Researchers are always looking for efficient and accurate ways to prevent catastrophic injuries in Thoroughbred racehorses. A team from the University of Kentucky recently completed a 2.5-year project measuring messenger RNA (mRNA) and identified three markers veterinarians might be able to use to identify at-risk horses before they race.

Allen Page, DVM, PhD, staff scientist and veterinarian at the university’s Gluck Equine Research Center, in Lexington, presented their findings at the 2020 American Association of Equine Practitioners virtual convention.

Understanding Gene Expression

Page’s team used gene expression analysis to identify and measure mRNA biomarkers in blood samples. “Gene expression analysis essentially means we are measuring differences in whole-blood mRNA gene copies—genes that are protein encoding,” he explained. “DNA is transcribed into mRNA, which is then translated by ribosomes into proteins.”

While several research groups have looked at protein changes to identify inflammation in horses, “we opted to go upstream of that and look at mRNA expression,” said Page, citing two reasons:

• Quantitative PCR analysis of mRNA expression is highly sensitive and specific (meaning it offers few false negatives and positives) for the markers they wanted to evaluate.
• To avoid supply chain issues with equine-specific protein quantification. “Horses are not high-priority targets for companies that develop protein quantification reagents, and as a result we’re limited in the number of proteins we can quantify from samples from horses,” Page explained. “But that’s not a problem we have with mRNA analysis.”

Exercise and Inflammation

Researchers have shown that mRNA expression of pro-inflammatory cytokines (proteins) increases in horses following exercise.

While some exercise-induced inflammation is natural and necessary for tissue healing and repair, said Page, exaggerated inflammatory responses could be associated with injuries.

“We know that most catastrophic injuries in racing occur in limbs with pre-existing pathology (disease or damage),” he said. “This pathology might stimulate the production of specific biomarkers/proteins or a nonspecific inflammatory response that could be the target of diagnostic testing.”

So, in their study, his team hypothesized that Thoroughbred racehorses that experience catastrophic injuries while racing would have evidence of increased inflammation when compared to noninjured controls.

They collected blood samples from September 2017 and May 2020 from 904 horses, including:

• 107 horses that experienced catastrophic injuries;
• 205 horses from the same races as catastrophically injured horses, as post-race controls;
• 374 random pre-race controls; and
• 218 horses they ended up excluding (e.g., catastrophic injuries due to clipped heels or noninjured controls that did not race again within 90 days of their sample collection).

The researchers conducted mRNA analysis on each sample for 21 genes known to play roles in inflammation, bone repair/remodeling, tissue repair, and/or injury response.

Study Results

After comparing blood samples, the researchers identified three promising mRNA markers for identifying horses at risk of catastrophic injury:

• Insulin-like growth factor 1 (IGF-1), a growth hormone that plays a role in bone development and repair. “IGF-1 has previously been shown to decrease during early inflammation before increasing, suggesting that increased IGF-1 expression might indicate chronic inflammation, particularly in regard to bone and joint pathology,” said Page.
• Matrix metalloproteinase-2 (MMP2), which plays an important role in tissue repair and fracture remodeling, also increased in horses with catastrophic injuries.
• Interleukin-1 receptor antagonist, a potent anti-inflammatory also known as IRAP, decreased in horses with catastrophic injuries, “again suggesting these horses had evidence of ongoing chronic inflammation,” Page said. “Those horses that had increased IRAP expression were seven times less likely to experience a catastrophic injury.”

“The mRNA expression of IGF-1, IRAP, and MMP2 may provide economical, effective, and noninvasive means of detecting horses at risk of catastrophic injury,” he concluded.

Future Applications

Page suggested veterinarians could use mRNA markers as a pre-race screening tool, collecting and testing blood samples three to five days before a race to identify horses that should be examined further with advanced imaging modalities. “We can run hundreds of samples per day and return those results within two to three days,” he said.

He added that his team is currently RNA-sequencing samples to identify even more markers of interest and is in the process of determining the effects of pre-race medications on gene expression. The team’s goal is to collect 10,000 pre-race samples to validate their findings.