In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

Sepsis is the leading cause of critical illness and mortality in human beings and animals. Neutrophils are the primary effector cells of innate immunity during sepsis. Besides degranulation and phagocytosis, neutrophils also release neutrophil extracellular traps (NETs), composed of cell-free DNA, histones, and antimicrobial proteins. Although NETs have protective roles in the initial stages of sepsis, excessive NET formation has been found to induce thrombosis and multiple organ failure in murine sepsis models. Since the discovery of NETs nearly a decade ago, many investigators have identified NETs in various species. However, many questions remain regarding the exact mechanisms and fate of neutrophils following NET formation. In humans and mice, platelet-neutrophil interactions via direct binding or soluble mediators seem to play an important role in mediating NET formation during sepsis. Preliminary data suggest that these interactions may be species dependent. Regardless of these differences, there is increasing evidence in human and veterinary medicine suggesting that NETs play a crucial role in the pathogenesis of intravascular thrombosis and multiple organ failure in sepsis. Because the outcome of sepsis is highly dependent on early recognition and intervention, detection of NETs or NET components can aid in the diagnosis of sepsis in humans and veterinary species. In addition, the use of novel therapies such as deoxyribonuclease and non-anticoagulant heparin to target NET components shows promising results in murine septic models. Much work is needed in translating these NET-targeting therapies to clinical practice.

Background

Functional Toll-like receptor 4 (TLR4) has been characterized in human and murine platelets indicating that platelets play a role in inflammation and hemostasis during sepsis. It is unclear whether canine platelets could express functional TLR4 by responding to its ligand, lipopolysaccharide (LPS). We sought to determine if dogs express functional TLR4 and if LPS-induced platelet activation requires co-stimulation with ADP or thromboxane A₂ (TxA₂). Canine platelets were unstimulated (resting) or activated with thrombin or ADP prior to flow cytometric or microscopic analyses for TLR4 expression. We treated resting or ADP-primed platelets with LPS in the absence or presence of acetylsalicylic acid (ASA) and inhibited TLR4 with function blocking antibody or LPS from Rhodobacter sphaeroides (LPS-RS).

Results

We discovered that dog platelets have variable TLR4 expression, which was upregulated following thrombin or ADP activation. LPS augmented P-selectin expression and thromboxane B₂ secretion in ADP-primed platelets via TLR4. Inhibition of cyclooxygenase by ASA attenuated LPS-mediated P-selectin expression demonstrating that TLR4 signaling in platelets is partially dependent on TxA₂ pathway.

Conclusion

Expression of functional TLR4 on canine platelets may contribute to hypercoagulability in clinical septic dogs. Cyclooxygenase and TxA₂ pathways in TLR4-mediated platelet activation may present novel therapeutic targets in dogs with sepsis.

Neutrophils release neutrophil extracellular traps (NETs), which are extracellular chromatin decorated with histones and antimicrobial proteins. Although known for antimicrobial properties, overzealous production of NETs (NETosis) may lead to cytotoxicity and multiple organ failure in sepsis. Pathogen-induced NETosis has been extensively studied in mice but its importance in dogs remains largely unknown. This study sought to characterize in vitro NETosis induced by E.coli LPS, including assessing the role of peptidylarginine deiminase (PAD) in canine NETosis. Neutrophils (1×10⁶ cells/ml) from healthy dogs were isolated and treated with 100μg/ml LPS, 100nM phorbol 12-myristate 13-acetate (PMA), or buffer for either 90 or 180min. NETs were assessed using fluorescence microscopy of living neutrophils and immunofluorescent microscopy. Supernatant and cellular debris were purified and cell-free DNA was quantified by spectrophotometry. The role of PAD was assessed by treating LPS- and PMA-activated neutrophils with 50, 100 or 200μM of the PAD inhibitor, Cl-amidine. In vitro NETosis was characterized by co-localization of cell-free DNA, citrullinated histone H3, and myeloperoxidase. LPS stimulation resulted in intracellular citrullination of histone H3. Compared to PMA chemically-induced NETosis, LPS resulted in smaller NETs with less extracellular citrullinated histone H3. Cl-amidine decreased citrullination of histones and NET production in either LPS- or PMA-stimulated neutrophils demonstrating that neutrophil PAD is essential for these cellular processes.

