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Background: The antiplatelet effect of clopidogrel can vary between patients. A modified thromboelastography (TEG) protocol (TEG-Platelet Mapping assay® [TEG-PM]) can be used for clopidogrel monitoring but is not widely available. Thrombin generation (TG) assays could offer a novel alternative. The main objective of this pilot study was to assess TG assay variables (lag time, peak, endogenous thrombin potential [ETP]) in dogs before and after 7 days of clopidogrel administration, and compare with TEG-PM variables (maximum amplitude [MA]-ADP and percentage (%) inhibition). Six healthy mix-breed dogs were enrolled in this pilot study. Blood samples for platelet count, TG assays, and TEG-PM were obtained at two time points, corresponding to baseline, and after 7 days of clopidogrel administration (mean 2.3 +/- 0.3 mg/kg PO q24 hours). Data were then compared with a Student’s t-test. Results There was no significant change in TG assay variables performed on platelet poor plasma after 7 days of clopidogrel administration: lag time (Day 1: 1.8 +/- 0.2 min, Day 7: 1.8 +/- 0.2 min, P = 0.42); Peak (Day 1: 76 +/- 7 nM, Day 7: 72 +/- 10 nM, P = 0.49); and ETP (Day 1: 399 +/- 27 nM*min, Day 7: 392 +/- 32 nM*min; P = 0.49). There were significant changes in TEG MA-ADP (Day 1: 19 +/- 8 mm, Day 7: 9 +/- 6 mm, P = 0.04) and % inhibition (Day 1: 58 +/- 27, Day 7: 99 +/- 0.3, P = 0.02) however over the course of the study. Conclusions Clopidogrel administration did not lead to changes in TG assay variables performed on platelet poor plasma samples, despite concomitant changes in TEG-PM variables consistent with platelet inhibition. Thrombin generation performed on platelet poor plasma does not appear to be a useful antiplatelet monitoring tool in dogs.

Objective To compare hemostatic variables performed on blood samples obtained from indwelling jugular catheters or direct venipuncture over a 72-hour period. Design Prospective experimental study. Setting University research laboratory. Animals Five healthy neutered male purpose-bred Beagle dogs. Interventions Each dog was sedated to facilitate placement of a long-stay 20-Ga polyurethane IV catheter into the jugular vein. Blood samples were obtained from the preplaced catheters at 4 time points corresponding to 0, 24, 48, and 72 hours relative to placement. Blood samples were also obtained by direct venipuncture of a peripheral vein using a 21-Ga butterfly catheter and evacuated blood tubes at the same time points. Platelet count, platelet closure time, prothrombin time, activated partial thromboplastin time, fibrinogen, and kaolin-activated thromboelastography were performed on these paired samples at each time point. The patency of the indwelling catheters was maintained by flushing every 6 hours with heparinized saline. Measurements and main results No significant differences were identified in any of the hemostatic variables obtained by either blood collection technique at any time point during the study (P > 0.05). There was also no significant day-to-day variation in any catheter-derived hemostatic variable obtained from individual dogs identified over the course of the study. Conclusions These data suggest that accurate hemostatic variables may be obtained using blood collected from indwelling jugular catheters, maintained with heparinized saline for at least 72 hours, in healthy dogs.

Background Vascular endothelial growth factor (VEGF) is a key molecular driver of angiogenesis and vascular permeability and is expressed by a wide variety of neoplasms. Although blood VEGF concentrations have been quantified in intracranial tumors of dogs, cerebrospinal fluid (CSF) VEGF concentration might be a more sensitive biomarker of disease. Objective Concentrations of VEGF in CSF are higher in dogs with central nervous system (CNS) neoplasia compared to those with meningoencephalomyelitis and other neurologic disorders. Animals One hundred and twenty‐six client‐owned dogs presented to a veterinary teaching hospital. Methods Case‐control study. Cerebrospinal fluid was archived from dogs diagnosed with CNS neoplasia and meningoencephalomyelitis. Control dogs had other neurological disorders or diseases outside of the CNS. A commercially available kit was used to determine VEGF concentrations. Results Detectable CSF VEGF concentrations were present in 49/63 (77.8%) neoplastic samples, 22/24 (91.7%) inflammatory samples, and 8/39 (20.5%) control samples. The VEGF concentrations were significantly different between groups (P

