Laser Capture Microdissection of Tertiary Lymphoid Structures from Formalin-Fixed Paraffin-Embedded Sections of Canine Cutaneous and Subcutaneous Sarcomas for NanoString Direct RNA Counting.

Laser capture microdissection (LCM) of formalin-fixed, paraffin-embedded sections is a way to analyze gene expression of morphologically distinct areas of tissue, as microscopically visualized with stained tissue sections. Herein, I describe a method for laser dissecting lymphoid aggregates in canine cutaneous and subcutaneous sarcomas and their adjacent sarcoma tissue to determine the differential expression of RNA as determined by NanoString nCounter technology. Canine soft tissue sarcomas (STS) are diversely derived mesenchymal neoplasms that, regardless of exact histogenesis, behave similarly and thus have been grouped together as a diagnostic entity.

Lymphoid Aggregates in Canine Cutaneous and Subcutaneous Sarcomas: Immunohistochemical and Gene Expression Evidence for Tertiary Lymphoid Structures.

Canine cutaneous/subcutaneous soft-tissue sarcomas (STS) are diversely derived mesenchymal neoplasms with a risk of recurrence and/or metastasis depending on the extent of surgical excision and histologic grade. Lymphoid aggregates (LAs) are often described in these tumours but not characterised. In humans, LA characterised as tertiary lymphoid structures (TLSs) improve the prognosis of many tumours, including sarcomas.

In-vitro exposure of feline red blood cells to rattlesnake venom causes echinocytosis.

The objective of this study was to investigate in-vitro exposure of healthy feline red blood cells to venom of four species of rattlesnakes and to evaluate the presence and type of echinocytes that form. Blood from 10 healthy felines was collected and within 30 min exposed to four species of rattlesnake venom. Cytologic evaluation for echinocytes was performed at 0-, 15-, and 30-min post-exposure to each of the species' venom.

Systemic iron reduction via an iron deficient diet decreases the severity of knee cartilage lesions in the Dunkin-Hartley guinea pig model of osteoarthritis.

Objective: Iron accumulation is emerging as a player in aging-related disorders due to its propensity for generating reactive oxygen species (ROS). Studies investigating the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. We designed a proof-of-principle study to determine the effect of systemic iron deficiency, via an iron deficient diet, on knee OA in an animal model.

Severe hyperferremia and hyperferritinemia in a dog with precursor-directed immune-mediated anemia.

A 4-year-old dog was evaluated at the Colorado State University Veterinary Teaching Hospital for lethargy and hyporexia of 24 hours duration. On presentation, she had a marked normocytic, normochromic, nonregenerative anemia (HCT 14%; RI 40-55). Her serum iron concentration (1651 μg/dL; RI 73-245) and serum ferritin concentration (1337 ng/mL; RI 89-489) were markedly elevated.

A safety evaluation of allogeneic freeze-dried platelet-rich plasma or conditioned serum compared to autologous frozen products equivalents in equine healthy joints.

Background: Hemoderivatives such as autologous conditioned serum (ACS) and platelet-rich plasma (PRP) have been used as potential disease-modifying therapies in musculoskeletal disorders such as osteoarthritis (OA). These therapies are based on the delivery of multiple growth factors and anti-inflammatory cytokines that are known to participate in inflammatory processes. The variability of cytokine content due to the autologous nature of the product, the non-availability for immediate use and need for storage at low temperatures are limitations for its use in the field.

Iron, hepcidin, and microcytosis in canine hepatocellular carcinoma.

Background: Erythrocyte microcytosis in some dogs with hepatocellular carcinoma (HCC) suggests a derangement in systemic iron. Hepcidin, the master regulator of iron, is secreted by the liver in response to interleukin 6 (IL-6) and/or bone morphogenetic protein 6 (BMP6) and can cause microcytosis.

Objectives: Pilot study to compare the quantities of hepcidin, IL-6, and BMP6 RNA molecules in archival tumoral (HCC) and adjacent peritumoral (non-HCC) hepatic tissue to determine if they are different between tissue types or associated with microcytosis.

Multiplate platelet aggregometry in dogs undergoing laparoscopic liver biopsy for diagnosis of chronic hepatopathy.

