Measurement of humeroradial and humeroulnar transarticular joint forces in the canine elbow joint after humeral wedge and humeral slide osteotomies

Objective— To determine the effect of humeral wedge and humeral slide osteotomies on force distribution between the articular surfaces of the humerus and the radius and ulna in normal canine thoracic limbs.
Study Design— In vitro mechanical testing.
Sample population— Cadaveric canine right thoracic limbs (n=12).
Methods— Transarticular elbow force maps were measured using a tactile array pressure sensor in elbow joints of axially aligned limbs under 200 N axial load before and after humeral wedge and humeral slide osteotomies.
Results— Loading induced 2 distinct areas of high forces that corresponded with the proximal articular surfaces of the radius and ulna. Mean force on the proximal articular surface of the ulna was reduced by 25% and 28% after 4 and 8 mm sliding osteotomies, respectively. Statistically significant differences were not observed for the wedge osteotomies.
Conclusion— Humeral slide osteotomy significantly decreases force on the proximal articular surface of the ulna.
Clinical Relevance— The proximal articular surface of the ulna contributes significantly to load transfer through the canine elbow joint. Abnormalities that significantly increase this force might contribute to canine elbow dysplasia, specifically fragmentation of the medial coronoid process and osteochondritis dissecans of the medial aspect of the humeral condyle. Under the conditions studied, the overall reduction in mean joint surface force across the proximal articular surface of the ulna after humeral slide osteotomy indicates that this technique merits further investigation for potential use in medial compartmental osteoarthritis of the canine elbow joint.     

The jump behavior of the humeroradial and tarsocrural joints of the horse

Osteoligamentous preparations of the elbow and hock joints of the horse show “jump” behavior when flexed and extended. This behavior is not seen in undissected anatomical specimens or in the live horse. The hypothesis is that energy is stored in the collateral ligaments during the middle range of joint displacement and quickly released nearing full flexion or extension. This energy storage mechanism has the attribute of negative work. In the undissected specimen and the live horse energy is stored in muscles and tendons as well as in the collateral ligaments, and the obvious jump phenomenon is not observed although the principle of negative work still pertains.     

In vitro susceptibility of canine and feline Escherichia coli to fosfomycin

Therapeutic options for multi-drug resistant (MDR) Escherichia coli in dogs or cats are limited. The objective of this study was to establish in vitro susceptibility of canine and feline E. coli to fosfomycin. Two sources of isolates were categorized based on susceptibility as to no resistance (NDR), single drug resistance (SDR), multidrug resistance (MDR) or extreme drug resistance (XDR). Clinical isolates were collected from throughout the US from dogs (n=157) or cats (n=43) with naturally occurring infection between March 2008 and January 2010. Experimental isolates were collected from fecal samples of dogs treated with no drug (NDR), amoxicillin (expressing SDR) or enrofloxacin (expressing MDR or XDR). Fosfomycin minimum inhibitory concentrations (MIC) were determined using E-Test(?). For clinical isolates, most (165/200) originated from the urinary tract, with the number of isolates per resistant category being: NDR (N=44, 22%), SDR (N=65, 32.5%), MDR (N=74, 37%), and XDR (N=17, 8.5%). Of these isolates, 99% (197/200) were susceptible to fosfomycin with the MIC(90) and MIC(50) being 2 and 1 μg/ml, respectively (range: 0.25-196 μg/ml). The number of experimental isolates in each category was NDR (3), SDR (23), MDR (38), and XDR (11) (29.3, 44, and 14.7%, respectively). Of these, 100% were susceptible to fosfomycin with MIC(90) and MIC(50) being 1.5 and 1 μg/ml (range: 0.38-4 μg/ml), respectively. The susceptibility of canine and feline MDR and XDR E. coli to fosfomycin at concentrations well below the susceptible breakpoint supports further investigation for its use when treating E. coli resistant to alternative antimicrobials.     

