Intramuscular alfaxalone with or without buprenorphine or hydromorphone provides sedation with minimal adverse effects in healthy rabbits (Oryctolagus cuniculus) in a randomized blinded controlled trial.

OBJECTIVE: To evaluate the effects of alfaxalone administered IM with or without buprenorphine or hydromorphone in healthy rabbits (Oryctolagus cuniculus). ANIMALS: 24 male rabbits undergoing elective orchiectomy between August 21, 2021, and November 6, 2021. PROCEDURES: In this controlled clinical trial, rabbits were randomly assigned to receive alfaxalone (4 mg/kg, IM) alone (group A; n = 8) or with buprenorphine (0.03 mg/kg, IM; group BA; 8) or hydromorphone (0.1 mg/kg, IM; group HA; 8). Vital signs and sedation scores were recorded immediately prior to (T0) and 10 minutes after (T1) treatment. Ease of IV catheter placement and pain scores were also evaluated. All rabbits received ketamine (2.5 mg/kg, IV), midazolam (0.13 mg/kg, IV), and meloxicam (0.5 mg/kg, SC) before orchiectomy but after IM treatments. Results were compared across groups with ANOVA or Fisher exact tests and across time with paired t tests. RESULTS: Sedation score, median time to recumbency, and ease of catheter placement did not differ among groups. Supraglottic airway device placement was possible for 1 rabbit in group A, 1 in group BA, and 2 in group HA. Mean respiratory rate at T1 versus T0 was significantly decreased for groups BA (63.8 vs 128.6 breaths/min) and HA (66.7 vs 123.2 breaths/min). Mean postoperative pain scores were significantly lower for rabbits in group HA (0.58), compared with those in groups A (2.25) and BA (2.06). CLINICAL RELEVANCE: All 3 treatments provided reliable sedation; however, alfaxalone (4 mg/kg, IM) combined with hydromorphone (0.1 mg/kg, IM) may be a better choice for painful procedures.

Insulin activation of the phosphatidylinositol 3-kinase/protein kinase B (Akt) pathway reduces lipopolysaccharide-induced inflammation in mice.

Insulin is used to control pro-inflammatory hyperglycemia in critically ill patients. However, recent studies suggest that insulin-induced hypoglycemia may negate its beneficial effects in these patients. It is noteworthy that recent evidence indicates that insulin has anti-inflammatory effects that are independent of controlling hyperglycemia. To date, the mechanism by which insulin directly reduces inflammation has not been elucidated. It is well established that insulin activates phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling in many cell types. We and others have shown that this pathway negatively regulates LPS-induced signaling and pro-inflammatory cytokine production in monocytic cells. We hypothesized that insulin inhibits inflammation during endotoxemia by activation of the PI3K/Akt pathway. We used a nonhyperglycemic mouse model of endotoxemia to determine the effect of continuous administration of a low dose of human insulin on inflammation and survival. It is noteworthy that insulin treatment induced phosphorylation of Akt in muscle and adipose tissues but did not exacerbate lipopolysaccharide (LPS)-induced hypoglycemia. Insulin decreased plasma levels of interleukin-6, tumor necrosis factor-alpha, monocyte chemotactic protein 1 (MCP1)/JE, and keratinocyte chemoattractant, and decreased mortality. The PI3K inhibitor wortmannin abolished the insulin-mediated activation of Akt and the reduction of chemokine and interleukin-6 levels. We conclude that insulin reduces LPS-induced inflammation in mice in a PI3K/Akt-dependent manner without affecting blood glucose levels.

Tissue factor activity is increased in a combined platelet and microparticle sample from cancer patients.

BACKGROUND: Cancer patients have an increased risk of thrombosis. Tissue factor (TF) antigen and TF activity associated with microparticles in plasma are elevated in patients with various types of cancer. Of these two measurements, TF activity is considered superior to TF antigen levels because the activity more closely reflects the ability of TF to initiate coagulation. Recent studies showed that platelets also express TF. OBJECTIVE: To determine the level of TF activity associated with a combined platelet and microparticle sample from cancer patients (n = 20) and healthy individuals (n = 23). METHODS: TF activity was measured using a two step chromogenic assay and soluble P-selectin was measured by ELISA in healthy controls and metastatic cancer patients. RESULTS: We determined the composition of a combined platelet and microparticle sample. The sample consisted of platelets, large microparticles (30-200 nm) and membrane debris. We compared the TF activity of a combined platelet and microparticle sample from cancer patients with that from healthy individuals. We found that TF activity in a combined platelet and microparticle sample from cancer patients was higher than in samples from healthy individuals (21.5+/-12.3 pM (n = 20) versus 8.6+/-6.8 pM (n = 23), mean+/-SD, p < 0.001). Cancer patients also had a higher level of soluble P-selectin compared with controls (18.9+/-5.5 ng/mL versus 13.2+/-2.3 ng/mL, p < 0.001). CONCLUSION: This study indicates that measurement of TF activity in a combined platelet and microparticle sample can be used as a simple assay to determine the level of circulating TF.

Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis.

Hemostasis requires both platelets and the coagulation system. At sites of vessel injury, bleeding is minimized by the formation of a hemostatic plug consisting of platelets and fibrin. The traditional view of the regulation of blood coagulation is that the initiation phase is triggered by the extrinsic pathway, whereas amplification requires the intrinsic pathway. The extrinsic pathway consists of the transmembrane receptor tissue factor (TF) and plasma factor VII/VIIa (FVII/FVIIa), and the intrinsic pathway consists of plasma FXI, FIX, and FVIII. Under physiological conditions, TF is constitutively expressed by adventitial cells surrounding blood vessels and initiates clotting. In addition so-called blood-borne TF in the form of cell-derived microparticles (MPs) and TF expression within platelets suggests that TF may play a role in the amplification phase of the coagulation cascade. Under pathologic conditions, TF is expressed by monocytes, neutrophils, endothelial cells, and platelets, which results in an elevation of the levels of circulating TF-positive MPs. TF expression within the vasculature likely contributes to thrombosis in a variety of diseases. Understanding how the extrinsic pathway of blood coagulation contributes to hemostasis and thrombosis may lead to the development of safe and effective hemostatic agents and antithrombotic drugs.

Tissue factor: a link between C5a and neutrophil activation in antiphospholipid antibody induced fetal injury.

Fetal loss in patients with antiphospholipid (aPL) antibodies has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically activation of complement with generation of the anaphylotoxin C5a, is an essential trigger of fetal injury. In this study, we analyzed the role of the procoagulant molecule tissue factor (TF) in a mouse model of aPL antibody-induced pregnancy loss. We found that either blockade of TF with a monoclonal antibody in wild-type mice or a genetic reduction of TF prevented aPL antibody-induced inflammation and pregnancy loss. In response to aPL antibody-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells but not fetal-derived cells (trophoblasts) was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL antibodies. The identification of TF as an important mediator of C5a-induced oxidative burst in neutrophils in aPL-induced fetal injury provides a new target for therapy to prevent pregnancy loss in the antiphospholipid syndrome.

Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenicity of human platelets.

Tissue factor (TF) is an essential cofactor for the activation of blood coagulation in vivo. We now report that quiescent human platelets express TF pre-mRNA and, in response to activation, splice this intronic-rich message into mature mRNA. Splicing of TF pre-mRNA is associated with increased TF protein expression, procoagulant activity, and accelerated formation of clots. Pre-mRNA splicing is controlled by Cdc2-like kinase (Clk)1, and interruption of Clk1 signaling prevents TF from accumulating in activated platelets. Elevated intravascular TF has been reported in a variety of prothrombotic diseases, but there is debate as to whether anucleate platelets-the key cellular effector of thrombosis-express TF. Our studies demonstrate that human platelets use Clk1-dependent splicing pathways to generate TF protein in response to cellular activation. We propose that platelet-derived TF contributes to the propagation and stabilization of a thrombus.

Genetic susceptibility to thrombosis.

Thrombosis is associated with atherosclerosis, sepsis, cancer, and numerous other inflammatory diseases. Complications of thrombosis, such as myocardial infarction, stroke, and venous thromboembolism, contribute significantly to morbidity and mortality. Susceptibility to thrombosis is conferred by both genetic and environmental factors. Tissue factor is the primary cellular initiator of blood coagulation and is a major contributor to thrombosis. In this review, we discuss the association between various polymorphisms and the risk for thrombosis.

Tissue factor in hemostasis and thrombosis.

Tissue factor (TF) plays an essential role in hemostasis. The tissue-specific pattern of TF expression is consistent with additional hemostatic protection in vital organs. An aberrant expression of TF within the vasculature occurs in a variety of diseases, including atherosclerosis, cancer, and sepsis. TF expression in these diseases is associated with thrombotic events. Future therapeutic strategies may prove beneficial in the treatment of thrombosis. However, these strategies should be designed to avoid compromising hemostasis.

Atherosclerosis in mice is not affected by a reduction in tissue factor expression.

