Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing.

The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis.

Hand-assisted laparoscopic ovariohysterectomy in the mare.

OBJECTIVE: To develop a minimally invasive, hand-assisted laparoscopic ovariohysterectomy (HALS-OHE) technique in the mare and to evaluate safety and any associated complications. STUDY DESIGN: Experimental study. ANIMALS: Eight, 11-24-year-old mares with anatomically normal urogenital tracts. METHODS: The surgical technique was developed in 2 non-survival mares and subsequently evaluated in 6 survival procedures. Food was withheld for 48 hours, then mares were anesthetized and positioned in dorsal recumbency for laparoscopic surgery. A hand access device (Omniport) was placed followed by 4 laparoscopic portals. Transection of the ovarian pedicles and broad ligaments was achieved using a combination of a laparoscopic stapling instrument (Endo GIA II), an ultrasonically activated instrument (Harmonic Scalpel), and endoscopic clips (Endo Clip II ML). The genital tract was exteriorized through the laparotomy, and the uterus transected and sutured in a conventional pattern. Horses were evaluated through postoperative day 14 when a necropsy was performed. RESULTS: Four mares recuperated well after surgery, 1 mare was euthanatized because of bilateral femur fracture during anesthetic recovery, and another developed severe pleuropneumonia. At necropsy all but 1 abdominal incision was healing routinely. One mare had abscessed along the celiotomy incision and developed visceral adhesions. Uncomplicated healing of transected mesovarial, mesometrial, and uterine remnants was observed. CONCLUSIONS: Ovariohysterectomy in horses can be accomplished using HALS technique. CLINICAL RELEVANCE: HALS-OHE technique represents a minimally invasive and technically feasible alternative for conventional OHE. Careful patient selection and preparation may reduce the complications observed. The HALS technique may be useful in other laparoscopic surgical procedures.

Elution of metronidazole and gentamicin from polymethylmethacrylate beads.

OBJECTIVE: To characterize the elution and bioactivity of metronidazole and gentamicin sulfate polymerized, individually and in combination, with polymethylmethacrylate (PMMA). STUDY DESIGN: In vitro experimental study. METHODS: PMMA beads containing metronidazole (3 concentrations), gentamicin sulfate, or metronidazole and gentamicin sulfate were immersed in 5 mL of phosphate-buffered saline in triplicate. Eluent was replaced at specified time intervals for 1 or 21 days, and antibiotic concentrations were measured by high-performance liquid chromatography. Changes in antibiotic bioactivity attributable to polymerization or copolymerization of the antibiotics with PMMA, ethylene oxide sterilization, and storage of AIPMMA beads containing metronidazole were evaluated. RESULTS: Antibiotic elution patterns were similar for all groups. Day 1 elution for groups containing metronidazole or gentamicin individually represented a mean 63%-66% and 79%, respectively, of the 21-day total. Approximately 50% of the day 1 elution occurred during the first hour. The elution of metronidazole was dose dependent. The elution of metronidazole (day 3-21) and gentamicin (all days) was significantly greater when metronidazole and gentamicin were combined (P <.05). The addition of metronidazole delayed polymerization of PMMA. Neither polymerization nor copolymerization of metronidazole and gentamicin with PMMA, gas sterilization, or 2-month storage of beads containing metronidazole significantly affected antimicrobial bioactivity. CONCLUSIONS: Metronidazole elution from PMMA was dose dependent. Copolymerization of metronidazole and gentamicin sulfate in PMMA resulted in increased rates of elution. Intraoperative preparation of metronidazole-impregnated PMMA beads is not practical, but sterilization and storage for 2 months should not affect efficacy. CLINICAL RELEVANCE: The local delivery of biologically active metronidazole and gentamicin by elution from PMMA is feasible.

Evaluation of the harmonic scalpel for laparoscopic bilateral ovariectomy in standing horses.

OBJECTIVE: To evaluate use of the Harmonic Scalpel (Ethicon Endo-Surgery Inc., Cincinnati, OH) for performing laparoscopic bilateral ovariectomy in standing horses. STUDY DESIGN: Experimental study. ANIMALS: Eight mares aged 2-20 years and weighing 410-540 kg. METHODS: Standing laparoscopic bilateral ovariectomy was performed in 8 mares with normal reproductive tract anatomy. The Harmonic Scalpel (an ultrasonically activated instrument) was used to simultaneously transect and obtain hemostasis of the ovarian pedicle. Necropsy was performed on 4 mares 3 days after surgery and 4 mares 30 days after surgery. Gross and histopathologic evaluation of the ovarian pedicles was performed to characterize tissue reaction. RESULTS: Complete hemostasis of the ovarian pedicles was obtained in all mares. Median transection time for the ovarian pedicle was 28 minutes. Postoperative complications included transient fever, moderate subcutaneous emphysema, and incisional seroma formation. On necropsy examination, there were no signs of generalized peritonitis, postoperative hemorrhage, or adhesion formation. Mild to moderate acute inflammation and scar formation with moderate chronic inflammation at the ovarian pedicle was found at 3 and 30 days. Median depth of coagulation necrosis at 3 days was 2.87 mm. CONCLUSIONS: The Harmonic Scalpel appears to provide reliable hemostasis of the ovarian pedicle during elective laparoscopic ovariectomy in horses. Clinical Relevance-The Harmonic Scalpel represents a safe alternative to other methods of hemostasis during elective laparoscopic ovariectomy in horses.

Experimental evaluation of urinary bladder marsupialization in male goats.

OBJECTIVE: To evaluate the outcome of urinary bladder marsupialization in male goats. STUDY DESIGN: Prospective, experimental study. ANIMALS: Six healthy mixed-breed male goats. METHODS: After experimentally induced urethral obstruction, 6 male goats had urinary bladder marsupialization. Renal ultrasonography, complete blood count, and serum biochemical analysis were evaluated preoperatively (day 0), at 7 postoperative days, and then at 30-day intervals until 180 days. Stomal diameter was recorded immediately postoperatively and at each postoperative interval. Necropsy examination was performed on day 180 or when stomal stricture or death occurred. RESULTS: Stomal stricture occurred in 1 goat at 120 days. Another goat was found dead at 150 days; severe, suppurative cystitis was identified on necropsy. All goats had mild urine scald dermatitis. Serum biochemical values remained within normal limits, but significant decreases in white blood cell count, serum creatinine concentration, and stomal diameter occurred. At necropsy, all bladders were tubular in shape. Histological evidence of chronic suppurative cystitis and chronic, mild lymphoplasmacytic pyelitis occurred in all goats. Bacterial culture of renal tissue yielded growth in 3 goats, and bladder mucosal swabs yielded bacterial growth in all goats. CONCLUSIONS: Although clinical signs of ascending urinary tract infection were not observed in goats with patent stomata, urinary bladder marsupialization may result in ascending urinary tract inflammation or infection. CLINICAL RELEVANCE: Based on our results, urinary bladder marsupialization should be recommended with caution as the primary method for management of urinary tract obstruction in clinical cases.