Equine skeletal scintigraphy: Comparing lesion detection ability of methylene diphosphonate and hydroxymethylene diphosphonate in the caudal cervical and proximal metacarpal/metatarsal regions.

BACKGROUND: Data regarding the lesion detection ability of different radiotracers are lacking in equine bone scintigraphy. METHODS: In this prospective study, hydroxymethylene diphosphonate (HMDP) and methylene diphosphonate (MDP) were compared in horses with increased radiopharmaceutical uptake either in the caudal cervical region (CS group) or in the proximal metacarpal/metatarsal region (PMR group). Region of interest analysis was used to determine normal bone-to-soft tissue ratios, lesion-to-normal bone ratios and lesion-to-soft tissue ratios. Qualitative scoring and total count rates were recorded for each image. RESULTS: A total of 213 scintigrams were included. Within the PMR group, there were significantly higher lesion-to-normal bone ratios for MDP compared with HMDP (p = 0.02). In the CS group, normal bone-to-soft tissue ratios were significantly higher for HMDP (p = 0.01). The interobserver agreement with regard to the qualitative assessment of the scintigrams was poor. LIMITATION: Paired studies, comparing the different radiotracers in the same patient, were not feasible. CONCLUSION: This study revealed minor differences between the two radiotracers, although these have no practical implications. Both radiopharmaceuticals are well suited for detecting lesions at the investigated sites using equine bone scintigraphy.

Thoracic processi spinosi findings agree among subjective, semiquantitative, and modified semiquantitative scintigraphic image evaluation methods and partially agree with clinical findings in horses with and without thoracolumbar pain.

Impinging processi spinosi in the equine thoracic spine are a common cause of poor performance in the horse. A modified semiquantitative scintigraphic image analysis has been proposed for the evaluation of equine processi spinosi. This technique showed a high inter- and intraobserver agreement when compared to subjective and semiquantitative image analysis. The aim of this retrospective, method comparison study was to evaluate the agreement of the modified semiquantitative scintigraphic image assessment with previous methods of interpretation and to compare these scintigraphic evaluation techniques with radiographic and clinical findings. Two hundred twenty-three Warmblood horses that underwent scintigraphic, radiographic, and clinical examination of the thoracic spine were included in the study. Scintigraphic images were assessed using subjective, semiquantitative, and modified semiquantitative techniques. Radiographs were subjectively graded and horses were assigned to a group with or without thoracolumbar pain. Total radiographic and total scintigraphic grades were higher in horses with thoracolumbar pain (P < 0.05). Both the semiquantitative and the modified semiquantitative uptake ratios did not differ significantly in horses with or without thoracolumbar pain. The kappa agreement showed a substantial agreement between the modified semiquantitative scintigraphic and the semiquantitative scintigraphic evaluation techniques. The agreement between subjective scintigraphic and modified semiquantitative scintigraphic image evaluations was fair. There was a slight agreement between all scintigraphic techniques and radiographic findings. Limitations were the definition of thoracolumbar pain and the image analysis being restricted to the caudal thoracic processi spinosi. In conclusion, the modified semiquantitative scintigraphic image assessment obtained consistent results but did not perform better than previous evaluation methods. Further comparison to a defined diagnosis is warranted.

Semi-quantitative methods yield greater inter- and intraobserver agreement than subjective methods for interpreting 99m technetium-hydroxymethylene-diphosphonate uptake in equine thoracic processi spinosi.

Scintigraphy is a standard diagnostic method for evaluating horses with back pain due to suspected thoracic processus spinosus pathology. Lesion detection is based on subjective or semi-quantitative assessments of increased uptake. This retrospective, analytical study is aimed to compare semi-quantitative and subjective methods in the evaluation of scintigraphic images of the processi spinosi in the equine thoracic spine. Scintigraphic images of 20 Warmblood horses, presented for assessment of orthopedic conditions between 2014 and 2016, were included in the study. Randomized, blinded image evaluation was performed by 11 veterinarians using subjective and semi-quantitative methods. Subjective grading was performed for the analysis of red-green-blue and grayscale scintigraphic images, which were presented in full-size or as masked images. For the semi-quantitative assessment, observers placed regions of interest over each processus spinosus. The uptake ratio of each processus spinosus in comparison to a reference region of interest was determined. Subsequently, a modified semi-quantitative calculation was developed whereby only the highest counts-per-pixel for a specified number of pixels was processed. Inter- and intraobserver agreement was calculated using intraclass correlation coefficients. Inter- and intraobserver intraclass correlation coefficients were 41.65% and 71.39%, respectively, for the subjective image assessment. Additionally, a correlation between intraobserver agreement, experience, and grayscale images was identified. The inter- and intraobserver agreement was significantly increased when using semi-quantitative analysis (97.35% and 98.36%, respectively) or the modified semi-quantitative calculation (98.61% and 98.82%, respectively). The proposed modified semi-quantitative technique showed a higher inter- and intraobserver agreement when compared to other methods, which makes it a useful tool for the analysis of scintigraphic images. The association of the findings from this study with clinical and radiological examinations requires further investigation.

An international multi-centre prospective study on the efficacy of an intraarticular polyacrylamide hydrogel in horses with osteoarthritis: a 24 months follow-up.

