Mathieu Spriet

Scintigraphic evaluation of intra‐arterial and intravenous regional limb perfusion of allogeneic bone marrow‐derived mesenchymal stem cells in the normal equine distal limb using 99mTc‐HMPAO

REASONS FOR PERFORMING STUDY Mesenchymal stem cells (MSCs) are commonly injected intralesionally for treatment of soft tissue injuries in the horse. Alternative routes of administration would be beneficial for treatment of lesions that cannot be accessed directly or to limit needle-induced iatrogenic damage to the surrounding tissue. OBJECTIVES The purpose of our study was to evaluate MSC distribution after intra-arterial (IA) and intravenous (IV) regional limb perfusions (RLP) using scintigraphy. We hypothesised that MSCs would persist in the distal limb after tourniquet removal and that both techniques would lead to diffuse MSC distribution. METHODS Six horses were used in the study. MSCs were labelled with hexamethyl propylene amine oxime (HMPAO) and technetium-99m. RLP was performed through the median artery of one forelimb and the cephalic vein of the opposite limb under general anaesthesia. The tourniquet was left in place for 45 min. Scintigraphic images were obtained at 0, 45, 75 min, 6 h and 24 h post injection. RESULTS Distribution of labelled MSCs through the entire distal limb was achieved with all 6 IA RLP, but 3 out of 6 IV RLP showed poor or absent uptake distal to the metacarpus. Mesenchymal stem cell persistence was 39% (30-60%) and 28% (14-50%) (median [minimum-maximum]) at 6 h for IA and IV RLP, respectively. Severe arterial thrombosis occurred in one horse after IA RLP. CONCLUSIONS Both IA and IV RLP of the distal limb result in MSC persistence in perfused tissues. The IA perfusion resulted in more reliable cell distribution to the pastern and foot area. POTENTIAL RELEVANCE Regional limb perfusion of MSCs might be used in cases where intralesional injection is not possible or in order to avoid iatrogenic needle damage. Further work is needed to assess the safety of IA RLP before its clinical use.

Distribution and persistence of technetium‐99 hexamethyl propylene amine oxime‐labelled bone marrow‐derived mesenchymal stem cells in experimentally induced tendon lesions after intratendinous injection and regional perfusion of the equine distal limb

REASONS FOR PERFORMING STUDY Intralesional (i.l.) injection is currently the most commonly used technique for stem cell therapy in equine tendon injury. A comparison of different techniques of injection of mesenchymal stem cells for the treatment of tendon lesions is required. OBJECTIVES We hypothesised that vascular perfusion of the equine distal limb with mesenchymal stem cells (MSCs) would result in preferential distribution of MSCs to acute tendon injuries. STUDY DESIGN In vivo experimental study. METHODS Lesions were surgically induced in forelimb superficial digital flexor tendons of 8 horses. Three or 10 days after lesion induction, technetium-99 hexamethyl propylene amine oxime-labelled MSCs were injected via i.v. or intra-arterial (i.a.) regional limb perfusion (RLP) at the level of the distal antebrachium and compared to i.l. injection. Mesenchymal stem cell persistence and distribution within the forelimb and tendon lesions was assessed with scintigraphy for 24 h. RESULTS Lesion uptake was higher with i.l. injection than with RLP, but MSC persistence decreased similarly over time in all 3 techniques. Intra-arterial RLP resulted in a better distribution of MSCs and a higher uptake at the lesion site than i.v. RLP. Limbs perfused i.a. on Day 10 showed greater accumulation of MSCs in the lesion than limbs perfused on Day 3. Arterial thrombosis occurred in 50% of the i.v. RLP limbs and in 100% of the i.a. RLP limbs, which led to clinical complications in one horse. CONCLUSIONS AND POTENTIAL RELEVANCE Compared with i.l. injection, RLP results in lower uptake but similar persistence of MSCs at the site of tendon lesions. A time dependent accumulation of MSCs was identified with i.a. RLP. The i.a. RLP appears more advantageous than the i.v. RLP in terms of distribution and uptake. However, the described i.a. technique produced arterial thrombosis and thus cannot currently be recommended for clinical use.

