Tracy Stokol

Concentrated Bone Marrow Aspirate Improves Full-thickness Cartilage Repair Compared with Microfracture in the Equine Model

BACKGROUND The purpose of this study was to compare the outcomes of treatment with bone marrow aspirate concentrate, a simple, one-step, autogenous, and arthroscopically applicable method, with the outcomes of microfracture with regard to the repair of full-thickness cartilage defects in an equine model. METHODS Extensive (15-mm-diameter) full-thickness cartilage defects were created on the lateral trochlear ridge of the femur in twelve horses. Bone marrow was aspirated from the sternum and centrifuged to generate the bone marrow concentrate. The defects were treated with bone marrow concentrate and microfracture or with microfracture alone. Second-look arthroscopy was performed at three months, and the horses were killed at eight months. Repair was assessed with use of macroscopic and histological scoring systems as well as with quantitative magnetic resonance imaging. RESULTS No adverse reactions due to the microfracture or the bone marrow concentrate were observed. At eight months, macroscopic scores (mean and standard error of the mean, 9.4 + or - 1.2 compared with 4.4 + or - 1.2; p = 0.009) and histological scores (11.1 + or - 1.6 compared with 6.4 + or - 1.2; p = 0.02) indicated improvement in the repair tissue in the bone marrow concentrate group compared with that in the microfracture group. All scoring systems and magnetic resonance imaging data indicated that delivery of the bone marrow concentrate resulted in increased fill of the defects and improved integration of repair tissue into surrounding normal cartilage. In addition, there was greater type-II collagen content and improved orientation of the collagen as well as significantly more glycosaminoglycan in the bone marrow concentrate-treated defects than in the microfracture-treated defects. CONCLUSIONS Delivery of bone marrow concentrate can result in healing of acute full-thickness cartilage defects that is superior to that after microfracture alone in an equine model. CLINICAL RELEVANCE Delivery of bone marrow concentrate to cartilage defects has the clinical potential to improve cartilage healing, providing a simple, cost-effective, arthroscopically applicable, and clinically effective approach for cartilage repair.

Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States

The objectives were to evaluate the effects of elevated pre- and postpartum nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) concentrations during the transition period on reproductive performance and milk production in dairy cattle. In a prospective cohort study of 91 freestall, total mixed ration-fed herds in the northeastern United States, blood samples were collected from approximately 15 prepartum and 15 different postpartum transition animals in each herd. All samples were stratified based on pre- or postpartum status at the time of sample collection, and 2,259 and 2,290 animals were used to evaluate reproductive and milk production performance, respectively. Reproductive performance was assessed by time to conception within 70 d post-voluntary waiting period (VWP) and milk production was assessed using mature-equivalent 305-d (ME305) milk yield estimated at 120 d in milk. While controlling for body condition score (BCS), calving season, median ME305 milk production, and parity, NEFA and BHBA concentrations were evaluated with time to event analysis to investigate reproductive performance. These same predictor variables were used to determine the effects of elevated NEFA and BHBA concentrations on ME305 milk yield with herd as a random effect. Heifers and cows were grouped in the final analyses if the results between groups were similar. In all animals sampled prepartum, the risk of pregnancy within 70 d post-VWP was reduced by 19% when NEFA concentrations were >or=0.27 mEq/L. In all animals sampled postpartum, those with NEFA concentrations >or=0.72 mEq/L had a 16% decrease in risk of pregnancy and those with BHBA concentrations >or=10mg/dL had a 13% decrease in risk. In cows and heifers, ME305 milk yield was decreased by 683 kg when prepartum NEFA concentrations were >or=0.33 mEq/L. In heifers sampled postpartum, ME305 milk yield was increased by 488 kg when NEFA concentrations were >or=0.57 mEq/L and increased by 403 kg when BHBA concentrations were >or=9 mg/dL. In cows sampled postpartum, ME305 milk yield was decreased by 647 kg when NEFA concentrations were >or=0.72 mEq/L and decreased by 393 kg when BHBA concentrations were >or=10mg/dL. With the exception of milk production in heifers, this study indicates that increased concentrations of serum NEFA and BHBA had a detrimental effect on reproductive performance and milk production.

Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.

In this study the herd alarm level was defined as the proportion of sampled transition cows per herd with increased prepartum nonesterified fatty acid (NEFA), postpartum beta-hydroxybutyrate (BHBA), or NEFA concentrations that were associated with herd-level incidence of displaced abomasum (DA) or clinical ketosis (CK), pregnancy rate (PR), and milk production. The objectives were to 1) identify the herd alarm level for excessive negative energy balance and 2) describe the herd-level prevalence of this proportion. This was a prospective cohort study of 60 free-stall herds fed total mixed rations in the northeast United States. Two cohorts of approximately 15 animals were assessed for prepartum NEFA and postpartum BHBA and NEFA. The herd alarm level (i.e., the proportion of sampled animals above a certain metabolite threshold) was as follows: 15% had prepartum NEFA of 0.27 mEq/L; 15 and 20% had BHBA of 10 and 12 mg/dL, respectively; and 15% had postpartum NEFA of 0.60 and 0.70 mEq/L. The different herd alarm levels correspond to differences between the metabolites and respective herd-level effect. The herd-level effects for herds above the herd alarm level for prepartum NEFA were 3.6% increase in DA and CK incidence, 1.2% decrease in PR, and 282 kg decrease in average mature equivalent 305-d (ME 305) milk. For BHBA, the herd-level effects were a 1.8% increase in DA and CK, 0.8% decrease in PR, and 534 and 358 kg decrease in projected ME 305 milk yield for heifers and cows, respectively. For postpartum NEFA, the herd-level effects were 1.7% increase in DA and CK, 0.9% decrease in PR, and 288 and 593 kg decrease in projected ME 305 milk yield for heifers and cows, respectively. The prevalence of herds in which more than 15% of animals sampled had prepartum NEFA concentration >or=0.30 mEq/L was 75%, BHBA >or=12 mg/dL was 40%, and postpartum NEFA >or=0.70 mEq/L was 65%. This study showed that there were detrimental herd-level effects if a large enough proportion of cows had increased metabolite concentrations, and further demonstrated that a high prevalence of herds have opportunity for improvement.

Hypercoagulability in Cats with Cardiomyopathy

BACKGROUND Arterial thromboembolism (ATE) is a common complication of feline cardiomyopathy; however, the pathogenesis of ATE is unknown. HYPOTHESIS Systemic activation of the coagulation cascade (hypercoagulability) and endothelial injury promote ATE in cardiomyopathic cats. ANIMALS Healthy cats (n = 30) and 3 groups of cardiomyopathic cats: Group (1) left atrial enlargement only (LAE [n = 11]), ie, left atrial to aortic ratio >1.4; Group (2) LAE with spontaneous echocardiographic contrast, atrial thrombi or both (SEC-T [n = 16]); and Group (3) acute ATE with LAE (n = 16). METHODS Hypercoagulability was defined by 2 or more laboratory abnormalities reflecting coagulation factor excess (high fibrinogen concentration or Factor VIII coagulant activity), inhibitor deficiency (low antithrombin activity), or thrombin generation (high thrombin-antithrombin complex [TAT] and d-dimer concentrations). High von Willebrand factor antigen concentration (vWF : Ag) was considered a marker of endothelial injury. Data were analyzed using nonparametric statistics. RESULTS The 3 groups of cats with cardiac disease had higher median fibrinogen concentrations than did the healthy cats. Criteria of hypercoagulability were found exclusively in cats with SEC-T (50%) and ATE (56%). Hypercoagulability was not associated with left atrial size or congestive heart failure (CHF). ATE cats had significantly higher median vWF : Ag concentration than did the other groups. CONCLUSION AND CLINICAL IMPORTANCE Systemic hypercoagulability is evident in many cardiomyopathic cats, often without concurrent CHF or overt ATE. Hypercoagulabilty may represent a risk factor for ATE. High vWF : Ag in ATE cats was attributed to downstream endothelial injury from the occlusive thrombus.