OBJECTIVE: Rivaroxaban is a new anticoagulant option for dogs, yet its reported oral bioavailability is as low as 60%. The objective of this study was to examine the influence of feeding and gastroprotectant medications on the bioactivity (anti‐Xa activity) of rivaroxaban in healthy dogs. DESIGN: Prospective experimental study. SETTING: University research laboratory. ANIMALS: Five healthy neutered male purpose‐bred Beagles. INTERVENTIONS: Dogs were administered a median dose of 1.8 mg/kg rivaroxaban (range, 1.6‐1.8 mg/kg) orally once daily for 2 consecutive days with either (1) no food, (2) food, (3) sucralfate 30 minutes before rivaroxaban, or (4) omeprazole at the same time as rivaroxaban. Blood was collected from preplaced jugular catheters immediately before and at 6 time points after rivaroxaban administration (2, 4, 8, 24, 36, and 48 hours). A rivaroxaban calibrated anti‐Xa activity assay (RIVA) was used to monitor anticoagulant effect. MEASUREMENTS AND MAIN RESULTS: Rivaroxaban administration resulted in significant increases in RIVA (P = 0.02), with peak activities occurring 2 to 4 hours after dosingduring each study arm. No feeding was associated with significantly higher RIVA at the 36‐hour time point compared to all other treatment arms (P

Sea turtles are frequently presented for rehabilitation with injuries for which analgesic treatment is warranted. Ketoprofen is a nonsteroidal anti-inflammatory drug (NSAID) widely used in clinical veterinary medicine for musculoskeletal pain relief. Pharmacokinetics of 2 mg/kg IM have been studied in loggerhead sea turtles (Caretta caretta) as a single and a repeated dose q24hr for 3 days. Safety of longer term administration has not been performed, however, and NSAID use carries a risk of potential complications, including gastrointestinal ulceration, kidney damage, and bleeding. The objective of the current study was to determine the effects of a 5-day course of ketoprofen on thromboelastography (TEG) and hematological (including thrombocytes) and plasma biochemical analytes in loggerheads. A secondary objective was to determine 24-hr trough concentrations of ketoprofen after 5 days of treatment. Eight loggerheads were treated with ketoprofen 2 mg/kg IM q24hr for 5 days, and TEG, hematology, and plasma biochemistry panels were performed before and at the conclusion of treatment. Eight controls were treated with an equivalent volume of saline intramuscularly. Virtually no changes were detected before and after treatment or between treatment and control groups in any of the 24 endpoints evaluated, and marginal differences were not considered clinically relevant. Plasma ketoprofen concentrations after 5 days of treatment indicated no accumulation over that duration. Ketoprofen at 2 mg/kg IM q24hr for up to 5 days in loggerheads appears safe with respect to blood clotting and blood data, although other potential effects were not evaluated.

BACKGROUND Glioblastoma patient survival statistics have remained unchanged for more than three decades. Despite tumor resection and chemoradiotherapy, recurrence is inevitable. Moreover, the invasive behavior of glioblastoma confounds treatment. To improve patient survival statistics, a targeted therapy that can home to distant tumor foci is desperately needed. Induced neural stem cells (iNSCs) armed with cytotoxic payloads have proven efficacious against human xenograft models of glioblastoma. To further propel iNSCs to human clinical trials, we investigated the safety, toxicity, and persistence of iNSCs in a canine model. METHODS Autologous iNSCs generated from the skin of four non-tumor-bearing, purpose-bred, male beagles were engineered to express TRAIL and thymidine kinase (TK). iNSCs were loaded with ferumoxytol to facilitate MRI-tracking. Canines were divided into two cohorts to denote iNSC administration route: scaffold encapsulation or intracerebroventricular (ICV). Two dose levels were investigated: 1′106 iNSCs/kg or 3′106 iNSCs/kg. The scaffold cohort received a single dose of iNSCs while the ICV cohort received three doses of iNSCs via a Rickham reservoir. To activate TK, canines were administered valganciclovir. Canine health was assessed via neurological exams, MRI, and serial blood, urine, and CSF analyses. RESULTS No acute injection reactions were observed. Three of four canines exhibited surgery-induced blindness. Urine and CSF analyses were unremarkable. Unexpectedly, blood analyses showed transient neutropenia. Hypodense signal was observed on all MRI sequences through endpoint. Post-mortem histopathology of the spleen, liver, and lung were unremarkable. As expected, brain tissues exhibited gliosis, fibrous thickening, and inflammation. Spinal cords exhibited acute hemorrhaging, attributed to perimortem CSF draws. Surprisingly, significant testicular degeneration was observed; this was confirmed to be caused by valganciclovir. In conclusion, iNSCs exhibit limited toxicity and warrant further exploration. FUTURE DIRECTIONS Prospective studies will investigate the efficacy of autologous iNSCs in a spontaneous canine glioma model in preparation for human clinical trials.

