SPECIES-SPECIFIC CHARACTERISTICS OF WIDELY USED LABORATORY ANIMAL MODELS AND THEIR IMPACT ON THE EFFICACY OF PRECLINICAL STUDIES FOR SMALL-DIAMETER BIODEGRADABLE VASCULAR PROSTHESES (A REVIEW)

Infectious diseases, which often result in deadly sepsis or septic shock, represent a major global health problem. For understanding the pathophysiology of sepsis and developing new treatment strategies, reliable and clinically relevant animal models of the disease are necessary. In this review, two large animal (porcine) models of sepsis induced by either peritonitis or bacteremia are introduced and their strong and weak points are discussed in the context of clinical relevance and other animal models of sepsis, with a special focus on cardiovascular and immune systems, experimental design, and monitoring. Especially for testing new therapeutic strategies, the large animal (porcine) models represent a more clinically relevant alternative to small animal models, and the findings obtained in small animal (transgenic) models should be verified in these clinically relevant large animal models before translation to the clinical level.

In this article, we reviewed the use of photodynamic therapy (PDT) for breast cancer (BC) in animal models. These in vivo models imitate the cancer disease progression, aid diagnosis, as well as create opportunities to assess treatment during the approval process for the new drug. BC ranks first among women's cancers. Nowadays, there are many diagnostic methods and therapy options for BC but the majority of them have severe side effects. This article discusses the advantages and some disadvantages of the use of small and large animals used for BC models. A literature review showed that the majority of studies have used large animal models, and recently there has been more interest in developing BC in small animal models. BC cell lines such as MCF-7, BT-474, MDA-MB-231, and 4T1 are commercially available for two-dimensional and three-dimensional in vitro cell cultures and subcutaneous models. The purpose of this article is to discuss the performance of PDT in animal models and its further clinical implications. PDT is known to be a non-invasive therapy, which uses monochromatic light and energy to excite photosensitizers (PSs) for the generation of reactive oxygen species as the required factors. Herein, we discuss the use of five photosensitizers in BC models such as chlorin e6 (Ce6), methylene blue, indocyanine green, 5-aminolevulinic acid, and meta-tetra(hydroxyphenyl)chlorin. The database PubMed and Scopus were searched for keywords: 'photodynamic therapy', 'breast cancer', 'animal model', 'clinical studies', and 'photosensitizer(s)'. The PDT search results in animal experiments and its effect on a living organism indicate the possibility of its application in clinical trials on women with local and disseminated BC. The availability and accessibility of small and large BC animal models enable the progress and trial of cancer drugs for innovative technologies and new diagnostics and treatments.

