Preclinical Large Animal Studies Research Articles

Conceptualizing Scaffold Guided Breast Tissue Regeneration in a Preclinical Large Animal Model.

Scaffold-guided breast tissue regeneration (SGBTR) can transform both reconstructive and cosmetic breast surgery. Implant-based surgery is the most common method. However, there are inherent limitations, as it involves replacement of tissue rather than regeneration. Regenerating autologous soft tissue has the potential to provide a more like-for-like reconstruction with minimal morbidity. Our SGBTR approach regenerates soft tissue by implanting additively manufactured bioresorbable scaffolds filled with autologous fat graft. A pre-clinical large animal study was conducted by implanting 100 mL breast scaffolds (n = 55) made from medical-grade polycaprolactone into 11 minipigs for 12 months. Various treatment groups were investigated where immediate or delayed autologous fat graft, as well as platelet rich plasma, were added to the scaffolds. Computed tomography and magnetic resonance imaging were performed on explanted scaffolds to determine the volume and distribution of the regenerated tissue. Histological analysis was performed to confirm the tissue type. At 12 months, we were able to regenerate and sustain a mean soft tissue volume of 60.9 ± 4.5 mL (95% CI) across all treatment groups. There was no evidence of capsule formation. There were no immediate or long-term post-operative complications. In conclusion, we were able to regenerate clinically relevant soft tissue volumes utilizing SGBTR in a pre-clinical large animal model.

Porcine Model of Cerebral Ischemic Stroke Utilizing Intracortical Recordings for the Continuous Monitoring of the Ischemic Area.

Our aim was to use intracortical recording to enable the tracking of ischemic infarct development over the first few critical hours of ischemia with a high time resolution in pigs. We employed electrophysiological measurements to obtain quick feedback on neural function, which might be useful for screening, e.g., for the optimal dosage and timing of agents prior to further pre-clinical evaluation. Micro-electrode arrays containing 16 (animal 1) or 32 electrodes (animal 2-7) were implanted in the primary somatosensory cortex of seven female pigs, and continuous electrical stimulation was applied at 0.2 Hz to a cuff electrode implanted on the ulnar nerve. Ischemic stroke was induced after 30 min of baseline recording by injection of endothelin-1 onto the cortex adjacent to the micro-electrode array. Evoked responses were extracted over a moving window of 180 s and averaged across channels as a measure of cortical excitability. Across the animals, the cortical excitability was significantly reduced in all seven 30 min segments following endothelin-1 injection, as compared to the 30 min preceding this intervention. This difference was not explained by changes in the anesthesia, ventilation, end-tidal CO2, mean blood pressure, heart rate, blood oxygenation, or core temperature, which all remained stable throughout the experiment. The animal model may assist in maturing neuroprotective approaches by testing them in an accessible model of resemblance to human neural and cardiovascular physiology and body size. This would constitute an intermediate step for translating positive results from rodent studies into human application, by more efficiently enabling effective optimization prior to chronic pre-clinical studies in large animals.

Abstract P2104: Adeno-associated Virus Serotype 5 Is A Suitable Vector For S100a1-based Gene Therapy Of Post-ischemic Chronic Cardiac Dysfunction