BACKGROUND Cerebrospinal fluid (CSF) lactate concentrations increase after seizure activity in many human patients independent of the underlying disease process. The effect of seizure activity on CSF lactate concentration in dogs is unknown. HYPOTHESIS/OBJECTIVES Cerebrospinal fluid lactate concentration is unaffected by seizure activity in dogs and is more dependent on the underlying disease process causing the seizures. ANIMALS One-hundred eighteen client-owned dogs with seizure disorders. METHODS Case series. Cerebrospinal fluid lactate concentration was determined using a commercially available lactate monitor. Seizure semiology, time from last seizure to CSF collection, number of seizures within the 72 hours preceding CSF collection, and clinical diagnosis were recorded. RESULTS Dogs with focal seizures had higher CSF lactate concentrations than did those with generalized seizures (P = .03). No differences in lactate concentrations were found among dogs with single seizures, cluster seizures or status epilepticus (P = .12), among dogs with CSF collection at different time points after the last seizure activity (P = .39) or among dogs having different numbers of seizures within the 72 hours preceding CSF collection (P = .42). A significant difference (P = .001) was found in CSF lactate concentrations among diagnostic groups, and dogs with inflammatory and neoplastic disease had higher concentrations than did dogs with idiopathic or unknown epilepsy. CONCLUSIONS AND CLINICAL IMPORTANCE Cerebrospinal fluid lactate concentration is minimally affected by seizure activity in dogs and increased concentrations are more likely associated with the underlying disease process.

In this study, we take an important step toward clinical translation by generating the first canine‐induced neural stem cells (iNSCs). We explore key aspects of scale‐up, persistence, and safety of personalized iNSC therapy in autologous canine surgery models. iNSCs are a promising new approach to treat aggressive cancers of the brain, including the deadly glioblastoma. Created by direct transdifferentiation of fibroblasts, iNSCs are known to migrate through the brain, track down invasive cancer foci, and deliver anticancer payloads that significantly reduce tumor burden and extend survival of tumor‐bearing mice. Here, skin biopsies were collected from canines and converted into the first personalized canine iNSCs engineered to carry TNFα‐related apoptosis‐inducing ligand (TRAIL) and thymidine kinase (TK), as well as magnetic resonance imaging (MRI) contrast agents for in vivo tracking. Time‐lapse analysis showed canine iNSCs efficiently migrate to human tumor cells, and cell viability assays showed both TRAIL and TK monotherapy markedly reduced tumor growth. Using intraoperative navigation and two delivery methods to closely mimic human therapy, canines received autologous iNSCs either within postsurgical cavities in a biocompatible matrix or via a catheter placed in the lateral ventricle. Both strategies were well tolerated, and serial MRI showed hypointense regions at the implant sites that remained stable through 86 days postimplant. Serial fluid sample testing following iNSC delivery showed the bimodal personalized therapy was well tolerated, with no iNSC‐induced abnormal tissue pathology. Overall, this study lays an important foundation as this promising personalized cell therapy advances toward human patient testing.

Stem cell transplantation, as used clinically, suffers from low retention and engraftment of the transplanted cells. Inspired by the ability of platelets to recruit stem cells to sites of injury on blood vessels, we hypothesized that platelets might enhance the vascular delivery of cardiac stem cells (CSCs) to sites of myocardial infarction injury. Here, we show that CSCs with platelet nanovesicles fused onto their surface membranes express platelet surface markers that are associated with platelet adhesion to injury sites. We also find that the modified CSCs selectively bind collagen-coated surfaces and endothelium-denuded rat aortas, and that in rat and porcine models of acute myocardial infarction the modified CSCs increase retention in the heart and reduce infarct size. Platelet-nanovesicle-fused CSCs thus possess the natural targeting and repairing ability of their parental cell types. This stem cell manipulation approach is fast, straightforward and safe, does not require genetic alteration of the cells, and should be generalizable to multiple cell types.

Cold-stunning in sea turtles is a frequent natural cause of mortality and is defined as a hypothermic state due to exposure to water temperatures12°C. Derangements of biochemistry and hematology data by cold stunning have been well documented, although the effects on coagulation have not yet been investigated. The objectives of this study were to characterize the hemostatic state of non-cold-stunned sea turtles and to compare cold-stunned sea turtles at admission and after successful rehabilitation via a sea turtle-specific thromboelastography (TEG) protocol. TEG enables evaluation of the entire coagulation process, and the methodology has recently been established in sea turtles. Initially, 30 wild and apparently healthy sea turtles were sampled as controls: loggerhead sea turtles (