In vitro properties of diffuse cytoplasmic esterase-positive canine mononuclear leukocytes

Depletion of cytoplasmic esterase-positive canine peripheral blood monocytes from mononuclear cell suspensions was attempted using plastic adherence, carbonyl iron ingestion and/or Sephadex G-10 filtration. An esterase-positive, nonadherent, nonphagocytic subpopulation was identified and further characterized by the presence or absence of cell membrane receptors for the Fc portions of immunoglobulin and the activated third component of complement. The majority of these nonadherent cells lacked these receptors. The data suggests that canine peripheral blood monocytes are a heterogenous cell population.     

In vitro function of canine neutrophils during experimental inflammatory disease

The effect of acute inflammation on neutrophil function in the dog was studied by measuring in vitro phagocytosis and killing of Staphylococcus aureus. Phagocytosis was not impaired after 30 or 60 minutes and bactericidal activity was not impaired after 60 minutes incubation. However, average bactericidal activity after 30 minutes incubation was diminished significantly (P less than 0.01). Wide variations in bactericidal activity after 30 minutes incubation during the course of the inflammation did not correlate with neutrophil count, number of toxic neutrophils, or clinical course of the inflammation. These results indicate that a defect in bactericidal activity can occur in dogs with severe inflammatory disease, and that repeated assays, rather than single determinations, may be needed to detect this dysfunction.     

In vitro effects of 2-methoxyestradiol on canine tumour cells

The in vitro antiproliferative, apoptotic and cell‐cycle effects of 2‐methoxyestradiol (2ME₂), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME₂. Inhibition of tumour cell proliferation was evaluated using a tetrazolium‐based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V‐fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell‐cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME₂ ranging from 0.88 to 7.67 µM, depending on the cell line tested. Profound G₂/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose‐dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.     

In vitro metabolism of progesterone by canine hair follicle cells

Dermal papilla cells (DPC) and dermal fibroblasts (DFB) derived from hair follicles from two different body sites (head, flank) of four male, castrated beagle dogs were incubated for 24 h with radioactive progesterone (P4). Thin-layer chromatography was used for separation and autoradiography for identification of the radioactive metabolites. In DFB the main metabolites were cortisol and 4-pregnene-11beta-ol-3,20-dione, whereas in DPC they were 5alpha-pregnane-3,20-dione and cortisol. The highest percentage of metabolism of P4 was found in DFB of the head. Smaller amounts of other metabolites were found in both cell types of both locations.     

In vitro evaluation of five canine tibial plateau leveling methods

OBJECTIVE: To compare application time, accuracy of tibial plateau slope (TPS) correction, presence and magnitude of rotational and angular deformities, and mechanical properties of 5 canine tibial plateau leveling methods. SAMPLE POPULATION: 27 canine tibial replicas created by rapid prototyping methods. PROCEDURE: The application time, accuracy of TPS correction, presence and magnitude of rotational and angular deformation, and construct axial stiffness of 3 internal fixation methods (tibial plateau leveling osteotomy, tibial wedge osteotomy, and chevron wedge osteotomy [CWO]) and 2 external skeletal fixation (ESF) methods (hinged hybrid circular external fixation and wedge osteotomy linear fixation [WOLF]) were assessed. RESULTS: Mean bone model axial stiffness did not differ among methods. Mean application time was more rapid for WOLF than for other methods. Mean TPSs did not differ from our 5 degrees target and were lower for ESF methods, compared with internal fixation methods. Mean postoperative rotational malalignment did not differ from our target or among groups. Mean postoperative medio-lateral angulation did not differ from our target, except for CWO. Internal fixation methods lead to axially stiffer constructs than ESF methods. Reuse of ESF frames did not lead to a decrease in axial stiffness. CONCLUSIONS AND CLINICAL RELEVANCE: The 5 tibial plateau leveling methods had acceptable geometric and mechanical properties. External skeletal fixation methods were more accurate as a result of precise data available for determining the exact magnitude of correction required to achieve a 5 degrees TPS.     

