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Abstract

Objective—To evaluate the use of serum concentrations of biochemical markers of bone metabolism (osteocalcin [OC], bone-specific alkaline phosphatase [BS-ALP], and deoxypyridinoline [DPYR]) to compare healing in infected versus noninfected fractures and in fractures with normal repair versus delayed (nonunion) repair in rabbits.

Animals—32 female 9- to 10-month-old New Zealand White rabbits.

Procedure—A femoral fracture defect was made in each rabbit. Rabbits were assigned to the following groups: the bone morphogenetic-2 gene treatment group with either noninfected nonunion or infected (ie, inoculation of defects with Staphylococcus aureus) nonunion fractures or the luciferase (control) gene treatment group with either noninfected nonunion or infected nonunion fractures. Serum samples were obtained before surgery (time 0) and 4, 8, 12, and 16 weeks after surgery. Callus formation and lysis grades were evaluated radiographically at 16 weeks.

Results—Serum OC and BS-ALP concentrations decreased from time 0 at 4 weeks, peaked at 8 weeks, and then decreased. Serum DPYR concentration peaked at 4 weeks and then decreased, independent of gene treatment group or fracture infection status. Compared with rabbits with noninfected fractures, those with infected fractures had lower serum OC and BS-ALP concentrations at 4 weeks, higher serum OC concentrations at 16 weeks, and higher serum DPYR concentrations at 4, 8, and 16 weeks. Combined serum OC, BS-ALP, and DPYR concentrations provided an accuracy of 96% for prediction of fracture infection status at 4 weeks.

Conclusions and Clinical Relevance—Measurement of multiple serum biochemical markers of bone metabolism could be useful for clinical evaluation of fracture healing and early diagnosis of osteomyelitis. ( J Am Vet Med Assoc 2003;64:727–735)

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To characterize the fecal microbiota of horses and to investigate alterations in that microbiota on the basis of sample collection site (rectum vs stall floor), sample location within the fecal ball (center vs surface), and duration of environmental exposure (collection time).

ANIMALS 6 healthy adult mixed-breed mares.

PROCEDURES From each horse, feces were collected from the rectum and placed on a straw-bedded stall floor. A fecal ball was selected for analysis immediately after removal from the rectum and at 0 (immediately), 2, 6, 12, and 24 hours after placement on the stall floor. Approximately 250 mg of feces was extracted from the surface and center of each fecal ball, and genomic DNA was extracted, purified, amplified for the V1-V2 hypervariable region of the 16S rDNA gene, and analyzed with a bioinformatics pipeline.

RESULTS The fecal microbiota was unique for each horse. Bacterial community composition varied significantly between center and surface fecal samples but was not affected by collection time. Bacterial community composition varied rapidly for surface fecal samples. Individual bacterial taxa were significantly associated with both sample location and collection time but remained fairly stable for up to 6 hours for center fecal samples.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, for horses, fecal samples for microbiota analysis should be extracted from the center of fecal balls collected within 6 hours after defecation. Samples obtained up to 24 hours after defecation can be analyzed with the realization that some bacterial populations may deviate from those immediately after defecation.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To evaluate effects of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors on the production of tumor necrosis factor-α (TNF-α) by interferon-γ (IFN-γ)– and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of horses as an in vitro model of inflammation in horses.

SAMPLE

1,440 samples of PBMCs from 6 healthy research horses.

PROCEDURES

From heparinized whole blood samples, PBMC cultures were obtained. An initial dose-response trial on 48 PBMC samples from 2 horses (24 samples each) was used to determine concentrations of IFN-γ and LPS for use as low- and high-level stimulation concentrations. Seventy-two PBMC samples from 6 horses were assigned equally to 1 of 4 PARP1 inhibition categories: no PARP1 inhibitor (PARP1 inhibition control); 2-((R)-2-methylpyrrolidin-2-yl)-1H-benzimidazole-4-carbozamide dihydrochloride (ABT888);4-(3-(1-(cyclopropanecarbonyl)piperazine-4-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (AZD2281); or N-(6-oxo-5,6-dihydrophenanthridin-2-yl) -N,N-dimethylacetamide hydrochloride (PJ34). Samples of PBMCs from each horse and each PARP1 inhibition category were then assigned to 1 of 3 levels of IFN-γ and LPS stimulation: none (control), low stimulation, or high stimulation. After a 24-hour incubation period, a TNF-α ELISA was used to measure TNF-α concentration in the supernatant. Results were compared across treatments and for each horse. Data were analyzed with repeated-measures ANOVA.

