Lise C. Berg

Serum amyloid A is expressed in histologically normal tissues from horses and cattle.

mRNA expression of the acute phase protein serum amyloid A (SAA) in histologically normal tissues derived from horses (n=13) and cattle (n=4) was investigated by quantitative reverse-transcriptase real-time polymerase-chain reaction. As expected, high constitutive SAA mRNA expression was demonstrated in hepatic tissue in both species. In horses, moderate (>1% of the hepatic expression) SAA mRNA expression was detected in the lung, mammary gland, pancreas, synovial membrane, thymus, thyroid gland and uterus. Other equine tissues and organs sampled included adipose tissue, adrenal gland, aorta, brain, different gastro-intestinal tissues, heart, kidney, lymph node, ovary, testis, prostate, skeletal and cardiac muscle, skin and spleen; all showed low (<1% of the hepatic expression) SAA mRNA expression. In cattle, SAA mRNA was expressed in moderate levels in adipose tissue, colon, jejunum, mammary gland, skeletal muscle, synovial membrane, thymus, thyroid gland, and uterus; expression was low in the remainder of the samples (same tissue panel as horses). The results confirm the liver as the main site of SAA production. Even though there was some inter-species variation in tissues expressing SAA mRNA, several organs communicating with the external environment (lung, mammary gland, uterus, and certain parts of the gastro-intestinal tract) showed SAA mRNA expression, which supports the hypothesis that SAA might possess a role in the innate defence against invading pathogens. The results of the study thus warrant further studies into functions of hepatically and extrahepatically produced SAA isoforms.

Ultrasonography of the equine cervical region: a descriptive study in eight horses

REASONS FOR PERFORMING STUDY In equine patients, the cause of clinical signs possibly related to the cervical region is often difficult to diagnose. Ultrasonography allows quick and noninvasive visualisation, but reference material of the normal equine neck is needed. OBJECTIVES To describe and document the normal ultrasonographic appearance of transverse scans in the cervical region with emphasis on the synovial articular facet joints, cervical vertebrae and paravertebral structures; and further, to provide images of frozen cross-sections for anatomical reference. METHODS A study describing the normal ultrasonographic appearance of the cervical anatomy was performed. Transverse scans were obtained from second cervical vertebra (C2) to first thoracic vertebra (T1). Post mortem photographs of frozen cross-sections were obtained as anatomical reference. RESULTS The structures were clearly visualised by ultrasonography and consistency was found between ultrasonographic images and corresponding cross-sectional anatomy. The articular facets varied between horses and facets (C2 to T1). Discrepancy in the existing anatomical descriptions was found. CONCLUSIONS AND POTENTIAL RELEVANCE The anatomical and ultrasonographic description provides a reference for ultrasonographic evaluation of equine cervical facet joints, vertebrae and paravertebral structures. The findings and variations found are considered to reflect the naturally occurring variations in horses.

Accuracy of ultrasound-guided intra-articular injection of cervical facet joints in horses: a cadaveric study.

REASONS FOR PERFORMING STUDY Intra-articular facet joint injection is an established diagnostic procedure in human medicine but there are no reports on its reliability in equine practice. OBJECTIVES To investigate the accuracy of ultrasound-guided intra-articular injections of the cervical facet joints and to estimate factors influencing the accuracy. METHODS Sixty injections with blue dye were performed on the facet joints between 2nd and 7th cervical vertebra (C2-C7) on horses subjected to euthanasia for nonorthopaedic reasons. The facet joints were subsequently dissected to verify accuracy of deposition. RESULTS Seventy-two percent of the injections were found to be intra-articular, 17% were intracapsular and a total of 98% were within 1 mm of the joint capsule. There was a marked effect of gained experience (P < 0.01), but not of other factors tested. CONCLUSIONS AND POTENTIAL RELEVANCE The results of the present study do not translate directly to injections performed in live horses, but they indicate that the method can be applied as a diagnostic as well as therapeutic procedure in C2 to C7 and that is advisable to practise injections on cadaver specimens before applying the technique.

Accuracy of ultrasound‐guided injections of thoracolumbar articular process joints in horses: A cadaveric study

REASONS FOR PERFORMING STUDY Arthrosis of the articular process joints (APJs) in the caudal thoracolumbar region of horses may cause back pain and subsequent reduced performance or lameness. Ultrasound-guided injections of the APJs of the equine back have been described only briefly in the literature. OBJECTIVES To evaluate factors affecting the accuracy of intra-articular injections of the APJs in the caudal thoracolumbar region. METHODS One-hundred-and-fifty-four injections with blue dye were performed on APJs including the T14-L6 region in 12 horses subjected to euthanasia for reasons unrelated to back problems. The backs were subsequently dissected to verify the location of the injectate in relation to the APJs. RESULTS Twenty-seven percent of the injections were found to be intra-articular and a total of 77% found to be within 2 mm of the joint capsule including the intra-articular deposits. Application of a medial approach and 18 gauge needle were significantly associated with an intra-articular injection or deposition close to the joint capsule. Operator, APJ (location) and back number (chronological) did not significantly affect the accuracy of injection. CONCLUSIONS AND POTENTIAL RELEVANCE Injection of the vertebral APJ in the thoracolumbar region using ultrasound guidance is a reliable method, as most of the injections were either in or within 2 mm of the joint. Based on the findings of this cadaver study, the medial approach is expected to be the most accurate in live horses. Further investigations are required to evaluate the diagnostic and therapeutic potential of this method in clinical practice.