OBJECTIVE To evaluate a panel of coagulation assays for their potential utility in rivaroxaban monitoring as alternatives to the rivaroxaban-specific anti-Xa activity (RIVA). DESIGN Prospective experimental study. SETTING University research laboratory. ANIMALS Five healthy neutered male Beagles. INTERVENTIONS Dogs were administered a median dose of 1.8 mg/kg rivaroxaban (range, 1.6-1.8 mg/kg) orally once daily for 2 consecutive days as part of a pharmacodynamic study. Blood was collected from a preplaced jugular catheter at time points relative to their rivaroxaban administration (0, 2, 4, 8, 24, 36, and 48 h) for measurement of RIVA, prothrombin time (PT), activated partial thromboplastin time, RapidTEG, and thrombin generation variables. MEASUREMENTS AND MAIN RESULTS One hundred forty data points were available for analysis. There was poor correlation between RIVA and RapidTEG variables: R time (R) (min) (r = 0.554, P < 0.0001), K time (K) (min) (r = -0.204, P = 0.016), alpha angle (degrees) (r = 0.152, P = 0.073), Maximum amplitude (MA) (mm) (r = 0.106, P = 0.215), and G value (G) (dynes/s) (r = 0.108, P = 0.205). A good correlation was noted between thrombin generation variables and RIVA: lag time (min) (r = 0.827, P < 0.0001), peak (nM) (r = -0.752, P < 0.0001), and endogenous thrombin potential (nM·min) (r = -0.762, P < 0.0001). There was an excellent correlation between PT and RIVA (r = 0.915, P < 0.0001) and a good correlation between activated partial thromboplastin time and RIVA (r = 0.772, P < 0 .0001). CONCLUSIONS Of all the coagulation tests investigated, the PT correlated best with RIVA. There is potential for PT being a convenient second-line monitoring option in dogs receiving rivaroxaban, but further work is necessary to validate other PT assays. Thromboelastography performed with strong activators correlated poorly with anti-Xa activity.

OBJECTIVE: Determine the correlation between kaolin‐activated thromboelastography (TEG) variables (R, K, angle, and maximum amplitude [MA]) and PCV, fibrinogen concentration (FC), and total fibrinogen (TF) in an ex vivo model. ANIMALS: Two healthy adult mixed‐breed dogs. PROCEDURES: Citrated whole blood was obtained and separated into packed red cells, platelet rich plasma, and platelet poor plasma (PPP). An aliquot of PPP was heated to denature heat labile proteins (fibrinogen, factor V, factor VIII). Blood components were recombined for analyses of 6 physiological scenarios: anemia with low fibrinogen; anemia with moderate fibrinogen; anemia with normal fibrinogen; anemia with normal saline; normal PCV and normal fibrinogen; and normal PCV and low fibrinogen. A Kruskal–Wallis test, along with linear regressions on pairwise combinations of TEG variables, was used to determine the correlation between TEG variables and PCV, FC, and TF. RESULTS: Maximum amplitude correlated with FC (R² 0.60, P < 0.001) and TF (R² 0.57, P < 0.001) but not PCV (R² 0.003, P = 0.7). Angle and K time were moderately correlated with FC ([angle: R² 0.53, P < 0.001]; [K: R² 0.55, P < 0.001]) and TF ([alpha angle: R² 0.52, P < 0.001]; [K: R² 0.51, P < 0.001]) but not PCV. The R time was weakly correlated with PCV (R² 0.15, P < 0.009) but not FC or TF. CONCLUSIONS AND CLINICAL RELEVANCE: In an ex vivo model, plasma proteins but not PCV impacted TEG variables. This suggests that TEG changes noted with anemia are imparted by changes in available fibrinogen in a fixed microenvironment rather than artifact of anemia.

Thromboelastography (TEG) provides a global evaluation of haemostasis. This diagnostic test is widely used in mammals but has not previously been performed in reptiles, mainly due to the limited availability of taxon-specific reagents. The objective of this pilot study was to establish a protocol to perform TEG in sea turtles. Pooled citrated plasma, stored at −80°C, from four green turtles (Chelonia mydas) was assayed on a TEG 5000. Several initiators were evaluated: kaolin (n=2), RapidTEG (n=2), fresh (n=2) and frozen (n=6) thromboplastin extracted from pooled brain tissue from several chelonian species, human recombinant tissue factor at 1:100 (n=1), Reptilase (n=2), and rabbit thromboplastin (n=1). Both fresh and frozen chelonian thromboplastin were superior in producing quantifiable TEG reaction time compared with all other reagents. These findings are consistent with the lack of an intrinsic pathway in turtles and confirmed a lack of coagulation in the turtle samples in response to mammalian thromboplastin. A TEG protocol was subsequently established for harvested species-specific frozen thromboplastin. The frozen thromboplastin reagent remained stable after one year of storage at −80°C. The developed protocol will be useful as a basis for future studies that aim to understand the pathophysiology of haemostatic disorders in various stranding conditions of sea turtles.

Cold-stunning in sea turtles is a frequent natural cause of mortality and is defined as a hypothermic state due to exposure to water temperatures