Immunohistochemical Localization of RANK, RANKL and OPG in Healthy and Arthritic Canine Elbow Joints

Objective— To determine if the receptor activator of nuclear factor-κB–receptor activator of nuclear factor-κB ligand–osteoprotegerin (RANK–RANKL–OPG) system is active in bone remodeling in dogs and, if so, whether differences in expression of these mediators occur in healthy and arthritic joints.
Study Design— Experimental study.
Sample Population— Fragmented processus coronoidei (n=20) were surgically removed from dogs with elbow arthritis and 5 corresponding healthy samples from dogs euthanatized for reasons other than elbow joint disease.
Methods— Bright-field immunohistochemistry and high-resolution fluorescence microscopy were used to investigate the distribution of RANK, RANKL, and OPG in healthy and arthritic joints.
Results— All 3 molecules were identified by immunostaining of canine bone tissue. In elbow dysplasia, the number of RANK-positive osteoclasts was increased. In their vicinity, cells expressing RANKL, a mediator of osteoclast activation, were abundant whereas the number of osteoblasts having the potential to limit osteoclastogenesis and bone resorption via OPG was few.
Conclusions— The RANK–RANKL–OPG system is active in bone remodeling in dogs. In elbow dysplasia, a surplus of molecules promoting osteoclastogenesis was evident and is indicative of an imbalance between the mediators regulating bone resorption and bone formation. Both OPG and neutralizing antibodies against RANKL have the potential to counterbalance bone resorption.
Clinical Relevance— Therapeutic use of neutralizing antibodies against RANKL to inhibit osteoclast activation warrants further investigation.     

Chlorhexidine Gluconate Versus Chloroxylenol for Preoperative Skin Preparation in Dogs

The efficacy of 3% chloroxylenol (PCMX) or 4% chlorhexidine gluconate (CG) for preoperative skin preparation was assessed in 100 dogs undergoing clean or clean-contaminated surgical procedures. Replication Organism Detection and Counting (RODAC) plates were used to quantify skin bacteria colony forming units (CFU) at the operative site before and after skin preparation and immediately postoperatively. Reduction of CFU after skin preparation and immediately postoperatively was significant for each agent. However, CFU levels were significantly lower in the CG group than in the PCMX group after surgical preparation, regardless of initial CFU numbers. No significant difference in CFU counts was observed between antiseptic groups postoperatively. Within-group comparisons showed PCMX to be significantly less efficacious when the prescrub CFU number was greater than 1,000. Bacterial reduction was similar in the CG group regardless of prescrub CFU levels. The number of negative cultures after skin preparation was significantly greater with CG than with PCMX. Chlorhexidine gluconate also had fewer cultures with heavy bacterial growth (>5 CFUs) after surgical preparation. There was no significant difference between antiseptics in the number of negative cultures or cultures with more than 5 CFUs immediately after surgery. The number of skin reactions and postoperative wound infections that occurred with each technique were similar. Three percent PCMX, as used in this study, was less effective than 4% CG in its immediate antimicrobial activity, however, this difference was not associated with an increased wound infection rate.     

Molecular variation within some Japanese isolates of Verticillium dahliae

Eight isolates of Verticillium dahliae from Japan, classified into four groups based on pathogenicity to differential hosts, were compared with isolates in previously defined RFLP groups. Within each of two of the pathogenicity groups (JB and JC) the pairs of haploid isolates were closely related but those in a third group (JA; isolates not pathogenic to sweet pepper or tomato) were not. Only one of the six haploid isolates (one of the two in the JA group) could be placed in an existing RFLP group. The two diploid isolates (the JD pathogenicity group) were similar to RFLP group D and only distantly related to the six haploid isolates. Of the Japanese pathogenicity groups, only JD corresponded to an existing RFLP group.     

COMPARISON OF THE ULTRASONOGRAPHIC APPEARANCE OF OSTEOCHONDROSIS LESIONS IN THE CANINE SHOULDER WITH RADIOGRAPHY, ARTHROGRAPHY, AND ARTHROSCOPY

Osteochondrosis lesions in 29 shoulder joints (from 20 dogs) were evaluated with ultrasound (US) and the results were compared with survey radiography, arthrography, and arthroscopy. US was performed with a 7-12 MHz linear matrix transducer which was placed in cranio-caudal direction just distally to the acromion while the joint was adducted and maximally endorotated to visualize the caudal aspect of the humeral head. With US, the subchondral defect was completely visible in 21 joints and partially visible in 8 joints. The length of the subchondral defect measured on US was comparable with the length measured on survey radiographs. In two joints, the cartilage flap was mineralized and thus already visible on survey radiographs. The mineralized flap was visible on US as a straight hyperechoic line above the subchondral defect. In the other joints, survey radiographs could not assess the status of the articular cartilage. In 17 joints, the presence of a cartilage flap or cartilage fissuring was suspected based on the presence of a second hyperechoic line at the base of the subchondral defect, and this suspicion was confirmed by arthroscopic examination in 16 joints and also by arthrographic examination in 15 joints. One joint that was suspected of having a cartilage flap on US was normal on arthroscopy and arthrography. When US revealed only focal thickening of the anechoic cartilage layer (5 joints), the joints appeared normal on arthroscopic and arthrographic examination. Of the four joints where the subchondral defect was irregular and covered by heteroechogeneous material on US, arthroseopy revealed the presence of a lesion resembling chondromalacia in two joints, the presence of a small cartilage flap in one joint and the presence of scar tissue underneath the flap at the level of the subchondral defect in one joint. In conclusion, US is a helpful imaging modality in the identification of osteochondritic lesions in the canine shoulder joint. US also appears to be a satisfactory imaging tool for identifying lesions such as joint mice, joint effusion, and distinct new bone formation.     

