Planar bone scintigraphy and CT findings in dogs with forelimb lameness

Scintigraphy has been used for many years in veterinary medicine, due to its high sensitivity, for the localization of lameness of unknown origin in horses and for the assessment of thyroid/skeletal neoplasia in dogs. In the last few years bone scintigraphy (BS) has become increasingly used in dogs for the localization of occult lameness, when clinical examination and radiographic exam are inconclusive [1]. This study describes BS and computed tomographic (CT) findings in dogs referred for monolateral forelimb obscure lameness, for which a precise localization had not been found by clinical examination nor radiographic exam (no abnormalities at all, mild radiological abnormalities which could not be related to the grade of lameness or symmetrical bilateral alterations). Eight dogs matched inclusion criteria: 3 mixed breed, 1 Bernese mountain dog, 1 Amstaff, 1 Labrador retriever, 1 Australian shepherd and 1 Boxer. BS images showed intense IRU (Increased Radiopharmaceutical Uptake) of elbow joint in 6 cases; these findings coincided to CT alterations of proximal ulna in 5 dogs (mostly located in the medial coronoid process - MCP region: bone density alterations, evidence of fragmentation, new bone formation). In one of these cases, an intense IRU was observed in correspondence of the region of the flexors attachment, with no concurrent abnormalities on CT examination. These findings were suggestive of an obscure form of flexor enthesopathy. In one case we observed diffuse and intense IRU of the carpus joint; this coincided with arthrosis and the presence of a subchondral cyst. One dog showed only mild IRU of the elbow joint, not compatible with the degree of lameness. Because of lack of significant IRU, CT and MRI examination were performed and revealed the presence of an expansive lesion in correspondence of the brachial plexus roots compatible with PNST (Peripheral Nerve Sheath Tumor). BS' high sensitivity allowed the localization of the lameness thanks to the assessment of functional bone state, as already stated in literature [2]. However, its low specificity required additional imaging (CT, MRI), targeted on the region identified on scintigraphic examination. In our experience, the combined use of functional and morphologic diagnostic imaging techniques (bone scintigraphy and computed tomography- magnetic resonance) has been helpful to reach a definitive diagnosis. Further studies, with an increased sample size, are needed to evaluate whether there is a correlation between bone density changes and grade of IRU in limbs affected by different pathologies

Cervical cystic lymphangioma in a young dog: CT findings

Lymphangioma is a rare lymphatic disorder; in veterinary medicine it is still considered a benign tumour, while in human medicine has been recently classified as a Lymphatic Malformation (LM), in fact it probably origins from a failure in development of connections between lympatic and venous system. Reported localizations of canine lymphangioma include skin, subcutaneous and fascial tissue of axilla, limbs, inguinal and mammary regions; lymph nodes; retroperitoneal space; nasopharynx. In the present report we describe the computed tomographic (CT) features of a cervical cystic lymphangioma in a young dog. A 1-year-old intact male Italian Shepherd dog was referred to the primary care veterinarian with a 1-month history of left ventrolateral neck swelling. No other clinical signs were present. Ultrasonographic (US) examination revealed a mass with hyperechoic thick wall, hypoechoic content with hyperechoic fluctuating areas. Fine needle aspiration biopsy (FNAB) revealed a cloudy pinkish fluid, citologically referable to serous-hematic fluid with chronic inflamation. For better assessment of the morphology and of the margins of the lesion, the dog was referred for CT examination. Pre- and post-contrast CT scan of head, neck and thorax were made. A mass located between the muscles of the caudal neck and thoracic/axillary regions (from the level of C4 to the level of T2) was found, which partially occupied the left visceral space of the neck and bulged into the thoracic inlet. The mass was ellipsoid-shaped (40x45x140 mm), with well-defined margins and heterogeneous soft tissue attenuation. It was apparently capsulated, with fluid-like content and soft tissue attenuating septa and small areas within the fluid. Adjacent to the mass three areas of soft tissue mineralization, smoothly marginated, were found. It was responsible for mild mass effect, without significant compression on the surrounding structures. Mild left axillary and left medial retropharyngeal lymphadenomegaly was noted, with normal shape and attenuation of the limph nodes. Post-contrast images showed moderate enhancement of the mass wall and the soft-tissue-attenuating areas/septa within it; no enhancement of the fluid was noted. No other abnormalities were found. The mass was completely surgically excised and submitted for histopathologic analysis, with a definitive diagnosis of cystic lymphangioma. In human literature CT features of lymphangioma are widely described, while in veterinary literature there are no reports about its CT appearance; moreover, human lymphangioma is described as preferentially located in the neck and axillary regions, while, to our knowledge, cervical localization has never been reported before for canine lymphangioma

