Histomorphological evaluation of thermal injury following palatoplasty performed with CO2 laser or LigaSure device in dogs with brachycephalic obstructive airway syndrome.

The elongated soft palate is an abnormality that characterizes most brachycephalic dogs and contributes to the brachycephalic obstructive airway syndrome (BOAS). Palatoplasty is routinely performed in brachycephalic dogs; several surgical techniques exist. The use of surgical instruments such as monopolar electrocoagulation, CO2 or diode laser, bipolar vessel sealing device and harmonic shears has become routine to reduce the operating time, the intraoperative risk of bleeding and the postoperative oedema. This prospective study aimed to compare the histomorphological effect of a CO2 laser and LigaSure device in palates of dogs undergoing palatoplasty. Twenty owned brachycephalic dogs were included, 10 palatoplasties were performed using CO2 laser and 10 using LigaSure™ device. The dogs were positioned in sternal recumbency. A transoral approach was performed: the elongated soft palate was grasped with Allis forceps and brought rostrally, the palatoplasty was performed using the tonsillar crypts as anatomical landmarks. Surgical specimens were routinely fixed in 10 % formalin. Two sections perpendicular to the surgical margins were trimmed from each sample, paraffin-embedded and stained with hematoxylin and eosin (H&E). Tissue damage induced by the two types of surgical devices was graded (1-4, from minimal to severe) and the depth of thermal injury measured in µm on captured images (using an image analysis program - ImageJ). Mean values and standard deviations (SD) were calculated based on six measurements for each sample. The tissue damage was graded 3.7±0.48 in group LigaSure™ and 2.8±1 in group Laser. The mean depth of thermal injury was 874.94±184.92 µm in the LigaSure™ group and 451,76±137,86 µm in the Laser group. The comparison between the two groups showed significant lower grade and extension of thermal injury in the palate samples obtained with CO2 laser (p<0.05). Additionally, there is a lack of literature that correlates the histological changes with the clinical outcomes of the different palatoplasty methods in brachycephalic dogs. By comparing histological changes and clinical outcomes, we aim to provide valuable insights for optimizing the surgical approach for palatoplasty in brachycephalic dogs, ultimately improving postoperative outcomes for these patients.

Unusual tongue metastasis from lung adenocarcinoma in a cat with feline lung-digit syndrome.

This report describes the pathological findings in a 15-year-old spayed female Domestic Shorthaired cat with a pulmonary adenocarcinoma characterized by feline lung-digit syndrome (FLDS) and unusual tongue metastasis. Felis catus papillomavirus type 3 (FcaPV-3) DNA was amplified from the lingual sample but not from samples of the pulmonary mass or digital or splenic metastatic lesions, indicating the presence of FcaPV-3 in the oral cavity but not suggesting a role for FcaPVs in tumour pathogenesis. FLDS is a clinical entity in which primary lung tumours present because of metastatic digital lesions. In humans, tongue metastasis may be a rare initial presentation of lung cancer, whereas, to the best of our knowledge, tongue metastasis of feline tumours has not been reported. Although lingual metastases are rare, the present findings serve to remind clinicians that metastatic manifestations of primary lung tumours in cats may involve multiple extrapulmonary sites, including the tongue.

p63 immunoexpression in hair follicles of normal and alopecia X-affected skin of Pomeranian dogs.

BACKGROUND: Alopecia X in Pomeranians is caused by a hair cycle deregulation, associated with downregulation of key regulatory genes of the Wnt and Shh pathways, and stem-cell markers. However, the pathogenesis remains unclear. p63 is an important transcription factor correlated with the aforementioned hair cycle modulating genes. HYPOTHESIS/OBJECTIVES: The aim of this study was to highlight possible changes of p63 immunohistochemical expression within the hair follicles in canine alopecia X compared with normal skin. ANIMALS: Skin biopsies from 19 alopecia X-affected and six control Pomeranians were analysed. MATERIALS AND METHODS: Serial histological sections of skin biopsies harbouring anagen, telogen and kenogen hair follicles were immunohistochemically evaluated for differences in p63 expression in the affected and control samples. RESULTS: Dogs with alopecia X had a significantly decreased immunoexpression of p63 in telogen and kenogen hair follicles. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease of p63 immunoexpression observed in canine alopecia X suggests an involvement of p63 in hair cycle.