Comparisons of computed tomography, contrast-enhanced computed tomography and standing low-field magnetic resonance imaging in horses with lameness localised to the foot. Part 2: Lesion identification

REASONS FOR PERFORMING STUDY No previous study compares computed tomography (CT), contrast-enhanced computed tomography (CECT) and standing low-field magnetic resonance imaging (LFMRI) to detect lesions in horses with lameness localised to the foot. This study will help clinicians understand the limitations of these techniques. OBJECTIVES To determine if CT, CECT and LFMRI would identify lesions within the distal limb and document discrepancies with lesion distribution and lesion classification. METHODS Lesions in specific structures identified on CT and MR images of feet (31 limbs) from the same horse were reviewed and compared. Distributions of lesions were compared using a Chi-squared test and techniques analysed using the paired marginal homogeneity test for concordance. RESULTS Lesions of the deep digital flexor tendon (DDFT) were most common and CT/CECT identified more lesions than LFMRI. Deep digital flexor tendon lesions seen on LFMRI only were frequently distal to the proximal extent of the distal sesamoid and DDFT lesions seen on CT/CECT only were frequently proximal to the distal sesamoid. Lesions identified on LFMRI only were core (23.3%) or splits (43.3%), whereas lesions identified only on CT were abrasions (29.8%), core (15.8%), enlargement (15.8%) or mineralisation (12.3%). Contrast-enhanced CT improved lesion identification at the DDFT insertion compared to CT and resulted in distal sesamoidean impar ligament and collateral sesamoidean ligament vascular enhancement in 75% of cases. Low-field MRI and CT/CECT failed to identify soft tissue mineralisation and bone oedema, respectively. CONCLUSIONS AND POTENTIAL RELEVANCE Multiple lesions are detected with CT, CECT and LFMRI but there is variability in lesion detection and classification. LFMRI centred only on the podotrochlear apparatus may fail to identify lesions of the pastern or soft tissue mineralisation. Computed tomography may fail to identify DDFT lesions distal to the proximal border of the distal sesamoid.

Influence of the position of the foot on MRI signal in the deep digital flexor tendon and collateral ligaments of the distal interphalangeal joint in the standing horse.

REASONS FOR PERFORMING STUDY Hyperintense signal is sometimes observed in ligaments and tendons of the equine foot on standing magnetic resonance examination without associated changes in size and shape. In such cases, the presence of a true lesion or an artifact should be considered. A change in position of a ligament or tendon relative to the magnetic field can induce increased signal intensity due to the magic angle effect. OBJECTIVES To assess if positional rotation of the foot in the solar plane could be responsible for artifactual changes in signal intensity in the collateral ligaments of the distal interphalangeal joint and in the deep digital flexor tendon. METHODS Six isolated equine feet were imaged with a standing equine magnetic resonance system in 9 different positions with different degrees of rotation in the solar plane. RESULTS Rotation of the limb induced a linear hyperintense signal on all feet at the palmar aspect of one of the lobes of the deep digital flexor tendon and at the dorsal aspect of the other lobe. Changes in signal intensity in the collateral ligaments of the distal interphalangeal joint occurred with rotation of the limb only in those feet where mediolateral hoof imbalance was present. CONCLUSIONS The position and conformation of the foot influence the signal intensity in the deep digital flexor tendon and in the collateral ligaments of the distal interphalangeal joint. POTENTIAL RELEVANCE The significance of increased signal intensity in the deep digital flexor tendon and in the collateral ligaments of the distal interphalangeal joint should be interpreted with regard to the position and the conformation of the foot.

Scintigraphic comparison of intra‐arterial injection and distal intravenous regional limb perfusion for administration of mesenchymal stem cells to the equine foot

REASONS FOR PERFORMING STUDY Intra-arterial (i.a.) and intravenous (i.v.) regional limb perfusions (RLP) through the median artery and cephalic vein, respectively, have been previously investigated for administration of mesenchymal stem cells (MSCs) to the equine distal limb. Limitations due to thrombosis of the arteries after i.a. RLP and poor distribution of MSCs to the foot with i.v. RLP were observed. These techniques need to be modified for clinical use. OBJECTIVES Evaluate the distribution, uptake and persistence of radiolabelled MSCs after i.a. injection through the median artery without a tourniquet and after i.v. RLP through the lateral palmar digital vein. STUDY DESIGN In vivo experimental study. METHODS (99m) Tc-HMPAO-labelled MSCs were injected through the median artery of one limb and the lateral palmar digital vein of the other limb of 6 horses under general anaesthesia. No tourniquet was used for the i.a. injection. A pneumatic tourniquet was placed on the metacarpus for i.v. injection. Scintigraphic images were obtained up to 24 h after injection. RESULTS Intra-arterial injection resulted in MSCs retention within the limb despite the absence of a tourniquet and no thrombosis was observed. Both i.a. injection and i.v. RLP led to distribution of MSCs to the foot. The i.a. injection resulted in a more homogeneous distribution. The MSC uptake was higher with i.v. RLP at the initial timepoints, but no significant difference was present at 24 h. CONCLUSIONS Both i.a. injection through the median artery without a tourniquet and i.v. RLP performed through the lateral palmar digital vein under general anaesthesia are safe and reliable methods for administration of MSCs to the equine foot. The i.a. technique is preferred owing to the better distribution, but is technically more challenging. The feasibility of performing these techniques on standing horses remains to be investigated.