Novel contributions in canine craniometry : anatomic and radiographic measurements in newborn puppies

The largest differences in intraspecific head shape among the Carnivora order are to be found in dogs. Based on their skull morphotypes, dog breeds are currently classified as dolichocephalic, mesaticephalic and brachycephalic. Due to the fact that some breeds have not been yet defined, this classification is incomplete; moreover, multi-breed studies on the skull morphology of puppies have never been performed. The aim of this work was to verify (i) whether differences in the skull conformation of purebred puppies are already present within the first week of age; (ii) whether radiographic and anatomic measures could be considered interchangeable, and (iii) to possibly classify puppies from non-categorized breeds thanks to their radiographic cranial measurements using neural nets. One hundred and thirty-seven dead puppies aged 0-7 days were examined considering their anatomic and radiographic measures. All linear measures and anatomic indices significantly differed among brachycephalic and non-brachycephalic puppies. Radiographic indices, with the exception of CI, identified the three skull morphotypes (p<0.05, for all comparisons). Radiographic and anatomic measures proved to be non-interchangeable in newborn puppies. Finally, nineteen puppies belonging to 5 non-categorized breeds could be classified thanks to neural nets in the three skull morphotypes with different probability (P between 0,66 and 0,95)

Cartilage canals in newborn dogs : histochemical and immunohistochemical findings

Cartilage canals (CCs) are microscopic structures involved in secondary ossification centers (SOCs) development. The features of CCs were investigated in the humeral and femoral proximal epiphyses of small-sized newborn dogs (from premature to 28 days after birth) with histochemical and immunohistochemical approaches. Masson's Trichrome revealed a ring-shaped area around CCs, which changes in colour from green (immature collagen) to red (mature collagen) as ossification progresses; perichondrium staining always matched the ring colour. Safranin-O was always negative. Immunohistochemical analysis revealed immunopositivity for both collagen type I and V around the CCs; collagen type II was negative. CCs count showed a tendency to be higher in the humerus than in the femur. This work enlightened for the first time changes in composition of CCs surrounding matrix during SOCs development in dogs, paving the way to further investigations

Meniscus maturation in the swine model: role of endostatin in cellular differentiation

The development of an engineered meniscus derives from the need to regenerate a tissue which is largely unable to self-repair with consequent loss of functionality. Hence a deeper knowledge of the native meniscus morphology and biomechanics in its different regions, including molecules involved in regulation of the maturation process, is essential. The meniscus is a complex tissue, displaying great regional variation in extracellular matrix components and in vascularization, as a result of several biomechanical stimuli. Its biochemical composition is modulated to adapt the tissue to the different functions that are required throughout growth, until a \u201cmature\u201d phase is reached in adulthood. The aim of this work is to evaluate the biological role of Endostatin in the regulation of angiogenesis as in the fibro-chondrogenic differentiation of neonatal meniscal cells in the pig. The swine is an attractive model for meniscal repair studies, as its knee joint is closely comparable to the human one in terms of anatomical structure, vascularization, and healing potential. Our preliminary data show that Endostatin contributes to the acquisition of chondrocyte phenotype in an undifferentiated but committed cellular population. Thus, a better understanding of the role of Endostatin in cell metabolism might lead to a deeper knowledge of the events regulating meniscus maturation. These findings may be crucial for the development of an engineered scaffold able to induce meniscal cell differentiation by releasing Endostatin-rich microspheres