Volumetric assessment and longitudinal changes of subcortical structures in formalinized Beagle brains.

High field MRI is an advanced technique for diagnostic and research purposes on animal models, such as the Beagle dog. In this context, studies on neuroscience applications, e.g. aging and neuro-pathologies, are currently increasing. This led to a need for reference values, in terms of volumetric assessment, for the structures typically involved. Nowadays, several canine brain MRI atlases have been provided. However, no reports are available regarding the measurements' reproducibility and little is known about the effect of formalin on MRI segmentation. Here, we assessed the segmentation variability of selected structures among operators (two operators segmented the same data) in a sample of 11 Beagle dogs. Then, we analyzed, for one Beagle dog, the longitudinal volumetric changes of these structures. We considered four conditions: in vivo, post mortem (after euthanasia), ex vivo (brain extracted and studied after 1 month in formalin, and after 12 months). The MRI data were collected with a 3 T scanner. Our findings suggest that the segmentation procedure was overall reproducible since only slight statistical differences were detected. In the post mortem/ ex vivo comparison, most structures showed a higher contrast, thereby leading to greater reproducibility between operators. We observed a net increase in the volume of the studied structures. This could be justified by the intrinsic relaxation time changes observed because of the formalin fixation. This led to an improvement in brain structure visualization and segmentation. To conclude, MRI-based segmentation seems to be a useful and accurate tool that allows longitudinal studies on formalin-fixed brains.

Strain and Shear-Wave Elastography and Their Relationship to Histopathological Features of Canine Mammary Nodular Lesions.

Mammary gland tumours have a significant impact on the health of dogs, requiring diagnostic tools to support clinicians to develop appropriate therapeutic strategies. Sonoelastography is an emerging technology that is able to define the stiffness of the tissue and has promising applications in the evaluation of mammary gland lesions. In the present study, strain elastography (STE) and shear-wave (SWE) elastography were compared in 38 mammary nodular lesions for their ability to define the histopathological features of canine mammary lesions. Among the techniques, SWE showed better repeatability (intraclass correlation coefficient: 0.876), whereas STE was found to be only acceptable (intraclass correlation coefficient: 0.456). Mammary nodular lesions showed a wide range of tissue stiffening with a similar mean value for STE and SWE in benign (4 ± 0.3 and 115.4 ± 12.6 kPa, respectively) and malignant lesions (3.8 ± 0.1 and 115.5 ± 4.5 kPa, respectively). A significant correlation was found between lesion fibrosis and STE (STE-I: r = 0.513, p < 0.001; STE-R: r = 0.591, p < 0.001) or SWE-S (r = 0.769; p < 0.001). In conclusion, SWE was reliable and correlated with fibrosis and was similar for both benign and malignant lesions, suggesting that other collateral diagnostic techniques should be considered in conjunction with SWE to characterize mammary nodular lesions in dogs.

Comparison between Image-Guided Transbronchial Cryobiopsies and Thoracoscopic Lung Biopsies in Canine Cadaver: A Pilot Study.

To date, the only method of sampling lung tissue with a high diagnostic yield is represented by surgical lung biopsies (SLB), which are highly invasive and have a high risk/benefit ratio. In humans, transbronchial lung cryobiopsies (TBLC) have recently been introduced, which are described to be less invasive and able to significantly increase diagnostic confidence in most patients with interstitial lung diseases. The aim of this study was to evaluate the feasibility and diagnostic yield of TBLC compared to SLB in small animals. A total of 21 pulmonary cryobiopsies under fluoroscopic and real-time CT fluoroscopic guidance and 21 video-assisted thoracoscopic surgery (VATS) lung biopsies were collected from three dog cadavers. Upon histological examination, cryobiopsy samples were smaller than VATS biopsies, but were still large enough to reach a specific diagnosis or to allow pattern recognition. Morphological features on TBLC and SLB were concordant in all cases. Cryobiopsy samples showed fewer artifacts and a higher percentage of alveolar tissue than VATS samples. TBLC is a feasible and useful alternative to SLB for lung histopathological examination in dogs. The effectiveness and reduced invasiveness of TBLC compared to SLB could represent many advantages in the diagnosis of diffuse lung diseases in small animals.

