Adipose-Derived Stem Cell-Incubated HA-Rich Sponge Matrix Implant Modulates Oxidative Stress to Enhance VEGF and TGF-ß Secretions for Extracellular Matrix Reconstruction In Vivo.

This study demonstrated both adipose-derived stem cells (ASCs) in vitro and in vivo combined with three-dimensional (3D) porous sponge matrices on implant wound healing. Sponge matrices were created from hyaluronic acid (HA), collagen (Col), and gelatin (Gel), constructing two types: HA-L (low content) and HA-H (high content), to be cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Fourier transform infrared spectroscopy method verified carboxyl groups of HA and amino groups of Col and Gel reacting between the raw materials and scaffolds to identify the successive cross-linking. The swelling ratios of two types of sponge matrices were analyzed by water absorption capabilities, and the results displayed both over 30-fold dry scaffold weight enhancements. In biodegradation tests, matrices were hydrolyzed over time by three cutaneous enzymes, hyaluronidase, lysozyme, and collagenase I. ASCs from rats were cultured within the HA-H scaffold, demonstrating higher antioxidative abilities and secretions on related genes and proteins compared to the other two groups. The ASC HA-H matrix promoted cell proliferation to stimulate capillary angiogenesis inducer secretions, including vascular endothelial growth factor (VEGF) and transforming growth factor-ß (TGF-ß). In vivo histological examinations showed ASCs from implanted HA-H implant transported into the subcutis, and rat skin cells also infiltrated into the original matrix zone to increase the extracellular matrix (ECM) reconstructions. Our experimental data revealed that the ASC HA-H sponge implant was effective in improving wound repair.

Morphometrics of nuchal ligament in greyhound in different neck and body positions.

This study was conducted to describe the morphometrics of nuchal ligament and investigate the effects of different neck and body positions on the nuchal ligament in greyhounds. Nine adult greyhounds cadavers without any locomotion abnormalities were dissected through the neck musculature on the left side to expose the nuchal ligament. Three pins were placed to mark regions of interest on the nuchal ligament: at one cm cranial to the site of origin (the most dorsal point of the spinous process of the first thoracic vertebra), at the midpoint of the nuchal ligament and one cm caudal to the nuchal ligament site of insertion (close to the caudal aspect of the spinous process of the axis). Each cadaver was positioned on a masonite board and placed on a table on the floor in their lateral recumbency and seven different standardized body positions; P1-P7 were mimicked using goniometers and metal wires. Photographs were taken by positioning and fixing the camera above the nuchal ligament region. The length and widths (W1, W2 and W3) of nuchal ligament were measured using Image Pro software (Image-Pro Express version 5.0) on standardized photographs of each of seven different body and neck positions. The length of nuchal ligament in relation to the neutral position (P1) was less (- 7%, p > 0·05) in P6 (neck elevated) and increased in all other positions (+1%, p > 0·05 for P2, +19%, p < 0·05 for P3, +37%, p < 0·05 for P4, +1%, p > 0·05 for P5, +40%, p < 0·05 for P7). Nuchal ligament width at the middle (W2) decreased significantly with P4 (- 26%, p < 0·05), and P7 (- 32%, p < 0·05). Also, nuchal ligament width at the site of origin (W3) decreased significantly with P4 (- 24%, p < 0·05) and P7 (-35%, p < 0·05). These findings reflect the need for clinical and biomechanical studies to describe in-depth the gross anatomy of the nuchal ligament in greyhounds. They suggest that different neck and body positions change the shape, and hence, the function of the nuchal ligament during movement.

Observations of different types of sacra in Greyhounds based on the occurrence of sacrocaudal fusion.

