Duration of in vivo endotoxin tolerance in horses.

Endotoxemia models are used to study mechanisms and treatments of early sepsis. Repeated endotoxin exposures induce periods of endotoxin tolerance, characterized by diminished proinflammatory responses to lipopolysaccharide (LPS) and modulated production of proinflammatory cytokines. Repeated measure designs using equine endotoxemia models are rarely performed, despite the advantages associated with reduced variability, because the altered responsiveness would confound study results and because the duration of equine endotoxin tolerance is unknown. We determined the interval of endotoxin tolerance, in vivo, in horses based on physical, clinicopathologic, and proinflammatory gene expression responses to repeated endotoxin exposures. Six horses received 30 ng/kg LPS in saline infused over 30 min. Behavior pain scores, physical examination parameters, and blood for complete blood count and proinflammatory gene expression were obtained at predetermined intervals for 24h. Horses received a total of 3 endotoxin exposures. The first exposure was LPS 1, followed 7 days later by LPS 7 or 14-21 days later by LPS 14-21. Lipopolysaccharide exposures were allocated in a randomized, crossover design. Lipopolysaccharide produced clinical and clinicopathologic signs of endotoxemia and increased expression of tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and IL-8, P<0.001. Horses exhibited evidence of endotoxin tolerance following LPS 7 but not following LPS 14-21. Horses had significantly lower pain scores, heart rates, respiratory rates and duration of fever, after LPS 7 compared to LPS 1 and LPS 14-21, P<0.001, and expression of TNFα was lower in the whole blood of horses after LPS 7, P=0.05. Clinical parameters and TNFα gene expression were similar or slightly increased in horses following LPS 14-21 compared to measurements made in horses following LPS 1, suggesting that endotoxin tolerance had subsided. A minimum of 3 weeks between experiments is warranted if repeated measures designs are used to assess in vivo response to endotoxin in horses.

Effects of buprenorphine and estrous cycle in a murine model of cecal ligation and puncture.

The effect of opioids on the immunopathology of sepsis models in mice has been controversial. In previous work, we showed that mortality and various inflammatory parameters did not differ between female mice given saline or buprenorphine after cecal ligation and puncture. To investigate further, we hypothesized that buprenorphine would not affect outcomes of sepsis at any stage of estrous. Female mice were allocated into 4 groups (n = 20 per group) according to stage of estrous. Mice then underwent cecal ligation and puncture and received either buprenorphine or saline. In 3-wk survival studies, overall survival did not differ between buprenorphine- and saline-treated mice. When mice were stratified according to stage of estrous, survival did not vary among saline-treated groups but was lower in buprenorphine-treated mice in metestrus compared with proestrus. To investigate inflammation as a potential mechanism for survival, we measured cell counts and cytokine levels in the peripheral blood and peritoneal lavage fluid at 12 and 24 h after cecal ligation and puncture. At 24 h, buprenorphine-treated mice in proestrus had more circulating neutrophils and monocytes than did saline-treated mice in proestrus and more circulating WBC than did mice in any other stage with or without buprenorphine. Our current results suggest that the effects of buprenorphine on a 50% survival model of sepsis in BALB/c female mice are minimal overall but that the stage of estrous has various effects in this model. Investigators should consider the effects of buprenorphine and estrous cycle when using female mice in sepsis research.

Analysis of the relationship of extrahepatic portosystemic shunt morphology with clinical variables in dogs: 53 cases (2009-2012).

OBJECTIVE: To investigate differences in clinical variables among dogs with extrahepatic portosystemic shunts (EHPSSs) of various morphologies. DESIGN: Retrospective case series. ANIMALS: 53 dogs with EHPSSs. PROCEDURES: Medical records of dogs undergoing preoperative CT angiography of an EHPSS over a 3-year period were reviewed. Analysis was performed to investigate relationships of clinical variables with shunt morphology. Morphologies were analyzed individually as well as in several groups. RESULTS: Shunt morphologies included 10 splenocaval, 9 splenophrenic, 11 splenoazygos, 10 right gastric-caval, 12 right gastric-caval with a caudal loop, and 1 right gastric-azygos with a caudal loop. Several biochemical variables associated with EHPSS were lowest in dogs with splenocaval shunts. Preoperative clinical signs were more common in dogs that had shunts with vena caval than right azygos vein insertion (36/41 [88%] vs 7/12 [58%]) and insertion caudal to the liver than diaphragmatic insertion (29/32 [91%] vs 14/21 [67%]). Neurologic signs were more common when shunts inserted into the vena cava caudal to the liver than in other locations (21/32 [66%] vs 6/21 [29%]) and were most frequent with splenocaval shunts. Urinary tract signs were more common when shunts had right gastric vein origin than gastrosplenic vein origin (14/23 [61%] vs 10/30 [33%]). CONCLUSIONS AND CLINICAL RELEVANCE: Splenocaval shunts caused more clinical abnormalities than did other shunt morphologies. Results suggested that dogs with shunt insertion in the caudal vena cava, especially caudal to the liver, were most likely to have clinical signs.