Comparisons of computed tomography, contrast enhanced computed tomography and standing low-field magnetic resonance imaging in horses with lameness localised to the foot. Part 1: anatomic visualisation scores.

REASONS FOR PERFORMING STUDY To date, few reports exist comparing magnetic resonance imaging (MRI) and computed tomography (CT) for imaging of the equine distal limb, yet clinicians are required to decide which modality to use regularly. OBJECTIVES To report and compare anatomic visualisation scores obtained for CT, contrast enhanced CT (CECT) and standing low-field MRI (LFMRI) in the equine foot. HYPOTHESIS Anatomic visualisation score discrepancies would exist between CT, CECT and LFMRI. METHODS Images of 22 lame horses (31 limbs) undergoing both CT and LFMRI of the foot were reviewed. When available, CECT images were reviewed. The deep digital flexor tendon (DDFT) was categorised into proximal to distal levels (A-D), structures were assigned visualisation scores (Grades 0-3) and technique comparisons were made using the paired marginal homogeneity test. RESULTS Computed tomography and LFMRI had similar visibility scores for the navicular bone, middle phalanx, DDFT-B, collateral ligaments of the distal interphalangeal joint and collateral sesamoidean ligament of the navicular bone. The proximal and distal phalanx had lower visibility scores with LFMRI. The distal DDFT (C-D), distal sesamoidean impar ligament and synovial structures had higher scores with LFMRI. Contrast enhanced CT lowered DDFT and collateral sesamoidean ligament scores and raised distal interphalangeal synovium CT visualisation scores. CONCLUSIONS AND POTENTIAL RELEVANCE Visualisation scores differ depending on imaging technique and anatomic structure of interest. This information increases our understanding of the limitations of CT, CECT and LFMRI to visualise anatomy in clinical cases.

Catastrophic scapular fractures in Californian racehorses: Pathology, morphometry and bone density

REASONS FOR PERFORMING STUDY To enhance understanding of the nature and pathogenesis of scapular fractures in racehorses. HYPOTHESIS Scapular fractures in racehorses have a consistent configuration related to sites of pre-existing stress modelling and remodelling. METHODS Fractured and intact scapulae collected post mortem were examined visually and with computed tomography (CT). Scapular fracture configuration, bone modelling changes and standardised CT morphometry and density measurements were recorded. Statistical comparisons were made between fractured, nonfractured contralateral and control scapulae. RESULTS Thirty-nine scapulae from 10 Thoroughbred (TB) and 10 Quarter Horse (QH) racehorses were obtained. All 14 fractured scapulae (from 12 horses) had a consistent comminuted fracture configuration. A complete fracture coursed transversely through the neck of the scapula at the level of the distal aspect of the spine (8.9 ± 0.9 cm proximal to the lateral articular margin of the glenoid cavity). The distal fragment of 13 fractured scapulae was split into 2 major fragments by a fracture in the frontal plane that entered the glenoid cavity (2.8 ± 0.4 cm caudal to the cranial articular margin). Focal areas of periosteal proliferation and/or radiolucency were present in the distal aspect of the scapular spine of all fractured and intact contralateral scapulae, but less commonly (P<0.01) in intact scapula from horses without a scapular fracture. Fractured scapulae had 7-10% lower mean density and 46-104% greater density heterogeneity in the spine adjacent to the transverse fracture compared to control scapulae (P<0.03). CONCLUSIONS AND CLINICAL RELEVANCE Thoroughbred and QH racehorses have a characteristic scapular fracture configuration that is associated with pre-existing pathology of the distal aspect of the spine. This location is consistent with scapular stress fractures diagnosed in lame TB racehorses. Catastrophic fracture is the acute manifestation of a more chronic process. Consequently, there are opportunities for early detection and prevention of fatalities.

