Endotoxin-induced changes in expression of cyclooxygenase isoforms in the lamellar tissue of extracorporeally haemoperfused equine limbs

Angiogenesis and sepsis-related equine laminitis have several features in common. Both events can be induced by endotoxin (lipopolysaccharide— LPS) and both are associated with increased expression of the enzyme cyclooxygenase (COX), of which two isoforms (COX-1 and COX-2) exist. To examine the causal relationship between LPS exposure and COX expression and to investigate the tissue distribution of COX in the LPS-exposed tissue, the technique of extracorporeal haemoperfusion of isolated equine forelimbs was utilized. Perfusion was performed for 10 hr under physiological conditions (control-perfused limbs, n = 5) and with addition of 80 ng/L of endotoxin (LPS-perfused limbs; n = 5). After perfusion, samples of lamellar tissue were collected from the dorsal aspect of the hoof wall. Additional control samples were collected from three non-perfused limbs. Immunohistochemical analysis was performed using antibodies against COX-1 and COX-2, and intensity of immunohistochemical staining was scored for each isoform. In the lamellar tissue of control- and LPS-perfused limbs, there was no significant difference in COX-1 staining intensity and distribution, whereas COX-2 expression was significantly increased in LPS-perfused limbs (especially in endothelial cells, fibroblasts and intravasal leucocytes as well as in epidermal basal cells at the base of the primary epidermal lamellae). These results suggest that COX-2 and its metabolites are involved in the initiation of pathological changes seen in sepsis-associated events such as sepsis-related laminitis. In such cases, COX-2 could therefore be an important therapeutic target; however, early therapy may be required as increase in COX-2 expression occurs within 10 hr after LPS exposure.     

Comparison of three dorsal techniques for arthrocentesis of the distal interphalangeal joint in horses

OBJECTIVE: To compare 3 dorsal techniques for arthrocentesis of the distal interphalangeal joint in horses with regard to ease of performing the technique and to determine the role of operator experience in ease of performing these techniques. DESIGN: Observational study. Sample Population-Forelimbs from 17 equine cadavers and 12 horses (16 joints) undergoing arthrocentesis for therapeutic or diagnostic purposes. PROCEDURES: In both forelimbs from 7 of the equine cadavers, 3 arthrocentesis techniques (dorsal perpendicular, dorsolateral, and dorsal inclined) were performed in random order by a single experienced individual, and number of attempts needed to successfully insert the needle into the joint was recorded. For the forelimbs from the remaining 10 cadavers, veterinary students without experience in arthrocentesis performed each of the 3 arthrocentesis techniques (2 limbs/student) in random order, and number of attempts was recorded. In the clinical patients, arthrocentesis was performed by means of the dorsal inclined technique. RESULTS: For both the experienced individual and the veterinary students, number of attempts needed was significantly lower with the dorsal inclined technique than with the dorsal perpendicular or dorsolateral technique. Arthrocentesis was successful with the dorsal inclined technique in all 16 joints in the clinical patients; synovial fluid was recovered from 14 of the 16 joints. The procedure was well tolerated in all horses, except one that reacted to needle insertion. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the dorsal inclined technique for arthrocentesis of the distal interphalangeal joint was easier to perform than was the dorsal perpendicular or dorsolateral technique, regardless of experience level of the operator.     

Desmotomy of the accessory ligament of the superficial digital flexor tendon in the horse with use of a tenoscopic approach to the carpal sheath

