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Abstract

Objective

To examine the site-specific and dose-dependent effects of insulin-like growth factor I (IGF-I) on normal equine tendon in vitro.

Samples

Superficial digital flexor tendon explants derived from a euthanatized 3-year-old horse.

Procedure

Explants in culture were treated with 0, 100, 250, or 500 ng of IGF-I/ml for 14 days with an end-stage radiolabel of 20 μCi of [3H]proline/ml or 5 μCi of [3H]thymidine/ml. The tendon tissues were then analyzed biochemically for hydroxyproline content by reverse-phase high-performance liquid chromatography, DNA content by fluorometry, and glycosaminoglycan content by the dimethylmethylene blue dye-binding assay. In addition, morphologic analysis of the explants comprised histologic examination, autoradiography, and immunohistochemistry.

Results

Hydroxyproline content was significantly increased in explants treated with 100 and 250 ng of IGF-I/ml. Additionally, the collagen synthetic rate, measured by incorporation of [3H]proline into hydroxyproline, was significantly increased for all treatment groups. On the basis of autoradiograms, fibroblast proliferation and collagen synthesis were predominantly confined to the endcap and adjacent endotenon of the explants. Enhanced immunoreactivity for type-I collagen, compared with type-III collagen, was evident in the treated explants, an observation supported by positive staining for type-I collagen with picrosirius red. Histologically, treated explants contained greater numbers of larger and more metabolically active fibroblasts, compared with untreated controls.

Conclusion

IGF-I enhances collagen synthesis in normal equine flexor tendon in a dose-dependent manner. IGF-I also exerts its primary effect on cell proliferation and collagen synthesis in the epitenon and adjacent endotenon and accompanying perivascular connective tissues, consistent with enhancement of intrinsic tendon metabolism.

Clinical Relevance

IGF-I may have a potential role in the treatment of tendinitis in horses. (Am J Vet Res 1997;58:103–109)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To characterize discrete palmar carpal osteochondral fragmentation in horses and to document the effect of osteoarthritis and surgical removal of these fragments on functional outcome.

Design—Retrospective case series.

Animals—25 horses.

Procedures—Medical records and radiographic views were reviewed to identify horses that had radiographic evidence of palmar carpal fragmentation, which was subsequently treated by arthroscopic removal. Information collected included cause of fracture, initial and long-term clinical and radiographic findings, and functional outcome.

Results—Palmar carpal fragmentation of 30 carpal bones was identified in 25 unilaterally affected horses. A known traumatic event was reported to cause the fragmentation in 17 of the 25 (68%) horses. Of the 25 horses, 17 (68%) had fragmentation involving the antebrachiocarpal joint, 7 (28%) had fragmentation involving the middle carpal joint, and 1 (4%) had fragmentation involving the carpometacarpal joint. The proximal aspect of the radial carpal bone was the most commonly affected site (12/30 fragments), followed by the accessory carpal bone (6/30). Of the 25 horses, 19 (76%) were not lame (sound) after surgery and returned to their intended use, 4 (16%) were considered pasture sound, and 2 were euthanized (because of severe postoperative osteoarthritis or long bone fracture during recovery from anesthesia). Eight of the 14 horses with preoperative evidence of osteoarthritis returned to function after surgery. Twelve of 17 horses with antebrachiocarpal joint fragments and 6 of 7 horses with middle carpal joint fragments returned to their previous use.

Conclusions and Clinical Relevance—Results indicated that the prognosis for horses after arthroscopic removal of palmar carpal osteochondral fragments is good. Early intervention, before the development of osteoarthritis, is recommended.

Full access
in Journal of the American Veterinary Medical Association

Abstract

Objectives—To define a portion of the nucleotide sequences of each of the 6 insulin-like growth factor (IGF) binding proteins (IGFBPs) in horses and describe patterns of messenger RNA (mRNA) and protein expression for IGFBPs in normal equine tendons.

Animals—7 horses.

Procedure—Total RNA was extracted from the tensile region of normal superficial digital flexor tendons and reverse transcribed into complimentary DNA (cDNA). The cDNA was amplified via PCR, and products representing portions of each IGFBP were cloned and sequenced. Nucleotide sequences were used to deduce the amino acid sequences, and both nucleotide and predicted amino acid sequences were compared with those published for bovine, human, mouse, and ovine IGFBPs. Gene expression was quantitated by real-time PCR assay, and protein expression was evaluated by western ligand blot (WLB).

Results—Clones ranged in size from 262 to 522 bp and had high degrees of sequence homology with other mammalian species. Sequence homology was highest between bovine and equine IGFBPs (86% to 95%) and amongst the IGFBP-5 sequences from the various species (92% to 95%). Message for IGFBP-2 to -6, but not IGFBP-1, was expressed in normal tendon. Protein expression for IGFBP-2, -3, and -4 was detected by WLB in normal tendon and markedly increased in damaged tendons.

