Characterization of canine microglial cells isolated ex vivo

Microglia are the principal immune effector elements of the brain sharing immunophenotypic and functional characteristics of macrophages as well as of antigen presenting cells (APCs). The purpose of this study was to isolate canine microglial cells and make them available for ex vivo characterizations of their functions and immunophenotype. After isolation, carried out by density gradient centrifugation, microglial cells accumulated on distinct interfaces of 1.077 and 1.066 g/ml of a Percoll gradient. Identification of microglial cells in other species is realized by their specific immunophenotype of CD11b/c+ and CD45low. Our results indicate, that expression of CD45 is very low or even absent in canine microglial cells. In addition, they expressed CD18 and CD11b/c+, as determined by flow cytometry and immunohistochemistry. Fourteen additional monoclonal antibodies (mAbs) were used to characterize and compare canine microglial cells with monocytes. Microglia and monocytes can be clearly distinguished by their differential expression intensity of surface antigens (CD45, CD44, CD14). Functional characterization was assessed by a reactive oxygen species (ROS)-generation test and phagocytosis assay using flow cytometry. In conclusion, ex vivo examination of microglia is possible in dogs and most probably reflects the conditions in vivo. The measurement of tissue culture artifacts can be largely avoided using this method.     

Effects of suture tension during unilateral cricoarytenoid lateralization on canine laryngeal resistance in vitro

Objective— To evaluate the effect of abduction suture tension for unilateral arytenoid lateralization on laryngeal resistance. Study Design— Experimental study. Animals— Canine cadaver larynges (n=16). Methods— Laryngeal resistance was calculated in all specimens with the epiglottis in open and closed positions. Left cricoarytenoid lateralization was performed under low or high suture tension, and laryngeal resistance was re‐calculated. The effects of suture tension on laryngeal resistance were evaluated by repeated measures ANOVA. Results— Cricoarytenoid lateralization under low or high suture tension significantly reduced laryngeal resistance with the epiglottis in an open or closed position. There was no difference in laryngeal resistance with an open epiglottis between the low‐tension (1.00±0.0001 cm H₂O/L/s) and high‐tension (1.10±0.35 cm H₂O/L/s) groups (P=.33). The low‐tension group (22.80±14.20 cm H₂O/L/s) had significantly greater laryngeal resistance than the high‐tension group (8.45±4.00 cm H₂O/L/s) with a closed epiglottis (P=.016). There was no difference in laryngeal resistance with a closed epiglottis for the low‐tension group before (34.30±36.50 cm H₂O/L/s) and after (22.80±14.20 cm H₂O/L/s; P=.42) arytenoid lateralization. Conclusions— Cricoarytenoid lateralization under low suture tension significantly reduced laryngeal resistance with an open epiglottis, but resulted in a significantly greater resistance with a closed epiglottis than cricoarytenoid lateralization under high suture tension. Clinical Relevance— Clinically, use of a low‐tension suture for cricoarytenoid lateralization may provide an adequate decrease in open‐epiglottis laryngeal resistance to alleviate clinical signs, while maintaining enough closed‐epiglottis laryngeal resistance to reduce the risk of postoperative aspiration pneumonia.     

An experimental evaluation of castellated laryngofissure and bilateral arytenoid literalisation for the relief of laryngeal paralysis in dogs

The aim of this study was to compare the efficacy of 2 surgical procedures for providing an adequate laryngeal airway in dogs after surgically induced laryngeal paralysis. The laryngeal function of 10 healthy, adult experimental dogs was assessed by clinical examination, laryngoscopy, arterial blood gas measurement and analysis of tidal breathing flow-volume loops before, and after, bilateral recurrent laryngeal neurectomy. A castellated laryngofissure with vocal fold resection was done on 5 dogs and bilateral arytenoid cartilage lateralisation performed on the remaining 5 dogs. Six weeks later, the laryngeal function of the 10 dogs was reassessed, before the dogs were killed and a necropsy performed. The visible changes in laryngeal structure were recorded. Both surgical procedures alleviated some of the airway obstruction caused by the bilateral recurrent laryngeal neurectomy, but bilateral arytenoid cartilage lateralisation produced more consistent clinical improvement, a wider rima glottidis, increased inspiratory air flow and a significant increase in post-operative arterial oxygen tension.     