Background

Canine neutrophils release neutrophil extracellular traps (NETs) in response to lipopolysaccharide but NETs from clinical septic dogs had not been identified. The primary aim is to describe the methodology of identifying and quantifying neutrophil extracellular traps (NETs) in cytology samples of septic foci in dogs with sepsis using immunofluorescence microscopy. Cytology samples including endotracheal tracheal wash (ETW), bronchoalveolar lavage (BAL), abdominal and pleural effusion collected from 5 dogs (3 septic, 2 non-septic) were fixed, permeabilized and stained for myeloperoxidase (MPO), citrullinated histone H3 (citH3) and cell-free DNA (cfDNA). Fluorescence microscopy was used to identify and quantify NETs in 10 random views at 40× magnification. NETs were identified based on co-localization of MPO, citH3 and cfDNA. NETs were quantified as a ratio (number of NETs: number of neutrophils). Neutrophils were identified based on cytoplasmic MPO, cellular diameter and nuclear morphology.

Results

NETs were identified and quantified in all cytology samples collected from septic dogs. A small number of NETs was documented in one dog with sterile chronic bronchitis. No NETs were found in sterile abdominal effusion collected from one dog with congestive heart failure.

Conclusions

Immunofluorescence microscopy could be a useful tool for the study of NETs in dogs with clinical sepsis.

Background

Equine whole blood collection and storage methods have been evaluated to assess red blood cell viability; however, platelet (PLT) viability has not been comprehensively assessed.

Objectives

The purpose of the study was to compare viability of PLTs collected in whole blood into 2 different anticoagulants.

Methods

Whole blood from 6 healthy adult Thoroughbred horses was collected into citrate-phosphate-dextrose-adenine (CPDA) or acid-citrate-dextrose (ACD). Platelet count, pH, and concentrations of glucose, lactate, carbon dioxide, oxygen, bicarbonate, sodium, potassium, and chloride were measured within 10 minutes of collection and then again one hour later at which time PLT aggregometry was performed to assess PLT function.

Results

Aggregometry mean amplitudes were significantly higher in CPDA compared to ACD. Blood glucose, pH, bicarbonate, sodium, and lactate concentrations were significantly higher in CPDA compared to ACD. Lactate concentration was higher following one hour in either anticoagulant. Potassium, oxygen, and carbon dioxide concentrations were significantly higher in ACD compared to CPDA at collection.

Conclusions

Platelet aggregometry results suggest that CPDA is superior to ACD for maintaining PLT viability following whole blood collection. This may be associated with the higher, more neutral pH as well as an increase in glucose available for metabolism. Although lactate was increased in the CPDA samples it was not high enough to decrease pH and therefore may not have been high enough to cause morphologic lesions and loss of PLT viability.

Objective

To compare fiber diameter, pore area, compressive stiffness, gelation properties, and selected growth factor content of platelet-rich fibrin gels (PRFGs) and conventional fibrin gels (FGs).

Sample

PRFGs and conventional FGs prepared from the blood of 10 healthy horses.

Procedures

Autologous fibrinogen was used to form conventional FGs. The PRFGs were formed from autologous platelet-rich plasma of various platelet concentrations (100 × 10³ platelets/μL, 250 × 10³ platelets/μL, 500 × 10³ platelets/μL, and 1,000 × 10³ platelets/μL). All gels contained an identical fibrinogen concentration (20 mg/mL). Fiber diameter and pore area were evaluated with scanning electron microscopy. Maximum gelation rate was assessed with spectrophotometry, and gel stiffness was determined by measuring the compressive modulus. Gel weights were measured serially over 14 days as an index of contraction (volume loss). Platelet-derived growth factor-BB and transforming growth factor-β1 concentrations were quantified with ELISAs.