Objectives: To assess platelet function via the Multiplate analyser in dogs undergoing laparoscopic liver biopsy for diagnosis of chronic hepatopathy.

Materials And Methods: Twenty-seven client-owned dogs were prospectively enrolled. Before laparoscopic liver biopsy, whole blood impedance platelet aggregometry via the Multiplate analyser was performed.

Systemic administration of a pharmacologic iron chelator reduces cartilage lesion development in the Dunkin-Hartley model of primary osteoarthritis.

Iron has been emerging as a key contributor to aging-associated, chronic disorders due to the propensity for generating reactive oxygen species. To date, there are a limited number of publications exploring the role of iron in the pathogenesis of primary/age-related osteoarthritis (OA). The objective of this study was to determine whether reduced iron via pharmacologic iron chelation with deferoxamine (DFO) affected the development and/or severity of cartilage lesions in a primary OA model.

Phase I clinical trial of an antithrombotic drug protocol combining apixaban and clopidogrel in dogs.

Introduction: Combining an antiplatelet drug, clopidogrel, with the direct oral Factor Xa inhibitor, apixaban, could provide an effective antithrombotic strategy in dogs. Thus, a limited 3 + 3 phase I dose-escalation clinical trial in healthy dogs was conducted to evaluate bleeding (primary end-point) and pharmacodynamic (PD)/pharmacokinetic (PK) parameters (secondary end-point).

Animals: Eleven beagle dogs, median body weight 10.

Coagulation status, fibrinolysis, and platelet dynamics in dogs with chronic inflammatory enteropathy.

Background: Coagulation status is poorly understood in dogs with chronic inflammatory enteropathy (CIE). Fibrinolytic activity and platelet dynamics have not been evaluated in CIE dogs.

Objectives: To assess coagulation status and fibrinolysis in normoalbuminemic CIE dogs (CIE-N) and CIE dogs with protein-losing enteropathy (CIE-PLE) compared to healthy controls (HC).

Variability in the cleavage of exosome-associated transferrin receptor questions the utility of clinically useful soluble transferrin receptor assays for dogs, cats, and horses.

Whole transferrin receptor (TfR) is present in reticulocyte exosomes. Soluble transferrin receptor (sTfR) is cleaved from whole TfR in human plasma, with the remnant cytoplasmic domain (cTfR) remaining membrane associated. In humans, sTfR is a biomarker that can detect iron deficiency in the presence of inflammatory disease.

Virtual Microscopy Is More Effective Than Conventional Microscopy for Teaching Cytology to Veterinary Students: A Randomized Controlled Trial.

Virtual microscopy (VM) using scanned slides and imaging software is increasingly used in medical curricula alongside instruction in conventional microscopy (CM). Limited reports suggest that VM is useful in the veterinary education setting, and generally well-received by students. Whether students can apply knowledge gained through VM to practical use is unknown.

Platelet-Rich Products and Their Application to Osteoarthritis.

Autologous platelet-rich plasma (PRP) is a biological preparation made from the patient's own plasma that contains a platelet concentration above the whole blood baseline. Owing to the release of growth factors and other cytokines after degranulation, platelets have a central role in inflammation and in different stages of the healing process. For this reason, PRP-derived products have been used to enhance healing of musculoskeletal injuries and modulate progression of inflammatory processes, including osteoarthritis (OA).

Platelet aggregometry testing during aspirin or clopidogrel treatment and measurement of clopidogrel metabolite concentrations in dogs with protein-losing nephropathy.

Background: Dogs with protein-losing nephropathy (PLN) are treated with antiplatelet drugs for thromboprophylaxis but no standardized method exists to measure drug response. It is also unknown if clopidogrel metabolite concentrations [CM] differ between healthy and PLN dogs.

Objectives: Assess response to aspirin or clopidogrel in PLN dogs using platelet aggregometry (PA) and compare [CM] between healthy and PLN dogs.

Carbon monoxide releasing molecule enhances coagulation and decreases fibrinolysis in canine plasma exposed to Crotalus viridis venom in vitro and in vivo.