Introduction: For S100A1-based heart failure gene therapies, AAV9 and 6 have shown efficacy in pre-clinical large animal studies. As AAV9 has shown concerning signs of toxicity in clinical studies and AAV6 displays poor production yields, there is need for a novel safe and cardiac-specific AAV serotype. Hypothesis: We hypothesized that in a pig model the safety proven and scalably manufacturable AAV5 may be a suitable vector for S100A1-based gene therapy of post-ischemic cardiac dysfunction. Methods: AAV production, 2h balloon-occlusion of the LCX, retrograde cardiac gene delivery, cardiac MRI, late gadolinium enhancement (LGE), global T1 relaxation, qPCR, RNA-Seq, WGCNA, KEGG, Reactome, LAD-ligation mouse model Results: In a comparative study of AAV5-, 6- and 9-luciferase (luc) in healthy farm pigs (n=5 each; 1x10 13 vgc/pig), AAV5 achieved a more homogeneous cardiac apical-basal transduction pattern than AAV6 with a higher luc activity than AAV9. In a clinically relevant endpoint driven study, we demonstrated a significant improvement in EF (+19 ± 5 %) 12 weeks after retrograde AAV5- S100A1 gene delivery compared to AAV5-luc in infarcted pigs (n=4 each; 1x10 13 vgc/pig). Moreover, S100A1 -treated pigs showed significantly less infarct extension (-0.5 ± 0.3 g vs. 5 ± 1.3 g (luc)) measured by cardiac MRI. There were no unfavorable alterations in blood chemistry or ECG. S100A1 expression was predominantly contained to the heart. The WGCNA unveiled a significant correlation between the improved EF and a suppression of inflammatory and immunological pathways (r=-0.96, p < 0.01) and between the absent infarct extension and enhanced activity of cardioprotective signaling (r=-0.82, p < 0.05). With injections of 2х10 11 vgc of AAV5- S100A1 or AAV5-gfp (n=4 each) into the remote myocardium in the mouse model, we confirmed a significant improvement in FS (+43.8 ± 8.8 %, vs. gfp) and suppression of inflammatory gene expression including i.e., IL1b or TNFa by S100A1. Conclusion: We conclude that AAV5 is suitable for S100A1-based gene therapy of post-ischemic cardiac dysfunction and that this vector/target combination can help accelerating the way towards a clinical trial. We also found novel signaling pathways that may be involved in S100A1’s therapeutic actions.

Glucosamine regulates macrophage function in heart failure.

Glucosamine regulates macrophage function in heart failure.

Hydroxylation of N-acetylneuraminic Acid Influences the in vivo Tropism of N-linked Sialic Acid-Binding Adeno-Associated Viruses AAV1, AAV5, and AAV6.

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy. However, a number of recent preclinical large animal studies failed to translate into the clinic. This illustrates the formidable challenge of choosing the animal models that promise the best chance of a successful translation into the clinic. Several of the most common AAV serotypes use sialic acid (SIA) as their primary receptor. However, in contrast to most mammals, humans lack the enzyme CMAH, which hydroxylates cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) into cytidine monophosphate-N-glycolylneuraminic acid (CMP-Neu5Gc). As a result, human glycans only contain Neu5Ac and not Neu5Gc. Here, we investigate the tropism of AAV1, 5, 6 and 9 in wild-type C57BL/6J (WT) and CMAH knock-out (CMAH−/−) mice. All N-linked SIA-binding serotypes (AAV1, 5 and 6) showed significantly lower transduction of the heart in CMAH−/− when compared to WT mice (5–5.8-fold) and, strikingly, skeletal muscle transduction by AAV5 was almost 30-fold higher in CMAH−/− compared to WT mice. Importantly, the AAV tropism or distribution of expression among different organs was also affected. For AAV1, AAV5 and AAV6, expression in the heart compared to the liver was 4.6–8-fold higher in WT than in CMAH−/− mice, and for AAV5 the expression in the heart compared to the skeletal muscle was 57.3-fold higher in WT than in CMAH−/− mice. These data thus strongly suggest that the relative abundance of Neu5Ac and Neu5Gc plays a role in AAV tropism, and that results obtained in commonly used animal models might not translate into the clinic.

The Application of Machine Learning to Identify Large Animal Blast Exposure Thresholds.

