Immune cell profiling in the joint following human and murine articular fracture.

OBJECTIVE: Human and in vivo animal research implicates inflammation following articular fracture as contributing to post-traumatic arthritis. However, relevant immune cell subsets present following injury are currently undefined. Immunophenotyping human and murine synovial fluid may help to identify immune cell populations that play key roles in the response to articular fracture. METHODS: Immunophenotyping by polychromatic flow cytometry was performed on human and mouse synovial fluid following articular fracture. Specimens were collected in patients with closed ankle fracture at the time of surgical fixation and from C57BL/6 mice with closed articular knee fracture. Immune cells were collected from injured and uninjured joints in mice via a novel cell isolation method. Whole blood samples were also collected. Immunohistochemistry (IHC) was performed on mouse synovial tissue to assess for macrophages and T cells. RESULTS: Following intra-articular fracture, the prominent human synovial fluid immune cell subset was CD3+ T cells, containing both CD4+ and CD8+ T cells. In mice, infiltration of CD45+ immune cells in synovial fluid of the fractured limb was dominated by CD19+ B cells and CD3+ T cells at 7 days after intra-articular fracture. We also detected adaptive immune cells, including macrophages, NK cells, dendritic cells and monocytes. Macrophage and T cell findings were supported by IHC of murine synovial tissue. CONCLUSIONS: Determining specific cell populations that mediate the immune response is essential to elucidating the chain of events initiated after injury and may be an important step in identifying potential immune signatures predictive of PTA susceptibility or potential therapeutic targets.

Global metabolic profiling of human osteoarthritic synovium.

Osteoarthritis (OA) is a debilitating disease associated with pain and loss of function in numerous diarthrodial joints of the body. Assessments of the severity and/or progression of OA are commonly based on radiographic stages and pain level, which aren't always correlated to severity of disease or joint dysfunction and may be confounded by other factors(1). There has been recent interest in identifying a biochemical signature of OA(1) that may be detected in serum, urine, and/or synovial fluid that would represent repeatable and predictable biomarkers of OA onset and/or progression. The objective of this study was to use global metabolic profiling to identify a distinct metabolic profile for cultured human synovial tissue from patients with end-stage OA compared to patients with little or no evidence of disease. While metabolic profiles from cultured tissues are not expected to reproduce in vivo profiles, it is expected that perturbations in metabolism caused by end-stage disease would result in differences in metabolic profiles in vitro compared to tissue with little or no evidence of disease. Because metabolomic perturbations often occur prior to alterations in the genome or proteome, metabolomic analysis possibly provides an earlier window to an altered biochemical profile for OA onset and/or progression, and may provide a unique set of potential drug targets. The synovium was targeted because it has been implicated in OA as a mediator of disease progression; osteoarthritic synovium has been demonstrated to express pro-inflammatory cytokines, such as Tumor Necrosis Factor - α (TNF-α), Interleukin-1 ß (IL-1ß), and IL-6(2), suggesting that a diseased synovial lining could produce an ideal set of biomarkers for diagnosing OA and/or monitoring disease progression. Media from the culture of synovial explants dissected from diseased human joints (early or end-stage OA) was subjected to global metabolic profiling with a liquid chromatography (LC)/and gas chromatography (GC)/mass spectrophotometry (MS)-based technology platform. Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries developed at Metabolon, Inc (Durham, NC). Global metabolic profiling resulted in the identification of 105 distinct compounds across all sample groups, with 11 compounds showing significantly different relative concentrations between end-stage and no/early disease groups. Metabolites specific to collagen metabolism, branched-chain amino acid metabolism, energy metabolism and tryptophan metabolism were amongst the most significant compounds, suggesting an altered metabolic state with disease progression.

Coronal plane ankle alignment, gait, and end-stage ankle osteoarthritis.