Little attention has been paid to the normal fusion of the vertebrae of greyhounds despite the common occurrence of sacrocaudal fusion. The current study aimed to investigate and provide data on the morphology of different types of fused sacra (B, C and D) in greyhounds and also to determine the potential association between the sex, body mass and morphology of fused sacra (S. Weight, S. Length and S. Width) in greyhounds. The sacra were collected from 171 greyhounds from Melbourne, Australia. After classifying the sacra based on the occurrence and types of the sacrocaudal fusion, they were measured for weight of the sacrum, length of sacrum and the width of sacrum. Multiple linear regression analyses were used to quantify the association between weight of the sacrum (as the outcome variable) type of sacrum (A, B, C and D), body mass and sex (as explanatory variables). The results proved that there are measurable differences between each type of fused sacra (B, C and D) and the standard sacra (A). In addition, this study showed that sex or body mass do not influence the occurrence of different types of fusion. The results of this study showed that the occurrence of sacrocaudal fusions was independent of body size in this population of greyhounds. Sacrocaudal fusion might affect the biomechanics in greyhounds independently of effects of body size.

Structural anatomy and morphometric analyses of sacra in greyhounds.

This study was conducted to provide structural and morphological data on the sacra of greyhounds. Descriptive quantitative investigation was carried out on 171 sacra of greyhound`s cadavers and then classified into standard and fused sacra based on the number of fused sacral vertebrae. The weight, length and width of sacrum of sacra were measured. Both standard (59%) and fused sacra (41%) were identified. The average length and width of the standard sacrum were found to be 46.14 ± 2.53 mm and 57.89 ± 3.54 mm, respectively. The sacral length was 1.61-mm longer in males (p < .01), and the sacral width was 0.46-mm shorter in males but not significant (p = .51). The average weight of a standard sacrum was 26.54 ± 4.55 g and was 1.18 g heavier in males but not statistically significant (p = .24). Results showed that one-kilogram increase in the body mass was associated with a 0.3 mm (p < .001) increase in sacral length, and a 0.54 mm (p < .001) increase in sacral width, respectively. The morphological data of the standard and fused sacra provided in this study might help the veterinary community to improve treatment and rehabilitation and help the trainer to design the right training protocol for racing greyhounds. In addition, the results of this study are a step to understand the sacrum's functions and how the greyhound's body functions and future studies are required to investigate the biological importance of these findings.

Characterisation of ovine lymphatic vessels in fresh specimens.

BACKGROUND AND AIM: The development and use of experimental models using lymphatic cannulation techniques have been hampered by the lack of high-quality colour imaging of lymphatic vessels in situ. Most descriptions of lymphatic anatomy in sheep have historically depended on schematic diagrams due to limitations in the ability to publish colour images of the lymphatic vessels with decent resolution. The aim of this work was to encourage more widespread use of the ovine cannulation model by providing clear photographic images identifying the location and anatomical layout of some major lymphatic ducts and their in situ relationship to surrounding tissues. METHODS: The cadavers of the sheep were collected after they had been euthanized at the end of animal trials not associated with this study. The lymphatics were dissected and exposed to show their appearance in the surrounding tissues and their relationship to other organs. Patent Blue was used to locate lymphatic vessels in exploratory preparations. However, in order to present the natural appearance of the vessels, we used minimal dissection and dye was not used for the photographed examples. Instead, we have indicated the course of the vessels with lines where their position is less clear. RESULTS AND CONCLUSION: In this paper, we have used sheep specimens as examples to show characteristic images of lymphatic vessels. The images of in situ lymphatics and lymph nodes combined with schematic summaries provide a concise illustration of the lymphatic drainage scheme in sheep.

Lymphatic cannulation models in sheep: Recent advances for immunological and biomedical research.

Lymphatic cannulation models are useful tools for studying the immunobiology of the lymphatic system and the immunopathology of specific tissues in diseases. Sheep cannulations have been used extensively, as models for human physiology, fetal and neonatal development, human diseases, and for studies of ruminant pathobiology. The development of new and improved cannulation techniques in recent years has meant that difficult to access sites, such as mucosal associated tissues, are now more readily available to researchers. This review highlights the new approaches to cannulation and how these, in combination with advanced omics technologies, will direct future research using the sheep model.

Development of an ovine efferent mammary lymphatic cannulation model with minimal tissue damage.