Objective— To describe a tenoscopic approach to the carpal sheath for desmotomy of the accessory ligament of the superficial digital flexor tendon. Study Design— The surgical procedure was developed with use of normal forelimbs from equine cadavers and experimental horses. Animals or Sample Population— Twelve equine cadaveric forelimbs, 4 forelimbs from 2 horses anesthetized for terminal surgical laboratories, and 10 forelimbs from five experimental horses were used. Methods— The limbs were positioned lateral side up with the carpus slightly flexed. After distention of the carpal sheath, a portal was made approximately 2 cm proximal to the distal radial physis for arthroscope insertion. An instrument portal was made approximately 0.2 cm proximal to the distal radial physis. After flexion of the limb to 90°, the accessory ligament of the superficial digital flexor tendon was palpated and desmotomy was performed. Cadaveric limbs were dissected to confirm complete desmotomy. Experimental horses were monitored for short- (perioperative) and long- (4 weeks) term postoperative complications. Results— A tenoscopic approach to the carpal sheath provided adequate surgical access to the accessory ligament of the superficial digital flexor tendon for desmotomy. Most of the accessory ligament of the superficial digital flexor tendon could be easily seen within the sheath, except for the proximal 2 cm that could be readily palpated and subsequently transected. Important technical considerations were location of the arthroscope portal, adequate sheath distention, limb flexion to 90°, and desmotomy location. It was beneficial, but apparently not essential, to avoid the proximal perforating vessel. Postoperatively, some horses had swelling but were not lame and had normal range of motion of the carpus. Conclusions— Desmotomy of the accessory ligament of the superficial digital flexor tendon could be performed by using a lateral tenoscopic approach to the carpal sheath. Clinical Relevance— Desmotomy of the accessory ligament of the superficial digital flexor tendon by using a tenoscopic approach to the carpal sheath is an alternative technique to the medial incisional approach.     

Endotoxemia in horses: protection provided by antiserum to core lipopolysaccharide

An equine antiserum to core lipopolysaccharide was produced by inoculation of 6 horses with a boiled cell bacterin made from the J-5 mutant of Escherichia coli O111:B4. The antiserum immunoglobulin G titer to J-5 mutant E coli, as determined by enzyme-linked immunosorbent assay, was 1:15,006. Pooled serum prepared before inoculation (preimmune serum) had a J-5 immunoglobulin G titer of 1:350. The J-5 antiserum was tested for its protective efficacy in sublethal endotoxemia in 14 horses. Four horses served as nontreated controls and were given nothing before endotoxin challenge exposure (10 micrograms/kg of body weight, IV). Pooled preimmune serum (3 ml/kg, IV) was administered to 5 horses and J-5 antiserum (3 ml/kg, IV) was administered to 5 other horses 2 to 15 hours before endotoxin challenge exposure. During the 24 hours postendotoxin challenge exposure, endotoxemia was accompanied by significant (P less than 0.05) time-related changes in temperature, heart rate, pulse character, respiratory rate and character, capillary refill time, mucous membrane color, fecal composition, attitude, PCV, total plasma protein, WBC count, platelet count, plasma fibrinogen, prothrombin time, activated partial thromboplastin time, fibrinolytic degradation products, plasma glucose, and plasma lactate in all horses. There were no apparent treatment vs time interactions (P greater than 0.05). Two horses (1 control and 1 given J-5 antiserum) died suddenly from unknown causes immediately after endotoxin challenge exposure. Seemingly, equine antiserum to core lipopolysaccharide did not provide protection from the adverse effects of experimental endotoxemia produced by bolus IV infusion of 10 micrograms of endotoxin/kg.     

Radiation exposure to personnel during examination of limbs of horses with a portable hand-held fluoroscopic unit.

OBJECTIVES: To determine radiation exposure to personnel during fluoroscopic imaging of limbs of horses with a portable unit and to determine distance from the c-arm at which radioprotective clothing is not required. DESIGN: Repeated-measures cohort study. SAMPLE POPULATION: Part 1, 1 forelimb and 1 hind limb from each of 5 equine cadavers; parts 2 and 3, personnel involved during imaging of limbs of 5 and 9 horses, respectively. PROCEDURE: Radiation exposure rates were mapped around the suspended c-arm of a portable fluoroscopy unit during imaging of various joints of equine cadaver limbs. During similar examinations in live horses, exposure rates to the fluoroscopist and assistant were measured. Mean duration for fluoroscopy of various joints was determined by observing an experienced fluoroscopist. Exposure to fluoroscopists and assistants per examination and per annum was estimated. RESULTS: Radiation exposure rates were dependent on distance and direction relative to the c-arm and consistently highest on the tube side of the unit. Exposure was significantly greater than background amounts until approximately 4.7 m from the c-arm. During examination of live horses, exposure was highest to the fluoroscopist's hand nearest the tube. Typically, exposure to the fluoroscopist and assistant during carpal examination was 25 to 40 times greater than that for comparable radiographic examination. Annual exposure for fluoroscopists was more than twice the recommended maximum permissible dose. CONCLUSIONS AND CLINICAL RELEVANCE: Fluoroscopic imaging of limbs of horses represents a major source of radiation exposure. Annual maximum permissible doses of radiation will be rapidly exceeded if required radioprotective clothing is not worn.     