The Office of Naval Research sponsored the Blast Load Assessment Sense and Test program to develop a rapid, in-field solution that could be used by team leaders, commanders, and medical personnel to make science-based stand-down decisions for service members exposed to blast overpressure. Toward this goal, the authors propose an ensemble approach based on machine learning (ML) methods to derive a threshold surface for potential neurological deficits that encompasses the intensity of the blast events, the number of exposures, and the period over which the exposures occurred. Because of collection challenges presented by human subjects, the authors utilized data representing a comprehensive set of measures, including structural, behavioral, and cellular changes, from preclinical large animal studies on minipig models. This article describes the development process used to procure the resulting methodology from these studies. Using an ensemble of ML methods applied to experimental data obtained from 71 Yucatan minipigs, the relationship between blast exposure and neurological deficits was delineated. Despite a relatively small sample size, ML methods with k-fold cross-validation (with k = 5) were justified because of the complexity of the dataset reflecting numerous nonlinear relationships between cellular, structural, and behavioral markers. Based on the physiological responses and environmental measures collected during the large animal study, two models were developed to investigate the relationship between multiple outcome measures and exposure to blast. The histological features model was trained on single-exposure animal data to predict a binary injury response (injured or not) using histological features. The environmental features model related the observed behavioral changes to the environmental parameters collected. The histological features model predicted a binary injury outcome from cellular and physiological measurements. Features identified in developing this classification model showed some level of correlation to observed behavioral changes, suggesting that glial activation inflammation and neurodegenerative responses occur even at the lowest levels of blast exposures tested. The results of the environmental features model, which estimated injury risk from environmental blast exposure characteristics, suggested that the observed changes are not just a function of impulse but an average dynamic impulse rate. Noticeable behavioral deficits were observed at loading rates of 100 kPa (impulse/positive duration) or peak pressures of 300-350 kPa, with an approximate positive phase duration of 3.4 ms for single exposure. Based on this analysis, a 3D threshold surface was developed to characterize the potential risk of neurological deficits. The ensemble approach facilitated the identification of a pattern of changes across multiple variables to predict the occurrence of changes in brain function. Many changes observed after blast exposure were subtle, making them difficult to measure in human subjects. ML methodologies applied to minipig data demonstrated the value of these techniques in analyzing complex datasets to complement human studies. Importantly, the threshold surface supports the development of science-based blast exposure guidelines.

Development of a Minipig Model of BINT From Blast Exposure Using a Repeatable Mobile Shock Expansion Tube.

The Office of Naval Research (ONR) sponsored the Blast Load Assessment Sense and Test (BLAST) program to provide an approach to operationally relevant monitoring and analysis of blast exposure for optimization of service member performance and health. Of critical importance in this effort was the development of a standardized methodology for preclinical large animal studies that can reliably produce outcome measures that cannot be measured in human studies to support science-based guidelines. The primary advantage of this approach is that, because animal studies report physiological measures that correlate with human neuropathology, these data can be used to evaluate potential risks to service members by accounting for the anatomical and physiological differences between humans and large animal models. This article describes the methodology used to generate a comprehensive outcome measure dataset correlated with controlled blast exposure. To quantify outcomes associated with a single exposure to blast, 23 age- and weight-matched Yucatan minipigs were exposed to a single blast event generated by a large-bore, compressed gas shock tube. The peak pressure ranged from 280 to 525kPa. After a post-exposure 72-hour observation period, the physiological response was quantified using a comprehensive set of neurological outcome measures that included neuroimaging, histology, and behavioral measures. Responses of the blast-exposed animals were compared to the sham-treated cohort to identify statistically significant and physiologically relevant differences between the two groups. Following a single exposure, the minipigs were assessed for structural, behavioral, and cellular changes for 3 days after exposure. The following neurological changes were observed: Structural-Using Diffusion Tensor Imaging, a statistically significant decrement (P < .001) in Fractional Anisotropy across the entire volume of the brain was observed when comparing the exposed group to the sham group. This finding indicates that alterations in brain tissue following exposure are not focused at a single location but instead a diffuse brain volume that can only be observed through a systematic examination of the neurological tissue. Cellular-The histopathology results from several large white matter tract locations showed varied cellular responses from six different stains. Using standard statistical methods, results from stains such as Fluoro-Jade C and cluster of differentiation 68 in the hippocampus showed significantly higher levels of neurodegeneration and increased microglia/macrophage activation in blast-exposed subjects. However, other stains also indicated increased response, demonstrating the need for multivariate analysis with a larger dataset. Behavioral-The behavior changes observed were typically transient; the animals' behavior returned to near baseline levels after a relatively short recovery period. Despite behavioral recovery, the presence of active neurodegenerative and inflammatory responses remained. The results of this study demonstrate that (1) a shock tube provides an effective tool for generating repeatable exposures in large animals and (2) exposure to blast overpressure can be correlated using a combination of imaging, behavioral, and histological analyses. This research demonstrates the importance of using multiple physiological indicators to track blast-induced changes in minipigs. The methodology and findings from this effort were central to developing machine-learning models to inform the development of blast exposure guidelines.