Conclusions and Clinical Relevance—Results provide basic information and tools needed for further characterization of the role of the IGF system in tendon healing and may lead to the ability to potentiate the response of healing tendon to exogenous IGF-I via concurrent manipulation of IGFBPs. (Am J Vet Res 2005;66:300–306)

Full access
in American Journal of Veterinary Research

Summary

Osteochondral fragments from the axial proximoplantar/proximopalmar region of the proximal phalanx were removed from 38 joints in 30 horses. Ninety-three percent of the horses were Standardbreds, and 28 of the 30 had a low-grade lameness. All but 1 of the horses had hind limb involvement.

A total of 43 fragments were removed. Most (71%) of the fragments involved the medial aspect of the joint and had to be dissected from a covering of synovial tissue. Histologically, the circumference of most fragments consisted of a transition zone at the attachment of the joint capsule, a region of nonarticular, non-weight-bearing cartilage, a region where organized, dense connective tissue, presumably remnants of the short sesamoidean ligament were attached, and a region consisting of irregular truncated bony surfaces covered by mature fibrous tissue, which appeared to be the result of healing of a chronic fracture. There were several areas of degenerate hyaline cartilage, but no areas of normal hyaline cartilage or areas containing retained cartilage cores or other evidence of delayed endochondral ossification. Immunohistochemical staining of 4 segments from 1 horse revealed sensory substance P immunoreactive nerves in the fibrous tissue surrounding the bony fragments and within the central cancellous spaces. The histologic appearance suggests that these osteochondral fragments may be a result of fracture, rather than a manifestation of osteochondrosis.

Free access
in Journal of the American Veterinary Medical Association

SUMMARY

Objective

To develop a high-performance liquid chromatography (HPLC) assay for insulin-like growth factor I (IGF-I) and to use it to quantify elution of IGF-I from polymerized fibrin in an in vitro system.

Sample Population

Equine fibrinogen and calcium-activated bovine thrombin were used to form fibrin containing human recombinant IGF-I.

Procedure

Multiple fibrin disks were formed from polymerized fibrinogen and thrombin; 6 disks were loaded with 25 μg of recombinant human IGF-I at the time of polymerization, and 4 remained as unladen controls. The resultant clots were incubated at 37 C and 90% humidity for 22 days. Phosphate-buffered saline solution in each well was replaced daily, and IGF-I content was assayed by HPLC. Solid-phase separation was used to assay free IGF-I peptide peaks. A scan was done to determine optimal wavelength for IGF-I absorbance. Commercially pure IGF-I was used to construct a standard curve, and the IGF-I content of medium removed from all 10 wells each day was assayed.

Results

Pure unbound IGF-I eluted from the fibrin polymers for 22 days; initial rapid daily release of 1.6 to 1.7 μg of IGF/ml of medium changed after day 3 to commence an asymptotic decrease to 110 ng of IGF/ml by day 22. The fibrin disks had dissolved by day 22, and the experiment was terminated. Control disks did not have detectable IGF-I content at any time. Limit of the HPLC assay was 25 ng of IGF-l/ml. Retention time for nonprotein-bound IGF-I was 10.3 ± 0.15 minutes.

Conclusion

IGF-I (25 μg) can be added to polymerized fibrin and eluted as free ligand over a 22-day period of culture at 37 C. Release of IGF-I was initially independent of fibrin dissolution, but later appeared to follow a pattern consistent with fibrin degradation.

Clinical Relevance

IGF-I can be incorporated as a depot form in polymerized fibrin and is released over time in sufficient concentration to effectively stimulate articular chondrocyte metabolic activity. (Am J Vet Res 1997;58:1431–1435)

Free access
in American Journal of Veterinary Research

SUMMARY

Objective

To clone and determine molecular structure of equine stromelysin 1 (matrix metalloproteinase 3) and examine stromelysin expression in articular cartilage.

Samples and Procedure

Total RNA was harvested from equine arthritic cartilage specimens and was used for reverse transcription and polymerase chain reaction amplification to develop overlapping complementary DNA (cDNA) clones. Four cDNA sequences were ligated into plasmid (pGEM3Z) constructs and subcloned into bacterial expression vectors, and sequence was determined by automated dye terminator sequencing. Stromelysin mRNA expression was assessed in normal and arthritic cartilage and synovium by northern blotting. Interleukin 1 (IL-1) regulation of stromelysin transcriptional activity in articular chondrocytes cultured in the presence of 0, 20, and 50 ng of IL-1α/ml was assessed by northern blotting of total RNA isolated from the cell layer and probed with 32P-labeled stromelysin cDNA.

Results

4 overlapping clones provided the full-length cDNA sequence of equine stromelysin, including portions of untranslated 5′ and 3′ regions, and the entire translated portion coding for the stromelysin prepropeptide. The coding region of 1,431 base pairs was well conserved between species, with 86, 83, and 78% sequence homology to that of human, rabbit, and mouse stromelysin, respectively. Predicted amino-acid (AA) sequence data indicated highly conserved features. Comparison of the equine AA sequence revealed 89, 88, and 84% homology to the AA structure in human, rabbit, and mouse stromelysin, respectively. Minimal stromelysin mRNA expression was evident in normal cartilage and synovium, and increased expression was evident in arthritic cartilage. Marked dose-dependent up-regulation of stromelysin transcriptional activity was evident in chondrocyte cultures exposed to 20 and 50 ng of IL-1/ml.

