western blot, and LVEF value was observed by cardiac color doppler ultrasound (before, after 1w, after 2w and after 3w, respectively).&#x0D; Results MEF2A factor, gata-4 expression and LVEF value of the three groups of samples were detected: (1) compared with MEF2A factor in general training group (ET) and quiet group (Q), gata-4 expression was slightly improved, but there was no significant difference (P&gt;0.05). After 3w, the increase of LVEF value presented significant differences (Pwhile 1w and 2w showed no significant differences compared with the quiet group. (2) comparison between the over-training group (OT) and the quiet group (Q) showed significant differences in MEF2A factor, gata-4 expression, and LVEF decreased value (P0.05) between the two groups after 2w and the quiet group (Q). Cardiac tissue was taken after 2w to observe the expression of MEF2A, and gata-4 was compared with the silent group (Q) without significant difference (P&gt;0.05).&#x0D; Conclusions (1) based on the test data of general training group (ET), reasonable and scientific aerobic exercise can effectively enhance the cardiac function and improve the cardiac activity ability. (2) according to the test data of over-training group (OT), overloading and long-term over-training can lead to hypoxia of heart function and decrease of vitality, resulting in hypoxia and ischemia of the motor heart and damage of cardiac function. (3) according to the observation and test data of the MSCMs transplant-over-training group (MOT), MSCMs transplantation can effectively improve the cardiac function of sports injuries, enhance the cardiac vitality, and repair damaged cells and tissues to a certain extent. It can effectively prevent and treat heart injury caused by overtraining. At the same time, it provides animal experimental research support for the research of sports heart in sports medicine.

Depression models in experimental animals

Depression is a mental disorder that is estimated by the World Health Organization to affect 350 million people worldwide. But its pathogenesis and underlying mechanisms have not been understood yet. To present a satisfying explanation for the causes and treatments of these sorts of diseases animal models can be a powerful model for the researchers. Experimental animal research has been frequently used, in related with clinical studies, to test a number of hypotheses regarding the etiology of depression and its related behaviors. In the literature, experimental animal models about depression were described. These are chronic mild stress, forced swimming test, learned helplessness, tail suspension test, psycho-stimulant drug withdrawal and olfactory bulbectomy. In the oral presentation, it was summarized the experimental animal models that are used most commonly for depression, and discussed their advantages and limitations. In conclusion, it seems that some experimental animal models such as chronic mild stress and forced swimming test in several experiments have been using for investigating depression etiology and treatment and the models are very useful for searching the disease.

Animal experimental research design in critical care

BackgroundLimited translational success in critical care medicine is thought to be in part due to inadequate methodology, study design, and reporting in preclinical studies. The purpose of this study was to compare reporting of core features of experimental rigor: blinding, randomization, and power calculations in critical care medicine animal experimental research. We hypothesized that these study design characteristics were more frequently reported in 2015 versus 2005.MethodsWe performed an observational bibliometric study to grade manuscripts on blinding, randomization, and power calculations. Chi-square tests and logistic regression were used for analysis. Inter-rater agreement was assessed using kappa and Gwet’s AC1.ResultsA total of 825 articles from seven journals were included. In 2005, power estimations were reported in 2%, randomization in 35%, and blinding in 20% (n = 482). In 2015, these metrics were included in 9, 47, and 36% of articles (n = 343). The increase in proportion for the metrics tested was statistically significant (p < 0.001, p = 0.002, and p < 0.001).ConclusionsOnly a minority of published manuscripts in critical care medicine journals reported on recommended study design steps to increase rigor. Routine justification for the presence or absence of blinding, randomization, and power calculations should be considered to better enable readers to assess potential sources of bias.

Reintroduction of the Rat for Experimental Subarachnoid Hemorrhage with Accelerated Clot Formation: A Low Mortality Model with Persistent Clots as a Precondition for Studies in Vasospasm.