BACKGROUND: Two mammary lymphatic cannulation models in sheep have been described with minimal use in the past 50 years. The purpose of this study was to investigate a new surgical technique to allow long term monitoring of mammary lymph flow and composition from the mammary glands, with rapid ewe recovery and minimal complications post-surgery. RESULTS: We developed a modified methodology for cannulating the efferent mammary lymphatic from the mammary lymph node with minimum tissue damage. Compared to the previous models, our method required only a small incision on the aponeurosis of the external abdominal oblique muscles and thus reduced the difficulties in suturing the aponeurosis. It allowed for lymph collection and assessment for at least one week post-surgery with concurrent milk collection. CONCLUSION: This method allows for good ewe recovery post-surgery and in vivo sampling of efferent mammary lymph from the mammary lymph nodes in real-time and comparison with milk parameters.

Techniques for thoracic duct cannulation without thoracotomy in piglets.

BACKGROUND: Pigs are the natural hosts of many zoonotic pathogens such as influenza viruses and Staphylococcus aureus and thus have advantages over non-natural hosts when studying these zoonotic diseases. In addition, pulmonary infections are a key issue in the pig industry, for example: porcine reproductive and respiratory syndrome virus infection. Exploration of the pathogenesis of swine pulmonary infections, in particular at the onset of disease, will provide valuable information for the development of vaccines against these diseases. Therefore, there is need to develop a methodology that allows for in vivo sampling of efferent pulmonary lymph with minimum damage to the target tissues for studying the pathogenesis of swine pulmonary infections. RESULTS: We introduce the surgical procedures for cannulating the thoracic duct at its point of entry into the external jugular vein cranial to the first rib on the left in pigs. Using this methodology, we monitored the amounts of triglyceride and cholesterol in the lymph collected from the thoracic duct following 30 h fasting and at multiple time points after meals. It was found that the levels of triglyceride rather than cholesterol corresponded to the milky appearance of the lymph samples. CONCLUSIONS: Our techniques provide a strategy for collecting lymph including pulmonary lymph from the thoracic duct without thoracotomy. A pig model for collecting in vivo, in situ efferent lymph draining the lower respiratory tract and its local lymph nodes in real-time with minimal tissue damage to the target tissues opens a new door for studying disease processes in pulmonary infections. Techniques described here are the key to this door.

Observations of sacrocaudal fusion in Greyhounds and other dogs.

OBJECTIVES: To describe the incidence and forms of nonpathological sacrocaudal fusion in racing Greyhounds and compare them with those in a variety of other domestic dog breeds. METHODS: This retrospective observational study used archived anatomical specimens from 81 racing Greyhounds and 10 Beagles, and archived clinical radiographs from 81 non-Greyhound dogs representing 37 other breeds. Dogs less than two years of age and dogs with evidence of soft tissue or osseous pathology involving the sacrocaudal region were excluded. The incidence of osseous sacrocaudal fusion (any type and complete fusion) was compared between Greyhounds and all of the other dogs combined, using the Fisher's exact test. RESULTS: Sacrocaudal fusion of some type was found in 33 (41%) of 81 Greyhounds but in only 14 (15%) of 91 non-Greyhound dogs (p <0.01). Complete fusion (osseous fusion of vertebral bodies and both transverse and articular processes) between the sacrum and the first caudal vertebra was the most common form in Greyhounds, found in 27 (33%) of 81 Greyhounds, but in only three (3.3%) of 91 non-Greyhound dogs (p <0.01). CLINICAL SIGNIFICANCE: Sacrocaudal fusion appears to be more prevalent in Greyhounds than in other domestic dog breeds and may be attributable to selection pressure for speed on a region of the spine that is naturally prone to variation. Its significance for performance and soundness requires further study.

Anatomical basis for the development of a thoracic duct cannulation model without thoracotomy in Large White pigs.