Cartilage-derived retinoic acid-sensitive protein in equine synovial fluid from healthy and diseased joints.

REASONS FOR PERFORMING STUDY More sensitive and specific diagnostic methods for early detection of changes in the joint cartilage are needed. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a potential marker of cartilage synthesis and regeneration. This is the first study on equine CD-RAP. OBJECTIVES To evaluate the ability of a commercially available human sandwich ELISA assay to detect equine CD-RAP in synovial fluid from healthy and diseased joints. METHODS Synovial fluid was collected from 28 horses with no signs of joint disease and from 5 with induced inflammatory arthritis. CD-RAP concentrations were measured using a human CD-RAP ELISA. Intra- and interassay imprecision of the assay were evaluated by multiple measurements on pools of equine synovial fluid. Assay inaccuracy was determined by linearity under dilution. RESULTS The assay showed moderate to large intra- and interassay variation when applied to equine synovial fluid. Equine CD-RAP was detected in synovial fluid from healthy horses ranged at 8.2-52 ng/ml. Repeated arthrocentesis (after injection of isotonic saline), age, joint or gender did not significantly affect CD-RAP concentrations. Twelve hours after intra-articular injection of lipopolysaccharide, concentrations of CD-RAP were significantly lower than after injection of isotonic saline and remained significantly lower until the end of the study at 144 h. CONCLUSION AND POTENTIAL RELEVANCE The assay is suitable for longitudinal monitoring of CD-RAP concentration in individual horses. Disease significantly influenced CD-RAP levels. Similar to previous results obtained in man, CD-RAP seems to be a marker of cartilage synthesis and/or regeneration in horses.

Production of serum amyloid A in equine articular chondrocytes and fibroblast-like synoviocytes treated with proinflammatory cytokines and its effects on the two cell types in culture

OBJECTIVE To investigate the role of the major equine acute phase protein serum amyloid A (SAA) in inflammation of equine intraarticular tissues. SAMPLE Articular chondrocytes and fibroblast-like synoviocytes (FLSs) from 8 horses (4 horses/cell type). PROCEDURES Chondrocytes and FLSs were stimulated in vitro for various periods up to 48 hours with cytokines (recombinant interleukin [IL]-1β, IL-6, tumor necrosis factor-α, or a combination of all 3 [IIT]) or with recombinant SAA. Gene expression of SAA, IL-6, matrix metalloproteinases (MMP)-1 and -3, and cartilage-derived retinoic acid-sensitive protein were assessed by quantitative real-time PCR assay; SAA protein was evaluated by immunoturbidimetry and denaturing isoelectric focusing and western blotting. RESULTS All cytokine stimulation protocols increased expression of SAA mRNA and resulted in detectable SAA protein production in chondrocytes and FLSs. Isoforms of SAA in lysed chondrocytes and their culture medium corresponded to those previously detected in synovial fluid from horses with joint disease. When exposed to SAA, chondrocytes and FLSs had increased expression of IL-6, SAA, and MMP3, and chondrocytes had increased expression of MMP-1. Chondrocytes had decreased expression of cartilage-derived retinoic acid-sensitive protein. CONCLUSIONS AND CLINICAL RELEVANCE Upregulation of SAA in chondrocytes and FLSs stimulated with proinflammatory cytokines and the proinflammatory effects of SAA suggested that SAA may be involved in key aspects of pathogenesis of the joint inflammation in horses.

Synovial folds in equine articular process joints.