Evaluation of cytokine production by equine alveolar macrophages exposed to lipopolysaccharide, Aspergillus fumigatus, and a suspension of hay dust

OBJECTIVE: To evaluate cytokine production by equine alveolar macrophages after exposure to lipopolysaccharide (LPS), Aspergillus fumigatus, and hay dust, and determine the effect of clenbuterol on the cytokine response. ANIMALS: 6 horses. PROCEDURE: Alveolar macrophages were exposed to PBS solution (negative control), LPS, hyphae and conidia of Aspergillus fumigatus (AF), or a suspension of hay dust (HDS) and incubated for 24 hours at 37 degrees C. Concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta were measured in the supernatant. The procedure was repeated with cells that were concurrently incubated with 0.5 microM clenbuterol. RESULTS: Exposure to HDS and AF significantly increased production of TNF-alpha by equine alveolar macrophages. The increase in TNF-alpha produced in response to HDS and AF was 5 and 7 times as great, respectively, as the increase measured in response to LPS. The concentration of IL-1beta in the supernatant was significantly increased after exposure of cells to AF. Clenbuterol was effective at inhibiting TNF-alpha production by cells exposed to LPS, HDS, or AF. CONCLUSIONS AND CLINICAL RELEVANCE: Increased production of TNF-alpha and IL-1 indicated that the pro-inflammatory cytokines produced by alveolar macrophages in response to allergens may play a role in recurrent airway obstruction (RAO) in horses. Equine alveolar macrophages are not only a primary pulmonary defense mechanism but may also influence the pathogenesis of equine RAO. The beta2-adrenoceptor agonist clenbuterol, a drug that is commonly used for treatment of equine RAO, promotes immediate bronchodilation and may also contribute to downward modulation of the inflammatory response.     

Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses

REASONS FOR PERFORMING STUDY: Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. HYPOTHESIS: Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. METHODS: Twelve healthy horses received an i.v. infusion either of saline or a phospholipid emulsion (100 mg/kg), followed immediately by E. coli 055:B5 LPS (30 ng/kg). Clinical parameters, haematological profiles, serum tumour necrosis factor (TNF) activity, serum lipid profiles, urine analyses and severity of haemolysis were monitored before and at selected times after LPS. Monocytes were also incubated in vitro with LPS in the presence or absence of emulsion, after which TNF and tissue factor activities were determined. RESULTS: Clinical signs of endotoxaemia were reduced in horses receiving the emulsion, including clinical score, heart rate, rectal temperature, serum TNF activity, and the characteristic leucopenic response to LPS, when compared to horses not receiving the emulsion. Three horses receiving the emulsion had none, 2 had mild and one had moderate haemolysis. There were no differences in urinalysis results and creatinine concentrations, either within the groups over time or between the groups. Serum concentrations of phosphatidylcholine, bile acids and triglycerides peaked immediately after the infusion; there were no significant changes in concentrations of nonesterified fatty acids or cholesterol. Incubation of equine monocytes with emulsion prevented LPS-induced TNF and tissue factor activities. CONCLUSIONS: Rapid administration of emulsion significantly reduced inflammatory effects of LPS in vivo and caused a clinically insignificant degree of haemolysis. The results of the in vitro studies indicate that emulsion prevents not only LPS-induced synthesis of cytokines, but also expression of membrane-associated mediators (i.e. tissue factor). POTENTIAL RELEVANCE: Rapid i.v. administration of emulsions containing phospholipids that bind endotoxin may provide a clinically useful method of treating endotoxaemia in horses.     