Plasma D-dimer for the diagnosis of thromboembolic disorders in dogs

D-dimer is formed during thrombus formation when factor XIIIa crosslinks the terminal D-domains of fibrin. The D-dimer epitope is exposed when the thrombus is lysed by plasmin. Thus, D-dimer represents both thrombin and plasmin activation and is specific for fibrinolysis. D-dimer concentrations are increased in dogs with DIC or other thromboembolic disorders, but because D-dimer is an indicator of physiologic or pathologic fibrinolysis, values are elevated in other conditions associated with fibrinolysis, including orthopedic surgery, neoplasia, and internal hemorrhage. It can be used as an ancillary test for the diagnosis of DIC but is not recommended as a sole test for this purpose. D-dimer has the potential to be a useful laboratory test for the detection of pulmonary thromboembolism in dogs. Further studies are needed to determine the appropriate applications for this test in veterinary patients to aid in clinical decision making, treatment, and patient care.

Using Nonesterified Fatty Acids and β-Hydroxybutyrate Concentrations During the Transition Period for Herd-Level Monitoring of Increased Risk of Disease and Decreased Reproductive and Milking Performance

Dairy cows visit a state of negative energy balance (NEB) as they transition from late gestation to early lactation. At the individual level, there are several metabolic adaptations to manage NEB, including mobilization of nonesterified fatty acids (NEFA) from body fat reserves and glucose sparing for lactogenesis. Based on current pen-level feeding and management practices, strategies to minimize excessive NEB in both the individual and herd should focus on herd-level testing and management. This article reviews strategies for testing and monitoring of excessive NEB at the herd level through individual testing of 2 energy markers: NEFA and β-hydroxybutyrate.

Clinical and clinicopathologic features of dogs that consumed foodborne hepatotoxic aflatoxins: 72 cases (2005–2006)

OBJECTIVE To characterize clinical signs, clinicopathologic features, treatments, and survival in dogs with naturally acquired foodborne aflatoxicosis. DESIGN Retrospective case series. ANIMALS 72 dogs that consumed aflatoxin-contaminated commercial dog food. PROCEDURES Medical records of affected dogs were reviewed. Between December 2005 and March 2006, dogs were identified as having foodborne aflatoxin hepatotoxicosis on the basis of the history of consumption of contaminated food or characteristic histopathologic lesions (subject dog or a recently deceased dog in the same household or kennel). Recorded information included signalment, clinical features, clinicopathologic test results, treatments, and survival. Data were analyzed by survival status. RESULTS Most dogs were of large breeds from breeding kennels. No significant differences were found in age or weight between 26 (36%) survivor dogs and 46 (64%) nonsurvivor dogs. Severity of clinical signs varied widely; 7 dogs died abruptly. In order of onset, clinical features included anorexia, lethargy, vomiting, jaundice, diarrhea (melena, hematochezia), abdominal effusion, peripheral edema, and terminal encephalopathy and hemorrhagic diathesis. Common clinicopathologic features included coagulopathic and electrolyte disturbances, hypoproteinemia, increased serum liver enzyme activities, hyperbilirubinemia, and hypocholesterolemia. Cytologic hepatocellular lipid vacuolation was confirmed in 11 dogs examined. In comparisons of clinicopathologic test results between survivor and nonsurvivor dogs, only granular cylindruria (7/21 dogs) consistently predicted death. Best early markers of aflatoxicosis were low plasma activities of anticoagulant proteins (protein C, antithrombin) and hypocholesterolemia. Despite aggressive treatment, many but not all severely affected dogs died. CONCLUSIONS AND CLINICAL RELEVANCE Serum liver enzyme activities and bilirubin concentration were unreliable early markers of aflatoxin hepatotoxicosis in dogs. Hypocholesterolemia and decreased plasma protein C and antithrombin activities may function as exposure biomarkers.