Implications of different degrees of arytenoid cartilage abduction on equine upper airway characteristics

Reason for performing study: The necessary degree of arytenoid cartilage abduction (ACA) to restore airway patency at maximal exercise has not been determined. Objectives: Use computational fluid dynamics modelling to measure the effects of different degrees of ACA on upper airway characteristics of horses during exercise. Hypothesis: Maximal ACA by laryngoplasty is necessary to restore normal peak airflow and pressure in Thoroughbred racehorses with laryngeal hemiplegia. Methods: The upper airway was modeled with the left arytenoid in 3 different positions: maximal abduction; 88% cross‐sectional area of the rima glottis; and 75% cross‐sectional area of the rima glottis. The right arytenoid cartilage was maximally abducted. Two models were assumed: Model 1: no compensation of airway pressures; and Model 2: airway pressure compensation occurs to maintain peak airflow. The cross‐sectional pressure and velocity distributions for turbulent flow were studied at peak flow and at different positions along the airway. Results: Model 1: In the absence of a change in driving pressure, 12 and 25% reductions in cross‐sectional area of the larynx resulted in 4.11 and 5.65% reductions in peak airflow and 3.68 and 5.64% in tidal volume, respectively, with mild changes in wall pressure. Model 2: To maintain peak flow, a 6.27% increase in driving tracheal pressure was required to compensate for a cross‐sectional reduction of 12% and a 13.63% increase in driving tracheal pressure was needed for a cross‐sectional area reduction of 25%. This increase in negative driving pressure resulted in regions with low intraluminal and wall pressures, depending on the degree of airway diameter reduction. Conclusion: Assuming no increase in driving pressure, the decrease in left ACA reduced airflow and tidal volume. With increasing driving pressure, a decrease in left ACA changed the wall pressure profile, subjecting the submaximally abducted arytenoid cartilage and adjacent areas to airway collapse. Clinical relevance: The surgical target of ACA resulting in 88% of maximal cross‐sectional area seems to be appropriate.     

Effects of suture tension and surgical approach during unilateral arytenoid lateralization on the rima glottidis in the canine larynx

OBJECTIVES: To evaluate the effect of abduction suture tension and cricothyroid (CT) joint disarticulation on the area, height, and width of the rima glottidis (RG) during unilateral arytenoid lateralization. STUDY DESIGN: Experimental study. ANIMALS: Nine canine cadaver larynges. METHODS: Left arytenoid lateralization was performed with high or low abduction suture tension. RG area, height, and width were measured by computerized planimetric analysis with the epiglottis in an open and closed position. The experiment was performed with the CT joint intact and disarticulated. The effects of suture tension, CT disarticulation, and their interaction on RG area with the epiglottis closed or open were evaluated by repeated measures analysis of variance (ANOVA). RESULTS: RG area increased by 82% and 129% (P <.0001) with low and high suture tension, respectively. The aperture not covered by the epiglottis in a closed position was 467% larger with high suture tension than with low tension (P <.0001). CT disarticulation had no significant effect on RG geometry with either low or high suture tension (P =.4970). CONCLUSIONS: Low suture tension increased RG area when the epiglottis was in an open position without increasing RG aperture when the epiglottis was closed. Suture tension had a significant effect on RG opening when the epiglottis was closed. CT disarticulation did not modify the geometry of the RG. CLINICAL RELEVANCE: Use of a low-suture tension should be considered during arytenoid lateralization because it has the potential to reduce the risk of aspiration pneumonia.     

In vitro effect of ventriculocordectomy before laryngoplasty on abduction of the equine arytenoid cartilage

Objective: To determine whether ventriculocordectomy (VCE) performed before prosthetic laryngoplasty (PL) results in increased rima glottidis size compared with PL alone. Study Design: Experimental study. Animals: Equine cadaver larynges (n=13). Methods: Right arytenoid cartilages were maximally abducted using a standard PL technique. Standard PLs were then performed on the left side and the force required to maximally abduct the left arytenoid cartilage recorded (F_max). Photographs were taken of the rima glottidis at zero force and at five equal levels of force up to F_max. The force applied was released, left VCE performed, and photographs repeated. Arytenoid left:right angle quotients (LRQ) and glottic cross‐sectional area ratios (CSAR) were calculated at each force level in each condition (PL and VCE‐PL). Results: Mean LRQ and CSAR for both PL and VCE‐PL increased with increasing force, initially rapidly before plateauing at ～50% of F_max. LRQ and CSAR were significantly greater for VCE‐PL than for PL (P<.001). When VCE was performed before PL, 12% less force was required to achieve an LRQ of 0.8, and 45% less for a CSAR of 0.8. Conclusions: In vitro, VCE performed before PL enables the arytenoid cartilage to be abducted to a greater degree for a given PL suture force.     