REASONS FOR PERFORMING STUDY Cervical synovial folds have been suggested as a potential cause of neck pain in humans. Little is known about the extent and characteristics of cervical synovial folds in horses. OBJECTIVES The objective of this explorative study was to determine the frequency of synovial folds in equine cervical articular process joints and to provide a characterisation of the size and morphology of the synovial folds. METHODS Equine cervical articular process joints from 6 horses were included in the study, ranging from cervical vertebra 2 (C2) to cervical vertebra 7 (C7) bilaterally. The articular process joints were dissected, and the cranial and caudal synovial folds of each joint were measured and embedded in paraffin. Synovial folds were analysed histologically and classified according to type, as adipose, fibrous and mixed type. Factors potentially influencing fold size were investigated, including joint number (from C2/C3 to C6/C7), fold type, position of fold within the joint (cranial or caudal) and side of neck (right or left). RESULTS Synovial folds were identified in 98% of cervical articular process joints examined. The width of the synovial folds varied from 4 to 41 mm, and the height from 1 to 17.8 mm. Thirty-eight per cent of the synovial folds were of adipose type, 41% of fibrous type and 21% of mixed type. Synovial fold size was significantly influenced by the side of the neck and fold type. CONCLUSIONS AND POTENTIAL RELEVANCE This study provides a characterisation of the frequency, size and morphology of equine cervical synovial folds in 6 horses. Synovial folds were present in 98% of the cervical articular process joints examined, and the size of the synovial folds indicates that they could be damaged by acute injury or chronic disease in the cervical articular process joints.

EFFECTS OF X-RAY BEAM ANGLE AND GEOMETRIC DISTORTION ON WIDTH OF EQUINE THORACOLUMBAR INTERSPINOUS SPACES USING RADIOGRAPHY AND COMPUTED TOMOGRAPHY-A CADAVERIC STUDY.

The widths of spaces between the thoracolumbar processi spinosi (interspinous spaces) are frequently assessed using radiography in sports horses; however effects of varying X-ray beam angles and geometric distortion have not been previously described. The aim of this prospective, observational study was to determine whether X-ray beam angle has an effect on apparent widths of interspinous spaces. Thoracolumbar spine specimens were collected from six equine cadavers and left-right lateral radiographs and sagittal and dorsal reconstructed computed tomographic (CT) images were acquired. Sequential radiographs were acquired with each interspinous space in focus. Measurements were performed for each interspinous space in the focus position and up to eight angled positions as the interspinous space moved away from focus (±). Focus position measurements were compared to matching sagittal CT measurements. Effect of geometric distortion was evaluated by comparing the interspinous space in radiographs with sagittal and dorsal reconstructed CT images. A total of 49 interspinous spaces were sampled, yielding 274 measurements. X-ray beam angle significantly affected measured width of interspinous spaces in position +3 (P = 0.038). Changes in width did not follow a consistent pattern. Interspinous space widths in focus position were significantly smaller in radiographs compared to matching reconstructed CT images for backs diagnosed with kissing spine syndrome (P < 0.001). Geometric distortion markedly affected appearance of interspinous space width between planes. In conclusion, X-ray beam angle and geometric distortion influence radiographically measured widths of interspinous spaces in the equine thoracolumbar spine, and this should be taken into consideration when evaluating sport horses.

Validation of the IDS Octeia ELISA for the determination of insulin-like growth factor 1 in equine serum and tendon tissue extracts.

BACKGROUND Insulin-like growth factor (IGF-1) is an important mediator of tissue repair in horses. OBJECTIVES The aim of the study was to evaluate whether IGF-1 could be measured reliably in equine serum and tendon tissue extracts, using an IGF-1 ELISA kit developed for human serum and plasma. METHODS A glycyl-glycine pretreatment protocol of samples was compared with the pretreatment procedure recommended by the manufacturer. Intra- and inter-assay imprecision were evaluated by repeated measurements of equine serum pools. Assay inaccuracy was determined based on the linearity of serially diluted equine serum samples and tendon tissue extracts. The recovery of IGF-1 was evaluated in serum and tendon tissue extracts spiked with known amounts of IGF-1. RESULTS The range of IGF-1 released using the manufacturers pretreatment was between 23% and 56% of the amount released using the gly-gly pretreatment in different equine samples. In serum pools with low, intermediate, and high IGF-1 concentrations, intra-assay imprecision was 4.0%, 4.0%, and 3.1%, respectively, and inter-assay imprecision was 13.9%, 7.3%, and 12.8%, respectively. The recovery of serially diluted serum was 96 ± 3% when diluted with serum, and 72 ± 15% when diluted with PBS. The recovery after dilution was 108 ± 17% in tendon tissue extracts. Recovery from serum spiked with a fixed amount of IGF-1 was 101 ± 5% and 99 ± 7% from tendon tissue samples. CONCLUSIONS The IDS Octeia IGF-1 ELISA kit can be used for measuring IGF-1 in equine serum and tendon tissue extract after pretreatment with glycyl-glycine.

Joint cartilage tissue engineering and pre-clinical safety and efficacy testing

Thomas G. Koch1,2, Lorenzo Moroni3, Younes Leysi-Derilou4 and Lise C. Berg5 1Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, 2Orthopaedic Research Lab, Aarhus University Hospital, Aarhus C, 3Tissue Regeneration Department, MIRA institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, 4Department of Biomedical Sciences, University of Guelph, Guelph, ON 5Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C 1,4Canada 2,5Denmark 3The Netherlands