Characterization of In Vitro Endothelial Linings Grown Within Microfluidic Channels

In vivo, endothelial cells grow on the inner surface of blood vessels and are shaped to conform to the vessels geometry. In the smallest vessels this shape entails substantial bending within each cell. Microfabricated channels can replicate these small-scale geometries, but endothelial cells grown within them have not been fully characterized. In particular, the presence of focal adhesions and adherens junctions in endothelial cells grown in microchannels with corners has not been confirmed. We have fabricated square microfluidic channels (50 μm wide, 50 μm deep) and semicircular microfluidic channels (60 μm wide, 45 μm deep) in polydimethylsiloxane and cultured human umbilical vein endothelial cells (HUVEC) within them. Immunofluorescent staining and three-dimensional reconstruction of image stacks taken with confocal microscopy confirmed that HUVEC are capable of forming adherens junctions on all channel walls in both channel geometries, including the sidewalls of square profile channels. The presence of shear stress is critical for the cells to form focal adhesions within both channel geometries. Shear stress is also responsible for the conforming of HUVEC to the channel walls and produces a square cross-sectional geometry of in vitro endothelial linings within square profile channels. Thus, geometry and applied shear stress are important design criteria for the development of in vitro endothelial linings of microvessels.

Clinical Efficacy and Safety of Recombinant Canine Erythropoietin in Dogs with Anemia of Chronic Renal Failure and Dogs with Recombinant Human Erythropoietin-Induced Red Cell Aplasia

The efficacy and safety of recombinant canine erythropoietin (rcEPO) therapy was evaluated in 19 dogs with anemia of chronic renal failure (group 1) and 6 dogs with chronic renal failure and recombinant human erythropoietin (rhEPO)-induced red cell aplasia (group 2). Hematocrit (Hct) and absolute reticulocyte count (ARC) were monitored weekly for the first 8 weeks, CBC (including ARC) and serum iron profiles were evaluated monthly, and serum biochemical analyses were performed every 2 months for 6 (group 2) to 12 (group 1) months. For group 1 dogs, median Hct and ARC increased significantly during the 1st week of rcEPO treatment, and median Hct was sustained at >35% after week 5. In contrast, median Hct and ARC for group 2 did not change significantly with rcEPO treatment, even with doses greater than those used in group 1. Nevertheless, 2 (33%) of the 6 dogs in group 2 developed erythroid hyperplasia, reticulocytosis, and increases in Hct with rcEPO treatment. Although median systolic blood pressure did not change significantly in either group, 5 dogs developed systolic blood pressures > or = 180 mm Hg during the study. Appetite and energy level improved in most group 1 dogs with increases in Hct. Recombinant cEPO stimulated erythrocyte production in dogs with nonregenerative anemia secondary to chronic renal failure without causing the profound erythroid hypoplasia that can occur in rhEPO-treated dogs. Unfortunately, rcEPO was not as effective in restoring erythrocyte production in dogs that had previously developed rhEPO-induced red cell aplasia.

Monoclonal antibodies to equine CD14.

CD14 is a receptor for the complex of lipopolysaccaride (LPS) and LPS-binding protein. Binding of this complex to CD14 in association with Toll-like receptor 4 provides a major pathway for the initiation of innate immune responses to bacterial pathogens. We used a mammalian expressed extracellular region of equine CD14 (rCD14) derived from an IgG fusion protein to produce monoclonal antibodies (mAbs) to CD14. Eight mAbs were tested by flow cytometric analysis of equine leukocytes and by immunoblotting using rCD14 indicating that the mAbs recognized at least three different epitopes on equine CD14. One mAb, clone 105, was used for further characterization of CD14+ cells in peripheral blood mononuclear cells (PBMC). Phenotyping indicated that the majority of the CD14+ PBMC were non-B/non-T-cells. Magnetic cell sorting enriched CD14+ cells to > 95% as detected by flow cytometry. Differential cell counts on Wrights-stained cytospin smears of CD14+ cell fractions demonstrated that 49-73% of them were monocytes. The discrepancy between CD14+ cells detected by flow cytometric analysis and monocytes based on morphologic criteria suggests that some of the equine CD14+ PBMC are lymphoid cells. The mAbs to equine CD14 provide new tools for cellular analysis and CD14+ cell isolation in horses.