Therapeutic Applications of Botulinum Neurotoxins in Veterinary Medicine.

Botulinum neurotoxins (BoNTs) are emerging as multipurpose therapeutic compounds for the treatment of several different syndromes involving peripheral and central nervous systems, and muscular and musculoskeletal disorders both in human and veterinary medicine. Therefore, the study of BoNTs is rapidly developing and identifying newly produced BoNT variants. Efforts should be made to clarify the biological and pharmacological characteristics of these novel BoNTs as well as the natural ones. The high potential of BoNTs as a therapeutic compound for medical syndromes lies in its ability to reach a specific cell type while bypassing other cells, thus having mild or no side effects. In this paper the recent developments in BoNTs are reviewed with the aim of analyzing the current knowledge on BoNTs' biological mechanisms of action, immunogenicity, formulations, and therapeutic applications in the veterinary field, highlighting advantages and drawbacks and identifying the gaps to be filled in order to address research priorities.

Evaluation of Three Methods of Sensory Function Testing for the Assessment of Successful Maxillary Nerve Blockade in Horses.

Maxillary nerve blocks (MNBs) commonly facilitate dental surgeries in standing horses. The goal of this prospective, blinded, cross-over design trial including 15 client-owned horses was to evaluate 3 methods of sensory function testing for confirming a successful MNB. Testing was performed bilaterally before sedation, 5 min after sedation, and 15 and 30 min after MNB with 0.5% bupivacaine and involved a needle prick dorsal to each naris, hemostat clamping of each nostril, and gingival algometry (measuring sensitivity to pain). Responses to stimulation were numerically scored and scores were summed up to a total score. Total score increases on the blocked side by ≥ 2 between baseline and 30 min Post MNB recordings signified a successful MNB. Sedation in the preceding 6 h, presence of sino-nasal disease, side of dental pathology, age, butorphanol administration, and detomidine dosing (µg/kg/min) throughout the tooth extraction procedure were recorded. In 73% of horses, MNB was successful. Sedation in the preceding 6 h (P = .732), age (P = .936), side of pathology (P = .516), and sino-nasal disease (P = .769) were not associated with total scores. Detomidine dosage and butorphanol use did not differ between horses in which the MNB was considered successful and for those in which it was not (P = .967 and P = .538, respectively). Scores obtained with gingival algometry were less closely associated with total scores (rho = .649) than those obtained with needle prick and nostril clamping (rho = .819 and .892, respectively). Therefore, needle prick and nostril clamping are considered the more reliable methods for use in clinical practice to determine the success of an MNB.

A critical evaluation for validation of composite and unidimensional postoperative pain scales in horses.

Proper pain therapy requires adequate pain assessment. This study evaluated the reliability and validity of the Unesp-Botucatu horse acute pain scale (UHAPS), the Orthopedic Composite Pain Scale (CPS) and unidimensional scales in horses admitted for orthopedic and soft tissue surgery. Forty-two horses were assessed and videotaped before surgery, up to 4 hours postoperatively, up to 3 hours after analgesic treatment, and 24 hours postoperatively (168 video clips). After six evaluators viewing each edited video clip twice in random order at a 20-day interval, they chose whether analgesia would be indicated and applied the Simple Descriptive, Numeric and Visual Analog scales, CPS, and UHAPS. For all evaluators, intra-observer reliability of UHAPS and CPS ranged from 0.70 to 0.97. Reproducibility was variable among the evaluators and ranged from poor to very good for all scales. Principal component analysis showed a weak association among 50% and 62% of the UHAPS and CPS items, respectively. Criterion validity based on Spearman correlation among all scales was above 0.67. Internal consistency was minimally acceptable (0.51-0.64). Item-total correlation was acceptable (0.3-0.7) for 50% and 38% of UHAPS and CPS items, respectively. UHAPS and CPS were specific (90% and 79% respectively), but both were not sensitive (43 and 38%, respectively). Construct validity (responsiveness) was confirmed for all scales because pain scores increased after surgery. The cut-off point for rescue analgesia was ≥ 5 and ≥ 7 for the UHAPS and CPS, respectively. All scales presented adequate repeatability, criterion validity, and partial responsiveness. Both composite scales showed poor association among items, minimally acceptable internal consistency, and weak sensitivity, indicating that they are suboptimal instruments for assessing postoperative pain. Both composite scales require further refinement with the exclusion of redundant or needless items and reduction of their maximum score applied to each item or should be replaced by other tools.