Tumor necrosis factor activity in the circulation of horses given endotoxin

Serum and plasma from horses injected with endotoxin was examined for cytotoxic activity. Each of the cell lines, L929 and WEHI 164 clone 13, was sensitive to the cytotoxic effects of equine serum; however, a precipitation artifact caused by the use of isopropanol in the WEHI assay limited the use of this assay to samples containing less than 2 mg of protein/ml. In foals treated with a sublethal IV bolus of 5 micrograms of lipopolysaccharide (LPS)/kg and in adult horses given a low-dose continuous infusion of LPS (30 ng/kg/h for 4 hours), cytotoxic activity was detected in all serum or plasma samples taken between 30 minutes and 4 hours after LPS infusion began. In horses given either continuous or bolus LPS infusions, circulating cytotoxic activity peaked at 1 to 2 hours before decreasing sharply. The onset of pyrexia after LPS infusion coincided with the appearance of circulating cytotoxic activity, but the temperature remained high, even after cytotoxic activity disappeared. Treatment of horses with flunixin meglumine (1 mg/kg) appeared to blunt the pyrexic effect of low-dose continuous LPS infusion, but had no significant effect on circulating cytotoxic activity. Incubation of serum samples with an antibody raised against a portion of human tumor necrosis factor (TNF) resulted in the removal of greater than 90% of serum cytotoxicity, suggesting strongly that the cytotoxic activity was attributable to TNF. These findings are consistent with the hypothesis that TNF is an early acting mediator of the effects of endotoxin in the horse.     

Endotoxin-induced production of interleukin 6 by equine peritoneal macrophages in vitro.

A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (IL-6) in vitro in response to endotoxin. Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages. Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5). Culture medium supernatants were collected after 3, 6, 12, and 24 hours' incubation and were frozen at -70 C until assayed for IL-6 activity. Supernatant IL-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B 13.29 clone B.9, which is dependent on IL-6 for survival. Results indicated that equine peritoneal macrophages produce IL-6 in vitro and that supernatant medium IL-6 activity was significantly (P less than 0.05) increased by exposure to endotoxin. Significant (P less than 0.05) time and treatment effects on macrophage IL-6 production were apparent. The IL-6 activity peaked at 6 or 12 hours' incubation, then remained high through 24 hours' incubation, regardless of endotoxin exposure. Medium IL-6 activity during 3 and 6 hours' incubation was significantly (P less than 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak IL-6 activity was similar among all endotoxin concentrations. Endotoxin concentration did not have an effect on medium IL-6 activity from macrophages exposed to endotoxin for 12 or 24 hours.     

ULTRASONOGRAPHY OF THE EQUINE SHOULDER: TECHNIQUE AND NORMAL APPEARANCE

This study was intended to document normal ultrasonographic appearance of the equine shoulder and anatomic landmarks useful in clinical imaging. Both forelimbs of five equine cadavers and both forelimbs of six live adult horses were used. To facilitate understanding of the images, a zoning system assigned to the biceps brachii and to the infraspinatus tendon was developed. Ultrasonography was performed with a real-time B-mode semiportable sector scanner using 7.5- and 5-MHz transducers. On one cadaver limb, magnetic resonance imaging (MRI) was performed using a system at 1.5 Tesla, T1-weighted spin-echo sequence. Ultrasonography images were compared to frozen specimens and MRI images to correlate the ultrasonographic findings to the gross anatomy of the shoulder. Ultrasonography allowed easy evaluation of the biceps brachii and the infraspinatus tendon and their bursae, the supraspinatus muscle and tendons, the superficial muscles of the shoulder, and the underlying humerus and scapula. Only the lateral and, partially, the caudal aspects of the humeral head could be visualized with ultrasound. Ultrasonographic appearance, orientation, and anatomic relationships of these structures are described. Ultrasonographic findings correlated well with MRI images and with gross anatomy in the cadavers' limbs.     

