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