In vitro activity of difloxacin against canine bacterial isolates.

The in vitro activity of difloxacin against canine bacterial isolates from clinical cases was studied in the United States and The Netherlands. Minimal inhibitory concentrations (MIC), the postantibiotic effect, the effect of pH on antimicrobial activity, and the bacterial killing rate tests were determined according to standard techniques. The MICs of American and Dutch isolates agreed in general. The MICs of the American gram-negative isolates ranged from 0.06 to 2.0 microg/ml, and the MICs of the Dutch gram-negative isolates ranged from 0.016 to 8.0 microg/ml. A few European strains of Proteus mirabilis and Klebsiella pneumoniae had relatively high MICs. Bordetella bronchiseptica also was less susceptible to difloxacin. The MICs of the American gram-positive cocci ranged from 0.125 to 4.0 microg/ml, and the MICs of Dutch isolates ranged from 0.125 to 2.0 microg/ ml. Difloxacin induced a concentration-dependent postantibiotic effect that lasted 0.2-3 hours in cultures with Escherichia coli, Staphylococcus intermedius, Streptococcus canis, Proteus spp., and Klebsiella pneumoniae. There was no postantibiotic effect observed against canine Pseudomonas aeruginosa. Decreasing the pH of the medium increased the MIC of Proteus mirabilis for difloxacin. The MICs of Escherichia coli and Klebsiella pneumoniae were lowest at neutral pH and were slightly increased in acid or alkaline media. At a neutral pH, most tested bacterial species were killed at a difloxacin concentration of 4 times the MIC. Similar results were obtained when these same bacteria were tested against enrofloxacin. A Klebsiella pneumoniae strain in an acidic environment was readily killed at difloxacin or enrofloxacin MIC, but at neutral pH the drug concentration had to be raised to 4 times the MIC for a bactericidal effect. After 24 hours of incubation at pH 7.1, difloxacin and enrofloxacin had similar bactericidal activity for all bacteria tested except Staphylococcus intermedius. Against S. intermedius, difloxacin was more bactericidal than enrofloxacin.     

Ultrastructure of canine articular cartilage: comparison of normal and degenerative (osteoarthritic) hip joints.

Normal canine hip cartilage was compared with cartilage from the degenerative lesions found in young dogs with canine hip dysplasia. The upper 0.5 mm of normal cartilage was characterized. Four distinct layers or zones were found: a layer of fine fibrous material covering the surface, a layer (surface layer) of small (32 nm diameter or less) collagen fibrils tightly packed in bundles and oriented parallel to the surface, a layer (upper layer) or less tightly packed collagen fibrils oriented mostly parallel to the surface with about 33% of the fibrils 64 nm or more, and a layer (intermediate layer) of randomly oriented fibrils with more than 50% of the fibrils 64 nm or larger. Fibril density was high in the surface layer and decreased with depth into the cartilage. In a moderately advanced lesion of degenerative cartilage, there was a layer of amorphous material over the surface. The tightly packed surface layer of small fibrils was absent. The surface itself was uneven and fissued. At depths from the surface comparable to the upper and the intermediate layers in normal cartilage, the proportion of large fibrils was less than in normal cartilage. The overall density of fibrils in degenerative cartilage increased with depth into the tissue. Cells flattened parallel to the surface, with relatively large nuclei, were found in the upper layer of normal cartilage. Cells in the intermediate layers were larger and round. The oblong cells of the upper layer of normal cartilage were not found in any layer of degenerative cartilage. Differences between cells in other layers of normal and degenerative cartilages were minimal. A model for the arrangement of chondrocytes and collagen fibrils for normal and degenerative cartilage was proposed. Ultrastructural changes in degenerative cartilage were prominent in the upper 0.5 mm of cartilage. These changes were changes in the number of collagen fibrils/mum-2 and a change from a characteristic pattern of collagen fibril diameters and orientation found in normal tissue.     