Imaging diagnosis-portal vein aplasia and interruption of the caudal vena cava in three dogs

Severe portal vascular anomalies have been reported previously accompanying azygos continuation of the caudal vena cava, polysplenia, and situs anomalies in dogs and people. Three dogs with portal vascular anomalies were identified by means of CT angiography as having portal vein aplasia with portal insertion into the caudal vena cava, azygos continuation of the caudal vena cava, and interruption of the pre-hepatic caudal vena cava. This information confirms that complex embryological defects may occur in patients presenting for congenital portosystemic shunt, and that CT angiography is a non-invasive method of completely evaluating these potentially non-surgical portal vascular anomalies.

Evaluation of a diode laser for use in induction of tendinopathy in the superficial digital flexor tendon of horses

OBJECTIVE To evaluate use of a diode laser to induce tendinopathy in the superficial digital flexor tendon (SDFT) of horses. ANIMALS 4 equine cadavers and 5 adult horses. PROCEDURES Cadaveric SDFT samples were exposed to a diode laser at various energy settings to determine an appropriate energy for use in in vivo experiments; lesion size was assessed histologically. In vivo experiments involved laser energy induction of lesions in the SDFT (2 preliminary horses [0, 25, 75, and 87.5 J] and 3 study horses [0 and 125 J]) and assessment of lesions. Study duration was 21 days, and lesions were assessed clinically and via ultrasonography, MRI, and histologic evaluation. RESULTS Lesion induction in cadaveric tissues resulted in a spherical cavitated core with surrounding tissue coagulation. Lesion size had a linear relationship (R2 = 0.9) with the energy administered. Size of in vivo lesions in preliminary horses indicated that larger lesions were required. In study horses, lesions induced with 125 J were ultrasonographically and histologically larger than were control lesions. At proximal and distal locations, pooled (preliminary and study horses) ultrasonographically assessed lesions were discrete and variable in size (mean ± SEM lesion percentage for control lesions, 8.5 ± 3%; for laser lesions, 12.2 ± 1.7%). Ultrasonography and MRI measurements were associated (R2 > 0.84) with cross-sectional area measurements. CONCLUSIONS AND CLINICAL RELEVANCE In vivo diode laser-induced lesions did not reflect cadaveric lesions in repeatable size. Further research is required before diode lasers can reliably be used for inducing tendinopathy.

SCINTIGRAPHIC TRACKING OF MESENCHYMAL STEM CELLS AFTER PORTAL, SYSTEMIC INTRAVENOUS AND SPLENIC ADMINISTRATION IN HEALTHY BEAGLE DOGS

Mesenchymal stem cells have been proposed to treat liver disease in the dog. The objective of this study was to compare portal, systemic intravenous and splenic injections for administration of mesenchymal stem cells to target the liver in healthy beagle dogs. Four healthy beagle dogs were included in the study. Each dog received mesenchymal stem cells via all three delivery methods in randomized order, 1 week apart. Ten million fat-derived allogeneic mesenchymal stem cells labeled with Technetium-99m (99mTc)-hexamethyl-propylene amine oxime(HMPAO) were used for each injection. Right lateral, left lateral, ventral, and dorsal scintigraphic images were obtained with a gamma camera equipped with a low-energy all-purpose collimator immediately after injection and 1, 6, and 24 h later. Mesenchymal stem cells distribution was assessed subjectively using all four views. Pulmonary, hepatic, and splenic uptake was quantified from the right lateral view, at each time point. Portal injection resulted in diffuse homogeneous high uptake through the liver, whereas the systemic intravenous injection led to mesenchymal stem cell trapping in the lungs. After splenic injection, mild splenic retention and high homogeneous diffuse hepatic uptake were observed. Systemic injection of mesenchymal stem cells may not be a desirable technique for liver therapy due to pulmonary trapping. Splenic injection represents a good alternative to portal injection. Scintigraphic tracking with 99mTc-HMPAO is a valuable technique for assessing mesenchymal stem cells distribution and quantification shortly after administration. Data obtained at 24 h should be interpreted cautiously due to suboptimal labeling persistence.