Results

Fiber diameters were significantly larger and mean pore areas were significantly smaller in PRFGs than in conventional FGs. Gel weight decreased significantly over time, differed significantly between PRFGs and conventional FGs, and was significantly correlated with platelet concentration. Platelet-derived growth factor-BB and transforming growth factor-β1 concentrations were highest in gels and releasates derived from 1,000 × 10³ platelets/μL.

Conclusions and clinical relevance

The inclusion of platelets in FGs altered the architecture and increased the growth factor content of the resulting scaffold. Platelets may represent a useful means of modifying these gels for applications in veterinary and human regenerative medicine.

Genetic bleeding disorders can have a profound impact on a horse's health and athletic career. As such, it is important to understand the mechanisms of these diseases and how they are diagnosed. These diseases include haemophilia A, von Willebrand disease, prekallikrein deficiency, Glanzmann's Thrombasthenia and Atypical Equine Thrombasthenia. Exercise-induced pulmonary haemorrhage also has a proposed genetic component. Genetic mutations have been identified for haemophilia A and Glanzmann's Thrombasthenia in the horse. Mutations are known for von Willebrand disease and prekallikrein deficiency in other species. In the absence of genetic tests, bleeding disorders are typically diagnosed by measuring platelet function, von Willebrand factor, and other coagulation protein levels and activities. For autosomal recessive diseases, genetic testing can prevent the breeding of two carriers.

Background

Platelet (PLT) concentrates (PC) can be produced via the buffy coat (BC) or platelet-rich plasma (PRP) protocols. The 2 methods have not been compared with canine blood.

Objectives

The aims of the study were to compare the PLT, WBC, and RBC concentrations, in vitro PLT function, and markers of platelet storage lesion (PSL) in canine PC generated by 2 different protocols, and determine microbial growth throughout storage.

Methods

PC from 8 healthy donor dogs were produced using 2 standard protocols, PRP and BC. PLT, WBC, and RBC counts, optical aggregometry assays, and PSL markers (pH, pCO2 , HCO3 , lactate and glucose concentrations, and LDH activity) were determined on storage days 0, 1, 3, 5, and 7. Aerobic and anaerobic bacterial cultures were also performed.

Results

Mean PLT counts were comparable between protocols and remained stable throughout storage up to day 7, while median WBC and RBC counts on day 0 were significantly higher in the BC-PC group (17,800 WBCs/μL; 195,000 RBCs/μL) than in the PRP-PC group (200 WBCs/μL; 10,000 RBCs/μL) (P = .012). In PRP-PC aggregometry, the median slope and amplitude in response to γ-thrombin and convulxin (+ ADP) were significantly decreased, and virtually absent in BC-PC during storage. PSL markers (lactate, LDH activity) were higher in BC-PC. Aerobic bacterial growth was observed in 2 PRP-PC and 1 BC-PC.

Conclusions

This in vitro study suggests that PRP-PC had lesser WBC and RBC contamination and superior PLT function compared with BC-PC. In vivo studies are required to address safety and efficacy of PRP-PC.

The aim of time-varying heart rate variability spectral analysis is to detect and quantify changes in the heart rate variability spectrum components during nonstationary events. Of the methods available, the nonparametric short-time Fourier Transform and parametric time-varying autoregressive modeling are the most commonly employed. The current study (1) compares short-time Fourier Transform and autoregressive modeling methods influence on heart rate variability spectral characteristics over time and during an experimental ozone exposure in mature adult spontaneously hypertensive rats, (2) evaluates the agreement between short-time Fourier Transform and autoregressive modeling method results, and (3) describes the advantages and disadvantages of each method. Although similar trends were detected during ozone exposure, statistical comparisons identified significant differences between short-time Fourier Transform and autoregressive modeling analysis results. Significant differences were observed between methods for LF power (p ≤ 0.014); HF power (p ≤ 0.011); total power (p ≤ 0.027); and normalized HF power (p = 0.05). Furthermore, inconsistencies between exposure-related observations accentuated the lack of agreement between short-time Fourier Transform and autoregressive modeling overall. Thus, the short-time Fourier Transform and autoregressive modeling methods for time-varying heart rate variability analysis could not be considered interchangeable for evaluations with or without interventions that are known to affect cardio-autonomic activity.