Ultrasonography of the equine elbow technique and normal appearance

This study was intended to document normal ultrasonographic appearance of the equine elbow and anatomic landmarks useful in clinical imaging. Both forelimbs of five equine cadavers and both forelimbs of six live adult horses were used (4 Arabian-Barbes, 3 Arabs, 2 Anglo-Arabs, 1 Selle Français, 1 Anglo-Hispano-Arab, three to 18 years old). To facilitate the reading of the scans, a zoning system was developed for some anatomic structures. Ultrasonography was performed with a real-time B-mode semi-portable sector scanner using 7.5 & 5 MHz transducers. On one cadaver limb, MRI was performed on a system at 1.5 Tesla, T1 weighted spin echo, TR of 475 msec, RE of 15 msec, image matrix size 179 × 256 pixels. Ultrasonography images were compared with gross anatomy and with MRI scans to provide the normal ultrasonographic representation of the equine elbow. The lateral collateral ligament, the triceps brachii tendon with its subtendinous bursa, the proximal tendon of the ulnaris lateralis and the articular cartilage of the humeral trochlea were easy to examine ultrasonographically. The medial collateral ligament and the distal biceps brachii tendon required more expertise to assess. Ultrasonographic appearance and course of these structures are described. The 7.5 MHz transducer was best to be used. Ultrasonographic findings correlated well with MRI scans and with gross anatomy in the cadavers' limbs.     

Depletion of pulmonary intravascular macrophages partially inhibits lipopolysaccharide-induced lung inflammation in horses

Horses are unique in their extreme sensitivity to endotoxin-induced cardio-pulmonary shock and mortality. The mechanisms behind increased sensitivity of the horse to endotoxin remain unknown. Pulmonary intravascular macrophages (PIMs) are pro-inflammatory cells occurring in horses. Because the functions of equine PIMs in endotoxemia remain unknown, we studied the role played by equine PIMs in endotoxin-induced pulmonary pathophysiology. We achieved this by using a recently developed protocol to deplete PIMs in order to compare lipopolysaccharide (LPS)-induced pulmonary responses in horses with or without PIMs. Horses treated with gadolinium chloride (GC; 10 mg/kg intravenous) to deplete PIMs or endotoxin-free saline (n = 4) were injected with Escherichia coli LPS (E. coli LPS; 50 ng/kg intravenously) 48 h after GC or saline. Control horses (n = 5) received two injections of endotoxin-free saline at 48 h intervals. All the horses were euthanized 2 h after LPS or saline challenge. Immunohistology for the PIMs showed their reduced numbers in GC-treated horses. The LPS treatment of normal and GC-treated horses increased diastolic and systolic pulmonary arterial pressures at 30 min compared to the saline-treated horses (P < 0.05). However, horses pre-treated with GC did not have an LPS-induced increase in mean pulmonary arterial pressure compared to the LPS-treated horses (P < 0.05). Light and electron microscopic immunocytochemistry detected extensive labeling for LPS in PIMs of LPS-treated horses. Both the LPS-treated groups had more alveolar septal cells positive for TNF-alpha and IL-1beta compared to control horses, which did not receive LPS (P < 0.05). However, GC-treated horses challenged with the LPS showed less IL-1beta-positive cells (P < 0.05). Immuno-electron microscopy localized TNF-alpha and IL-1beta in PIMs. These new data show that PIMs endocytose LPS and contain TNF-alpha and IL-1beta and their depletion partially inhibits LPS-induced pulmonary inflammatory responses.     