Swine cortical and cancellous bone: histomorphometric and densitometric characterisation

Introduction: Swine bone morphology, composition and remodelling are similar to humans\u2019, therefore they are considered good models in bone-related research. They have been used for several studies involving bone growth, bone and cartilage fractures and femoral head osteonecrosis. Nevertheless, the literature about pig normal bone features is incomplete. This work aims to fill the literature gaps on the microarchitecture and Bone Mineral Density (BMD) of swine femoral diaphysis and distal epiphysis and tibial plateau and diaphysis. Materials and methods: Five hind limbs were collected from slaughtered 80-100 kg pigs. Microscopic analysis of cortical and cancellous bone from middle/distal femur and proximal/middle tibia was performed to determine basic histomorphometric parameters at different sites. Dual-energy X-Rays Absorptiometry was also employed to evaluate BMD. ANOVA and correlation between BMD, bone area (BA) and cortical thickness were performed. Results and discussion: Diaphyseal cortical bone was mostly plexiform both in the tibia and the femur; primary/secondary osteons without clear organization were also found. Mean values for bone area, bone perimeter, trabecular width, number and separation and BMD at different anatomical sites were defined. No significant difference was found for these values at different anatomical sites. BMD proved to be positively correlated with cortical thickness (r=0,80; p<0,01). Despite the small sample size, these results seem homogeneous. They could therefore represent reference values for normal bone parameters in pigs. Applied anatomy and regenerative medicine, in fact, demand very precise information about bone micromorphology, composition and density to provide reliable indication in bone substitutes building. Moreover, since the interpretation of bone abnormalities is based on mastering normal bone characteristics, the definition of reference parameters is mandatory to avoid misinterpretation and allow comparative evaluation. Conclusion: The results of this study, although preliminary, may be considered a dependable starting point for the definition of normal bone features in pigs

Ultrastructural and matrix evaluation of morpho-functional age-related changes in dog meniscus

Menisci are essential structures for the knee joint. Different attempts were made trying to replace or regenerate the meniscus after its tear, but the perfect solution is still far away. A better knowledge of the physiologic development of this structure through time could be useful to understand its behavior in the light of the tissue bio-engineering. In this study, the changes in canine meniscal morphology were evaluated to assess how it varies among diverse age stages. The fibers arrangement and matrix deposition in canine menisci from neonatal (died at birth), 10-days, 30-days and adult dogs, dead for causes not related to the present study, were evaluated by means of histochemistry (safranin-O and Sirius red staining), polarized light microscopy, immunofluorescence (collagen I and II) and Scanning Electron Microscopy (SEM). Moreover, quantitative measurements of glycosaminoglycans (GAGs), DNA and GAGs/DNA ratio were performed. The \u201cknotty\u201d structure of neonatal meniscus is probably due to balls of collagen fibres that are not completely stretched until the 30-days stage (Fig 1). The stretching of the fibres starts from the inner portion that is probably the first and the most compressed zone. Safranin-O staining shows how matrix composition vary during growth. Neonatal meniscus is characterized by a huge number of elongated cells (fibroblast-like) and GAGs, features that characterized a still afunctional tissue. With growth, more and more cells assumed a rounded shape. The end-point of the maturation process is represented by the adult meniscus: it is characterized by almost only rounded cells (fibro-chondrocyte-like), in small number, and surrounded by matrix (Fig 1). Nevertheless, 10-30 days interval could be considered the starting point of the meniscus specialization and maturation. Fibres arrangement starts like balls of collagen fibres that follow a disorganized pattern in the neonatal meniscus (Fig 1). In 10-days meniscus, these balls of fibres tend to disappear starting from the meniscus\u2019 inner portion, in association with an initial organization of the fibres according to the longitudinal and radial axes of the meniscus. The organization of fibres network is almost complete at 30-days of life, when all the fibres follow the two main axes of the meniscus and show a well-organized disposition, as seen in adult meniscus. Through the double immunofluorescence it is possible to recognized different aspect of maturation (Fig 3). Neonatal meniscus shows almost only collagen type I fibres. Collagen type I and II co-expression starts at 10 days (yellow) and become more evident in 30-days meniscus in which even a differentiation of the inner and the outer zone starts. The same differentiation persist in adult meniscus that is characterized by a frankly fibro-chondrocitic-like cellular phenotype. Biochemical analysis confirmed that cellularity decrease over the time starting from neonatal to adult (Fig 3). The same decreasing trend is observed in GAGs deposition. Even if 30-days meniscus present a lot of common characteristics with the adult one, the GAGs/DNA ratios show how the latter is the only that present a maturely functional tissue in which a small number of cells is able to produce a matrix rich of GAGs. Meniscal structure changes during growth. The starting point is represented by the neonatal tissue, rich of immature cells and with poor expression of matrix components. The end-point is the adult tissue, characterized by phenotypically mature cells, which assure a functional matrix deposition. Ten-thirty days interval seems to be the turning point of this developmental process. This work highlights how dog meniscal structure changes its morphology among different age stages; this fact may suggest a role of the biomechanical forces, physiologically acting on meniscus, in the development of its ultimate shape and functions. The knowledge of the developmental process of a structure has a capital importance to comprehend its physiologic anatomy and function