Thoracoabdominal aortic replacement, necessary in case of injuries, aneurysms and dissections, shows a high complication rate as a consequence of the perioperative ischemia / reperfusion-sequence (I/R). Clamping above and below the lesion leads to the spinal cord suffering from ischemia. Clamping times of less than 30 minutes show only a small risk of neurological deficit, while longer periods increase paraplegia rates disproportionately. The subsequent reperfusion as a second hit causes additional damage to the spinal cord. Up to 30 % of all patients who require being treated in the thoracoabdominal part of the aorta suffer from paraplegia within the first 24 postoperative hours (Kahn et al., 2012). While paraplegia following ischemia can be explained by the consequent death of motor neurons, reperfusion period is still poorly studied and understood. High metabolic activity and a need for substrate of the motor neurons aggravate I/R damage to the spinal cord (Sakurai et al., 1998). One of the triggers is free radicals that consume the available buffer enzymes (Gelman, 1995). Due to the loss of cellular energy (ATP), mediated by inhibition of mitochondrial phosphorylation, membrane pumps are inhibited. This in turn leads to a disequilibrium of the Na+ / K+ balance within the cell. What follows is an intracellular hyperkalemia with cellular edema and intracellular acidosis. Via other cellular mechanisms, this results in programmed cell death, namely apoptosis (Abe et al., 1995). Currently, there is no prophylaxis to avoid this damage in clinical practice of thoracic or abdominal aortic reconstruction. Although there are different approaches to this, no method has been implemented yet (Zvara, 2002). Hence, there is a need for better experimental approaches that can support the clinical treatment, in order to reduce the complication rate for thoracoabdominal aortic replacement. A crucial part in planning such an experimental project is to choose the best fitting model. It should be a combination of being as near to humans and to the clinical setting as possible. Furthermore it should give answers to present questions and upcoming more specific questions in the future. One animal model cannot serve all this demand so that several models are needed in order to choose the best fitting one for each experimental question. Therefore, inspired by experimental set-ups described in literature, our study group established three animal models to elucidate I/R of the spinal cord resulting in paraplegia. The large animal model was developed to offer the opportunity to map pathophysiology of aortic clamping in a clinical relevant porcine model to test potentially protective substances during I/R of the spinal cord. In this setting pigs were anesthetized and mechanically ventilated. In order to introduce two inflatable balloon catheters, femoral arteries were prepared via inguinal surgical cut downs. One of these balloon catheters was placed at the height of the left subclavian artery and the second one directly upstream of the aortic bifurcation (Figure 1). By inflating the balloons the blood flow was stopped by means of aortic occlusion in order to mimic an aortic crossclamping.Figure 1: Aortic anatomy with clamping locations.(1) Large animal model: One balloon catheter was placed at the height of the left subclavian artery and the second one directly upstream of the aortic bifurcation. (2) Medium sized animal model: Because of the strict segmental blood supply to the spinal cord in rabbits only infrarenal aortic crossclamping is needed. (3) Small animal model: Clamping of the aorta and left subclavian artery.When using such a model permission by the local authorities to let animals wake up again is not necessarily given, so other solutions need to be found in order to be able to control spinal cord function during anesthesia. Therefore motor evoked potentials (MEP) were recorded, reflex status was tested and histological staining was performed after harvesting of the spinal cord. The recording of the MEPs of the lower extremities presented potential damage of the spinal cord while the upper limbs served as technical proof. Corresponding to the low ischemic tolerance of the spinal cord, aortic occlusion was limited to 30 minutes and the follow-up period was extended up to 10 hours. Tissue damage was evaluated using hematoxylin and eosin, Nissl, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. In addition several plasma parameters for inflammation and oxidative stress were taken during the experiments (Simon et al., 2008; Simon et al., 2011). The advantages of such an animal model are plain to see, because there are two complementary aspects. On the one hand, anatomy and physiology of these animals are close enough to mimic the clinical situation of human patients and on the other hand one can use clinical equipment without special adjustments because of animal size. As anatomy is a very important factor in animal studies the pig is one of the most ideal animals to study ischemia and reperfusion of the spinal cord. Like humans, these animals provide blood to the spinal cord via many vessels of different origin. In humans the spinal cord receives blood supply through the unpaired anterior spinal artery and the two posterior spinal arteries. Both originate from the vertebral arteries. The anterior spinal artery also receives blood from segmental arteries of the aorta. Perfusion in pigs differs slightly from humans as the anterior spinal artery gets additional blood influx by an anastomosis with the median sacral artery that is much more pronounced in pigs than in humans. Therefore, as pigs are concerned, terminology is "aortic trifurcation" rather than bifurcation. Another advantage of a large animal model is that enough tissue and/or blood samples can be taken for several examinations whereas in small animal models these options are much more limited. At the same time, there are also clear disadvantages when pigs or sheep are being used for experimental models. Expenses for animals and for experimental set-up are much higher and it takes a lot more of manpower to realise such elaborate projects. Compared to mouse models costs can be several hundred times higher for each experiment. The required aortic occlusion near to the heart causes major hemodynamic imbalances and ischemia of all abdominal organs. Therefore, the large animal model should be carried out as a terminal test. For a better observation of the postoperative outcome and the effectiveness of various neuroprotective substances a rabbit model was established in which postoperative observation times up to 96 hours were performed. Even longer