Evaluation of the induction of vasoactive mediators from equine digital vein endothelial cells by endotoxin

OBJECTIVE: To determine the effect of endotoxin (lipopolysaccharide [LPS]) on vasoactive mediator production by cultured equine digital vein endothelial cells (EDVECs). SAMPLE POPULATION: EDVECs obtained from forelimb digital veins of 7 healthy adult horses. PROCEDURES: EDVECs were incubated with or without LPS (1 microg/mL) for 0, 2, 4, 6, 22, and 24 hours. The EDVECs were incubated for 18 hours with LPS (10 pg/mL to 1 microg/mL) with or without ibuprofen, cycloheximide, or L-nitroarginine methyl ester. Medium concentrations of prostacyclin, cyclic guanosine monophosphate, endothelin-1, and thromboxane A(2) were determined. Changes in inducible nitric oxide synthase and cyclooxygenase-2 expression were determined. RESULTS: LPS stimulated mean 4.2- and 14.1-fold increases in EDVEC prostacyclin and cyclic guanosine monophosphate production, respectively, after 22 hours. These effects were LPS concentration-dependent (LPS concentrations that induced a response halfway between the maximum response and baseline of 1.50 and 1.22 ng/mL, respectively). The LPS-induced cyclic guanosine monophosphate production was significantly inhibited (to basal concentrations) by L-nitroarginine methyl ester, and prostacyclin production was inhibited by cycloheximide and ibuprofen. Production of thromboxane A(2) by EDVECs was not detected. Endothelin-1 accumulated in the medium, but LPS did not enhance its production. Inducible nitric oxide synthase expression in EDVECs was not detected with the available antibodies, whereas LPS stimulated cyclooxygenase-2 expression in a time- and concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: LPS stimulated vasoactive mediator production by equine endothelial cells, which may play a role in LPS-induced digital hypoperfusion.     

Ultrastructure of equine endothelial cells exposed to endotoxin and flunixin meglumine and equine neutrophils

An in vitro system of cultured equine endothelial cells was evaluated as a model for endotoxin (ET) exposure in the horse. Primary cell lines from pulmonary vessels and aortas were cultured from tissues of 6 horses. Effects of ET alone with and without serum and in combination with the cyclo-oxygenase inhibitor flunixin meglumine and isolated equine neutrophils were evaluated by transmission electron microscopy. Cells plus serum were incubated with 10, 25, 50, or 100 micrograms of ET/ml of incubation medium for 1, 3, 8, or 24 hours. Cells without serum were cultured for 1 and 3 hours. Flunixin meglumine was used at a concentration of 20 micrograms/ml. Cells also were incubated in the presence of 1,000, 5,000, or 20,000 neutrophils/ml plus ET and in the presence of a combination of ET and flunixin meglumine for 1 or 3 hours. Endotoxin alone did not cause cell damage, and the only evidence of an effect was an increased number of secondary lysosomes at incubation hour 8. At incubation hour 24, cells appeared normal. Endotoxin plus neutrophils caused cells to become round and detach from the growth substrate. Cell pathologic changes included swollen and distorted mitochondria and cytoplasmic vacuolization. Response to the ET plus neutrophil combination was variable and ranged from 5% to 50% of the cells being affected. The variability appeared to have some correlation with cell age, as well as individual preparation of neutrophils.     

Limb loading activity of adult horses confined to box stalls in an equine hospital barn

OBJECTIVE: To determine a range of limb loading activity for healthy adult horses confined to box stalls in an equine veterinary teaching hospital and determine the effects of hospital environmental factors on load rates and daily limb loading patterns. ANIMALS: 6 mature healthy horses of various ages, breeds, and sexes, and 1 horse with a repaired metatarsal fracture. PROCEDURE: Step monitors were placed on 2 limbs of adult horses confined to box stalls. Relocation steps and weight shifts were recorded, as loading events, for 24 hours. Influence of forelimb versus hind limb and environmental factors on load rate (loading events per hour) were assessed with repeated-measures ANOVA. RESULTS: Loading activity was greater for the forelimb than the hind limb and was greater during the day than the night. Loading activity differences were not associated with daytime environmental factors. CONCLUSIONS AND CLINICAL RELEVANCE: Horses with normal locomotor activity appear to have higher load rates for forelimbs compared with hind limbs and higher load rates during the day compared with night. Knowledge of influence of environmental factors and mechanical restraint on limb loading activity may be useful in management of horses with musculoskeletal disorders. This information may also be used for in vitro simulation of in vivo loading of limbs during cyclic biomechanical investigations.     