In vitro effects of poly(ADP-ribose) polymerase inhibitors on the production of tumor necrosis factor-α by interferon- γ - and lipopolysaccharide-stimulated peripheral blood mononuclear cells of horses.

OBJECTIVE: To evaluate effects of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors on the production of tumor necrosis factor-α (TNF-α) by interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of horses as an in vitro model of inflammation in horses. SAMPLE: 1,440 samples of PBMCs from 6 healthy research horses. PROCEDURES: From heparinized whole blood samples, PBMC cultures were obtained. An initial dose-response trial on 48 PBMC samples from 2 horses (24 samples each) was used to determine concentrations of IFN-γ and LPS for use as low- and high-level stimulation concentrations. Seventy-two PBMC samples from 6 horses were assigned equally to 1 of 4 PARP1 inhibition categories: no PARP1 inhibitor (PARP1 inhibition control); 2-((R)-2-methylpyrrolidin-2-yl)-1H-benzimidazole-4-carbozamide dihydrochloride (ABT888);4-(3-(1-(cyclopropanecarbonyl)piperazine-4-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (AZD2281); or N-(6-oxo-5,6-dihydrophenanthridin-2-yl) -N,N-dimethylacetamide hydrochloride (PJ34). Samples of PBMCs from each horse and each PARP1 inhibition category were then assigned to 1 of 3 levels of IFN-γ and LPS stimulation: none (control), low stimulation, or high stimulation. After a 24-hour incubation period, a TNF-α ELISA was used to measure TNF-α concentration in the supernatant. Results were compared across treatments and for each horse. Data were analyzed with repeated-measures ANOVA. RESULTS: Median TNF-α concentration was significantly lower for PJ34-treated, high-level stimulated PBMCs than for PARP1 inhibition control, high-level stimulated PBMCs; however, no other meaningful differences in TNF-α concentration were detected among the inhibition and stimulation combinations. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that PJ34 PARP1 inhibition may reduce TNF-α production in horses, a potential benefit in reducing inflammation and endotoxin-induced damage in horses.

Neuropathic pain management in chronic laminitis.

Managing pain in horses afflicted by chronic laminitis is one of the greatest challenges in equine clinical practice because it is the dreadful suffering of the animals that most often forces the veterinarian to end the battle with this disease. The purpose of this review is to summarize our current understanding of the complex mechanisms involved in generating and amplifying pain in animals with laminitis and, based on this information, to propose a modified approach to pain therapy. Furthermore, a recently developed pain scoring technique is presented that may help better quantify pain and the monitoring of responses to analgesic treatment in horses with laminitis.

Sensory nerve conduction and nociception in the equine lower forelimb during perineural bupivacaine infusion along the palmar nerves.

The purpose of this investigation was to study lateral palmar nerve (LPN) and medial palmar nerve (MPN) morphology and determine nociception and sensory nerve conduction velocity (SNCV) following placement of continuous peripheral nerve block (CPNB) catheters along LPN and MPN with subsequent bupivacaine (BUP) infusion. Myelinated nerve fiber distribution in LPN and MPN was examined after harvesting nerve specimens in 3 anesthetized horses and processing them for morphometric analysis. In 5 sedated horses, CPNB catheters were placed along each PN in both forelimbs. Horses then received in one forelimb 3 mL 0.125% BUP containing epinephrine 1:200 000 and 0.04% NaHCO(3) per catheter site followed by 2 mL/h infusion over a 6-day period, while in the other forelimb equal amounts of saline (SAL) solution were administered. The hoof withdrawal response (HWR) threshold during pressure loading of the area above the dorsal coronary band was determined daily in both forelimbs. On day 6 SNCV was measured under general anesthesia of horses in each limb's LPN and MPN to detect nerve injury, followed by CPNB catheter removal. The SNCV was also recorded in 2 anesthetized non-instrumented horses (sham controls). In both LPN and MPN myelinated fiber distributions were bimodal. The fraction of large fibers (>7 µm) was greater in the MPN than LPN (P < 0.05). Presence of CPNB catheters and SAL administration did neither affect measured HWR thresholds nor SNCVs, whereas BUP infusion suppressed HWRs. In conclusion, CPNB with 0.125% BUP provides pronounced analgesia by inhibiting sensory nerve conduction in the distal equine forelimb.