observation times are possible when needed for answering specific questions. Due to its special anatomy with strict segmental blood supply to the spinal cord the rabbit is an easy to use experimental model for studying clinical spinal ischemia-reperfusion injury (DeGirolami et al., 1982). Because of this segmental blood supply only infrarenal aortic crossclamping is needed to gain neuronal damage in the spinal cord level comparable to the thoracic crossclamping of the aorta in humans and pigs (Figure 1). The surgical impact can therefore be reduced to the size of a laparotomy. Due to the limited surgical trauma and the avoided ischemia of the kidney and visceral organs blood pressure could be kept stable and postoperative awakening was possible due to intact visceral organ function. The neurological examinations were done at 0, 6, 24, 36, 48, 60, 72, 84 and 96 hours postoperatively, using a modified Tarlov score. After 96 hours, the spinal cord was harvested for histopathological examination like hematoxylin-eosin staining. In literature different clamping times were mentioned, so, in this case, clamping times between 17 and 25 minutes were tested. Here, 22 minutes of aortic clamping was found to be the perfect ischemia duration to gain reproducible paraplegia. The rabbit model provides, in contrast to terminal experiments on large animals, important statements concerning the clinical effectiveness of pharmacological conditioning. Clear benefits of such a model using medium sized animals are the possibility for long reperfusion observation, relatively low costs in combination with reduced manpower needed. This model offers the opportunity to gain enough tissue and/or blood for examinations. Another positive effect is that administration of drugs that have to be given intravenously can be applied much easier than in animals of smaller size. Best vein in most of the cases is certainly one of the ear veins that can be cannulised with human peripheral venous catheter. Furthermore, more abstract results can be found than in a large animal model. Reasons for this can certainly be found at DNA level. One can see the differences among the species already in macroscopic anatomy as already described above, e.g. of the blood supply of the spinal cord. Additionally, when wanting to perform immunohistological stainings, it is very complicated to find working antibodies. The reason is that many antibodies are produced in rabbits and therefore are not specific working on rabbit tissue. So laboratory possibilities are limited by using this kind of animal model (Simon et al., 2015). Mouse models have some important advantages. The vascular anatomy in mice is more similar to humans than in other animals such as rabbits. In mice there is one anterior and two posterior spinal arteries responsible for the spinal cord blood supply (Lang-Lazdunski et al., 2000) while e.g., in rabbits there is a strict segmental blood supply. The anatomical vessel structure in mice is the reason for the clamping of the thoracic aorta that induces an ischemia of the associated spinal cord sections, accordingly to humans and the clinical problems. Despite in large animal models clamping procedure in mice only needs 7 minutes to produce paraplegia, which causes less damage to the visceral organs compared to 30 minutes occlusion procedure in pigs. Due to the size of the animals only direct clamping of the aorta via a thoracotomy is possible, since murine blood vessels are too small for balloon occlusion (Figure 1). After the clamping procedure (including closing chest and skin), it is possible to awaken these animals and to observe the experiment for several days. Furthermore, mouse models offer also the advantage of the variety of genetically altered mouse strains. This is a clear advantage of mouse models for human diseases compared to rabbits or pigs and, therefore, should be taken into account. An additional factor in planning experimental models, although of only from a financial viewpoint, is, that animal models in mice are ten times cheaper and even more when considering following costs like keeping of animals. A single mouse (weight 20–25 g; C57BL/6J mice) costs approx. € 24, while a New Zealand White Rabbit (weight 2.5–3 kg) of the same company costs about €204 each. Finally, it is rather obvious that small animal models have model typical disadvantages. As the animal is of smallest size your experiment is limited if it is based on many or large amounts of blood or tissue samples. As already mentioned above, a small animal size needs special equipment or procedures. For example when performing a thoracotomy, intubation and ventilation is a challenge for the inexperienced scientist and takes time to be learned. Special equipment is not only needed for doing anesthesia like e.g., special inhaling units etc., but also offers a broad spectrum of experimental possibilities like e.g., whole body laser doppler imaging, which becomes the more difficult the larger the animal is. All together, there are several aspects one should consider if thinking about building up an animal model. First of all it is important to understand that experimental models are only an attempt to mimic clinical situation and require a lot of discussion when conclusions are to be drawn with a view to humans. There will never be an ideal model. For a deeper understanding of a clinic problem or side-effect several models are needed so that the best fitting one can be chosen. This indicates that a precise formulation of the question to be answered is needed in advance. It requires a rather big budget, a lot of time and manpower to realise a large animal model that should take place in an animal intensive care. At the same time this has the benefit of more clinical relevant data acquisition. For those needing a simple model that is easy to learn and only needs an animal operation theatre but no animal intensive care unit, the rabbit model might be the right choice. It combines the small surgical impact with the possibility of long-time postoperative observation. When looking at questions dealing with DNA focus or needing animals of genetically altered strains the mouse model will fit best as it offers these advantages that normally cannot be found in other animal models. Eventually, when building up a new model one should take into account that research in literature will not replace teamwork with already experienced scientists in this field. Results will always differ from literature of other groups, because micromanagement differs. So each new experiment, especially when using a new animal model, needs a learning curve for building up one's own know-how.