BACKGROUND: To collect lymph draining the lungs provides a useful strategy for tracing pulmonary microvascular fluid and protein biology. A methodology that allows for in vivo sampling of efferent pulmonary lymph in real-time in sheep by cannulating the thoracic duct without entering the thoracic cavity was previously established. To develop a similar thoracic duct cannulation model without thoracotomy in pigs, we investigated the anatomy of the left cervico-thoracic regions of 15 Large White (Yorkshire or Yorkshire-dominated) piglets (aged 4-7 weeks). RESULTS: The thoracic duct, together with the left tracheal trunk, joined the cardiovascular system (the ampulla of the thoracic duct) at a site located craniomedial to the first rib on the left in 80 % (12/15) of the piglets. CONCLUSIONS: As the location of the ampulla of the thoracic duct was consistent in most of the piglets, Large White piglets appear to be suitable for the development of a thoracic duct cannulation model without thoracotomy. The anatomical findings in this study will enable the development of further surgical procedures for cannulating the thoracic duct without thoracotomy, with minimal damage to local tissue, and without transecting any major blood vessels, nerves or muscle bellies. The establishment of a thoracic duct cannulation model for collecting in vivo, in situ efferent lymph, including pulmonary lymph, in pigs without entering the thoracic cavity would be invaluable for many immunological studies, studies on pulmonary immune responses in particular.

Biological activity of ovine IL-23 expressed using a foot-and-mouth disease virus 2A self-cleaving peptide.

Hetero-dimeric cytokines often require equi-molar expression of both subunits to achieve biological activity. Previously, we expressed ovine IL-12 p40 and p35 linked using a self-cleaving 2A peptide from foot-and-mouth disease virus (FMDV). We now generated a new improved vector for the expression of hetero-dimeric cytokines and demonstrate the more general applicability of this strategy by cloning and expressing ovine IL-23 using the 2A peptide to link IL-12/IL-23 p40 and p19. The resulting protein was shown to be biologically active when expressed in mammalian COS cells. IL-23 plays a significant role in the differentiation of Th17 cells as well as autoimmunity and the regulation of inflammatory processes. As such this reagents will be invaluable in the unravelling of regulation of the ovine immune system for both veterinary and human animal model applications.

Mucosal vaccination: lung versus nose.

The induction of potent mucosal immune responses able to prevent the establishment of infection at the onset of mucosal pathogen colonisation represents a desirable but challenging goal for vaccine development. Here we compare nasal vaccine delivery with intra-pulmonary vaccination using a sheep lymphatic cannulation model. Our results demonstrate that nasal delivery of a non-infective ISCOMATRIX(®) influenza vaccine does not induce primary immune responses in the lymph draining the nasal lymph nodes, suggesting that local immune responses in the lymph nodes draining the nasal cavity are relatively weak. However, this mode of delivery can boost existing immunity in the nasal lymph. Using the same adjuvant we were able to induce very potent immune responses in both blood and bronchoalveolar lavage (BAL), following intra-pulmonary delivery of ISCOMATRIX(®) influenza vaccine, even when very small doses of antigen were employed. Lung delivery could also induce comparable immune responses against other recombinant antigens mixed with ISCOMATRIX(®) adjuvant and could therefore become a method of choice for the induction of immunity to mucosal pathogens infecting the lower respiratory tract.

Thoracic duct cannulation without thoracotomy in sheep: a method for accessing efferent lymph from the lung.

We have developed and validated a novel method to access efferent lymph draining the lung and gut of sheep. In this model, efferent lymph derived from the lung could be collected via cannulation of the thoracic duct just prior the thoracic duct-jugular vein junction. The thoracic duct was accessed in the neck region without needing to broach the thoracic cavity, thus avoiding extensive tissue damage to the animal and need for ventilation during surgery. In addition, this surgical approach allows for a second cannulation of an adjacent lymphatic draining the head/neck region, providing for an 'in-built' internal control with which to compare lymph parameters. To test the verity of cannulation procedure, a test protein ovalbumin (OVA) was infused into the left and right lungs via bronchoscopy. We found that OVA was recovered almost exclusively in the lymph draining the lungs compared to the lymph draining the head/neck where it was essentially non-existent. The method described here will be invaluable for optimizing intra-lung delivery of drugs or vaccines. In addition, access to lymph will also allow for analysis of immune responses to infections originating at this site.