Age‐related differences in prostaglandin E2 synthesis by equine cartilage explants and synoviocytes

Briston, L., Dudhia, J., Lees, P. Age‐related differences in prostaglandin E₂ synthesis by equine cartilage explants and synoviocytes. J. vet. Pharmacol. Therap. 33 , 268–276. Time‐ and concentration‐related actions of lipopolysaccharide (LPS) on the synthesis of prostaglandin E₂ (PGE₂) were investigated in cartilage explants and synoviocytes harvested from 3 age groups of horses, all with clinically normal joint function: group A <10 years; group B 11–20 years and group C >20 years. Cartilage explants from group A horses were least and those from group C were most sensitive to LPS. Significant increases in PGE₂ concentration (P ≤ 0.01) were obtained in group C horses in response to LPS concentrations of 1.0 μg/mL (and higher) after exposure for 24, 36 and 48 h, whereas explants from group A horses failed to respond to LPS at concentrations up to 100 μg/mL after exposure times up to 48 h. In contrast, synoviocytes from group A horses were most and those from group C horses were least sensitive to LPS stimulation. Synoviocytes from group A horses responded to LPS concentrations of 1 μg/mL (and higher) with significantly increased concentrations of PGE₂ at 24 and 36 h. Significant but numerically smaller increases in PGE₂ concentration were induced by LPS in synoviocytes from groups B and C. As the effects of high PGE₂ concentrations are catabolic for cartilage, these observations suggest that both synoviocytes and chondrocytes might exert roles in the degenerative changes which occur in cartilage in horses with osteoarthritis.     

A biomechanical comparison of headless tapered variable pitch compression and ao cortical bone screws for fixation of a simulated midbody transverse fracture of the proximal sesamoid bone in horses

OBJECTIVE: To compare mechanical properties and failure characteristics of 2 methods of fixation for repair of a transverse, midbody fracture of the proximal sesamoid bone (PSB): 4.5-mm AO cortical bone screw (AO) placed in lag fashion and 4/5-mm Acutrak (AT) self-compressing screw. STUDY DESIGN: An in vitro biomechanical evaluation of intact forelimb preparations and forelimb preparations with a simulated midbody PSB fracture stabilized by a bone screw. SAMPLE POPULATION: Sixteen paired and 8 unilateral cadaveric equine forelimbs. METHODS: A midbody transverse osteotomy was created in the medial PSB of bilateral forelimbs of 8 equine cadavers. The osteotomized PSB in 1 forelimb from each cadaver was repaired with an AO screw. The osteotomized PSB in each contralateral limb was repaired with an AT screw. Eight unilateral intact control limbs were also studied. Mechanical properties were determined from axial compression, single cycle to failure, load-deformation curves. Failure characteristics were determined by evaluation of video images and radiographs. RESULTS: No statistically significant differences were found between repair groups. Both AO and AT groups had significantly lower mechanical properties than intact limbs except for stiffness. CONCLUSION: AO and AT constructs were mechanically comparable when used to stabilize a simulated midbody fracture of the medial PSB. Both constructs were mechanically inferior to intact limbs. Clinical Relevance- The AT screw should be considered for clinical use because of the potential for less soft tissue impingement and superior biocompatibility compared with the stainless-steel AO screw. However, postoperative external coaptation is necessary to augment initial fracture stability for either fixation method, and to maintain a standing metacarpophalangeal joint dorsiflexion angle between 150 degrees and 155 degrees.     