Cerebral vasospasm as a delayed, possibly treatable sequel of subarachnoid hemorrhage (SAH) is a focus of experimental animal research. For this purpose, the rat is not a good model because of the difficulty creating a stable subarachnoid clot that persists > 1 to 2 days and could induce vasospasm. Only in rat models with a high mortality of ∼ 50% or more can SAH and its effects be investigated. Therefore, other animals than rodents are used for investigating the delayed effects of SAH. Only animal studies addressing the acute effects of SAH use rats. We designed a model that allows intensive clot formation combined with low mortality to facilitate studies on the delayed effects of experimental SAH, for example, delayed vasospasm or other alterations of vessels. After in vitro acceleration of the clotting process in the rats' blood by tissue factor and preliminary in vivo testing, we induced a SAH by injecting blood together with tissue factor in 22 rats. We analyzed clot expansion, length of clot persistence, chronic alterations, and histologic changes. The injection of blood supplemented by tissue factor led to persistent voluminous blood clots in the subarachnoid space close to the large arteries. Despite the pronounced SAH, all animals survived, allowing investigation of delayed SAH effects. All animals killed within the first 7 days after surgery had extensive clots; in some animals, the clots remained until postoperative day 12. During further clot degradation connective tissue appeared, possibly as a precursor of SAH-related late hydrocephalus. The injection of blood together with tissue factor significantly improves SAH induction in the rat model. This rat model allows studying delayed SAH effects as found in humans.

Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine

BackgroundEpstein-Barr virus (EBV) can cause cancer in people from around the world. There is no EBV vaccine available for use on a global scale. However, emerging evidence suggests that the epitope on the gp350/220 capsid protein may be developed into an EBV vaccine. Nevertheless, the production of small, single epitope is challenging of stability issues and possible alteration of peptide structure. In this study, a tandem epitope was developed consisting of three single epitopes, aimed to improve stability, antigenicity and preserve epitope structure. Materials and methodsA tandem epitope was designed using bioinformatics based on the epitope structure of the gp350/220 protein. The tandem epitope structure was analyzed using a protein folding method with Abalone software, which was further refined via YASARA force field and molecular repairing using a FoldX method. Immunogenicity was examined with Epitopia software, whereas allergen properties were tested using AlgPred. The pattern of the tandem epitope binding with anti-gp350/220 antibodies was performed using Z-dock and snugDock. The tandem epitope was then overproduced in E. coli strain BL21 as a host cell. ResultOur model demonstrated a successfully designed and overproduced tandem epitope. The tandem epitope demonstrated a similar structure compared with the epitope of whole protein gp350/220. Our epitope also demonstrated non-allergen and antigenicity properties, and possessed antibody binding patterns consistent with whole protein gp350/220. Conclusion and recommendationThese data suggest a novel tandem epitope composed of three similar epitopes demonstrates antigenicity, structure, and binding properties consistent with whole protein gp350/220. We also demonstrate successful production of the tandem epitope using E. coli strain BL21 as a host. Future in vivo experimental animal research is necessary to test the ability of this tandem epitope to stimulate antibody production.

Therapeutic efficacy of microtube-embedded chondroitinase ABC in a canine clinical model of spinal cord injury.

