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OBJECTIVETo compare concentrations of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in aqueous humor from ophthalmologically normal dogs and dogs with naturally occurring primary angle-closure glaucoma (cPACG).SAMPLEAqueous humor samples from 12 eyes with cPACG and 18 ophthalmologically normal eyes of dogs.PROCEDURESA multiplex fluorescence-based ELISA was used to measure concentrations of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1, TIMP-2, and TIMP-4. Results for eyes with versus without cPACG were compared.RESULTSSignificantly higher mean concentrations of MMP-1 (45% higher), MMP-2 (55% higher), MMP-3 (39% higher), MMP-8 (79% higher), MMP-9 (29% higher), MMP-10 (60% higher), TIMP-1 (63% higher), and TIMP-2 (136% higher) were detected in aqueous humor from eyes with cPACG, compared with ophthalmologically normal eyes.CLINICAL RELEVANCEMMPs and TIMPs have pivotal roles in extracellular matrix turnover and homeostasis in the outflow pathways of the eye. Results of the present study documented higher concentrations of MMPs and TIMPs in aqueous humor samples from dog eyes with late-stage cPACG. Although, to our knowledge, TIMPs have not previously been evaluated in the context of cPACG, the markedly higher concentration of TIMPs in eyes with cPACG suggested that inhibition of proteolysis and extracellular matrix turnover might be a factor in the development of glaucoma in susceptible individuals. However, because the present study used samples from dogs with late-stage cPACG, further work is required to characterize the temporal relationship between MMP and TIMP concentration changes and onset or progression of disease.

OBJECTIVE To evaluate whether mesenchymal stem cells (MSCs) can be safely administered IV to dogs with congestive heart failure (CHF) secondary to myxomatous mitral valve disease (MMVD) to improve cardiac function and prolong survival time. ANIMALS 10 client-owned dogs with CHF secondary to MMVD. PROCEDURES Dogs with an initial episode of CHF secondary to MMVD were enrolled in a double-blind, placebo-controlled clinical trial. Five dogs in the MSC group received allogeneic Wharton jelly–derived MSCs (2 X 106 cells/kg, IV), and 5 dogs in the placebo group received a 1% solution of autologous serum (IV) for 3 injections 3 weeks apart. Cell-release criteria included trilineage differentiation, expression of CD44 and CD90 and not CD34 and major histocompatability complex class II, normal karyotype, and absence of contamination by pathogenic microorganisms. Patients were followed for 6 months or until death or euthanasia. Echocardiographic data, ECG findings, serum cardiac biomarker concentrations, CBC, and serum biochemical analysis results were obtained prior to and 4 hours after the first injection and every 3 months after the final injection. RESULTS Lymphocyte and eosinophil counts decreased significantly 4 hours after injection, and monocytes decreased significantly only in dogs that received an MSC injection. No significant differences were seen in the echocardiographic variables, ECG results, serum cardiac biomarker concentrations, survival time, and time to first diuretic drug dosage escalation between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE This study showed that MSCs can be easily collected from canine Wharton jelly as an allogeneic source of MSCs and can be safely delivered IV to dogs with CHF secondary to MMVD.

Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of in vitro models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 μm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.

Dogs with cancer treated with chemotherapy agents such as doxorubicin (DOX) develop cardiovascular toxicity, providing an opportunity to evaluate cardioprotective strategies in the setting of cancer treatment translatable to human disease. However, due to the lack of a suitable approach to culture primary adult canine cardiomyocytes, mechanistic interrogation of cardiotoxicity after cancer therapy remains a challenge. Our study thus aims to validate a canine myocardial slice culture model to study autophagy modulation and the role of extracellular vesicle (EV) associated miRNA in the setting of DOX induced cardiotoxicity. We hypothesize that induction of autophagy in canine myocardial tissue will reduce apoptosis, exert early changes to EVs, and ameliorate DOX cardiotoxicity. Left ventricular tissue from client-owned donated adult dog hearts was sectioned with a vibratome and viability of the cultured myocardial slices was evaluated by histology and MTT assay. Apoptosis was quantified by TUNEL, and autophagy by fluorescent LC3 protein puncta. Secreted EVs were isolated from cultured tissue by size exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblot. Canine myocardial slices are consistently viable for 7 days in culture - cardiomyocyte morphology is maintained with low levels of apoptosis, and baseline autophagy is observed. Induction of autophagy with rapamycin treatment results in reduced apoptosis. Cardiac tissue derived extracellular vesicles showed typical size, morphology and enrichment of proteins including tetraspanin CD9 and will be evaluated for changes to EV miRNA profile with autophagy modulation. The canine myocardial slice model allows, for the first-time, the elucidation of complex cross-talk among apoptosis, autophagy, and EVs with molecular and cellular resolutions. Detecting early changes in canine cardiomyocyte autophagy to halt cardiac damage rather than managing its consequences is a paradigm shift translatable towards preventing chemotherapy associated cardiotoxicity.