Conclusions

Stromelysin DNA sequence in horses is similar to that in people and rodents. Constitutive stromelysin message amounts in normal cartilage and synovium are low, but considerably increased in arthritic cartilage and in chondrocytes exposed to IL-1. (Am J Vet Res 1998;59:30–36)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate potential stimulatory or matrixsparing effects of insulin-like growth factor 1 (IGF-1 ), alone or in combination with a corticosteroid, in an interleukin 1 (IL-1)-induced model of cartilage degradation.

Samples

Cartilage from the weightbearing surfaces of trochlea and condyles of clinically normal 2-year-old male horses.

Procedure

Triamcinolone acetonide and IGF-1 effects were evaluated by assessing: matrix responses by sulfated glycosaminoglycan (GAG) assay and [35S]sulfated GAG synthesis; collagen content by hydroxyproline assay; and mitogenic response by [3H]thymidine incorporation into DNA and fluorometric assay of total DNA concentration.

Results

Conditioning of cartilage expiants with 10 ng of human recombinant IL-1α increased degradation and decreased synthesis of matrix proteoglycans (PG), without affecting matrix collagen content. Human recombinant IGF-1 decreased PG loss and reversed the reduction of PG synthesis in cartilage expiants conditioned with IL-1. Given alone, steroids decreased PG concentration and synthetic rate in normal cartilage. However, the previously diminished PG content, attributable to IL-1 conditioning, was not further exacerbated by steroid administration in IL-1-conditioned expiants. Combined treatment of normal cartilage expiants with IGF-1 and steroids resulted in PG preservation and increase in collagen content. Similar PG and collagen effects were not evident when treating IL-1-conditioned cartilage with IGF-1/steroid combinations. Decrease in chondrocyte proliferation was associated with steroid administration. Exposure to IGF and steroids prevented the decrease in mitogenesis that could lead to cellular loss, particularly in IL-1-conditioned expiants.

Conclusion

Combination IGF-1 and steroid treatment of normal cartilage cultures indicated substantial ability to override the anabolic suppression associated with steroids alone. Potentially, administration of corticosteroids, followed by IGF-1, may act to decrease propagation of detrimental mediator release while allowing appreciation of the chondroenhancing effects of IGF-1. These beneficial effects were considerably reduced in IL-1-induced cartilage damage. (Am J Vet Res 1997;58:524–530)

Free access
in American Journal of Veterinary Research

Summary

The genual joint in horses is complex, making synovial fluid aspiration and injection of the femoropatellar joint difficult. Horses commonly have signs of resentment to needle penetration at this site. We compared the safety and efficacy of a new technique, using a lateral approach to the femoropatellar joint, with that of the standard cranial approach in 12 horses. A significantly greater amount of fluid was obtained with the lateral approach (2.0 ± 0.5 ml, mean ± sem) than with the cranial approach (0.9 ± 0.2 ml). Significant differences were not observed in color, nucleated cell count, rbc count, or total protein of the fluid. Mean articular cartilage injury score was significantly lower with the lateral approach (0.3 ± 0.3), compared with that from the cranial approach (1.3 ± 0.4). Only 8% of the joints (1/12) in which the lateral approach was used were injured, compared with 67% (8/12) in the cranial approach. The lateral approach yielded more fluid and was less likely to result in injury to the articular surface than was the cranial approach.

Free access
in Journal of the American Veterinary Medical Association

Summary

Serial sections of bone and soft tissue from the metacarpophalangeal joints of 2 mature and 2 immature horses were evaluated for substance P immunoreactive sensory nerve fibers. Formalin-fixed specimens were sectioned, either nondemineralized or demineralized with formic acid or edta. Rabbit antiserum to substance P (sp) was used in the streptavidin-biotin-peroxidase complex method for immunolocalization of sp antigen, and staining with 3,3′- diaminobenzidine was used for permanent identification of sp fibers. Abundant sensory nerve fibers were identified in the joint capsule, synovial membrane subintimal layers, collateral ligaments, suspensory ligament and distal sesamoidean ligament attachments to the sesamoid bones, and the periarticular periosteal layers. Sparse sp-immunoreactive nerve fibers were found in subchondral bone plates of the metacarpus, proximal first phalanx, and dorsal articular surface of the sesamoid bones. Most sp fibers were associated with blood vessels in the small cancellous spaces and haversian canals of the subchondral bone. The deeper marrow spaces contained increased numbers of sp sensory fibers; a few appeared in small groups and as several sp-immunoreactive fibers in a larger nerve. Cortical bone contained only a few sp fibers in the haversian canals. Substance P fibers were not identified in the osteocytic lacunae, canaliculi, or the bony lamellae of the haversian systems of the subchondral bone plate, and its extension to the metaphyseal and diaphyseal cortical bone. Equine metacarpophalangeal joint soft tissues have an abundant sensory nerve supply, similar to that of other species. However, the subchondral bone plate also has sparse sensory nerve fibers, which is a unique finding, and may help explain signs of bone pain associated with disease states of the fetlock.

Free access
in American Journal of Veterinary Research
in Journal of the American Veterinary Medical Association