Review of Vascular Administration of Mesenchymal Stem Cells in the Equine Distal Limb

Intravenous regional limb perfusions result in heterogeneous and asymmetric distribution of stem cells. Furthermore, serious limitations related to poor foot perfusion and tourniquet failure are identified in standing horses. Intra-arterial injections should not be performed with a tourniquet due to a risk of arterial thrombosis. Intra-arterial injections without a tourniquet lead to homogeneous and diffuse distribution and are considered the preferred technique. Although more challenging, intra-arterial injections can be performed in the standing horse. Authors’ address: School of Veterinary Medicine, University of California, Davis, One Shields Ave, Davis, CA 95616; e-mail: mspriet@ucdavis.edu.

1. Introduction

Vascular administration of mesenchymal stem cells (MSCs) to the equine distal limb has been proposed as an alternative to direct injection into lesions. The main advantages of such techniques are the treatment of lesions that cannot be reached directly with a needle (for example, lesions within the hoof) or diffuse lesions that would require multiple injection sites. Another advantage of the technique is to avoid iatrogenic damage created by needles in the structures adjacent to lesions. Both intravenous and intra-arterial injection techniques have been used in laboratory animals^1,2  and in human patients.³  The intra-arterial technique seems beneficial regarding the perfusion of peripheral tissues.^1–3  In the horse, techniques of vascular administration can be subdivided according to the type of vessel used (vein or artery), the exact site of injection, and the use or not of a tourniquet. The outcome might also be affected by the use of standing sedation or general anesthesia. 

The use of an intracellular radioactive label (^99mTc-HMPAO) allows scintigraphic tracking of the MSCs after injection. Different injection techniques have recently been evaluated with scintigraphy. The goal of this review is to summarize the pros and cons of each technique and issue a recommendation regarding the clinical use of vascular administration of MSCs. The different techniques that have been investigated include cephalic vein and palmar digital vein regional limb perfusion (RLP) and median artery injection. The arterial injections have been assessed both with and without the use of a tourniquet. All techniques have been performed both in anesthetized and in standing horses.
 

2. Cephalic Vein Regional Limb Perfusion in Anesthetized Horses

Cephalic vein RLP under general anesthesia was the first technique investigated.^4,5 A pneumatic tourniquet inflated to 400 to 500 mm Hg was placed on the forearm just proximal to the cephalic vein catheter for 30 min. Twenty-five to 45 million radiolabeled MSCs suspended in 2 mL of saline were injected and flushed with 20 mL of saline. All of the radiolabeled MSCs remained in the limb while the tourniquet was in place and no major loss of radioactive signal was observed immediately after removal of the tourniquet. Only 3 of the 6 injected limbs had radioactive signal in the entire distal limb, whereas the other 3 limbs only had uptake in the carpal and proximal metacarpal areas.⁴ The maximal signal intensity was mostly in the area of the larger vessels (Fig. 1A). In horses with induced lesions in the superficial digital flexor tendon, the distribution and quantification of the radioactive signal was not different from the control horses.⁵

3. Lateral Palmar Digital Vein Regional Limb Perfusion in Anesthetized Horses

The lateral palmar digital vein RLP was assessed in a later study with the main objective to improve distribution of MSCs to the foot when compared with cephalic vein RLP.⁶ A pneumatic tourniquet inflated at 450 mm Hg was placed for 30 min in the metacarpal region, proximal to the injection site. Approximately 35 million MSCs suspended in 10 mL of saline and flushed with an additional 10 mL of saline were injected. Similar to the cephalic vein RLP in the anesthetized patient, the tourniquet effectively retained all radioactivity within the limb. The radioactive signal was present within the foot in all 6 injected limbs; however, a marked asymmetry was identified with low to absent radioactive signal at the medial aspect of the foot and pastern. Most of the radioactive signal was located in the area of the lateral palmar digital vein and coronary band vascular plexus (Fig. 1C).⁶

4. Cephalic Vein Regional Limb Perfusion in Standing Sedated Horses

For this study, horses were sedated with 0.01 mg/kg detomidine IV and 0.01 mg/kg butorphanol IV and a pneumatic tourniquet inflated at 225 mm Hg was placed on the forearm proximal to the injection site for 30 min.⁷ Approximately 40 million radiolabeled MSCs suspended in 2 mL of saline were injected and flushed with 20 mL of saline. Only 3 of 6 horses showed a radioactive signal distal to the proximal metacarpus (Fig. 1B). The quantification of the signal revealed a much lower uptake than on the initial study under general anesthesia, suggesting failure of the tourniquet.

5. Lateral Palmar Digital Vein Regional Limb Perfusion in Standing Sedated Horses

Horses were sedated similarly as for the cephalic vein RLP study.⁷ A pneumatic tourniquet inflated to 450 mm Hg was placed on the metacarpus for 20 min. Forty million radiolabeled MSCs in 10 mL of saline and flushed with an additional 10 mL of saline were used.⁷ Despite all horses tolerating the tourniquet well, very little radioactive signal was present distal to the injection site in 3 of the horses. The quantification of the signal revealed a wide range of uptake. The dynamic acquisition at the time of injection demonstrated failure of the tourniquet with the radioactive signal proximal to the tourniquet. In the 3 horses with a better distal radioactive signal, uptake within the hoof was lower than in the anesthetized horses, suggesting poor perfusion related to the weight-bearing position (Fig. 1D).⁷ [...]