Unilateral arytenoid lateralisation for the treatment of laryngeal paralysis in four cats

A unilateral arytenoid lateralisation procedure was successfully performed in four cats with either unilateral or bilateral laryngeal paralysis. All of the cats were presented for the investigation of signs of exercise-related respiratory embarrassment and stridor. Other clinical signs seen included altered vocalisation, an inability to purr, coughing and lethargy. A diagnosis of either bilateral or left or right unilateral laryngeal paralysis was made in each case following visual inspection of the vocal folds with the aid of a laryngoscope under a light plane of anaesthesia. In two cases the paralysis was bilateral and the condition was considered to be either congenital or idiopathic. In the other two cases the condition was unilateral and was considered to be iatrogenic as a result of previous surgical procedures. The results of this technique were considered excellent in all cases.     

Staphylococccus pseudintermedius surface proteins SpsD and SpsO mediate adherence to ex vivo canine corneocytes

The Gram-positive bacterium Staphylococcus pseudintermedius is regarded as the major cause of canine bacterial pyoderma. Despite its clinical importance, there is only very limited knowledge about the pathogenesis of S. pseudintermedius infection and the specific bacterial virulence factors involved in causing disease. Using a whole-genome approach, we have previously identified 18 predicted cell-wall-anchored surface proteins representing possible virulence factors in a clinical isolate of S. pseudintermedius (strain ED99). They were designated S. pseudintermedius surface proteins A-R (SpsA-SpsR). The present study tested three of the putative Sps proteins (SpsD, SpsL and SpsO) for their ability to mediate adherence of bacteria to canine corneocytes. The three proteins were expressed on the surface of the nonpathogenic surrogate host Lactococcus lactis, a Gram-positive bacterium that does not adhere to canine corneocytes. Adherence assays were performed using corneocytes from different healthy canine donors (n = 5), and bacterial cells were quantified using computerized image analysis. Two of the proteins, SpsD and SpsO, mediated adherence of L. lactis to canine corneocytes, suggesting that they contribute to S. pseudintermedius pathogenesis and may represent novel therapeutic targets to combat infection.     

The role of rituximab in the treatment of canine lymphoma: an ex vivo evaluation

Targeting the CD20 receptor that is common to many B-cell Non-Hodgkin's Lymphoma subtypes in people, rituximab is a chimeric monoclonal antibody which has significantly improved disease-free survival rates compared with the use of cytotoxic agents alone. This study evaluated ex vivo canine B cell binding and depletion by rituximab with flow cytometric technique as possible proof of concept for treatment of canine lymphoma. Despite immunohistochemistry supporting CD20 expression, rituximab did not bind or deplete canine B cells and it is unlikely that it will be added to the armamentarium of treatment options for canine lymphoma.     

Biomechanical characterisation of equine laryngeal cartilage

Reasons for performing the study: Upper airway obstruction is a common problem in the performance horse as the soft tissues of the larynx collapse into the airway, yet there is a paucity of information on biomechanical properties for the structural cartilage components. Objective: To measure the geometry and compressive mechanical properties of the hyaline cartilage to improve understanding of laryngeal function and morphology. Methods: A total of 11 larynges were harvested from Thoroughbred and Standardbred racehorses. During gross dissection, linear dimensions of the cricoid were obtained. From both the cricoid and arytenoid, specimens were cored to obtain 6 mm disc samples from 3 sites within the dorsal cricoid (caudal, middle and rostral) and 2 central sites in the arytenoids (inner, outer). The specimens were mechanically tested using radial confined compression to calculate the aggregate modulus and permeability of the tissue. The biomechanical data were analysed using a nested mixed effects model. Results: Geometrically, the cricoid has relatively straight walls compared to the morphology of human, ovine and canine larynges. There were significant observations of higher modulus with increasing age (0.13 MPa per year; P = 0.007) and stiffer cricoid cartilage (2.29 MPa) than the arytenoid cartilage (0.42 MPa; P<0.001), but no difference was observed between the left and right sides. Linear contrasts showed that the rostral aspect (2.51 MPa) of the cricoid was 20% stiffer than the caudal aspect (2.09 MPa; P = 0.025), with no difference between the arytenoid sites. Conclusions: The equine larynx is a well supported structure due to both the geometry and material properties of the cricoid cartilage. The hyaline structure is an order of magnitude higher in compressive modulus compared to the arytenoids and other hyaline‐composed tissues. Potential relevance: These characterisations are important to understand the biomechanics of laryngeal function and the mechanisms involved with surgical interventions.     