THE BONE TISSUE: MULTIDISCIPLINARY CHARACTERISATION IN EXPERIMENTAL ANIMALS AND SKELETAL DEVELOPMENT IN DOGS

Animals are considered sentient beings by law and the notion of animal welfare, both physical and psychological, is well-established (\u201cFive Freedoms\u201d). As a result, regulation on animal testing puts researchers through highly regulated and restrained protocols (to reduce the number of animals included in in vivo experiments and replace them with alternative procedures whenever possible. This project revolves around the study of the bone tissue in different species, grounding on the idea of cadaver \u201crecycle\u201d applied to bone-related research. The bone, in fact, is particularly easy to study in cadavers since it suffers the early consequences of post-mortal decay to a lesser extent compared to other tissues. The bone is a highly specialized connective tissue characterized by a mineralized extracellular matrix, specifically designed for structural, mechanical and metabolic functions. Despite general shared features, interspecific differences exist in bone microscopic organisation, which have not yet been completely elucidated. Because of its peculiarities, a comprehensive evaluation of the bone includes several different techniques, such as histology, densitometry and biomechanics. Histologic techniques are particularly common, especially when it is necessary to evaluate tissue biocompatibility and integration in animal models of bone healing and regeneration. However, due to highly mineralized nature of its matrix, several problems arise during bone processing, especially during decalcification (the removal of mineral from the bone while preserving all the essential microscopic elements and tissue antigenicity), a process that is yet far from being standardized, but is necessary to prepare paraffin-embedded specimens. The first two aims of this project were the definition of species-specific and site-specific guidelines for bone specimens\u2019 decalcification and the characterization of swine and ovine bone tissue with a multidisciplinary approach. Basic protocols for trabecular bone decalcification in different species with different solutions were provided, showing that excellent morphologic results can be achieved with solution containing strong acids in rodents, sheep and pig. As a general rule, hydrochloric acid solution proved to be too aggressive, as previously reported. On the other hand, the combined use of hydrochloric acid and formic acid proved to be a very good compromise, shortening experimental time and preserving morphology. Finally, a combination of citric acid formic acid provided excellent morphology, but at the expenses of decalcification time. Collagen I was preserved in swine, canine and ovine samples that proved to have good/excellent morphology, while it wasn\u2019t preserved in rats or mice, despite their excellent morphology. A multidisciplinary approach to the study of the bone in pigs and sheep was developed, evaluating several different parameters with different techniques on the same samples, also providing standard reference parameters about swine and ovine bone histology, densitometry and biomechanics, to be compared with future research results, to deepen the knowledge on the bone tissue and facilitate biomedical research on animal models. The histogenesis of bone can be divided in two modalities: endochondral and intramembranous, which share a common sequence of events. Bone growth starts during the fetal stage, and then continues after birth together with the growth of the entire individual. Special structures, called cartilage canals, are involved in the process of endochondral ossification, with nutrients supply to the growing cartilage and waste products elimination being their primary functions. Cartilage canals have been described in several species, but little information is known about them in dogs. The development of a newborn into an adult implies dramatic changes in both body size and shape. The dog, in particular, exhibits the highest intraspecific differences in body shape and size among mammals, and this might be reflected by different growth patterns. This is especially true when the skull is considered, as it is the most variable body part among different breeds. Little information is known about skull development in dogs in relation to their adult craniometric category (brachycephalic, mesaticephalic, dolychocephalic). Due to the extreme plasticity of young bones, moreover, skeletal development could potentially be influenced by the presence of orthopaedic diseases, which could modify the normal shape and size of the future adult bones. The last three aims of this project were the study of endochondral ossification in long bones, with particular emphasis on the role role of cartilage canals; the study of the effects of pathology (quadriceps contracture) on hindlimb development; and the study of skull development and its compared features in brachycephalic, mesaticephalic and dolychocephalic breeds. New insights on cartilage canals in small-sized dogs were provided, which confirmed their involvement in bone extracellular matrix production in dogs, as in other species, laying the basis for more extensive studies on the delicate mechanism of the formation of ossification centres and the regulation of ossification. The abnormalities induced by quadriceps contracture on hind limb skeletal development, mainly consisted of volume reduction and abnormal shape of several centres, were described in a litter of Dobermann. These alterations that should be carefully considered when evaluating puppies affected by the disease to plan a therapy. Finally, novel contribution in canine craniometry and skull development were provided, documenting morphometric differences among dolichocephalic, mesaticephalic and brachycephalic purebred puppies in the early neonatal period through a radiographic and anatomic study. Cadavers invariably proved to be an invaluable source of research material, providing a valid alternative to in vivo experiments and contributing to the reduction of the use of experimental animals