OBJECTIVE: To determine whether tissue factor (TF) contributes to the progression of atherosclerotic lesions in mice. METHODS AND RESULTS: We determined the effect of a 50% reduction of TF levels in all cells on atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. No differences were observed in the extent of atherosclerosis in apoE(-/-)/TF(+/+) and apoE(-/-)/TF(+/-) mice fed regular chow for 34 weeks. Atherosclerosis could not be analyzed in apoE(-/-) mice expressing low levels of TF because of premature death of these mice. Macrophages are a major source of TF in atherosclerotic plaques. Therefore, in a second series of experiments, we investigated the effect on atherosclerosis of selectively reducing hematopoietic cell-derived TF by transplanting bone marrow from mice expressing low levels of TF into low-density lipoprotein receptor deficient (LDLR(-/-)) mice. Atherosclerosis within the arterial tree and aortic root were similar in LDLR(-/-) mice with low-TF bone marrow compared with control bone marrow (TF(+/+) or TF(+/-)) after 4 and 16 weeks on an atherogenic diet. Furthermore, the cellular composition of the aortic root lesions was similar between the 2 groups. CONCLUSIONS: Our data indicate that either a 50% reduction of TF in all cells or a selective reduction in hematopoietic cell-derived TF does not affect the development of atherosclerotic lesions in mice.

Postprandial elevation of tissue factor antigen in the blood of healthy adults.

Atherosclerosis is a dynamic disease involving lipid metabolism, inflammation and thrombosis. A key factor in thrombosis is tissue factor, a small transmembrane glycoprotein. Tissue factor binds FactorVIIa, and this complex converts Factor X to Factor Xa, leading to thrombin generation and fibrin formation. Inhibition of this pathway is by tissue factor pathway inhibitor (TFPI). Tissue factor is found sequestered within atherosclerotic plaques, and plaque rupture allows tissue factor exposure to the circulation, leading to formation of a thrombus. Tissue factor is also associated with membrane microparticles in the circulation, most likely released from monocytes activated by an inflammatory event. We hypothesize that consumption of a typical western diet that is moderate in fat content leads to elevated levels of circulating tissue factor that may act as a marker of a prothrombotic state. Healthy volunteers, aged 18-55, consumed a moderate (40%) fat meal, with blood taken before and 3.5 and 6 h after the meal. Plasma was isolated and assayed for plasma triglycerides, tissue factor, thrombin antithrombin (TAT) complexes, TFPI and TNFalpha. The levels of circulating tissue factor increased 56% (from 78 pg/ml to 120 pg/ml) 3.5 h after the meal. Levels decreased, but had not returned to baseline 6 h postprandially. No significant differences in TAT, TFPI and TNFa levels were observed postprandially. These results demonstrate increased tissue factor levels in individuals who consumed a moderate fat diet. This suggests that the typical western diet may play a larger role in cardiovascular disease than merely altering lipid profiles.

Determinants of meat quality: tenderness.

Meat quality is a term used to describe a range of attributes of meat. Consumer research suggests that tenderness is a very important element of eating quality and that variations in tenderness affect the decision to repurchase. The present paper highlights recent information on the factors that affect tenderness. While the precise aetiology is not fully understood, a number of factors have been shown to affect tenderness. Of these factors, postmortem factors, particularly temperature, sarcomere length and proteolysis, which affect the conversion of muscle to meat, appear most important. However, it is now becoming clear that variation in other factors such as the muscle fibre type composition and the buffering capacity of the muscle together with the breed and nutritional status of the animals may also contribute to the observed variation in meat tenderness.

Isolation of two cytochrome P450 cDNAs, CYP1A1 and CYP1A2, from harp seal (Phoca groenlandica) and grey seal (Halichoerus grypus).

Two cytochrome P450 (CYP), CYP1A1 and CYP1A2, cDNA sequences have been isolated and cloned from harp seal (Phoca groenlandica) and grey seal (Halichoerus grypus). EROD, a model substrate for CYP1A, and heterologous antibodies have been employed as a biomarker in marine mammals, however the CYP1A sequences have not been characterised in these two seal species. mRNA was used as the template in RT-PCR, rather than DNA as this indicates transcription of the CYP1A gene in these seal species exposed to environmental contaminants. Harp and grey seal CYP1A1 amino acid sequences exhibited >99% identity and the CYP1A2 sequences were >98% identical. Phylogenetic analyses of the two seal species with other mammalian, and avian CYP1A sequences, showed the CYP1A1 and CYP1A2 sequences clustered with corresponding sequences in other mammalian species. The closest sequences to the seal CYP1As was dog CYP1A. The CYP1A sequence information presented in this study has provided the necessary data for the future production of species-specific probes for the use as biomarkers of environmental contaminant exposure.