OBJECTIVE: Unilateral ankle osteoarthritis (OA) is a debilitating condition which may lead to limb deformity, severe pain, and functional disability due to tibiotalar malalignment and gait dysfunction. The purpose of this study was to determine if coronal plane alignment (varus, valgus, or neutral) of the ankle resulted in different spatial-temporal gait mechanics, clinically-assessed function, and self-reported function in patients with end-stage ankle OA. METHODS: Following informed consent, 96 patients with end-stage unilateral ankle OA were radiographically categorized as having varus, valgus, or neutral tibiotalar alignment. Each subject completed the foot and ankle disability index (FADI) questionnaire to assess self-reported function. The spatial-temporal parameters of interest (stance time, step length, stride length, stride width, single-support time, double support time, and walking speed) were assessed while the subject walked at a self-selected speed. RESULTS: The varus group performed the timed up and go test significantly faster than the other groups (P=0.05). All other variables were similar between the three alignment groups. CONCLUSION: There was little difference in gait mechanics and function between patients with end-stage OA based on coronal plane ankle alignment suggesting that factors other than coronal plane alignment contribute to diminished function.

Arthrodesis of the talocalcaneal joint for the treatment of two horses with talocalcaneal osteoarthritis.

Two horses with lameness due to tolocalcaneal osteoarthritis that failed to respond to conservative management were treated by tolocalcaneal arthrodesis using cortex screws placed in lag fashion. The affected joint of one horse was arthrodesed by compressing and stabilizing the medial facet of the joint with three screws. In the second horse, the medial and lateral facets of the affected tolocalcaneal joint were each compressed and stabilized with two screws. The lameness of both horses improved markedly following surgery.

Surgical implantation of ultrafiltration probes in ovine bone and muscle.

It may be desirable to collect compounds directly from sites of interest if blood concentrations do not reflect tissue levels. Ultrafiltration and microdialysis probes may be used to do this, but the hollow fibers of these probes are quite fragile. For this reason, we developed a pull-through technique that allows their implantation into the ovine quadriceps muscle and femur. The sheep is placed under anesthesia in lateral recumbency. An incision is made midway between the patella and greater trochanter directly over the lateral femur. A hand drill is used to make a 4.5-mm hole into the medullary cavity through the lateral cortex of the distal femur. A second incision is then made over the greater trochanter. The drill bit is inserted into the trochanteric fossa and a hole is drilled distally through the medullary cavity of the femur to the level of the first hole. A looped 20-gauge wire is then inserted into the femur and removed through the distal hole. Suture is attached, and the wire is withdrawn, leaving the suture in place. The suture is tied to the ultrafiltration probe tubing, allowing the probe to be carefully drawn into position. For implantation into the muscle, a 10-gauge introducer is used. The introducer is placed through the quadriceps muscle and the probe is then threaded through it. This technique has been successfully performed on 18 sheep. All sheep tolerated the procedure well. Up to 2.0 mL/day of interstitial fluid was recovered from each site. The average lifetimes of the bone and muscle probes were 35 and 40 days, respectively.

Diagnosis of sporotrichosis in a donkey using direct fluorescein-labeled antibody testing.

A 4-year-old female donkey residing in an open field in Indiana was admitted for evaluation of facial lesions of 2 years duration. Cytologic and histologic examination of exudate and tissue from the lesions revealed a pyogranulomatous inflammatory reaction with numerous yeasts. Sporothrix schenckii was suspected to be the infectious agent; however, multiple culture attempts did not provide positive identification of the organism. Serologic examination supported infection with S. schenckii. A specific direct immunofluorescent antibody test performed on paraffin-embedded tissue sections confirmed the organism as S. schenckii. Clinical signs resolved after appropriate iodide therapy.

Neostigmine methylsulfate delays gastric emptying of particulate markers in horses.

Eight horses were allotted to 2 groups, each of 4 horses. All horses were given 100 plastic markers intragastrically via a nasogastric tube. One group of animals (control group) was not given medication after marker administration. The other group (test group) was given neostigmine methylsulfate (0.022 mg/kg of body weight) in the subcutaneous tissue at the time of marker administration and 30, 60, and 90 minutes later. All horses were killed 135 minutes after marker administration to locate the beads in the gastrointestinal tract. Gastric emptying of the markers was significantly delayed (P less than 0.05) in horses given neostigmine methylsulfate.