BACKGROUND: Polyacrylamide hydrogel (PAAG) was evaluated recently to treat osteoarthritis (OA) in horses with highly encouraging results; however no long term field-study was done to explore its clinical efficacy and lasting effect. The objective of this study was to evaluate the efficacy of PAAG in improving clinical signs of OA in horses. We hypothesized that lameness grade would significantly improve and the effect would last at least 2 years in osteoarthritic joints treated with PAAG. Forty three horses older than 2 years with OA in only one joint based on clinical evaluation, intra-articular anaesthesia and imaging (radiography) were included in this study. Horses were injected with 2 ml of PAAG into the affected joint and were followed up at 1, 3, 6, 12 and 24 months. Efficacy of PAAG was evaluated by blinded clinical assessment of lameness. Adverse reactions to joint injection were assessed. Data relating to case details, type of activity, joint and limb involved, lameness duration, lameness grading, previous joint treatment, joint effusion grading, radiographic grading, and owner assessment were recorded. Factors associated with the outcome measure "lameness grading" were analyzed using generalized linear mixed model for logistic regression. RESULTS: At 1, 3, 6, 12 and 24 months follow-up, 59%, 69%, 79%, 81/% and 82.5% of horses were non-lame respectively. Reduction of joint effusion was observed over time. No side effect was observed in the treated joints. There was a significant decrease in lameness grade from baseline to 1, 3, 6, 12 and 24 months (P < 0.0001) and a significant positive association with joint effusion (P < 0.0001). Estimates for odds ratio (OR) showed that the effect of treatment increased over time (OR for lower lameness from month 1 to month 24 relative to baseline increased from 20 to 58). CONCLUSIONS: PAAG significantly alleviated lameness and joint effusion in osteoarthritic joints. PAAG is a safe and lasting (at least 24 months) OA treatment in horses. PAAG is a promising new treatment for OA in horses.

The association of actin and myosin in the presence of gamma-amido-ATP proceeds mainly via a complex with myosin in the closed conformation.

The interaction of gamma-amido-ATP (ATPN) and its 2'(3')-O-methylanthraniloyl derivative (mantATPN) with skeletal myosin subfragment 1 (S1) and actomyosin (actoS1) was studied in stopped-flow experiments. Tryptophan fluorescence and fluorescence of the mant label or light scattering were measured simultaneously. Information about the binding of mant nucleotides was obtained from the quenching of tryptophan fluorescence by the mant label. The parameters of various kinetic models were fitted to the experimental traces. The high-fluorescence state of S1 forms with ATPN at a rate of 95 s-1 ("open-closed" transition); the transition is only slowly reversible, in contrast to the very fast equilibrium seen with its better known isomer AMPPNP [Urbanke, C., and Wray, J. (2001) Biochem. J. 358, 165-173]. The stabilization of the closed state of myosin by ATPN may be due to the formation of a complex with a pentacoordinated amido-gamma-phosphate, from which ATPN can dissociate at a rate of 0.005 s-1 or be hydrolyzed by cleavage of the beta-gamma bond at a rate of 2.5 x 10(-4) s-1. A corresponding actoS1-ATPN complex with myosin in the "closed" conformation is the first detectable intermediate in the association of actin and S1-ATPN, giving an experimental access to a state analogous to a key intermediate in the cross-bridge cycle.

Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide.

Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the "power stroke", which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.

Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPgammaNH(2) and GTPgammaF.

Guanosine triphosphate nucleotide analogues such as GppNHp (also named GMPPNP) or GTPgammaS are widely used to stabilize rapidly hydrolyzing protein-nucleotide complexes and to investigate biochemical reaction pathways. Here we describe the chemical synthesis of guanosine 5'-O-(gamma-amidotriphosphate) (GTPgammaNH(2)) and a new synthesis of guanosine 5'-O-(gamma-fluorotriphosphate) (GTPgammaF). The two nucleotides were characterized using NMR spectroscopy and isothermal titration calorimetry. Chemical shift data on (31)P, (19)F and (1)H NMR resonances are tabulated. For GTPgammaNH(2) the enthalpy of magnesium coordination is DeltaH degrees = 3.9 kcal.mol(-1) and the association constant K(a) is 0.82 mm(-1). The activation energy for GTPgammaNH(2).Mg2+ complex formation is DeltaH++ = 7.8 +/- 0.15 kcal.mol(-1), similar to that for the natural substrate GTP. For GTPgammaF we obtained a similar enthalpy of DeltaH degrees = 3.9 kcal.mol(-1) while the magnesium association constant is only K(a) = 0.2 mm(-1). The application of both guanine nucleotide analogues to the GTP-binding protein Ras was investigated. The rate of hydrolysis of GTPgammaNH(2) bound to Ras protein lay between the rates found for Ras-bound GTPgammaS and GppNHp, while Ras-catalysed hydrolysis of GTPgammaF was almost as fast as for GTP. The two compounds extend the variety of nucleotide analogues and may prove useful in structural, kinetic and cellular studies.

Gamma-amido-ATP stabilizes a high-fluorescence state of myosin subfragment 1.

On binding to myosin subfragment 1 (S1), the gamma-amido derivative of ATP (ATPgammaNH2), an isomer of adenosine 5'-[beta,gamma-imido]-triphosphate (AMPPNP), induces a larger increase in the intrinsic (tryptophan) fluorescence than is seen with ATP. A binding constant of 1.7x10(7) M(-1) was measured for ATPgammaNH2, compared to 2.1-2.4x10(7) M(-1) for AMPPNP. ATPgammaNH2 was hydrolyzed only very slowly by S1. ATPgammaNH2 appears to stabilize the 'closed' conformation of S1, and does so without cleavage of the beta-gamma phosphate bond. The dissociation of actin-S1 by ATPgammaNH2 and that of S1.ATPgammaNH2 by actin are both strikingly slow.