In vitro and in vivo bioactivity of recombinant canine hepatocyte growth factor

Hepatocyte growth factor (HGF) is crucial for the development and regeneration of the liver and offers a possible new therapeutic strategy for the treatment of canine liver disease. In this study, the in vitro and in vivo bioactivity of recombinant canine HGF (rcHGF) produced with a baculoviral expression system in insect cells was measured. In vitro rcHGF induced mitogenesis, motogenesis, and phosphorylated the HGF receptor c-MET and its downstream mediators PKB and ERK1/2 in two canine epithelial cell lines. After a partial hepatectomy (phx) in dogs, rcHGF increased phosphorylation of c-MET, PKB and ERK1/2. A moderate increase was seen with the cell cycle protein PCNA in rcHGF treated livers, but no HGF-induced increase in liver weight was seen 7 days after phx. Furthermore, rcHGF treated livers showed lower levels of the key mediator of apoptosis, caspase-3, at 7days after phx. It is concluded that rcHGF is a biologically active protein in vitro and in vivo and the baculoviral expression system supplies sufficient amounts of rcHGF for future clinical studies in dogs.     

In vitro metabolism of dehydroepiandrosterone and testosterone by canine hair follicle cells

The metabolism of radioactive dehydroepiandrosterone (DHEA) and testosterone was studied in dermal papilla cells (DPC) and dermal fibroblasts (DFB) derived from hair follicles from two different body sites (head, flank) of four male, castrated beagle dogs. Thin layer chromatography was used for separation, and autoradiography for identification of the radioactive metabolites. DHEA was metabolized mainly to 11 alpha-OH-testosterone and only to a minor extent to 11 alpha-OH-androstenedione and another unidentified metabolite. The highest percentage of metabolization of DHEA was found in DFB of the head. Testosterone was metabolized only to a minor extent (less than 10%) to 5 alpha-dihydrotestosterone and epiandrosterone and there was no significant difference between either the two cell types or the two locations. These results clearly show that the metabolization of androgens in canine DPC and DFB is different from that observed in cells from the human hair follicle.     

In vitro retinoid-induced growth inhibition and morphologic differentiation of canine osteosarcoma cells

OBJECTIVE: To determine differentiation and growth inhibition effects of retinoids on canine osteosarcoma cells. SAMPLE POPULATION: 3 osteosarcoma cell lines established from osteosarcomas in dogs. PROCEDURE: Osteosarcoma cells were incubated with various concentrations of all-trans-retinoic acid and 9-cis-retinoic acid or control medium, counted daily for 10 days, and evaluated for morphologic changes. Synthesis of DNA was measured by use of a cell proliferation ELISA. To analyze effect of retinoids on colony formation on plastic dishes, cells were cultured for 14 days, fixed, and stained; number of colonies was counted. RESULTS: In a dose-dependent manner, both retinoids induced morphologic differentiation and growth inhibition in the 3 osteosarcoma cell lines and inhibited each cell's ability to form anchorage-dependent colonies. CONCLUSION AND CLINICAL RELEVANCE: Retinoids induced differentiation of osteosarcoma cells of dogs, resulting in altered expression of their malignant phenotype. Induction of differentiation by retinoids may have potential as an adjunctive treatment for osteosarcoma in dogs.     