Non-syndromic mitral valve prolapse (MVP), or Barlow’s Disease, is a valvular heart disease with a prevalence of 2-3% in the human adult population. Consequences of MVP include mitral regurgitation that without surgical treatment can progress to congestive heart failure, arrhythmias and sudden death. Since MVP is a degenerative disease that worsens with age, surgical risk is of concern with an older population, necessitating the development of alternative strategies for early detection and effective medical intervention. Similar to humans, canine MVP is age-related with a lifetime prevalence close to 90%, providing a large population for study. Canine MVP represents a relevant spontaneous large animal model to dissect the mechanistic underpinnings of disease pathogenesis and evaluate novel treatment. We hypothesize that extracellular vesicles (EVs) and their associated non-coding RNA (ncRNA) may play a role in this disease by enabling propagation of the fibroblast-to-myofibroblast phenotypic switch in mitral valvular interstitial cells (VICs). VICs were isolated from 12 normal and 14 diseased canine mitral valves and cultured in defined chemical media for 48 hours. EVs were isolated from conditioned media using size exclusion chromatography. Total RNA was isolated from EVs with the Qiagen miReasy Plasma/Serum kit, and from VIC cells with the mirVana miRNA Isolation Kit. Sequencing libraries were created using QIAseq miRNA Library Kit, and RNAseq was performed with Illumina HiSeq 2500 sequencer with single read 100 base format. Reads were mapped to canine ncRNA databases (miRbase, RFam). VICs from diseased valves had higher expression of αSMA than cells from normal valves (p = 0.002) based on RT-qPCR. Sequence analysis showed decreased content of tRNA (p = 0.023) and miRNA precursor (p = 0.036) in EVs from diseased VICs, in addition to decreased expression of let-7f. Cells from diseased valves had lower Y-RNA content (p = 0.007). miR-8859a and miR-451 were preferentially packaged into EVs from both normal and diseased VICs. Preferential packaging of miR-155 was seen in normal VIC EVs and miR-191 in diseased VIC EVs. Functional roles of these miRs will need to be confirmed to determine their therapeutic potential for treatment of MVP.

Mesenchymal stem cells (MSCs) are widely investigated as potential therapeutic agents due to their potent immunomodulatory capacity. Although specific mechanisms by which MSC acts on immune cells are emerging, many questions remain, including the potential of extracellular vesicles (EVs) to mediate biological activities. Canine MSCs are of interest for both veterinary and comparative models of disease and have been shown to suppress CD4

Objective To evaluate hemostatic changes following experimental acute hemorrhage in dogs using traditional coagulation tests (eg, platelet count, prothrombin time [PT], and activated partial thromboplastin time [aPTT]), kaolin-activated thromboelastography (TEG), and whole blood multiple electrode impedance platelet aggregometry. Design Prospective study. Setting Research laboratory. Animals Five Beagles. Interventions Dogs were anesthetized prior to obtaining blood samples for baseline PCV, total plasma protein (TPP), arterial blood-gas, platelet count, PT, aPTT, TEG, fibrinogen, and aggregometry. Blood was obtained at 4 additional time points, following 20% blood volume loss, 40% blood volume loss, 60 minutes of sustained hypotension, and after autologous blood transfusion. In addition, heart rate and direct arterial blood pressure were measured at each time point. Measurements and main results Significant decreases were noted for PCV (P = 0.048), TPP (P Conclusion Platelet dysfunction occurred following hemorrhage in this model, despite a stable platelet count. Additionally, significant changes associated with hemorrhage were documented in aPTT, fibrinogen, and MA. Platelet function testing in dogs with naturally occurring hemorrhage warrants further investigation.