Histological Evaluation of Long-Term Collagen Type I Culture.

Histological approach to long-term culture on collagen type I permits the evaluation of vasculogenic mimicry morphological features and the identification of endothelial-like cell-specific antigens. Here, we show the preparation of collagen type I solution, the embedding and the sections cutting of D17 osteosarcoma cells long-term culture, and then the hematoxylin and eosin (H&E) staining to identify endothelial-like structure. Moreover, we provide the protocols for periodic acid-Schiff (PAS) staining to evidence glycoproteins and CD31 immunohistochemistry to exclude the presence of this endothelial marker, as per definition by vasculogenic mimicry concept. This method allows to consider long-term culture as tissue, promoting the deeper study of vascular-like structures in vitro.

A Magnetic Resonance-Relaxometry-Based Technique to Identify Blood Products in Brain Parenchyma: An Experimental Study on a Rabbit Model.

Magnetic resonance relaxometry is a quantitative technique that estimates T1/T2 tissue relaxation times. This has been proven to increase MRI diagnostic accuracy of brain disorders in human medicine. However, literature in the veterinary field is scarce. In this work, a T1 and T2-based relaxometry approach has been developed. The aim is to investigate its performance in characterizing subtle brain lesions obtained with autologous blood injections in rabbits. This study was performed with a low-field scanner, typically present in veterinary clinics. The approach consisted of a semi-automatic hierarchical classification of different regions, selected from a T2 map. The classification was driven according to the relaxometry properties extracted from a set of regions selected by the radiologist to compare the suspected lesion with the healthy parenchyma. Histopathological analyses were performed to estimate the performance of the proposed classifier through receiver operating characteristic curve analyses. The classifier resulted in moderate accuracy in terms of lesion characterization.

New insights about Haplosporidium pinnae and the pen shell Pinna nobilis mass mortality events.

Since early autumn 2016, Mass Mortality Events (MME) have drastically impacted the population of the fan mussel Pinna nobilis in the Mediterranean Sea. Haplosporidium pinnae, a newly described Haplosporidian species, has been considered the causative agent of the mortality outbreaks in association to opportunistic bacterial pathogens. In the present study, we first reported a cytological description of H. pinnae in moribund specimens of P. nobilis which were collected in the Gulf of Taranto (Ionian Sea, Italy) during summer 2018. Different life-cycle stages of the parasite, including uni- and binucleate cells, small plasmodia, big multinucleate plasmodia and sporocysts with spores, were detected in all the examined animals and most of the parasite cells were present in gills, mantle and digestive gland, while the spores were found only in the latter organ. Histology and molecular biology were also performed, confirming the nature of the infectious agent, as already reported in the area. Additionally, molecular study revealed the presence of bacteria from the Mycobacterium ulcerans - M. marinum complex but no evident macroscopical or microscopical lesions, just as no bacteria referred to Mycobacterium were observed by histology. In conclusion, the present study aimed to provide further contributions to the understanding of the mortality of P. nobilis, pointing to the role of the cytological method of investigation both for diagnostic and epidemiological purposes, and discussing the current epidemic situation in the Adriatic sea.

Health and Mortality Monitoring in Threatened Mammals: A First Post Mortem Study of Otters (Lutra lutra L.) in Italy.