Motility of the distal portion of the jejunum and pelvic flexure in ponies: effects of six drugs.

Bipolar stainless steel electrodes were surgically implanted in 4 ponies to record myoelectrical and mechanical activity of the distal portion of the jejunum and pelvic flexure. After determining normal activity, the effects of neostigmine, xylazine, flunixin meglumine, dipyrone, panthenol, and atropine sulfate were determined. Flunixin meglumine, dipyrone, and panthenol had no effect on the motility of the jejunum or pelvic flexure. Xylazine and atropine sulfate decreased motility of the distal portion of the jejunum and pelvic flexure, with atropine sulfate having a greater effect and lasting longer. Neostigmine stimulated propulsive motility in the pelvic flexure only.

Impedance monitoring of equine intestinal motility.

Myoelectrical and myomechanical activities of the distal portion of the jejunum and pelvic flexure were studied in 7 ponies, using permanently implanted monopolar and bipolar stainless steel electrodes. Dental acrylic embedded recording electrodes were surgically sutured to the serosal surface of the distal portion of the jejunum and pelvic flexure. Myoelectrically, regular spike bursts and irregular spike bursts were observed in the jejunum. Short spike bursts and long spike bursts were recorded and associated with spike potentials, using impedance recording techniques. Electrical and mechanical data could be monitored simultaneously from the same recording electrode, using separate channels on a physiograph. This method proved effective to monitor periodically myomechanical activity at the same time that myoelectrical activity was being evaluated. The recording system required fewer recording devices to be attached to the intestinal tract, was an inexpensive method of obtaining myomechanical recordings, and did not alter markedly the myoelectrical activity when mechanical activity was being monitored.

Effect of butorphanol, pentazocine, meperidine, or metoclopramide on intestinal motility in female ponies.

Effect of butorphanol, pentazocine, meperidine, and metoclopramide on jejunal and pelvic flexure myoelectric and mechanical activity in 4 female ponies was investigated. The agent to be tested or saline solution was administered IV at the start of a 6-hour recording trial. In the jejunum, duration between activity fronts of regular spiking activity, defined as the length of the migrating myoelectric complex (MMC), was measured. The average duration of the MMC during control trials was 150 +/- 46 minutes. The average duration of the MMC after meperidine, butorphanol, pentazocine, and metoclopramide administration was 295 +/- 70 minutes, 260 +/- 60 minutes, 275 +/- 60 minutes, and 163 +/- 64 minutes, respectively. Meperidine, butorphanol, or pentazocine significantly increased the MMC duration (P less than 0.05), and did not significantly alter the pelvic flexure activity. Seemingly, meperidine, butorphanol, and pentazocine inhibited cyclic myoelectric activity in the jejunum. Metoclopramide had no effect on jejunal or pelvic flexure motility.

Electromyographic, myomechanical, and intraluminal pressure changes associated with acute extraluminal obstruction of the jejunum in conscious ponies.

Bipolar electrodes, strain gauge force transducers, intraluminal pressure recording catheters, and extraluminal intestinal obstructors were surgically implanted in 4 ponies to record myoelectrical and mechanical activity of the distal portion of the jejunum and ileum. After determining normal intestinal activity and pressures, the distal portion of the jejunum was obstructed with an extraluminal obstructor. Myoelectrical and mechanical activity recorded from jejunal segments proximal to the obstruction increased significantly (P less than 0.01), whereas activity distal to the obstruction remained unchanged. Intraluminal pressure increases were recorded during periods of intestinal spasm. Obstruction pressures remained unchanged from preobstruction pressures.

Continuous infusion of gentamicin into the tarsocrural joint of horses.