Carbon monoxide releasing molecule-2 (CORM-2), an emerging therapeutic in human medicine, enhances plasmatic coagulation and attenuates fibrinolysis in vitro in human, rabbit and horse plasma and ameliorates hypocoagulation and hyperfibrinolysis secondary to venom exposure in human plasma in vitro. Fibrinogenases in rattlesnake venom cause decreased clot strength, and in the presence of tissue plasminogen activator (tPA) in vitro, a markedly increased rate of clot lysis. CO interacts with a haem group on fibrinogen, changing its configuration so that the fibrin clot is strengthened and more resistant to fibrinolysis.

Effects of storage conditions on two platelet agonists for whole blood impedance platelet aggregometry in dogs.

Background: Whole blood impedance platelet aggregometry (Multiplate-) can be performed with different agonists to evaluate platelet function. Although the manufacturer recommends disposal of stored reagents after 1 month at -20°C or 24 hours at 4°C, reagent integrity after reconstitution under different storage conditions is unknown. If reagent integrity is stable for longer periods, assay costs could decrease dramatically.

Reference intervals for coagulation variables in healthy adult domestic goats (Capra aegagrus hircus).

Background: Coagulation has not been studied extensively in adult goats, and reference intervals (RIs) for coagulation variables have not yet been established and would be clinically useful.

Objectives: The goal of this study was to establish coagulation variable RIs in adult goats for future coagulation interpretation.

Methods: Blood samples from 40 healthy adult goats were collected, and assays for PT, APTT, D-dimers, antithrombin III, and fibrinogen were performed.

Variability of tissue factor-activated thromboelastography and whole blood impedance platelet aggregometry in healthy dogs.

Objective: To assess interindividual (CV ) and intraindividual (CV ) variability over time for results of thromboelastography (TEG) and whole-blood impedance platelet aggregometry in healthy dogs.

Animals: Six healthy Beagle dogs.

Measurements And Main Results: Tissue factor (TF)-activated TEG and adenosine diphosphate (ADP) and arachidonic acid (AA)-induced whole blood impedance platelet aggregometry were performed at 3 different time points (days 1, 4, and 6).

Effects of Low-level Brodifacoum Exposure on the Feline Immune Response.

Anticoagulant rodenticides have been implicated as a potential inciting factor in the development of mange in wild felids, but a causative association between anticoagulant rodenticide exposure and immune suppression has not been established. Specific-pathogen-free domestic cats were exposed to brodifacoum over a 6-week period to determine whether chronic, low-level exposure altered the feline immune response. Cats were vaccinated with irrelevant antigens at different points during the course of the experiment to assess recall and direct immune responses.

Characteristics of hemostasis during experimental Ehrlichia canis infection.

Background: Ehrlichia canis infection in dogs can cause thrombocytopenia and clinical evidence of bleeding. It is unknown why some dogs show signs of bleeding whereas others do not despite clinically relevant thrombocytopenia.

Hypothesis/objectives: Activated platelets, decreased fibrinolysis or both mitigate bleeding tendency.

Carbon Monoxide-Releasing Molecule Enhances Coagulation and Decreases Fibrinolysis in Normal Canine Plasma.

The dog is an important companion animal and also purpose-bred for research studies. Coagulopathies in dogs are common, although the availability of blood products for therapy is inconsistent throughout the profession. A pro-coagulant therapeutic that is readily available and easily stored would be useful for the treatment of coagulopathies.

Iron protects porcine plasma coagulation kinetics from degradation by Crotalus atrox venom.

While the administration of antivenom to treat hemotoxic snake bite injury remains the gold standard of therapy, we have demonstrated that modifying human fibrinogen with iron and carbon monoxide renders it resistant to fibrinogenolytic snake venom enzymes. In order to translate these findings into a possible biometal-based therapy complementary to antivenom administration, a preclinical model that possesses fibrinogen that closely mimics the human molecule in response to iron and carbon monoxide needed to be identified. The goal of this investigation was to determine if a swine model could serve in this capacity by assessing the thrombelastographic response of porcine plasma to iron and carbon monoxide exposure, without or with further exposure to the fibrinogenolytic venom of the viper Crotalus atrox.

Clinically healthy overweight and obese dogs differ from lean controls in select CBC and serum biochemistry values.

Background: Obesity is a global disease, affecting nearly half a billion people. Unfortunately, this trend is mirrored in our canine population.

Objectives: As obesity is a complex inflammatory disease, there is a need to determine whether routine medical screening tests may indicate, or be influenced by, its presence.