Dead specimens provide valuable data for the conservation of threatened species, allowing investigations of mortality, health conditions, and demographic parameters. The Eurasian otter (Lutra lutra) is a semiaquatic carnivore listed as endangered in Italy. In 2009, we started the first post mortem (PM) study of otters in Italy, through collaborative research between mammal ecologists and veterinary pathologists, using standardized protocols. Twenty-eight otters, mostly collected between 2009 and 2017, were examined. Most otters were males (67%), between 1 and 3 years old (64%), and predominantly in good nutritional condition. Adult males were significantly larger than adult females (p < 0.02), as expected for the species, although both sexes appeared to be smaller than otters examined in Central−northern Europe. The youngest sexually mature female was 3 years old. Road traffic collisions were the major cause of death, especially in young individuals, and mainly occurred in autumn−winter, particularly for females. Investigations of the scene of death contributed to revealing factors forcing otters to travel out of the water and move over the road, suggesting appropriate measures to reduce vehicle collision risk. Other causes of death included blunt chest trauma of uncertain origin, dog and conspecific attacks, or diseases of infectious or non-infectious origin, such as ulcerative gastritis, pleuropneumonia and peritonitis. Other diagnosed diseases included lymphoma. Ecto- and endoparasites were rarely detected, although we report the first documentation of heartworm and Ixodes hexagonus infestation in Italian otters. It is important to continue comprehensive, standardized PM investigations of otters in Italy to define baseline health, biometric and demographic parameters, collect biological samples for comparative analyses, and to reduce road-kill mortality. The present study suggests that the timely collection of carcasses and collaborative and coordinated research efforts are essential for obtaining useful data for the conservation of otters.

An Update on Molecular Pathways Regulating Vasculogenic Mimicry in Human Osteosarcoma and Their Role in Canine Oncology.

Canine tumors are valuable comparative models for human counterparts, especially to explore novel biomarkers and to understand pathways and processes involved in metastasis. Vasculogenic mimicry (VM) is a unique property of malignant cancer cells which promote metastasis. Thus, it represents an opportunity to investigate both the molecular mechanisms and the therapeutic targets of a crucial phenotypic malignant switch. Although this biological process has been largely investigated in different human cancer types, including osteosarcoma, it is still largely unknown in veterinary pathology, where it has been mainly explored in canine mammary tumors. The presence of VM in human osteosarcoma is associated with poor clinical outcome, reduced patient survival, and increased risk of metastasis and it shares the main pathways involved in other type of human tumors. This review illustrates the main findings concerning the VM process in human osteosarcoma, search for the related current knowledge in canine pathology and oncology, and potential involvement of multiple pathways in VM formation, in order to provide a basis for future investigations on VM in canine tumors.

Hypertrophic gastropathy associated with gastric sarcoma in a dog.

A 14-y-old spayed female Labrador Retriever was presented with an 8-mo history of chronic vomiting. Abdominal ultrasound and gastrointestinal endoscopy revealed a mass protruding into the gastric lumen, with cytologic features suggestive of sarcoma. A partial gastrectomy was performed; the gastric body and antrum were thickened, with a cerebriform appearance of the mucosal surface. Histologic examination revealed a submucosal neoplastic proliferation of fusiform cells variably arranged in irregular bundles and scattered whorls. Fusiform cells strongly reacted to antibodies against vimentin, S100, and neuron-specific enolase; glial fibrillary acidic protein was moderately and multifocally expressed. Pancytokeratin, KIT, α-smooth muscle actin, and desmin were nonreactive. Histologic and immunohistochemical findings suggested a diagnosis of gastric sarcoma with features referable to a non-GIST (gastrointestinal stromal tumor), non-smooth muscle NIMT (non-angiogenic, non-lymphogenic intestinal mesenchymal tumor). The overlying gastric mucosa was thickened by elongated and dilated gastric glands, predominantly lined by intensely periodic acid-Schiff-stained mucous cells. This altered mucosal architecture was suggestive of Ménétrier-like disease. Although this disease has been hypothesized to predispose to gastric adenocarcinoma in dogs, an association with gastric sarcoma has not been documented previously in the veterinary literature, to our knowledge.

Superficial angiomyxoma in a pregnant cow.