Effect of Fixation Pin Insertion on the Bone-Pin Interface

Smooth and partially threaded 3.12 mm (1/8 inch) trochar-tipped Steinmann pins were inserted transversely through both diaphyseal cortices of eight mature canine tibias using five methods. Angular velocity (revolutions per minute) during insertion and temperature elevation due to friction during penetration of the second cortex were recorded. The force required for extraction of the pins from the bone and the histologic appearance of the bone-pin interface were determined for one-half of the pins 2 days after insertion and for one-half of the pins 56 days after insertion. The increase in temperature was similar for all methods of insertion except high speed power, which was significantly greater (p < 0.05). The force required for axial pin extraction was similar for pins inserted by hand chuck, predrilled, and low speed power methods after both 2 and 56 days. Pins inserted by high speed power and hand drill required force similar to the others for extraction after 2 days but significantly less force (p < 0.05) for extraction after 56 days. The partially threaded pins required significantly greater force (p < 0.01) extraction after both 2 and 56 days. Histologic examination revealed increased mechanical bone damage surrounding hand chuck inserted pins, increased bone necrosis surrounding high speed power inserted pins, and increased inflammatory changes surrounding hand drill inserted pins.     

CONTRAST HARMONIC IMAGING OF THE NORMAL CANINE SPLEEN

The purpose of this study was to assess the perfusion pattern and perfusion dynamics in the normal canine spleen using contrast harmonic imaging. Twenty-five dogs without clinical or ultrasonographic evidence of splenic disease were studied. Twenty-three dogs were scanned with only manual restraint; two dogs were sedated with buprenorphin. All dogs received an intravenous bolus of a microbubble contrast medium (SonoVue). The perfusion pattern during the blood pool phase represented a skewed bell-shaped curve. A tissue-specific late phase, similar to humans, was not observed. Time/intensity curves were generated for a selected region. Mean average-derived peak intensity (PI) was 6.6dB, mean time to peak intensity calculated from the initial rise (TTP) was 25.6 s and mean area under the curve (AUC) was 523.6 dBs. If dogs were divided into two body weight groups (< or =15 and >15 kg body weight), average derived peak intensity area, time to peak intensity, and area under the curve were lower for the smaller dogs than for the larger animals. However, differences were not statistically significant (P = 0.2, 0.05, and 0.08, respectively). No significant association was found between hematocrit, hemoglobin concentration, red blood cell count, blood pressure, heart rate, age, gender, and the perfusion variables. In conclusion, these baseline data may prove useful in the evaluation of dogs with diffuse or focal splenic disease.     

Differentially expressed genes in dicamba‐resistant and dicamba‐susceptible biotypes

In an ongoing effort to investigate the mechanism of auxinic herbicide resistance in Kochia scoparia (kochia), polymerase chain reaction‐based cDNA suppression subtractive hybridization was used to identify genes that are differentially expressed between dicamba‐resistant (HRd) and dicamba‐susceptible (S1) kochia biotypes in response to herbicide treatment. Both the HRd and S1 adaptor‐ligated cDNAs were used in separate hybridizations in order to generate biotype‐specific clones. A total of 710 cDNAs, representing putative differentially expressed mRNAs, were isolated and subjected to further screening. The false‐positive cDNAs were removed by conducting two colony hybridizations and at least one Northern hybridization. Differential or biotype‐specific expression was confirmed for six clones each from the HRd and S1 plants. The S1‐related genes were constitutively expressed at higher levels than in the HRd plants, but none had significant sequence similarity to known genes. Among the HRd‐related genes, HRd‐88 had 42% amino acid sequence identity to a conserved domain within thiol peptidases, which might be involved in auxin‐regulated gene expression. The constitutively expressed and inducible (by the dicamba treatment) HRd‐39 had 40% identity and 60% similarity to a domain from the Fe(II)/α‐ketoglutarate‐dependent hydroxylase superfamily. The HRd‐39 gene product had the characteristics of an enzyme that is able to detoxify dicamba via oxidative hydroxylation and thus its overexpression might confer the dicamba resistance phenotype.