Assessment of Electrospun Pellethane-Based Scaffolds for Vascular Tissue Engineering.

We examined the physicochemical properties and the biocompatibility and hemocompatibility of electrospun 3D matrices produced using polyurethane Pellethane 2363-80A (Pel-80A) blends Pel-80A with gelatin or/and bivalirudin. Two layers of vascular grafts of 1.8 mm in diameter were manufactured and studied for hemocompatibility ex vivo and functioning in the infrarenal position of Wistar rat abdominal aorta in vivo (n = 18). Expanded polytetrafluoroethylene (ePTFE) vascular grafts of similar diameter were implanted as a control (n = 18). Scaffolds produced from Pel-80A with Gel showed high stiffness with a long proportional limit and limited influence of wetting on mechanical characteristics. The electrospun matrices with gelatin have moderate capacity to support cell adhesion and proliferation (~30–47%), whereas vascular grafts with bivalirudin in the inner layer have good hemocompatibility ex vivo. The introduction of bivalirudin into grafts inhibited platelet adhesion and does not lead to a change hemolysis and D-dimers concentration. Study in vivo indicates the advantages of Pel-80A grafts over ePTFE in terms of graft occlusion, calcification level, and blood velocity after 6 months of implantation. The thickness of neointima in Pel-80A–based grafts stabilizes after three months (41.84 ± 20.21 µm) and does not increase until six months, demonstrating potential for long-term functioning without stenosis and as a suitable candidate for subsequent preclinical studies in large animals.

Large Animal Models in Regenerative Medicine and Tissue Engineering: To Do or Not to Do.

Rapid developments in Regenerative Medicine and Tissue Engineering has witnessed an increasing drive toward clinical translation of breakthrough technologies. However, the progression of promising preclinical data to achieve successful clinical market authorisation remains a bottleneck. One hurdle for progress to the clinic is the transition from small animal research to advanced preclinical studies in large animals to test safety and efficacy of products. Notwithstanding this, to draw meaningful and reliable conclusions from animal experiments it is critical that the species and disease model of choice is relevant to answer the research question as well as the clinical problem. Selecting the most appropriate animal model requires in-depth knowledge of specific species and breeds to ascertain the adequacy of the model and outcome measures that closely mirror the clinical situation. Traditional reductionist approaches in animal experiments, which often do not sufficiently reflect the studied disease, are still the norm and can result in a disconnect in outcomes observed between animal studies and clinical trials. To address these concerns a reconsideration in approach will be required. This should include a stepwise approach using in vitro and ex vivo experiments as well as in silico modeling to minimize the need for in vivo studies for screening and early development studies, followed by large animal models which more closely resemble human disease. Naturally occurring, or spontaneous diseases in large animals remain a largely untapped resource, and given the similarities in pathophysiology to humans they not only allow for studying new treatment strategies but also disease etiology and prevention. Naturally occurring disease models, particularly for longer lived large animal species, allow for studying disorders at an age when the disease is most prevalent. As these diseases are usually also a concern in the chosen veterinary species they would be beneficiaries of newly developed therapies. Improved awareness of the progress in animal models is mutually beneficial for animals, researchers, human and veterinary patients. In this overview we describe advantages and disadvantages of various animal models including domesticated and companion animals used in regenerative medicine and tissue engineering to provide an informed choice of disease-relevant animal models.

Requirements for Proper Immunosuppressive Regimens to Limit Translational Failure of Cardiac Cell Therapy in Preclinical Large Animal Models

Various cell-based therapies are currently investigated in an attempt to tackle the high morbidity and mortality associated with heart failure. The need for these therapies to move towards the clinic is pressing. Therefore, preclinical large animal studies that use non-autologous cells are needed to evaluate their potential. However, non-autologous cells are highly immunogenic and trigger immune rejection responses resulting in potential loss of efficacy. To overcome this issue, adequate immunosuppressive regimens are of imminent importance but clear guidelines are currently lacking. In this review, we assess the immunological barriers regarding non-autologous cell transplantation and immune modulation with immunosuppressive drugs. In addition, we provide recommendations with respect to immunosuppressive regimens in preclinical cardiac cell-replacement studies.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Perspectives on synthetic pharmacotherapy for the treatment of nosocomial pneumonia