In vitro and ex vivo inhibition of canine cyclooxygenase isoforms by robenacoxib: A comparative study

In vitro whole blood canine assays were used to quantify the inhibitory actions of the novel non-steroidal anti-inflammatory drug (NSAID) robenacoxib on the cyclooxygenase (COX) isoenzymes, COX-1 and COX-2, in comparison with other drugs of the NSAID class. COX-1 activity was determined by measuring serum thromboxane (Tx)B₂ synthesis in blood samples allowed to clot at 37 °C for 1 h. COX-2 activity was determined by measuring prostaglandin (PG)E₂ synthesis in blood samples incubated at 37 °C for 24 h in the presence of lipopolysaccharide. The rank order of selectivity for inhibition of COX-2 versus COX-1 (IC₅₀ COX-1:IC₅₀ COX-2) for veterinary drugs was highest with robenacoxib (128.8) compared to deracoxib (48.5), nimesulide (29.2), S+ carprofen (17.6), meloxicam (7.3), etodolac (6.6), R? carprofen (5.8) and ketoprofen (0.88). Selectivity expressed as the clinically relevant ratio IC₂₀ COX-1:IC₈₀ COX-2 was highest for robenacoxib (19.8) compared to deracoxib (2.3), S+ carprofen (2.5), R? carprofen (2.1), nimesulide (1.8), etodolac (0.76), meloxicam (0.46) and ketoprofen (0.21).An in vivo pharmacokinetic ex vivo pharmacodynamic study in the dog established dosage and concentration–effect relationships for single oral doses of robenacoxib over the dosage range 0.5–8.0 mg/kg. Values of C_max and AUC were linearly related to dosage over the tested range. Robenacoxib did not inhibit serum TxB₂ synthesis (COX-1) ex vivo at dosages of 0.5–4.0 mg/kg and produced only transient inhibition (at the 1 h and 2 h sampling times) at the 8 mg/kg dosage. All dosages of robenacoxib (0.5–8 mg/kg) produced marked, significant and dose related inhibition of PGE₂ synthesis (COX-2) ex vivo.The data demonstrate that in the dog robenacoxib is a highly selective inhibitor of the COX-2 isoform of COX, and significantly inhibits COX-2 and spares COX-1 in vivo when administered orally over the dosage range 0.5–4.0 mg/kg.     

Laryngeal paralysis in dogs: An evaluation of the bilateral arytenoid lateralisation procedure

Within the past decade, the number of reported cases of laryngeal paralysis in dogs has increased markedly. The disease is characterised by laryngeal obstruction caused by the inadequate abduction and instability of the arytenoid cartilages, aryepiglottic folds and vocal cords. Therefore, surgery performed to alleviate the clinical signs should ideally aim to reduce the airway obstruction and to prevent dynamic collapse. This paper presents the effectiveness of bilateral arytenoid lateralisation in 19 dogs with bilateral laryngeal paralysis. The procedure was effective in alleviating the clinical signs of laryngeal obstruction in all 19 dogs, with most surviving for several years. Postoperative laryngoscopic observations showed that the arytenoid cartilages were held in a satisfactory abducted position. A comparison of PaO₂ values before and after surgery demonstrated a significant (P 0·001) fall in the PaO₂ measurement between the pre- and postoperative blood samples. Postoperative complications included suture prosthesis failure in two dogs and a single non-fatal incident of aspiration pneumonia in two dogs.