Investigating biomarkers of meniscal injury in canine stifle joint synovial fluid

11 April 2023

Investigating biomarkers of meniscal injury in canine stifle joint synovial fluid

In 2020, Professor Mandy Peffers, together with Professor Eithne Comerford and Dr Marie Phelan, from the University of Liverpool, received BSAVA PetSavers funding for a master’s degree by research project entitled Determining predictive metabolomic biomarkers for meniscal injuries in dogs with cranial-cruciate ligament rupture using stifle joint synovial fluid. Student Christine Pye describes the study and its findings.

The menisci are a pair of C-shaped fibrocartilaginous structures within the stifle joint, situated on the peripheral weight-bearing aspects of the proximal tibia between the tibial and femoral condyles. They have several important functions in the stifle joint, including being involved in load bearing, load distribution and shock absorption, as well as contributing to joint stability, proprioception and joint lubrication. Injuries to the menisci in dogs typically occur as a consequence of instability in the stifle joint secondary to cranial cruciate ligament rupture (CCLR). The reported prevalence of meniscal injuries at the time of surgery for CCLR varies from 20% to 77% depending on the study. Importantly, meniscal injuries can occur postoperatively, weeks or months after surgery, due to residual instability in the joint or misdiagnosis of meniscal tears at the time of surgery. These late meniscal injuries can cause ongoing pain and lameness in the affected joint and have a reported incidence of approximately 3% to 15% of dogs after CCLR surgery.

Currently, diagnosis of late meniscal injuries can be challenging. Diagnostic imaging techniques such as computed tomography with arthrography, ultrasonography, and magnetic resonance imaging are available, but can lack sensitivity and require either expensive specialized equipment and/or advanced technical expertise. Surgical diagnosis by means of arthroscopy and arthrotomy (Figure 1) are more commonly used for diagnosis and subsequent treatment of late meniscal injuries. However, these surgical diagnostic techniques are more invasive than imaging and have inherent risks, including the safety implications of the animal going through a potentially unnecessary surgical procedure if no meniscal injury is found.

What if there was a way of diagnosing meniscal injuries in dogs by the means of a simple joint tap? If biomarkers of meniscal injury could be found within the stifle joint synovial fluid of dogs with CCLR, then this could ultimately lead to the development of a simple, inexpensive, minimally invasive test, one that is more sensitive than current non-surgical diagnostic techniques.

What interested you in getting involved in this project?

Like many other veterinary surgeons, I have come across the dilemma faced when a dog that seems to be doing well post-operatively after surgery for CCLR suddenly becomes lame again on the operated limb. After ruling out infection and implant problems, it is common for veterinary surgeons to be left considering whether this could be a case of a late meniscal injury.

I have always thought of veterinary research as being an important aspect of our profession, as only with good research can we gain the evidence to improve treatments and diagnostic techniques, and potentially help provide better outcomes for animals in our care and their owners. Having the opportunity to help to find solutions for this problem was something that I was eager to be involved with. It was the fact that the outcomes of this research could ultimately lead to a diagnostic test for meniscal injuries by means of a joint tap, therefore helping veterinary surgeons in practice, that really excited me about the project.

What were the aims of the project?

The aims of the project were to collect samples of stifle joint synovial fluid from dogs undergoing stifle joint surgery for either CCLR (with or without meniscal injuries) or medial patella luxation. These samples then underwent a technique called nuclear magnetic resonance (NMR) spectroscopy, which is a method of analysing small molecule metabolites within a biological sample. Statistical and mathematical analysis was then undertaken to analyse the differences in metabolites between those dogs with and without meniscal injuries, to ultimately determine whether any metabolites significantly changed depending on the meniscal injury status of the dog. Any metabolite differences could lead to the development of a minimally invasive, simple, inexpensive diagnostic test for meniscal injuries in dogs.

What were the biggest challenges and obstacles of the project?

One of the biggest obstacles faced was collecting control samples from dogs without either CCLR or meniscal injuries. Due to ethical reasons, we were unable to collect synovial fluid from completely healthy stifle joints, and so decided to collect samples from dogs undergoing surgery for other stifle joint disorders, such as medial patella luxation. As those dogs that are undergoing surgery for patella luxation tend to be smaller breed, younger dogs, the signalment of this group differed from the dogs with CCLR, limiting our ability to also investigate biomarkers of CCLR. Another challenge was the number of samples we needed to collect. Sample size calculations based on a previous small cohort study identified that at least 60 samples for each group were required. Ultimately, we managed to include 65 samples from dogs with CCLR and no meniscal injury, 72 samples from dogs with CCLR and a meniscal injury, and 17 control samples. It was thanks to our collaborating veterinary surgeons and veterinary nurses in this study that we were able to collect so many samples in a limited time window.

What were your main findings?

Using NMR spectroscopy, we found that areas on the spectra related to lipid resonances termed ‘mobile lipids’ were significantly higher in the group with meniscal injury compared to those without meniscal injury. Mobile lipids are NMR lipid resonances that arise from molecules such as methyl and methylene resonances belonging to lipid acyl chains. These are primarily from triglycerides, fatty acids and cholesteryl esters in lipid droplets, and also from phospholipidic acyl chains if not embedded in lipid membrane bilayers. Lipids serve various important functions in biological systems, including being components of cell membranes and other cellular organelles, acting as an energy source, and having a crucial role in signalling and regulation of cellular processes. An increase in lipids in synovial fluid of dogs with meniscal injury could potentially be due to damage to cellular phospholipid membranes, release of lipids from cell necrosis or apoptosis, or related to inflammatory changes or joint lubrication. We aim to publish these findings in the near future in a peer reviewed journal.

What are the next stages for this research?

Further work to identify the specific lipid species involved could lead to the discovery of biomarkers of meniscal injury in dogs and potentially be translatable to other species, such as humans.

What advice would you give to someone thinking of applying for a BSAVA PetSavers research grant?

For me personally, the funding from BSAVA PetSavers gave me the opportunity to take a step away from clinical practice and fulfil my ambition to be involved in veterinary research. Having completed my PetSavers-funded MPhil, I have now gone on to stay in academia undertaking a PhD. For the project as a whole, this work would not have been possible without the funding received from BSAVA PetSavers. I would whole-heartedly recommend to any veterinary surgeon interested in research to consider applying for a BSAVA PetSavers research grant.

About the author

Christine Pye graduated from the University of Bristol Veterinary School in 2011, having also completed an intercalated degree in Veterinary Conservation Medicine at the University of Liverpool. After 10 years in small animal clinical practice, including completing a Postgraduate Certificate in Small Animal Surgery, she joined the University of Liverpool to undertake an MPhil investigating biomarkers of meniscal injury in dogs, funded by BSAVA PetSavers. She is currently continuing her work in the field of biomarker analysis as a PhD student within the Department of Musculoskeletal Biology and Ageing Science at the University of Liverpool.