The purpose of this review is to evaluate the effectiveness of large animal models in gynecology research and provide future perspectives. Gynecological diseases are diverse and pose a serious threat to women's physical and mental health. In addition to the commonly used small animal models, large animal models have gradually entered the field of gynecological research. Results suggest that large animal models offer significant advantages in simulating human physiological processes, despite ethical and practical challenges. This paper reviews the application of large animal models in the study of gynecological diseases, provides a summary of the research characteristics of large animal models, analyses the advantages and challenges of these models in disease research, and compares the research differences between large and small animal models. It also discusses the relationship between these models and new alternative models, with a view to providing more new ideas for the selection of animal models in the study of gynecological diseases.

Dental implants and bone augmentation are among dentistry's most prevalent surgical treatments; hence, many dental implant surfaces and bone grafts have been researched to improve bone response. Such new materials were radiologically, histologically, and histomorphometrically evaluated on animals before being used on humans. As a result, several studies used animals to evaluate novel implant technologies, biocompatibility, surgical techniques, and osseointegration strategies, as preclinical research on animal models is essential to evaluate bioactive principles (on cells, compounds, and implants) that can act through multiple mechanisms and to predict animal behavior, which is difficult to predict from in vitro studies alone. In this study, we critically reviewed all research on different animal models investigating the osseointegration degree of new implant surfaces, reporting different species used in the osseointegration research over the last 30 years. Moreover, this is the first study to summarize reviews on the main animal models used in the translational research of osseointegration, including the advantages and limitations of each model and determining the ideal location for investigating osseointegration in small and large animal models. Overall, each model has advantages and disadvantages; hence, animal selection should be based on the cost of acquisition, animal care, acceptability to society, availability, tolerance to captivity, and housing convenience. Among small animal models, rabbits are an ideal model for biological observations around implants, and it is worth noting that osseointegration was discovered in the rabbit model and successfully applied to humans.

A significant hurdle for potential cell-based therapies is the subsequent survival and regenerative capacity of implanted cells. While many exciting developments have demonstrated promise preclinically, cell-based therapies for intervertebral disc (IVD) degeneration fail to translate equivalent clinical efficacy. This work aims to ascertain the clinical relevance of both a small and large animal model by experimentally investigating and comparing these animal models to human from the perspective of anatomical scale and their cellular metabolic and regenerative potential. First, this work experimentally investigated species-specific geometrical scale, native cell density, nutrient metabolism, and matrix synthesis rates for rat, goat, and human disc cells in a 3D microspheroid configuration. Second, these parameters were employed in silico to elucidate species-specific nutrient microenvironments and predict differences in temporal regeneration between animal models. This work presents in silico models which correlate favorably to preclinical literature in terms of the capabilities of animal regeneration and predict that compromised nutrition is not a significant challenge in small animal discs. On the contrary, it highlights a very fine clinical balance between an adequate cell dose for sufficient repair, through de novo matrix deposition, without exacerbating the human microenvironmental niche. Overall, this work aims to provide a path towards understanding the effect of cell injection number on the nutrient microenvironment and the "time to regeneration" between preclinical animal models and the large human IVD. While these findings help to explain failed translation of promising preclinical data and the limited results emerging from clinical trials at present, they also enable the research field and clinicians to manage expectations on cell-based regeneration. Ultimately, this work provides a platform to inform the design of clinical trials, and as computing power and software capabilities increase in the future, it is conceivable that generation of patient-specific models could be used for patient assessment, as well as pre- and intraoperative planning.

Chapter 14 - Molecular Network for Management of Neurodegenerative Diseases and their Translational Importance using Animal Biotechnology as a Tool in Preclinical Studies

Assessing bone quality--animal models in preclinical osteoporosis research.

Nanofiber vascular grafts have been shown to create neovessels made of autologous tissue, by in vivo scaffold biodegradation over time. However, many studies on graft materials and biodegradation have been conducted in vitro or in small animal models, instead of large animal models, which demonstrate different degradation profiles. In this study, we compared the degradation profiles of nanofiber vascular grafts in a rat model and a sheep model, while controlling for the type of graft material, the duration of implantation, fabrication method, type of circulation (arterial/venous), and type of surgery (interposition graft). We found that there was significantly less remaining scaffold (i.e., faster degradation) in nanofiber vascular grafts implanted in the sheep model compared with the rat model, in both the arterial and the venous circulations, at 6months postimplantation. In addition, there was more extracellular matrix deposition, more elastin formation, more mature collagen, and no calcification in the sheep model compared with the rat model. In conclusion, studies comparing degradation of vascular grafts in large and small animal models remain limited. For clinical translation of nanofiber vascular grafts, it is important to understand these differences.