OBJECTIVE: To develop a method for continuous infusion of gentamicin into the tarsocrural joint of horses, to determine pharmacokinetics of gentamicin in synovial fluid of the tarsocrural joint during continuous infusion, and to evaluate effects of continuous infusion of gentamicin on characteristics of the synovial fluid. ANIMALS: 12 healthy adult horses. PROCEDURE: An infusion catheter consisting of flow control tubing connected to a balloon infuser was used. Gentamicin solution (100 mg/ml) was infused in the right tarsocrural joint and balanced electrolyte solution was infused in the left tarsocrural joint for 5 days. Synovial fluid and serum gentamicin concentrations were measured by use of a fluorescence polarization immunoassay. RESULTS: 17 of the 24 (71%) infusion catheters initially placed functioned without complications for the entire 5-day infusion period. Median gentamicin concentration in synovial fluid from treated joints during the 5-day infusion period ranged from 2875 to 982 microg/ml. Median serum gentamicin concentration during this period ranged from 2.31 to 2.59 microg/ml. Mean (+/- SD) elimination half-life and total clearance of gentamicin from the synovial fluid were 6.25+/-1.01 hours and 1.52+/-0.96 ml/min, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: An infusion catheter can be used for continuous infusion of gentamicin into the tarsocrural joints of horses for up to 5 days. At a gentamicin dosage of 0.17+/-0.02 mg/kg/h, continuous intra-articular infusion results in synovial fluid gentamicin concentrations greater than 100 times the minimal inhibitory concentration reported for common equine pathogens.

Thromboxane, prostaglandin I2 (epoprostenol), and the hemodynamic changes in equine endotoxin shock.

This study had 2 objectives: (i) to correlate plasma thromboxane and prostaglandin I2 (epoprostenol) concentrations with hemodynamic changes occurring in equine endotoxin shock, and (ii) to determine the effects of flunixin meglumine on plasma concentrations of these prostaglandins relative to hemodynamic changes. Shock was induced in 2 groups, each of 4 anesthetized ponies, and in a 3rd group of 2 ponies. Group A ponies were given endotoxin only (and were not treated), and group B ponies were given endotoxin and then treated with flunixin meglumine. Group C ponies were treated with flunixin meglumine 5 minutes before they were fiven endotoxin. Arterial, pulmonary arterial, and central venous pressures were measured and blood samples were collected at 0, 0.1, 0.25, 0.5, 1, 1, 3, and 4 hours after ponies were given the endotoxin. The plasma thromboxane and prostaglandin I2 concentrations were increased in equine endotoxic shock. Increased thromboxane concentration was associated with the high pulmonary arterial and central venous pressures and low arterial blood pressure in the minutes immediately after the ponies were given endotoxin. The increased prostaglandin I2 concentration was associated with systemic hypotension at 1 to 2 hours after endotoxin. Treatment of ponies with flunixin meglumine after endotoxin was given (group B) prevented the prostaglandin I2 rise and the associated hypotension. Treatment with fluixin meglumine before endotoxin was given prevented the increase of the plasma thromboxane and prostaglandin I2 values, along with the associated hemodynamic changes.

Surgical repair of a supraglenoid tubercle fracture in a horse.

A fractured right supraglenoid tubercle in a 15-month-old Thoroughbred gelding was repaired by partial biceps brachii tenotomy, interfragmentary compression, and tension band wiring. The horse is currently being ridden daily and has remained sound for 4 years.

Umbilical cord remnant infections in foals: 16 cases (1975-1985).

Infection of the umbilical cord remnants was diagnosed in 16 foals. The infection was found in the urachus alone in 3 foals, the umbilical arteries in 2 foals, the urachus and umbilical arteries in 3 foals, the urachus and umbilical vein in 2 foals, and the urachus, umbilical vein, and umbilical arteries in 2 foals. The specific location of the infection was not determined in 4 foals. Eleven foals developed an acquired patent urachus between 4 and 16 days of age. Seven foals had one or more joints infected. All foals with umbilical cord remnant infections were treated with orally, intramuscularly, or intravenously administered antibiotics and 9 foals had the umbilical cord remnants excised. Nine foals survived, 6 of which had had excision of the infected remnants. Only 2 foals with joint infections survived.