RESULTS

Median TNF-α concentration was significantly lower for PJ34-treated, high-level stimulated PBMCs than for PARP1 inhibition control, high-level stimulated PBMCs; however, no other meaningful differences in TNF-α concentration were detected among the inhibition and stimulation combinations.

CONCLUSIONS AND CLINICAL RELEVANCE

Findings suggested that PJ34 PARP1 inhibition may reduce TNF-α production in horses, a potential benefit in reducing inflammation and endotoxin-induced damage in horses.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate use of technetium Tc 99m disodium hydroxymethylene diphosphonate (99m-Tc- HDP) for assessing fracture healing and 99m-Tc-HDP and technetium Tc 99m ciprofloxacin (99m-Tc-CIPRO) for early diagnosis of osteomyelitis in rabbits.

Animals—32 skeletally mature New Zealand White rabbits.

Procedure—A femoral fracture defect stabilized with bone plates and cortical screws was used. Scintigraphy was performed 4, 8, 12, and 16 weeks after surgery. The 99m-Tc-CIPRO scan was performed 48 hours after the 99m-Tc-HDP scan. The uptake ratio of the experimental limb to the normal limb was calculated by use of multiple regions of interest. Results of radiography performed to determine external callus and lysis grade and percentage defect ossification at 16 weeks were compared with scintigraphy results.

Results—Infected fractures had a higher uptake ratio for 99m-Tc-HDP and 99m-Tc-CIPRO than noninfected fractures. Infected fractures could be differentiated from noninfected fractures late in healing by use of 99m-Tc-HDP. Although 99m-Tc-CIPRO was better than 99m-Tc-HDP for identifying infection, there was a high incidence of false positive and negative results with 99m-Tc-CIPRO. There was an association between 99m-Tc-HDP uptake ratio and callus formation and a good correlation between 99m-Tc-HDP uptake ratio and defect ossification after 4 weeks.

Conclusions and Clinical Relevance—99m-Tc-HDP and 99m-Tc-CIPRO may be useful for diagnosing osteomyelitis late in fracture healing; however, false positive and false negative results occur. Technetium Tc 99m disodium hydroxymethylene diphosphonate may be useful for evaluating fracture healing. ( Am J Vet Res 2003;64:736–745)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate the use of hydrothermal ablation of articular cartilage for arthrodesis in horses through investigation of the effects of joint lavage with physiologic saline (0.9% NaCl) solution (80°C) for various treatment times on chondrocyte viability in the articular cartilage of the metacarpophalangeal and metatarsophalangeal joints of cadaveric horse limbs.

Sample Population—7 pairs of metacarpophalangeal and 8 pairs of metatarsophalangeal joints from 8 Thoroughbreds.

Procedure—The horses were euthanatized for reasons unrelated to musculoskeletal disease. On a random basis, 1 joint of each pair underwent intra-articular lavage for 5, 10, or 15 minutes with heated saline solution (80°C); the other joint underwent sham treatment of similar duration with saline solution at 22°C (control). Cartilage samples from the distal articular surface of metacarpus III (or metatarsus III), the proximal surface of the proximal phalanx, and the lateral and medial proximal sesamoid bones were assessed for chondrocyte viability via confocal microscopy and viability staining following enzymatic digestion.

Results—Compared with the control joints, findings of both viability assays indicated that the percentage of sites containing viable chondrocytes in heat-treated joints was decreased. Treatment hazard ratios of 0.048 (confocal microscopy) and 0.2 (digestion assay) were estimated. Histologically, periarticular soft tissues had minimal detrimental effects after heat treatment.

Conclusions and Clinical Relevance—Ex vivo intraarticular lavage with saline solution at 80°C resulted in the death of almost all articular chondrocytes in the joint. This technique may be a satisfactory method for extensive cartilage ablation when performing arthrodesis by minimally invasive techniques. (Am J Vet Res 2005;66:36–42)

Full access
in American Journal of Veterinary Research