Epsilon Archive for Student Projects

Abstract

Osteoartrit (OA) är ett mycket vanligt problem hos hästar och har i många fall sitt ursprung från felaktig belastning av leden, blir ofta kroniska och omfattar inflammation i ledens samtliga ingående delar. Resultatet är bland annat nedbrytning av ledbrosk och matrixkomponeneter. Idag baseras diagnostik och framtida prognos mestadels på den kliniska bilden och/eller bilddiagnostiska hjälpmedel.
I denna studie har två inflammationsmediatorer; interleukin 1β (IL-1β), high mobility group box protein 1 (HMGB-1), och ett enzym; matrixmetallopeptidas 13 (MMP-13) mätts i ledvätska och serum med hjälp av kommersiell enzyme-linked immunosorbent assay (ELISA)-teknik. Hypotesen är att dessa är förhöjda i ledvätska från leder med konstaterad hälta.
Tjugonio ledvätskeprov och 11 serumprov från 11 hästar som sökt till Universitetsdjursjukhuset i Uppsala för hälta analyserades i denna studie. I samband med diagnostisk ledbedövning samlades ledvätska och blodprov, och utifrån veterinärbedömning samt undersökning med det objektiva rörelseanalyssystemet Lameness Locator™ (LL) bedömdes leden som positiv eller negativ för hälta. IL-1β fanns i detekterbara nivåer i 2 av 29 ledvätskeprov samt i 7 av 11 serumprov. De hästarna med högst serumnivåer hade även förhöjda nivåer i ledvätska. HMGB-1 var detekterbart i 17 ledvätskeprov och ansågs förhöjt i fem av dessa. HMGB-1 var detekterbart i 2 av 11 serumprov. MMP-13 kunde ej analyseras tillfredsställande med den teknik som fanns tillgänglig.
Underlaget var litet och heterogent, och därför svårt att analysera statistiskt. De hästar med leder som bedömts positiva för hälta visade en högre genomsnittskoncentration av HMGB-1 (11,1ng/ml) jämfört med negativa leder (4,5ng/ml). Denna skillnad var ej statistiskt signifikant. De två leder med detekterbara nivåer av IL-1β tillhörde gruppen positiva leder (n=10). En statistiskt signifikant korrelation mellan HMGB-1 och leukocytantal i ledvätska kunde ses (p=0,02). Mycket forskning kvarstår innan man kan använda dessa biomarkörer kliniskt för diagnostik och prognos för OA på häst. Analysmetoderna behöver utvecklas och det saknas än så länge kunskap om normalnivåer hos friska hästar och hur dessa markörer förändras med tid och grad av OA. Det insamlade materialet kommer att tillföras en påbörjad biobank av ledvätskor och serum från kliniskt halta hästar som undersökts med LL. Denna biobank som nu innehåller prover från ca 50 hästar, kommer att ligga till grund för fortsatta studier av tänkbara biomarkörer för OA på häst.