Evaluation of electrical nerve stimulation for epidural catheter positioning in the dog.

OBJECTIVE: To evaluate the accuracy of epidural catheter placement at different levels of the spinal cord guided solely by electrical nerve stimulation and resultant segmental muscle contraction. STUDY DESIGN: Prospective, experiment. ANIMALS: Six male and two female Beagles, age (1 ± 0.17 years) and weight (12.9 ± 1.1 kg). METHODS: Animals were anesthetized with propofol and maintained with isoflurane. An insulated epidural needle was used to reach the lumbosacral epidural space. A Tsui epidural catheter was inserted and connected to a nerve stimulator (1.0 mA, 0.1 ms, 2 Hz) to assess positioning of the tip at specific spinal cord segments. The catheter was advanced to three different levels of the spinal cord: lumbar (L2-L5), thoracic (T5-T10) and cervical (C4-C6). Subcutaneous needles were previously placed at these spinal levels and the catheter was advanced to match the needle location, guided only by corresponding muscle contractions. Catheter position was verified by fluoroscopy. If catheter tip and needle were at the same vertebral body a score of zero was assigned. When catheter tip was cranial or caudal to the needle, positive or negative numbers, respectively, corresponding to the number of vertebrae between them, were assigned. The mean and standard deviation of the number of vertebrae between catheter tip and needle were calculated to assess accuracy. Results are given as mean ± SD. RESULTS: The catheter position in relation to the needle was within 0.3 ± 2.0 vertebral bodies. Positive predictive values (PPV) were 57%, 83% and 71% for lumbar, thoracic and cervical regions respectively. Overall PPV was 70%. No significant difference in PPV among regions was found. CONCLUSION AND CLINICAL RELEVANCE: Placement of an epidural catheter at specific spinal levels using electrical nerve stimulation was feasible without radiographic assistance in dogs. Two vertebral bodies difference from the target site may be clinically acceptable when performing segmental epidural regional anesthesia.

Distribution of venous remodeling in exercise-induced pulmonary hemorrhage of horses follows reported blood flow distribution in the equine lung.

Exercise-induced pulmonary hemorrhage (EIPH), which has been reported in humans and a variety of domestic animals following strenuous exercise, is most often documented in racehorses. Remodeling of pulmonary veins (VR) in equine EIPH was recently described, suggesting that it contributes to the pathogenesis of the disease. The cause of VR is unknown. We tested the hypothesis that the development of VR follows pulmonary blood flow distribution, preferentially occurring in the caudodorsal lung region. Furthermore, we hypothesized that VR underpins development of the other lesions of EIPH pathology. The lungs of 10 EIPH-affected horses and 8 controls were randomly sampled for histopathology (2,520 samples) and blindly scored for presence and severity of VR, hemosiderin (H), and interstitial fibrosis (IF). Mean sample score (MSS), mean lesion score, and percent samples with lesions were determined in four dorsal and three ventral lung regions, and the frequency, spatial distribution, and severity of lesions were determined. MSS for VR and H were significantly greater dorsally than ventrally (P < 0.001) and also decreased significantly in the caudocranial direction (P < 0.001). IF decreased only in the caudocranial direction. The percent samples with lesions followed the same distribution as MSS. VR often was accompanied by H; IF never occurred without VR and H. Similarity of the distribution of EIPH lesions and the reported fractal distribution of pulmonary blood flow suggests that VR develops in regions of high blood flow. Further experiments are necessary to determine whether VR is central to the pathogenesis of EIPH.

Influence of corticosteroids on interleukin-1ß-stimulated equine chondrocyte gene expression.

OBJECTIVE: To compare the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on expression of selected chondrocyte genes in recombinant equine interleukin-1ß (reIL-1ß) stimulated articular cartilage explants. DESIGN: In vitro experiment. ANIMALS: Horses (n = 6). PROCEDURES: Articular cartilage explants from 2- to 3- year-old horses were exposed to reIL-1ß in the presence and absence of TA and MPA at 10(-7) and 10(-6) M. Resting levels of mRNA of anabolic and catabolic genes of chondrocyte origin were quantified using qPCR after 6- and 12-hour incubations. Genes of interest included aggrecan interglobular domain, aggrecan, and collagen II, matrix metalloproteinases 3 and 13 (MMP3, MMP 13), aggrecanase 1, tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP 1, TIMP 2), BCL 2, vascular endothelial growth factor, and cyclooxygenase 2 (COX 2). RESULTS: IL-1ß significantly influenced the expression of most transcripts. MPA and TA inhibited the induction of MMP 13 at 6 and 12 hours; an effect that was significant at 6 hours with MPA at 10(-7) M and TA at 10(-6) M. Similarly, COX 2 was induced by reIL-1ß and MPA and TA significantly inhibited its upregulation. TIMP 2 expression was reduced by reIL-1ß, an effect that was significantly abrogated by MPA and TA. There were no significant differences observed between glucocorticoids for any gene studied. CONCLUSIONS: No differential effects of MPA or TA on chondrocytic gene expression were identified suggesting that any divergent influences of these glucocorticoids on chondrocyte metabolism are posttranslational.