ABSTRACTIntroduction: Nosocomial pneumonia is the second most common infection in hospital settings, resulting in substantial increases in morbidity, mortality, and length of hospital stay. The rapid increase in resistance of nosocomial pathogens to many antibiotics and the high dissemination of resistance genes highlight the need for innovative approaches to combat difficult-to-treat nosocomial respiratory infections.Areas covered: This review summarizes the synthetic antimicrobials that are currently in development for the treatment of nosocomial pneumonia, focusing on antibiotics in the final phases of clinical development and on the strategies employed by novel synthetic antimicrobial peptides.Expert opinion: Several novel synthetic antimicrobials are currently in the pipeline, and it appears that new antimicrobial peptides or mimetics will soon be made available, expanding the opportunities to treat nosocomial pneumonia. However, the approval process for use in the treatment of nosocomial pneumonia is arduous. Given that significant investments by pharmaceutical companies have ended in failure to obtain the approval of regulatory agencies, novel platforms for antimicrobial discovery are needed. The identification of new and fully synthetic chemical structures with activity against nosocomial pathogens needs to be followed by preclinical studies in large animals and by pharmacokinetic and pharmacodynamic studies in specific critically ill populations to assess lung penetration.

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach.

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies. This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

Diffuse Correlation Spectroscopy Analysis Implemented on a Field Programmable Gate Array.

Diffusive correlation spectroscopy (DCS) is an emerging optical technique that measures blood perfusion in deep tissue. In a DCS measurement, temporal changes in the interference pattern of light, which has passed through tissue, are quantified by an autocorrelation function. This autocorrelation function is further parameterized through a non-linear curve fit to a solution to the diffusion equation for coherence transport. The computational load for this non-linear curve fitting is a barrier for deployment of DCS for clinical use, where real-time results, as well as instrument size and simplicity, are important considerations. We have mitigated this computational bottleneck through development of a hardware analyzer for DCS. This analyzer implements the DCS curving fitting algorithm on digital logic circuit using Field Programmable Gate Array (FPGA) technology. The FPGA analyzer is more efficient than a typical software analysis solution. The analyzer module can be easily duplicated for processing multiple channels of DCS data in real-time. We have demonstrated the utility of this analyzer in pre-clinical large animal studies of spinal cord ischemia. In combination with previously described FPGA implementations of auto-correlators, this hardware analyzer can provide a complete device-on-a-chip solution for DCS signal processing. Such a component will enable new DCS applications demanding mobility and real-time processing.

Intravenous and Intra-amniotic &lt;em&gt;In Utero&lt;/em&gt; Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Differences in clotting parameters between species for preclinical large animal studies of cardiovascular devices

Several species of domestic animals are used in preclinical studies evaluating the safety and feasibility of medical devices; however, the relevance of animal models to human health is often not clear. The purpose of this study was to compare the clotting parameters of animal models to determine which animals most adequately mimic human clotting parameters. The clotting parameters of the different species were assessed in whole blood by in vitro thromboelastography using the clotting activators, such as tissue factor (extrinsic clotting screening test, EXTEM®) and partial thromboplastin phospholipid (intrinsic clotting screening test, IINTEM®). The measurements were performed using normal blood samples from humans (n = 13), calves (n = 18), goats (n = 56) and pigs (n = 8). Extrinsic clotting time (CT) and the intrinsic CT were significantly prolonged in calves compared to humans (249.9 ± 91.3 and 376.4 ± 124.4s vs. 63.5 ± 11.8 and 192.5 ± 29.0s, respectively, p < 0.01). The maximum clot firmness (MCF) in domestic animals (EXTEM®: 77-87mm, IINTEM®: 66-78mm) was significantly higher than that of humans (EXTEM®: 59.1 ± 6.0mm, IINTEM®: 58.8 ± 1.5mm, p < 0.01), and calves and goats exhibited longer time to MCF (MCF-t) than did humans and pigs (p < 0.01). Our results show that there are relevant differences in the four species' extrinsic and intrinsic clotting parameters. These cross-comparisons indicate that it is necessary to clarify characteristics of clotting properties in preclinical animal studies.