Age assessment in puppies : coming to terms with forensic requests

Age estimation in growing dogs is crucial not only in clinical practice but increasingly so in forensic practice as well. In the last few years, it has assumed great importance for correctly identifying the age of puppies illegally imported to Italy as well as to other European countries. Puppies are, in fact, transported when they are too young to be moved, which can cause both animal/public health and animal welfare issues. Therefore, the movement of animals within the European Community is governed by strict rules, and veterinarians are often required to evaluate the age of the imported puppies in a forensic scenario as accurately as possible. To date, X-ray evaluation of limb bones ossification centers (OCs) is generally accepted as a valid tool to assess the age of puppies. A wealth of information exists on this topic but it is not always easily available. This work is a historical review of the existing literature and proposes two tables illustrating the timelines of limb OCs appearance and closure, coming to terms with forensic requests to evaluate the age of a puppy. The timelines reported indicate the need to improve methodology to enhance the accuracy and to reduce the error in age estimation

Dynamics and regulatory mechanisms involved in immature oocyte chromatin remodeling

During meiotic arrest, and particularly during the oocyte growth phase leading to the formation of fully-grown oocyte, the chromatin enclosed within the oocyte nucleus, also known as Germinal Vesicle (GV), is subjected to several levels of regulation controlling both its structure and function. These events include mechanisms acting both locally, on specific loci, and on a large scale to remodel wide portions of the oocyte genome. Morphologically, the chromosomes lose their individuality as well as their characteristic appearance and form a loose chromatin mass, which in turn undergoes profound and dynamic rearrangements within the GV before the meiotic resumption. These \u2018large-scale chromatin configuration changes\u2019 are temporally correlated with the process of transcriptional silencing in the oocyte nucleus as well as with epigenetic modifications such as histone tail modifications and changes in the global level of DNA methylation. Moreover, chromatin configuration rearrangements are tightly associated with the acquisition of meiotic and developmental competence. The molecular mechanisms governing changes in large-scale chromatin configuration still remain largely unknown. Most likely, strategies set in place for the control and coordination of these events are part of a complex physiological process that ultimately confers the oocyte with meiotic and developmental competence. Here, we summarize some studies intended to explain the mechanism(s) regulating this complex process