Bilateral ureterocystostomy in a 450-kg horse with ectopic ureters.

A 450-kg yearling Clydesdale filly was determined to have bilateral ectopic ureters. The resulting incontinence caused severe malodorous perineal dermatitis. Bladder capacity was measured at 800 ml. The urethral sphincter lacked tone, and the horse was seen to urinate in a normal manner only 2 or 3 times a week. A midline celiotomy was performed, and the ureters were identified by cannulation from the ectopic openings. The ureters were ligated, and the cut ends were anastomosed to the dorsal bladder surface by an extravesicular end-to-side technique. A partial thickness seromuscular layer of the bladder was sutured over the ureters in a cranial direction from the anastomosis site for 15 mm. This fixed the ureters to the dorsal surface of the bladder and protected the anastomosis site from tension. After surgery, the horse urinated n a normal manner many times a day. Urinary incontinence continued, but gradually improved. Bladder capacity increased over 13 months to 4.3 L. Surgical (urethral extension) and medical (phenylpropanolamine and estrogen) treatments were instigated to increase urethral sphincter tone. Urinary incontinence continued to improve and, at 11 months after surgery, incontinence was negligible, and the perineal dermatitis had healed.

Treatment of radial-ulnar and tibial fractures in cattle, using a modified Thomas splint-cast combination.

In a retrospective study involving 15 cattle, radial-ulnar or tibial fractures were repaired with a modified Thomas splint combined with plaster cast. The cattle ranged in age from 1 day to 5 years and weighed from 46 to 775 kg. Eight cattle had radial-ulnar fractures and 7 had tibial fractures. The fractures healed in 14 cattle. The most frequent long-term complication was deviation of the involved long bones after healing. The degree of deviation decreased over several months' time in most cases. Thirteen of the 14 cattle with healed fractures were used for breeding or milking and their capacity for those functions was not diminished.

Bilateral wedge ostectomy on an 18-month-old llama with severe bilateral carpal valgus.

An 18-month-old llama was admitted with severe (45 degrees and 40 degrees) bilateral carpal valgus. The llama had grown normally until it was 6 months old, when the carpal deviations had commenced. Radiography revealed abnormalities at the distal ulnar physes. Premature closure of these physes or abnormal distal ulnar growth may have been the cause of the carpal valgus. The owners requested surgical correction of the condition. Medial wedge osteotomies of each radius were performed 3 weeks apart. Internal fixation of the osteotomized radial bones was accomplished with small right-angled T plates, and the limbs were placed in full-limb casts for 3 weeks and tube casts for 3 more weeks. Both limbs healed in straight alignment, and the llama was able to walk and run normally. Dorsal subluxation of the right radiocarpal joint was noticed after the operation. This was believed to be caused by the uncorrected dorsal bowing of the distal portion of the radius, which had occurred secondary to the severe carpal valgus.

Postpartum partial cystectomy through the vagina in a mare with everted partially necrotic bladder.

The bladder of a 750-kg Clydesdale mare had everted through the urethra into the vagina immediately after parturition. The bladder was reinverted into the peritoneal cavity by an attending veterinarian, but 4 days later, the bladder was everted again in the vagina. The mare was able to void urine through both ureters, which could be seen in the mucosal surface of the bladder. The everted bladder had become edematous and could not be reinverted through the urethra. A considerable portion of the fundus was necrotic. The mare was administered xylazine epidurally to induce perineal analgesia, and the necrotic portion of the bladder was resected and healthy bladder tissue was opposed with a double layer of simple continuous sutures. The urethral sphincter was longitudinally incised through the vaginal mucosa to allow reinversion of the bladder through the urethra. A purse-string suture inserted in the urethral opening decreased the urethral diameter and prevented recurrence of the condition. An inflated Foley catheter was maintained in the bladder for 5 days. The mare recovered normal urination after the catheter was removed.