Tracking Stem Cells in Lower Legs After Injection

Tracking Stem Cells in Lower Legs After Injection

A stem cell injection using regional limb perfusion.

Photo: Mathieu Spriet, DVM, MS, Dipl. ACVR, ECVDI/UC Davis

Whether it's an intravenous medication, a corticosteroid shot, or a vaccination, injections are commonplace in equine medicine. Stem cells, the key component in a popular new type of regenerative therapy, are also administered via injection. But where do the cells go once they've left the syringe and entered the horse's body? And do they stay where they need to stay, or go where they need to go?

Mathieu Spriet, DVM, MS, Dipl. ACVR, ECVDI, assistant professor in the Department of Surgical and Radiological Sciences at the University of California (UC), Davis, School of Veterinary Medicine, and a team of researchers recently set out to find an easy, noninvasive method of tracking stem cells injected into a horse's lower leg, and to see if different methods of administration had a different effect on where the cells travel once administered. He presented the findings at the North American Veterinary Regenerative Medicine Conference, held June 2-4 in Lexington, Ky.

"Basically, we wanted to know where the cells are going, whether they stay (where they were injected), and how many of them stay there; Ideally we want the cells to ... where the lesion is and stay there," Spriet explained.

Stem cells have been tracked using a material called green fluorescent protein; however, this technique requires a terminal study (i.e., the test horses are euthanized during the process of the study) to observe the results. Spriet et al. wanted to see if using a different material and method--a radioactive mixture of  the tracing compounds hexamethylpropyleneamine oxime (HMPAO) and Technetium-99m (TcHMPAO) introduced within the stem cells and tracked using a gamma camera (scintigraphic imaging)--would allow for in vivo (in the live horse) stem cell tracking. The TcHMPAO material and aforementioned method had previously been proven effective in rodents, but had never been tested in horses. If successful, "the technique is a noninvasive approach and allows multiple assessments over time for up to 24 hours after cell injections," Spriet noted.

The team designed an experiment to test TcHMPAO's efficacy and to compare the length of time the stem cells remained in the lower (distal) limb after either intravenous (IV, in the vein) or intra-arterial (IA, in the artery) injection, both making use of regional limb perfusion (a procedure in which a veterinarian places a tourniquet on the limb and injects the stem cells below the tourniquet, isolating the blood supply to the distal limb for a brief period).

"Most commonly, for tendon lesions, cells are injected directly into the lesions," Spriet noted. "The problems with direct intralesional injection is that it might be difficult to reach the lesion with a needle in some cases (lesions within the hoof, for example), it might requires multiples injections at different site (if the lesion is large), and the needle itself might cause damage to the tendon. This is why we are looking for alternative ways of administration, and this is where the regional limb perfusion (RLP) comes into play."

Spriet and colleagues employed six healthy adult horses with no musculoskeletal lesions.

Researchers applied the tourniquet to the horse's leg just before the stem cells were injected either in the cephalic vein or the median artery (both of which run along the horse's inner forearm) and left it in place for 45 minutes. Scintigraphic images (the technology used in bone scans, however in this case used to track stem cells rather than produce images of bones) were taken at the time of injection and again after 45 minutes, 75 minutes, six hours, and 24 hours. Spriet and his team measured the percent persistence of the stem cells (the amount of radioactivity of the stem cells in the limb at the time of imaging compared with the stem cell radioactivity activity immediately after injection; the higher the persistence, the easier the cells are to track via gamma camera) from each scintigraphic image. 

"We wanted to know if this would be an appropriate way to get the cells to reach a lesion that would be in this area," he added. "We were looking for good diffusion in the distal limb, but we didn't want diffusion in the entire body as we want the cells to remain concentrated in the area of interest."

Key findings of the study included:

  • Scintigraphic assessment using TcHMPAO was effective in comparing different methods of stem cell administration;
  • 100% persistence was noted with both techniques when the tourniquet was on the limb;
  • Six hours after injection, the persistence was 39% and 28% for the IA and IV techniques respectively;
  • The IA regional limb perfusion technique had good stem cell distribution in the distal limb in all six horses; and
  • The IV regional limb perfusion had poor or no diffusion to the pastern and hoof area in three of six horses.

"Using this technique we have demonstrated that both IA and IV regional limb perfusions lead to persistence of stem cells in the equine distal limb," Spriet concluded. "A difference in the distribution of cells exists between the two techniques; the IA regional limb perfusion is more reliable for diffuse distribution throughout the distal limb."

Spriet concluded that TcHMPAO is an effective method for tracking stem cells in the lower leg, and that RLP might be used as an alternative to intralesional injection in scenarios where stem cells are needed in the lower limbs.

He also added that "more work is still needed in order to further assess the safety of the procedure before broad clinical use." During the study one horse developed lameness and skin lesions after injections, and Spriet and his team are currently working to improve the technique and try to ensure the adverse reactions are minimal to nonexistent.

About the Author

Erica Larson, News Editor

Erica Larson, news editor, holds a degree in journalism with an external specialty in equine science from Michigan State University in East Lansing. A Massachusetts native, she grew up in the saddle and has dabbled in a variety of disciplines including foxhunting, saddle seat, and mounted games. Currently, Erica competes in eventing with her OTTB, Dorado.

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