See Moon and Bradbury (doi:10.1093/brain/awy067) for a scientific commentary on this article.Many hundreds of thousands of people around the world are living with the long-term consequences of spinal cord injury and they need effective new therapies. Laboratory research in experimental animals has identified a large number of potentially translatable interventions but transition to the clinic is not straightforward. Further evidence of efficacy in more clinically-relevant lesions is required to gain sufficient confidence to commence human clinical trials. Of the many therapeutic candidates currently available, intraspinally applied chondroitinase ABC has particularly well documented efficacy in experimental animals. In this study we measured the effects of this intervention in a double-blinded randomized controlled trial in a cohort of dogs with naturally-occurring severe chronic spinal cord injuries that model the condition in humans. First, we collected baseline data on a series of outcomes: forelimb-hindlimb coordination (the prespecified primary outcome measure), skin sensitivity along the back, somatosensory evoked and transcranial magnetic motor evoked potentials and cystometry in 60 dogs with thoracolumbar lesions. Dogs were then randomized 1:1 to receive intraspinal injections of heat-stabilized, lipid microtube-embedded chondroitinase ABC or sham injections consisting of needle puncture of the skin. Outcome data were measured at 1, 3 and 6 months after intervention; skin sensitivity was also measured 24 h after injection (or sham). Forelimb-hindlimb coordination was affected by neither time nor chondroitinase treatment alone but there was a significant interaction between these variables such that coordination between forelimb and hindlimb stepping improved during the 6-month follow-up period in the chondroitinase-treated animals by a mean of 23%, but did not change in controls. Three dogs (10%) in the chondroitinase group also recovered the ability to ambulate without assistance. Sensitivity of the dorsal skin increased at 24 h after intervention in both groups but subsequently decreased to normal levels. Cystometry identified a non-significant improvement of bladder compliance at 1 month in the chondroitinase-injected dogs but this did not persist. There were no overall differences between groups in detection of sensory evoked potentials. Our results strongly support a beneficial effect of intraspinal injection of chondroitinase ABC on spinal cord function in this highly clinically-relevant model of chronic severe spinal cord injury. There was no evidence of long-term adverse effects associated with this intervention. We therefore conclude that this study provides strong evidence in support of initiation of clinical trials of chondroitinase ABC in humans with chronic spinal cord injury.

The Experimental Animal Research of Peroral Endoscopic Tunnel Technique in Diagnosis and Treatment of the Diseases Around Abdomen Aorta

Introduction: The emergence of endoscopic tunnel technique makes many diseases which used to need surgical or laparoscopic surgical treatments entered into the endoscopic therapy. The tunnel technique could well prevent the communication between the intra-luminal and the extra-luminal space by sealing the entry incision of the tunnel, gas or fluid within the lumen was prevented from entering the extra-luminal space, which could ensure the endoscopic therapy free of perforation. The procedure is endoscope penetrate from body's natural orifices such as esophagus, gastric, rectum, vagina and so on to body cavity for treatment. But there are few bottlenecks in the development of NOTES. First, it is difficult to close the entrance of natural orifices. Second, the infection which is caused by liquid and air of natural orifices enter into body cavity would occur. Third, it would be lost in body cavity. This research use endoscopic tunnel techniques to perform NOTES in experimental pigs, The tunnel will resolve the problem of closing entrance and infection. Methods: Established an esophagus submucosal tunnel about 5cm long to cardia at the back wall in pigs, Full-thickness myotomy performed and endoscope entered into abdominal cavity through the muscle incision. Exhaust air by puncturing into abdominal cavity. Partial hepatectomy and splenectomy, around the celiac artery ganglion neurolysis, partial tissue resection in the aera of posterior peritoneum were performed. Experimental pigs were prohibited water and diet intake and took antibiotics for 5 days. Their survival situations were observed. Three days later, experimental pigs were executed and were opened to observe the structures of abdominal cavity. Results: Experimental pigs were died after operating splenectomy. Experimental pigs were alive after successfully operating other surgeries. Three days later experimental pigs were executed and were found no injuries in other organs, but adhesion to different degrees of tissues in surgery position. Extensive intra-abdominal endoscopic therapy couldn't be carried out with limit of current endoscopic equipment. Conclusion: Partial hepatectomy and splenectomy, around the celiac artery ganglion neurolysis and partial tissue resection in the area of posterior peritoneum were all successfully performed in experimental animals through endoscopic tunnel technique and the complications were under controlled by internal medicine treatments.

* The Chorioallantoic Membrane Assay for Biomaterial Testing in Tissue Engineering: A Short-Term In Vivo Preclinical Model.