Objective To investigate the effects of in vitro hemodilution with lactated Ringers solution (LRS), hetastarch (HES), and fresh frozen plasma (FFP) on whole blood coagulation in dogs as assessed by kaolin-activated thromboelastography. Design In vitro experimental study. Setting University teaching hospital. Animals Six healthy client-owned dogs. Interventions Whole blood was collected and diluted in vitro at a 33% and 67% dilution with either LRS, HES, or FFP. Measurements and Main Results Kaolin-activated thromboelastography was performed on each sample as well as a control. Thromboelastographic parameters R (min), alpha (deg), K (min), and MA (mm) were measured and compared to the sample control for each dilution using mixed model methodology. Prolongation in coagulation times were seen at both dilutions with LRS and HES. There was no significant difference in R times at the 33% dilution, but R time was significantly prolonged at the 67% dilution with HES (P = 0.004). MA was significantly decreased for LRS at both dilutions (P = 0.013, P < 0.001) and more profoundly decreased for HES (P < 0.001, P = 0.006). No significant difference in any parameter was found for FFP. Conclusions In vitro hemodilution of whole blood with both LRS and HES but not FFP resulted in significant effects on coagulation with HES having a more profound effect. In vivo evaluation of changes in coagulation with various resuscitation fluids is warranted and may be clinically relevant.

Objectives To determine thawing times of fresh frozen plasma (FFP), and to evaluate the activity of hemostatic proteins (coagulation factors V, VII, VIII, IX, X, and fibrinogen), clotting times (prothrombin time and activated partial thromboplastin time), and sterility of canine plasma stored refrigerated. Design Prospective laboratory-based study. Setting Veterinary teaching hospital blood bank. Interventions Phase 1: Six units of canine FFP were retrieved from the blood bank and thawed individually in a warm water bath. Time for thaw was recorded in minutes and reported as mean ± SD. Phase 2: One unit of fresh whole blood was collected from 9 dogs and processed routinely. Resulting plasma was divided into 2 aliquots, 1 stored as refrigerated plasma (RP) and 1 as frozen plasma. Samples from the RP were taken at 0, 1, 5, 7, and 14 days and from the FFP at days 0 and 14 for determination of clotting factor activity (V, VII, VIII, IX, and X and fibrinogen) and clotting times. Coagulation factors and clotting times were analyzed using a mixed effects linear model for ANOVA, comparing changes over time as well as differences between groups. For all comparisons, a P value of

OBJECTIVE: To characterize hemostatic profiles in dogs with acute pancreatitis. DESIGN: Prospective and observational study. SETTING: Tertiary referral centers. ANIMALS: Fifteen client‐owned dogs with acute pancreatitis enrolled between December 1, 2011 and June 1, 2012. MATERIALS AND METHODS: Blood samples were collected on admission for measurement of platelet count, PCV, thromboelastography (TEG), antithrombin, prothrombin time, activated partial thromboplastin time, D‐dimer, von Willebrand factor, and fibrinogen values, which were compared to reference intervals derived from healthy dogs. The Wilcoxon rank‐sum test was used to test for differences in continuous variables between study subjects and reference intervals. MEASUREMENTS AND MAIN RESULTS: Dogs with acute pancreatitis were globally hypercoagulable using TEG when compared with reference intervals. Dogs with acute pancreatitis had significantly higher D‐dimers (1,144 μg/L vs 251 μg/L [6264.5 vs 1374.5 nmol/L]; P = 0.001), fibrinogen (837 vs 232 mg/dL [8.37 vs 2.32 g/L]; P