In vitro susceptibilities of canine urinary bacteria to selected antimicrobial agents

Antimicrobial susceptibility tests were conducted on bacteria that were isolated from urine specimens collected by antepubic cystocentesis from dogs with urinary tract infections. Antimicrobics to which greater than or equal to 90% of these urinary bacteria were susceptible in vitro included trimethoprim-sulfamethoxazole (TMP-SMZ), nitrofurantoin, cephalexin, nalidixic acid, and gentamicin for isolates of Escherichia coli; ampicillin, TMP-SMZ, cephalexin, nalidixic acid, and gentamicin for isolates of Proteus mirabilis; ampicillin chloramphenicol, TMP-SMZ, nitrofurantoin, cephalexin, kanamycin, and gentamicin for isolates of coagulase-positive staphylococci; cephalexin, nalidixic acid, and gentamicin for isolates of Klebsiella pneumoniae; ampicillin, TMP-SMZ, and gentamicin for isolates of Streptococcus faecalis, Str faecium, and Str zymogenes; ampicillin, chloramphenicol, TMP-SMZ, and gentamicin for isolates of Str viridans; and ampicillin, chloramphenicol, TMP-SMZ, nitrofurantoin, cephalexin, kanamycin, and gentamicin for isolates of Str canis. No antimicrobial agent tested was effective in vitro at the 90% level for isolates of Pseudomonas aeruginosa, but gentamicin was closest, at 89%.     

In vitro effects of Yunnan Baiyao on canine hemangiosarcoma cell lines

Yunnan Baiyao is a Chinese herbal medicine that has been utilized for its anti‐inflammatory, haemostatic, wound healing and pain relieving properties in people. It has been utilized in the veterinary profession to control bleeding in dogs with hemangiosarcoma (HSA) and has been anecdotally reported to prolong survival times in dogs with this neoplasm. This study evaluated the in vitro activity of Yunnan Baiyao against three canine HSA cell lines after treatment with increasing concentrations of Yunnan Baiyao (50, 100, 200, 400, 600 and 800 µg mL^?1) at 24, 48 and 72 h. Mean half maximum inhibitory concentration (IC₅₀) at 72 h for DEN, Fitz, SB was 369.9, 275.9 and 325.3 µg mL^?1, respectively. Caspase‐3/7 activity increased in correlation with the IC₅₀ in each cell line which was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, APO‐BRDU Kit; BD Biosciences, San Jose, CA, USA) assay. VEGF in cell supernatant was also quantified. Overall, the study found that Yunnan Baiyao causes dose and time dependent HSA cell death through initiation of caspase‐mediated apoptosis, which supports future studies involving Yunnan Baiyao.     

In vitro effects of glucosamine and acetylsalicylate on canine chondrocytes in three-dimensional culture

OBJECTIVE: To determine effects of glucosamine and acetylsalicylate on canine chondrocytes in 3-dimensional culture. SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of 2 adult female dogs recently euthanatized for reasons unrelated to orthopedic abnormalities. PROCEDURE: Chondrocytes were cultured in a 3-dimensional agarose-based medium alone (control), with glucosamine (100 microg/ml; GL), or with acetylsalicylate (18 microg/ml; AS). Supernatant and agarose plugs from 4 wells/group/d were collected on days 3, 6, and 12 of culture. Agarose plugs were evaluated for percentage of viable cells, percentage of cells producing pericellular or territorial matrix, glycosaminoglycan (GAG) concentration, and type-II collagen production. Prostaglandin E2 concentration in supernatants was determined. RESULTS: Chondrocytes in all groups had characteristics indicative of viability and differentiation; however, on day 12, a lower percentage of viable cells was detected in the AS group, compared with the other 2 groups. On day 6, GAG concentration in the AS group was significantly greater than concentrations in the other 2 groups. On day 12, GAG concentrations in the GL and AS groups were significantly less than in the control group. Within the GL and AS groups, cell viability was significantly less on day 12, compared with day 3. Significant differences in PGE2 concentration among or within groups and evidence of type II collagen production were not detected. CONCLUSIONS: 3-dimensional culture of canine chondrocytes allows for production of hyaline cartilage matrix constituents and growth of cells with morphologic characteristics similar to those of articular cartilage. Acetylsalicylate and glucosamine, at the single concentration evaluated, had detrimental effects on chondrocyte viability, GAG production, or both.