The fields of regenerative medicine and tissue engineering offer significant promise to address the urgent unmet need for therapeutic strategies in a number of debilitating conditions, diseases, and tissue needs of an aging population. Critically, the safety and efficacy of these pioneering strategies need to be assessed before clinical application, often necessitating animal research as a prerequisite. The growing number of newly developed potential treatments, together with the ethical concerns involved in the application of in vivo studies, requires the implementation of alternative models to facilitate such screening of new treatments. The present review examines the current in vitro and in vivo models of preclinical research with particular emphasis on the chorioallantoic membrane (CAM) assay as a minimally invasive, short-term in vivo alternative. Traditionally used as an angiogenic assay, the CAM of the developing chick embryo provides a noninnervated rapidly growing vascular bed, which can serve as a surrogate blood supply for organ culture, and hence a platform for biomaterial testing. This review offers an overview of the CAM assay and its applications in biomedicine as an in vivo model for organ culture and angiogenesis. Moreover, the application of imaging techniques (magnetic resonance imaging, microcomputed tomography, fluorescence labeling for tracking) will be discussed for the evaluation of biomaterials cultured on the CAM. Finally, an overview of the CAM assay methodology will be provided to facilitate the adoption of this technique across laboratories and the regenerative medicine community, and thus aid the reduction, replacement, and refinement of animal experiments in research.

Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System.

The use of animal models in chemical safety testing will be significantly limited due to the recent introduction of the 3Rs principle of animal experimentation in research. Although several in vitro assays to predict the sensitizing potential of chemicals have been developed, these methods cannot distinguish chemical respiratory sensitizers and skin sensitizers. In the present study, we describe a novel in vitro assay that can discriminate respiratory sensitizers from chemical skin sensitizers by taking advantage of the fundamental difference between their modes of action, namely the development of the T helper 2 immune response, which is critically important for respiratory sensitization. First, we established a novel three-dimensional (3D) coculture system of human upper airway epithelium using a commercially available scaffold. It consists of human airway epithelial cell line BEAS-2B, immature dendritic cells (DCs) derived from human peripheral blood CD14+ monocytes, and human lung fibroblast cell line MRC-5. Respective cells were first cultured in individual scaffolds and subsequently assembled into a 3D multi-cell tissue model to more closely mimic the in vivo situation. Then, three typical chemicals that are known respiratory sensitizers (ortho-phthaldialdehyde, hexamethylene diisocyanate, and trimellitic anhydride) and skin sensitizers (oxazolone, formaldehyde, and dinitrochlorobenzene) were added individually to the 3D coculture system. Immunohistochemical analysis revealed that DCs do not migrate into other scaffolds under the experimental conditions. Therefore, the 3D structure was disassembled and real-time reverse transcriptase-PCR analysis was performed in individual scaffolds to analyze the expression levels of molecules critical for Th2 differentiation such as OX40 ligand (OX40L), interleukin (IL)-4, IL-10, IL-33, and thymic stromal lymphopoietin. Both sensitizers showed similarly augmented expression of DC maturation markers (e.g., CD86), but among these molecules, OX40L expression in DCs was most consistently and significantly enhanced by respiratory sensitizers as compared to that by skin sensitizers. Thus, we have established a 3D coculture system mimicking the airway upper epithelium that may be successfully applied to discriminate chemical respiratory sensitizers from skin sensitizers by measuring the critical molecule for Th2 differentiation, OX40L, in DCs.

Regulation of microRNA-17-92 cluster on bone development, remodeling, and metabolism

To review the regulation of microRNA-17-92 cluster on bone development, remodeling, and metabolism. The related literature was reviewed. The clinical genetic phenotype, animal experiment, and cell research were illustrated so as to explore the possible regulatory mechanisms. MicroRNA-17-92 cluster is involved in physiological normal organs development, pathological neoplasm occurrence, and development. Recently, studies have shown that microRNA-17-92 cluster constitutes an intricate molecular signaling network with its upstream transcription factors and downstream targeting proteins, which controls bone development, remodeling, and metabolism exquisitely. Present fundamental researches have certain understanding of the regulatory mechanisms of microRNA-17-92 cluster on bone development, remodeling, and metabolism. However, the exact mechanisms under these processes remain unknown.