Co-delivery of plasmid-encoded cytokines modulates the immune response to a DNA vaccine delivered by in vivo electroporation.

In this study, in vivo electroporation of a DNA vaccine adjuvanted with plasmids encoding different cytokines was investigated in large animals. Sheep were injected intramuscularly with a DNA vaccine encoding an antigen of Haemonchus contortus (pNPA) and plasmids encoding different cytokines followed by in vivo electroporation. Plasmids (pCI) carrying the genes of different cytokines including ovine IL-4(pCI-IL4), IL-10(pCI-IL10), GM-CSF(pCI-GMCSF), and MCP-1alpha(pCI-MCP1alpha), and pCI-IL4+pCI-GMCSF were co-delivered with pNPA. The results showed that co-delivery of pCI-GMCSF or pCI-IL4+pCI-GMCSF significantly enhanced both antibody responses and T cell proliferation responses to the antigen after two DNA immunisations compared to co-delivery of pCI. In contrast, antibody responses of the sheep that received pCI-IL10 were decreased significantly. Other cytokine expressing plasmids did not significantly alter the measured immune responses. Furthermore, co-delivery of pCI-GMCSF increased IgG2 response more than IgG1 responses, suggesting a Th1 bias. However, the increase in IgG2 over IgG1 was less apparent when co-delivery of pCI-IL4 with pCI-GMCSF. Interestingly, the co-delivery of pCI-IL4 alone did not increase the IgG1 titre, suggesting that both pCI-GMCSF and pCI-IL4 are required for optimal IgG1 production. Thus, co-delivery of plasmid-encoded cytokine genes with in vivo electroporation has the ability to effectively modulate immune responses to a DNA vaccine in a large animal.

Local immune responses following nasal delivery of an adjuvanted influenza vaccine.

A key barrier to producing effective nasal immunisations is the low efficiency of uptake of vaccines across the nasal mucosa. Using a recently developed cannulation system, we examined the antibody response induced by nasal immunisation with an ISCOMATRIX influenza vaccine. This showed for the first time, that following nasal vaccination, specific antibodies enter the circulation of primed animals via the draining lymphatics as a wave that peaks approximately 5-6 days after vaccination. These antibodies included some of the IgA isotype and possessed functional haemagglutination inhibition activity. These responses, though small, were induced using a very simple delivery system, emphasising the applicability of this cannulation model for evaluation of excipients and adjuvants aimed at improving intranasal vaccine efficacy.

A sheep cannulation model for evaluation of nasal vaccine delivery.

We have developed and validated a novel model to investigate the efficacy of nasal vaccine delivery. Based on lymphatic cannulation of the tracheal lymph trunk of sheep, the model allows collection of lymph draining from the Nasal Associated Lymphoid Tissue. The model is suitable for determining both the amount of material that is absorbed into the lymphatic system, following intra-nasal delivery and the immune response that occurs following vaccination into the nasal area. The cell populations that track in this duct were phenotyped and found to be similar to those previously reported to be present in efferent lymph draining from peripheral lymph nodes. Following intra-nasal spray, we demonstrated that the amount of material recovered in draining lymph is only a very small fraction of the total delivered. Nevertheless, intra-nasal spraying of a vaccine could activate local immune cells. The method described will be invaluable for optimising intra-nasal delivery systems by allowing a separate optimisation of vaccine uptake and immune responses induction.

Veterinary applications of cytokines.

In the recent past a large variety of cytokines have been cloned for most important veterinary species and more is planned with development of a coordinated approach to cytokine reagents production. Application of these cytokines in veterinary species can be found in the development of effective diagnostics, with the IFN-gamma-based detection of tuberculosis as a prime example. In addition, cytokines have been used to determine which immune responses are essential for immune protection with flow-on effects for the development of novel ways to induce these specific immune responses. The realisation that the murine immune system is quite different from the human, together with the increased availability of cytokine reagents for many large animals plus unique experimental approaches only available in these animals, has lead to an explosion in the use of veterinary species as models for human diseases.