Minimum inhibitory concentration and postantibiotic effect of amikacin for equine isolates of methicillin-resistant Staphylococcus aureus in vitro.

OBJECTIVE: To report the minimum inhibitory concentration (MIC) of amikacin sulfate for equine clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and characterize the initial kill and duration of the postantibiotic effect (PAE) for selected strains. STUDY DESIGN: Experimental study. METHODS: Isolates of MRSA (n=35) had their amikacin MIC determined using the E-test agar diffusion method. Two isolates with MICs>256 microg/mL limit were further characterized using broth macrodilution. Six distinct isolates with amikacin MICs of 32, 48, 128 (2 isolates) and 500 (2 isolates) microg/mL had PAE determinations made over a range of amikacin concentrations from 31.25-1000 microg/mL using standard culture-based techniques. RESULTS: Median MIC of the 35 isolates was 32 microg/mL (range 2 to >256 microg/mL). Mean PAE of selected MRSA strains had an overall mean (all amikacin doses) of 3.43 hours (range 0.10-9.57 hours). PAE for MRSA exposed to amikacin at 1000 microg/mL was 6.18 hours (range 3.30-9.57 hours), significantly longer than that for all other concentrations (P<.0001). There was no statistically significant effect of isolate MIC on PAE. CONCLUSIONS: Isolates had a wide range of MIC; however, growth of all 6 selected strains were inhibited within the range of concentrations tested, including 2 strains with MICs of 500 microg/mL. PAE duration was not influenced by the MIC of amikacin but was significantly longer with treatment at 1000 microg/mL than at lower concentrations. CLINICAL RELEVANCE: Clinical isolates of MRSA are susceptible to amikacin at concentrations achieved by regional perfusion: however, the modest duration of PAE observed suggest that further laboratory and in vivo evaluation be conducted before recommending the technique for clinical use.

Technique and outcome of nares amputation (Trader's technique) in immature shih tzus.

Dogs with brachycephalic conformation frequently suffer from stenotic nares. This study investigated the clinical effects of stenotic nares resection in shih tzu puppies using an amputation technique first described in 1949. The authors hypothesized that surgical correction of the stenotic nares would result in improvement of nasal discharge/noise, breathing difficulty (dyspnea), and exercise limitations (exercise intolerance), and that the technique would provide a cosmetic result. Based on the results of this study, the authors recommend early surgical correction (amputation) of stenotic nares in shih tzu puppies as an effective and cosmetic technique.

Serum concentrations of keratan sulfate, osteocalcin, and pyridinoline crosslinks after oral administration of glucosamine to standardbred horses during race training.

OBJECTIVE: To determine the effects of orally administered glucosamine on concentrations of markers of bone and cartilage metabolism in Standardbred horses during race training. ANIMALS: Twenty 16- to 20-month-old Standardbreds beginning race training. PROCEDURE: Horses were randomly assigned to 2 groups. One group received glucosamine hydrochloride (4 g, PO, q 12 h), and the second (control) group received glucose (4 g, PO, q 12 h). Serum samples were obtained prior to onset of the study (baseline) and at regular intervals for 48 weeks for determination of concentrations of keratan sulfate (KS), osteocalcin (OC), and pyridinoline crosslinks (PYD). RESULTS: Osteocalcin concentrations changed significantly with time; mean serum concentrations were significantly higher than baseline values for samples obtained at 24 to 48 weeks after onset of the study. Although a significant effect of time was observed for mean concentration of KS, concentrations did not differ significantly from baseline values at any time during the study when groups were analyzed separately. However, pooled analysis revealed significant increases of mean serum KS concentration at weeks 24 and 30. Significant changes in serum PYD concentrations were not detected. Oral administration of glucosamine did not significantly affect serum concentrations of any of the markers. CONCLUSIONS AND CLINICAL RELEVANCE: Increased serum OC in clinically normal Standardbreds during race training may reflect bone formation that accompanies adaptive remodeling of the appendicular skeleton. For these experimental conditions, glucosamine did not appear to exert a detectable influence on serum concentrations of these 3 markers of connective tissue metabolism.