Evaluation of computed tomographic anatomy of the equine metacarpophalangeal joint

OBJECTIVE: To determine the detailed computed tomography (CT) anatomy of the metacarpophalangeal (MCP) joint in healthy horses. SAMPLE POPULATION: 10 cadaveric forelimbs from 10 adult horses without orthopedic disease. PROCEDURES: CT of the MCP joint was performed on 4 forelimbs. In 1 of the limbs, CT was also performed after intra-articular injection of 30 mL of contrast medium (40 mg of iodine/mL). Transverse slices 1-mm thick were obtained, and sagittal and dorsal planes were reformatted with a slice thickness of 2 mm. The CT images were matched with corresponding anatomic slices from 6 additional forelimbs. RESULTS: The third metacarpal bone, proximal sesamoid bones, and proximal phalanx could be clearly visualized. Common digital extensor tendon; accessory digital extensor tendon; lateral digital extensor tendon; superficial digital flexor tendon (including manica flexoria); deep digital flexor tendon; branches of the suspensory ligament (including its attachment); extensor branches of the suspensory ligament; collateral ligaments; straight, oblique, and cruciate distal sesamoidean ligaments; intersesamoidean ligament; annular ligament; and joint capsule could be seen. Collateral sesamoidean ligaments and short distal sesamoidean ligaments could be localized but not at all times clearly identified, whereas the metacarpointersesamoidean ligament could not be identified. The cartilage of the MCP joint could be assessed on the postcontrast sequence. CONCLUSIONS AND CLINICAL RELEVANCE: CT of the equine MCP joint can be of great value when results of radiography and ultrasonography are inconclusive. Images obtained in this study may serve as reference for CT of the equine MCP joint.     

USE OF A BARIUM/GELATIN MIXTURE TO STUDY EQUINE VASCULATURE WITH POTENTIAL APPLICATION IN FREE-FLAP TRANSFER

The purposes of this study were: 1) to evaluate a barium (30%) and gelatin (4%) solution as an intravascular radiographic contrast medium when perfused into intact equine cadavers, 2) to determine the most appropriate skin site to harvest a flap with direct cutaneous vasculature, and 3) to identify potential recipent vessels for free-flap transfer.
Heparinized, aspirin-treated horses were euthanatized, exsanguinated, and perfused with the contrast media. After 24 hours refrigeration, the cadvers were dissected and radiographed. The contrast medium remained solid for the duration of studies (approximately 4 hours), and provided excellent visualization of the arterial vasculature and smaller venous vessls, but did not adequately fill large veins. Due to its location and consistent vascular supply, the skin perfused by branches of the cudal superficial epigastric artery and vein is an appropriate skin-flap donor site. Multiple potential recipent vessels were identified.     

Net joint moments and joint powers in horses with superficial digital flexor tendinitis

OBJECTIVE: To determine whether analysis of net joint moments and joint powers is a suitable technique for evaluation of mechanics and energetics of lameness in horses and to measure effects of superficial digital flexor tendinitis. ANIMALS: 6 sound horses. PROCEDURE: Horses were evaluated before (sound evaluation) and after (lame evaluation) induction of superficial digital flexor tendinitis in 1 forelimb by injection of collagenase. Recordings were made with an optoelectronic system and a force plate as horses trotted. Net joint moments and joint powers in the sagittal plane at each joint in the forelimbs during the stance phase were determined. Peak values were determined, and mechanical energy absorbed and generated at each joint was calculated. Comparisons were made between contralateral limbs during sound and lame evaluations. RESULTS: Lame limbs had significant reductions in peak values for net joint moments on the palmar aspect of metacarpophalangeal (fetlock), carpal, and humeroulnar joints. Total energy absorbed was significantly lower at every joint in lame limbs, compared with compensating limbs. CONCLUSIONS AND CLINICAL RELEVANCE: Horses with superficial digital flexor tendinitis had significant differences between lame and compensating limbs for net joint moments and joint powers at all joints, indicating that the gait of horses with superficial digital flexor tendinitis is energetically inefficient. Assessment of net joint moments and joint powers is a useful tool in evaluating equine lameness.