Osteoarthritis (OA) with subsequent lameness is a common problem among horses. Inflammation of the joint, often initialized by abnormalities in joint workload, tends to become chronic and includes the tissues of the entire joint (articular cartilage, subchondral bone and synovial membrane) and its adjacent structures (ligaments and synovial capsule). The joint inflammation results in degradation of articular cartilage with chondrocyte necrosis and fragmentation of its matrix components. Today, diagnosis and prognosis of OA in horses is mostly based upon clinical and/or radiological findings.
In the presented study, the presence of two inflammatory mediators; interleukin 1β (IL-1β), high mobility group box protein 1 (HMGB-1), and one enzyme; matrix metallopeptidase 13 (MMP-13) in synovial fluid and serum have been analyzed using commercial enzyme-linked immunosorbent assay (ELISA) technology. The hypothesis being that these substances are elavated in synovial fluid in joints with clinical lameness.
Twenty-nine synovial samples and 11 serum samples were collected from 11 horses. The horses were presented with lameness at the horse clinic Universitetsdjursjukhuset in Uppsala. Samples of synovial fluid and blood samples were collected during diagnostic intra-articular anesthesia. Based on subjective veterinary examination and objective examination with the lameness evaluation system Lameness Locator™ (LL) the joint was determined as being positive or negative regarding lameness. Detectable levels of IL-1β were present in 2 out of 29 synovial fluid samples and in 7 out of 11 serum samples. The horses with highest serum-levels of IL-1β also had elevated synovial fluid levels. HMGB-1 was detectable in in 17 out of 27 synovial samples and considered elevated in five of those. HMGB-1 was detected in 2 out of 11 serum samples. MMP-13 could not be analyzed in synovial fluid or serum with the methods available.
The number of horses and samples included in the presented study were few and heterogeneous, which made it difficult to analyze the results statistically. Samples from joints that were determined positive regarding lameness, had higher concentrations of HMGB-1 (mean = 11.1 ng/ml) than negative (non-lame) joints (mean = 4.5 ng/ml), but the difference was not statistically significant. The two joints that had detectable levels of IL-1β, were found in the 10 joints determined as positive (lame). The joints that had elevated levels of IL-1β, were also determined as positive (lame) joints. A statistically significant correlation could be seen between HMGB-1 and leukocyte count in synovial fluid (p=0,02). Additional research is still needed before any of these substances can be used as clinically valid biomarkers. More knowledge about normal variations and how these substances vary with duration and severity of OA is required, and the biochemical analyzes needs further development. The synovial fluids and sera, sampled in this study, will be included in a bio bank, where material from lame horses, evaluated by the LL system is stored. Samples from this bio bank will be used in the future research for evaluation of potential biomarkers in equine OA.SUMMARY
Osteoarthritis (OA) with subsequent lameness is a common problem among horses. Inflammation of the joint, often initialized by abnormalities in joint workload, tends to become chronic and includes the tissues of the entire joint (articular cartilage, subchondral bone and synovial membrane) and its adjacent structures (ligaments and synovial capsule). The joint inflammation results in degradation of articular cartilage with chondrocyte necrosis and fragmentation of its matrix components. Today, diagnosis and prognosis of OA in horses is mostly based upon clinical and/or radiological findings.
In the presented study, the presence of two inflammatory mediators; interleukin 1β (IL-1β), high mobility group box protein 1 (HMGB-1), and one enzyme; matrix metallopeptidase 13 (MMP-13) in synovial fluid and serum have been analyzed using commercial enzyme-linked immunosorbent assay (ELISA) technology. The hypothesis being that these substances are elavated in synovial fluid in joints with clinical lameness.
Twenty-nine synovial samples and 11 serum samples were collected from 11 horses. The horses were presented with lameness at the horse clinic Universitetsdjursjukhuset in Uppsala. Samples of synovial fluid and blood samples were collected during diagnostic intra-articular anesthesia. Based on subjective veterinary examination and objective examination with the lameness evaluation system Lameness Locator™ (LL) the joint was determined as being positive or negative regarding lameness. Detectable levels of IL-1β were present in 2 out of 29 synovial fluid samples and in 7 out of 11 serum samples. The horses with highest serum-levels of IL-1β also had elevated synovial fluid levels. HMGB-1 was detectable in in 17 out of 27 synovial samples and considered elevated in five of those. HMGB-1 was detected in 2 out of 11 serum samples. MMP-13 could not be analyzed in synovial fluid or serum with the methods available.
The number of horses and samples included in the presented study were few and heterogeneous, which made it difficult to analyze the results statistically. Samples from joints that were determined positive regarding lameness, had higher concentrations of HMGB-1 (mean = 11.1 ng/ml) than negative (non-lame) joints (mean = 4.5 ng/ml), but the difference was not statistically significant. The two joints that had detectable levels of IL-1β, were found in the 10 joints determined as positive (lame). The joints that had elevated levels of IL-1β, were also determined as positive (lame) joints. A statistically significant correlation could be seen between HMGB-1 and leukocyte count in synovial fluid (p=0,02). Additional research is still needed before any of these substances can be used as clinically valid biomarkers. More knowledge about normal variations and how these substances vary with duration and severity of OA is required, and the biochemical analyzes needs further development. The synovial fluids and sera, sampled in this study, will be included in a bio bank, where material from lame horses, evaluated by the LL system is stored. Samples from this bio bank will be used in the future research for evaluation of potential biomarkers in equine OA.