Electrospun vascular scaffold for cellularized small diameter blood vessels: A preclinical large animal study.

This study demonstrates the utility of tissue engineering to provide platform technologies for rehabilitation of patients recovering from severe, devastating cardiovascular diseases. The long-term goal is to provide alternatives to vascular grafting using bioengineered blood vessels derived from an autologous cell source with a functionalized vascular scaffold. This novel bilayered vascular construct for engineering blood vessels is designed to offer "off-the-shelf" availability for clinical translation.

Vector serotype screening for use in ovine perinatal lung gene therapy

PurposeSuccessful in utero or perinatal gene therapy for congenital lung diseases, such as cystic fibrosis and surfactant protein deficiency, requires identifying clinically relevant viral vectors that efficiently transduce airway epithelial cells. The purpose of the current preclinical large animal study was to evaluate lung epithelium transduction of adeno-associated viral (AAV) vector serotypes following intratracheal delivery. MethodsSix different AAV vector serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVrh10) expressing the green fluorescent protein (GFP) as the transgene were injected into the right upper lobe of perinatal sheep via bronchoscopy. At 1 week, samples were harvested, analyzed by fluorescent stereomicroscopy and immunohistochemistry, and quantified using a radial grid and quantitative real-time polymerase chain reaction (qPCR). ResultsFluorescent stereomicroscopy demonstrated GFP expression in the right upper lobe following injection of all AAV serotypes assessed except AAV5. Immunohistochemistry analysis confirmed GFP expression in small- and medium-sized airways following intratracheal injection of AAV1, 6, 8, 9, and rh10. However, only AAV8 and AAVrh10 resulted in transgene expression in large airways. These results were confirmed by qPCR, yet, after 40 cycles, AAV1 did not show GFP gene amplification. ConclusionAdeno-associated viral vector serotypes 6, 8, 9, and rh10 demonstrated efficient GFP transgene expression at early time points, and AAV8 demonstrated efficient transduction of all airway sizes with high pulmonary GFP expression tested using qPCR.

Developmental origins of metabolic disorders: The need for biomarker candidates and therapeutic targets from adequate preclinical models

The investigation on obesity and associated disorders have changed from an scenario in which genome drove the phenotype to a dynamic setup in which prenatal and early-postnatal conditions are determinant. However, research in human beings is difficult due to confounding factors (lifestyle and socioeconomic heterogeneity) plus ethical issues. Hence, there is currently an intensive effort for developing adequate preclinical models, aiming for an adequate combination of basic studies in rodent models and specific preclinical studies in large animals. The results of these research strategies may increase the identification and development of contrasted biomarkers and therapeutic targets.

A preclinical large animal study on a novel intervertebral fusion cage covered with high porosity titanium sheets with a triple pore structure used for spinal fusion.

To evaluate the osteoconductivity and the bonding strength of the newly developed interbody cage covered with the porous titanium sheet (porous Ti cage) to vertebral bodies in a sheep model. Twelve sheep underwent anterior lumbar interbody fusion at L2-3 and L4-5 using either the new porous Ti cages (Group-P) or conventional Ti cages with autogenous iliac bone (Group-C). The animals were euthanized at 2 or 4 months postoperatively and subjected to radiological, biomechanical, and histological examinations. Computed tomography analyses showed that the ratio of bone contact area in Group-P was significantly increased at 4 months compared with that at 2 months (p = 0.01). Although the ratio of bone contact area in Group-C was significantly higher than Group-P at 2 months (p < 0.001), there was no statistically significant difference between the two groups at 4 months. Biomechanical test showed that there was no significant difference in bonding strength between the two groups at either 2 or 4 months. Histological analyses revealed that the bone apposition ratio increased significantly with time in Group-P (p < 0.001). Although Group-C showed significantly higher bone apposition ratio than Group-P at 2 months (p = 0.001), there was no statistical difference between the two groups at 4 months. There was bone ingrowth into the porous Ti sheet, and bonding capacity of the porous Ti cage to the host bone increased with time. However, the speed of union to the bone with a porous Ti cage was marginally lower than a conventional cage along with an autogenous bone graft. Although it needs further experiment with a larger sample size, the results of the current study suggested that this material could achieve interbody fusion without the need for bone grafts.