A 3-year-old, pregnant, Alpine Brown cow showed a rapidly growing, pedunculated, skin mass located at the umbilical region, reaching 8 kg in weight over a 3-month period after its initial detection. Six days after parturition, the mass was completely surgically excised. During the follow-up period, the cow remained in good health, without signs of recurrence, and showed increased milk production. Histological examination of the mass revealed a loose proliferation of spindle-shaped or stellate cells, immersed in an abundant myxoid matrix with admixed numerous thin-walled blood vessels. Immunohistochemically, the tumour cells were positive for vimentin, α-smooth muscle actin, and desmin. Gross and histopathological features were compatible with superficial angiomyxoma, a subtype of angiomyxoma rarely described in humans, but not in the veterinary literature. The tumour did not infiltrate into the surrounding tissues, and there was no post-excision recurrence after 3 months. The possibility of hormonal dependence of the tumour during pregnancy is discussed based on such findings in some human cases.

Left atrial appendage rupture due to blunt chest trauma in an Eurasian otter (Lutra lutra).

An adult male Eurasian otter, found dead on the roadside, was submitted for post-mortem examination in April 2014 at the Veterinary Pathology Unit of the Faculty of Veterinary Medicine of Teramo, as part of the RECAL [RECovery and post mortem Analysis of Eurasian otters (Lutra lutra) in the National Park of Cilento, Vallo di Diano and Alburni (Salerno, Italy), and surrounding areas] project. Necropsy revealed an abundant hemothorax associated with multifocal, bilateral pulmonary contusions and lacerations, and a severe hemopericardium characterised by the presence of a wide blood clot in the intact pericardial sac. Two small laceration wounds of the left auricle were found at the base, along the atrioventricular groove, and on the outer free wall. Since myocardial and endocardial tissues showed no other gross and histopathological abnormalities, a left atrial appendage rupture resulting from a blunt chest trauma was diagnosed. Blunt traumatic cardiac rupture is a rarely reported, life-threatening condition in humans. To the best of our knowledge, this is the first report on a left atrial appendage rupture due to blunt chest trauma in veterinary literature. The possible occurrence of a cardiac rupture following a blunt thoracic injury should be taken into consideration in veterinary emergency care.

Inflammatory myofibroblastic tumor of the pancreas in a dog.

A large, ill-defined, firm, multinodular mass involving the pancreas was confirmed on postmortem examination of a 5-y-old, male Rottweiler that died following acute respiratory distress syndrome, after a period of anorexia and lethargy. Histologically, the mass consisted of plump spindle cells admixed with a variable number of macrophages, lymphocytes, plasma cells, and neutrophils. Foci of coagulative necrosis and hemorrhage were also observed. Spindle cells strongly reacted to antibodies against vimentin, α-smooth muscle actin, and calponin, whereas desmin was expressed only mildly and focally. Pan-cytokeratin, KIT, glial fibrillary acidic protein, and S100 protein were nonreactive. Variable numbers of MAC 387-positive cells, CD3+ lymphocytes, and numerous blood vessels were also detected throughout the mass. Histologic and IHC findings were consistent with a diagnosis of inflammatory myofibroblastic tumor of the pancreas.

Toxicological, gene expression and histopathological evaluations of environmentally realistic concentrations of polybrominated diphenyl ethers PBDE- 47, PBDE-99 and PBDE-209 on zebrafish embryos.