Vascular access animal models used in research

Experimental Heart Failure Models and Their Pathophysiological Characterization

Acute respiratory distress syndrome (ARDS) is a life-threatening, high mortality pulmonary condition characterized by acute lung injury (ALI) resulting in diffuse alveolar damage. Despite progress regarding the understanding of ARDS pathophysiology, there are presently no effective pharmacotherapies. Due to the complexity and multiorgan involvement typically associated with ARDS, animal models remain the most commonly used research tool for investigating potential new therapies. Experimental models of ALI/ARDS use different methods of injury to acutely induce lung damage in both small and large animals. These models have historically played an important role in the development of new clinical interventions, such as fluid therapy and the use of supportive mechanical ventilation (MV). However, failures in recent clinical trials have highlighted the potential inadequacy of small animal models due to major anatomical and physiological differences, as well as technical challenges associated with the use of clinical co-interventions [e.g., MV and extracorporeal membrane oxygenation (ECMO)]. Thus, there is a need for larger animal models of ALI/ARDS, to allow the incorporation of clinically relevant measurements and co-interventions, hopefully leading to improved rates of clinical translation. However, one of the main challenges in using large animal models of preclinical research is that fewer species-specific experimental tools and metrics are available for evaluating the extent of lung injury, as compared to rodent models. One of the most relevant indicators of ALI in all animal models is evidence of histological tissue damage, and while histological scoring systems exist for small animal models, these cannot frequently be readily applied to large animal models. Histological injury in these models differs due to the type and severity of the injury being modeled. Additionally, the incorporation of other clinical support devices such as MV and ECMO in large animal models can lead to further lung damage and appearance of features absent in the small animal models. Therefore, semi-quantitative histological scoring systems designed to evaluate tissue-level injury in large animal models of ALI/ARDS are needed. Here we describe a semi-quantitative scoring system to evaluate histological injury using a previously established porcine model of ALI via intratracheal and intravascular lipopolysaccharide (LPS) administration. Additionally, and owing to the higher number of samples generated from large animal models, we worked to implement a more sustainable and greener histopathological workflow throughout the entire process.

Recommendations of the National Heart, Lung and Blood Institute Heart and Lung Tolerance Working Group.

Increased life expectancy has led to an increase in the use of bone substitutes in numerous nations, with over two million bone-grafting surgeries performed worldwide each year. A bone defect can be caused by trauma, infections, and tissue resections which can self-heal due to the osteoconductive nature of the native extracellular matrix components. However, natural self-healing is time-consuming, and new bone regeneration is slow, especially for large bone defects. It also remains a clinical challenge for surgeons to have a suitable bone substitute. To date, there are numerous potential treatments for bone grafting, including gold-standard autografts, allograft implantation, xenografts, or bone graft substitutes. Tricalcium phosphate (TCP) and hydroxyapatite (HA) are the most extensively used and studied bone substitutes due to their similar chemical composition to bone. The scaffolds should be tested in vivo and in vitro using suitable animal models to ensure that the biomaterials work effectively as implants. Hence, this article aims to familiarize readers with the most frequently used animal models for biomaterials testing and highlight the available literature for in vivo studies using small and large animal models. This review summarizes the bioceramic materials, particularly HA and β-TCP scaffolds, for bone defects in small and large animal models. Besides, the design considerations for the pre-clinical animal model selection for bone defect implants are emphasized and presented.

Sensorineural hearing loss (SNHL), caused by pathology in the cochlea, is the most common type of hearing loss in humans. It is generally irreversible with very few effective pharmacological treatments available to prevent the degenerative changes or minimise the impact. Part of this has been attributed to difficulty of translating “proof-of-concept” for novel treatments established in small animal models to human therapies. There is an increasing interest in the use of sheep as a large animal model. In this article, we review the small and large animal models used in pre-clinical hearing research such as mice, rats, chinchilla, guinea pig, rabbit, cat, monkey, dog, pig, and sheep to humans, and compare the physiology, inner ear anatomy, and some of their use as model systems for SNHL, including cochlear implantation surgeries. Sheep have similar cochlear anatomy, auditory threshold, neonatal auditory system development, adult and infant body size, and number of birth as humans. Based on these comparisons, we suggest that sheep are well-suited as a potential translational animal model that bridges the gap between rodent model research to the clinical use in humans. This is especially in areas looking at changes across the life-course or in specific areas of experimental investigation such as cochlear implantation and other surgical procedures, biomedical device development and age-related sensorineural hearing loss research. Combined use of small animals for research that require higher throughput and genetic modification and large animals for medical translation could greatly accelerate the overall translation of basic research in the field of auditory neuroscience from bench to clinic.