OBJECTIVE To compare effectiveness of glycerol, dimethyl sulfoxide (DMSO), and hydroxyethyl starch (HES) solutions for cryopreservation of avian RBCs. SAMPLE RBCs from 12 healthy Ameraucana hens (Gallus gallus domesticus). PROCEDURES RBCs were stored in 20% (wt/vol) glycerol, 10% (wt/vol) DMSO freezing medium, or various concentrations of HES solution (7.5%, 11.5%, and 20% [wt/vol]) and frozen for 2 months in liquid nitrogen. Cells were then thawed and evaluated by use of cell recovery and saline stability tests, cell staining (7-aminoactinomycin D and annexin V) and flow cytometry, and scanning electron microscopy. RESULTS Percentage of RBCs recovered was highest for 20% glycerol solution (mean ± SE, 99.71 ± 0.04%) and did not differ significantly from the value for 7.5% HES solution (99.57 ± 0.04%). Mean saline stability of RBCs was highest for 10% DMSO (96.11 ± 0.25%) and did not differ significantly from the value for 20% HES solution (95.74 ± 0.25%). Percentages of cells with 7-aminoactinomycin D staining but without annexin V staining (indicating necrosis or late apoptosis) were lowest for 10% DMSO freezing medium (3%) and 20% glycerol solution (1%) and highest for all HES concentrations (60% to 80%). Scanning electron microscopy revealed severe membrane changes in RBCs cryopreserved in 20% HES solution, compared with membrane appearance in freshly harvested RBCs and RBCs cryopreserved in 10% DMSO freezing medium. CONCLUSIONS AND CLINICAL RELEVANCE Cryopreservation of avian RBCs with HES solution, regardless of HES concentration, resulted in greater degrees of apoptosis and cell death than did cryopreservation with other media. Transfusion with RBCs cryopreserved in HES solution may result in posttransfusion hemolysis in birds.

Canine myxomatous mitral valve disease (MMVD) resembles the early stages of myxomatous pathology seen in human non-syndromic mitral valve prolapse, a common valvular heart disease in the adult human population. Canine MMVD is seen in older subjects, suggesting age-related epigenetic dysregulation leading to derangements in valvular cell populations and matrix synthesis or degradation. We hypothesized that valvular interstitial cells (VICs) undergo disease-relevant changes in miRNA expression. In primary VIC lines from diseased and control valves, miRNA expression was profiled using RT-qPCR and next generation sequencing. VICs from diseased valves showed phenotypic changes consistent with myofibroblastic differentiation (vimentinlow+, α-SMAhigh+), increases in senescence markers (p21, SA-β-gαl), and decreased cell viability and proliferation potential. RT-qPCR and miRNA sequencing analyses both showed significant (p

To determine thawing times of fresh frozen plasma (FFP), and to evaluate the activity of hemostatic proteins (coagulation factors V, VII, VIII, IX, X, and fibrinogen), clotting times (prothrombin time and activated partial thromboplastin time), and sterility of canine plasma stored refrigerated.Prospective laboratory-based study.Veterinary teaching hospital blood bank.Phase 1: Six units of canine FFP were retrieved from the blood bank and thawed individually in a warm water bath. Time for thaw was recorded in minutes and reported as mean ± SD. Phase 2: One unit of fresh whole blood was collected from 9 dogs and processed routinely. Resulting plasma was divided into 2 aliquots, 1 stored as refrigerated plasma (RP) and 1 as frozen plasma. Samples from the RP were taken at 0, 1, 5, 7, and 14 days and from the FFP at days 0 and 14 for determination of clotting factor activity (V, VII, VIII, IX, and X and fibrinogen) and clotting times. Coagulation factors and clotting times were analyzed using a mixed effects linear model for ANOVA, comparing changes over time as well as differences between groups. For all comparisons, a P value of0.05 was considered significant. Batch bacterial aerobic and anaerobic cultures of the RP samples were submitted on days 7 and 14 and from the frozen plasma on day 14.Time to thaw for FFP units was 34.7 ± 1.38 minutes. Refrigerated storage resulted in significant decreases in the activity of all clotting factors and a subsequent prolongation in clotting times. However, no values were outside of the reference interval. All bacterial cultures yielded no growth.Refrigerated storage results in only minor loss of coagulation factor activity in canine plasma. The use of RP, therefore, may be a viable option in high-volume veterinary hospitals for rapid correction of coagulopathy in critical care patients.