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants. Biomonitoring studies have shown widespread presence of PBDEs in humans and their accumulation in food chain cause concern to human health, especially for foetus and infant development. The early-life stages are generally considered more sensitive to exposure to toxic compounds than juvenile or adults. For this reason the aim of this study was to evaluate the effects of the three most environmentally relevant BDE (BDE- 47, 99 and 209) on zebrafish embryos. The fish embryo toxicity (FET) OECD tests on zebrafish were performed followed by histopathogical examination to assess morphological changes. The gene expression of the thyroid stimulating hormone ß (Tshß), the transport proteins transthyretin (Ttr) and thyroxine-binding globulin (Tbg) as well as the enzyme iodothyronine deiodinase 1 (Dio1) was also assessed by Real-time PCR. BDE-47 and BDE-99 showed an increase of the severity of the effects at the lower concentrations while for the BDE-209 the effects were higher to the high concentrations. Although all compounds did not show any acute toxicity for none of the concentrations tested, they reported interesting sub-acute lesions, including yolk and pericardial edema, tail and head malformation, reduced and extremely reduced heart beat rate, blood stasis and spinal curvature, with the highest percentage recorded for BDE-209. Cardiac edema, damage of eye structure and hydrocephaly were confirmed also by histophatological examination. Furthermore, a toxic and dose-dependent liver vacuolization in BDE-209 was observed in all experimental groups. Although no statistically significant difference in gene expression was observed, BDE-209 up-regulated only Dio1 while the other congeners induced Tshß, Ttr, Tbg and Dio1. Overall, this research highlighted that exposure to BDE-47, BDE-99 and BDE-209 at realistic concentrations caused lethal and sub-lethal alterations and impaired genes involved in thyroid hormones homeostasis leading to abnormal development of zebrafish embryos.

Establishment of three-dimensional canine osteosarcoma cell lines showing vasculogenic mimicry and evaluation of biological properties after treatment with 17-AAG.

Vasculogenic mimicry (VM) is an alternative type of blood perfusion characterized by formation of non-endothelial cell-lined microcirculatory channels and is responsible for aggressive tumour biology and increased tumour-related mortality. VM-correlated genes are associated with vascular endothelial grown factor receptor 1 (VEGFR1), and hypoxia-related (hypoxia inducible factor 1 α-HIF1α) signalling pathways, whose molecules are client proteins of Hsp90 (heat shock protein 90) and are potential therapeutic targets. This pilot study was aimed to investigate vasculogenic mimicry in a three-dimensional (3D) cell culture system of two aggressive canine osteosarcoma (OSA) cell lines (D22 and D17), and to evaluate the response of these cells to 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) treatment in relation to tubular-like structure formation in vitro. Only D17 cell line formed hollow matrix channels in long-term 3D cultures and assumed endothelial morphology, with cells expressing both Hsp90 and VEGFR1, but lacking expression of endothelial marker CD31. 17-AAG treatment inhibited migration of D17 OSA cells, also decreasing VM markers in vitro and inducing a reduction of HIF1α transcript and protein in this cell line. Taken together, these preliminary data indicate that the biological effects of 17-AAG on D17 3D culture and on HIF1α regulation can provide interesting information to translate these findings from the basic research to clinical approach for the treatment of canine OSA as a model in comparative oncology.

Nectin-4 and p63 immunohistochemical expression in canine prostate tumourigenesis.

Nectin-4 is an E-cadherin-based adherens junction protein of normal epithelial cells, as well as a potent mediator of anchorage-independent cancer colony formation. It is considered a tumour-associated histological and serological marker in various human cancers. The transcription factor p63 is a basal cell marker in the normal prostate, involved in cell adhesion, as well as in the formation and survival of circulating tumour cell clusters. The aim of this study was to evaluate Nectin-4 and p63 immunohistochemical expression in 42 canine prostate tissues including 2 normal prostates, 10 benign prostatic hyperplasias (BPHs), 30 prostatic carcinomas (PCs), 1 pulmonary and 1 lymph node metastasis. From normal to neoplastic tissues, Nectin-4 showed a progressive switching from membranous (m-Nectin-4) to cytoplasmic (c-Nectin-4), regardless of the histological subtypes, except for lack of expression in solid PCs. Metastatic cells exhibited both strong membranous and cytoplasmic positivity. c-Nectin-4 expression was significantly (P < 0.0001) increased in PCs/metastasis compared to BPHs cases and a decrease (P < 0.05) of nuclear p63 immunostaining was also detected in the two groups. Furthermore, data showed a significant association (P < 0.05) between p63 and m-Nectin-4 distribution, although their colocalization was detected only in scattered cells by double immunofluorescence. Our results suggest the involvement of m-Nectin-4 in canine prostate tumourigenesis and metastatic potential, while the exact role of c-Nectin-4 expression detectable in primary PCs requires further investigations.