Development of a technique for continuous perineural blockade of the palmar nerves in the distal equine thoracic limb.

OBJECTIVE: To develop a technique for placing continuous peripheral nerve block (CPNB) catheters adjacent to palmar nerves in horses and to evaluate the effect of low-volume local anesthetic (LA) infusion on nociception in the distal equine thoracic limb. STUDY DESIGN: In vitro and in vivo laboratory investigation. STUDY MATERIAL AND ANIMALS: Forty-two thoracic limbs from 22 equine cadavers and five horses. METHODS: Thoracic limb specimens were dissected to find landmarks for catheter insertion adjacent to medial and lateral palmar nerves. Based on the anatomy of the proximal metacarpus, a technique for placing palmar CPNB catheters was developed and the potential for catheter dislodgement studied in vitro by fluoroscopic visualization during passive carpal flexion and dye injection following simulated limb motion. The feasibility of CPNB catheter instrumentation in standing, sedated horses was tested in five animals, with ultrasound control. Electrical and mechanical stimulation thresholds and response latencies for hoof withdrawal responses (HWR) were determined following saline or LA infusion. RESULTS: Medial and lateral CPNB catheters were inserted percutaneously 2 and 4-5 cm, respectively, distal to the accessory carpal bone and advanced for approximately 7 and 10 cm, respectively, to place the tip just proximal to the communicating branch of the nerves. Catheters were placed correctly in 88% and 85% of cadaver limbs. In the standing horses, LA infusion not only increased HWR thresholds and latencies to noxious mechanical or electrical stimulation but also caused vasodilation and limb swelling over time. CONCLUSION: The technique, developed in vitro, for placing and maintaining palmar CPNB catheters in the equine thoracic limb was successfully applied in vivo. Catheters were well tolerated but LA infusion may cause limb swelling, suggesting a need for further exploration of drug and infusion regimens. CLINICAL RELEVANCE: Continuous perineural LA infusion along palmar nerves may develop into an effective analgesic technique in horses suffering from lower limb pain.

Effects of low-volume hemoglobin glutamer-200 versus normal saline and arginine vasopressin resuscitation on systemic and skeletal muscle blood flow and oxygenation in a canine hemorrhagic shock model.

OBJECTIVE: To test the hypothesis that low-volume resuscitation with hemoglobin glutamer-200 improves hemodynamic function and tissue oxygenation, whereas arginine vasopressin resuscitation improves blood pressures more than low-volume saline or hemoglobin glutamer infusion but compromises systemic and muscle blood flow and oxygenation. DESIGN: Randomized laboratory investigation. SETTING: University research facility. SUBJECTS: Nineteen dogs. INTERVENTIONS: Dogs were instrumented to determine heart rate; arterial, central venous, pulmonary arterial, and pulmonary arterial occlusion pressures; cardiac output; and quadriceps muscle blood flow and oxygen tension (PMo2). Total and plasma hemoglobin, oxygen content, lactate, pH, standard base excess, and arginine vasopressin levels were determined, and systemic oxygen delivery (Do2I) and extraction ratio were calculated. Measurements were made before and 30 mins following hemorrhage. Dogs were resuscitated over 60 mins with saline (8.5 mL/kg), arginine vasopressin (0.4 IU/kg bolus plus 0.08 IU x kg x min), or 1:1 diluted hemoglobin glutamer-200. Recordings were then repeated. Subsequently, animals received 30 mL/kg shed blood (60 mL x kg x hr), and recordings were repeated immediately and 1 hr later. MEASUREMENTS AND MAIN RESULTS: Hemorrhage ( approximately 52 mL/kg) caused characteristic changes in hemodynamic, hematologic, systemic PMo2, and acid-base variables. Saline resuscitation increased both Do2I and muscle perfusion by 42% and 51%, while arginine vasopressin treatment reduced heart rate by 31% and increased mean arterial pressure by 22% but not cardiac output, Do2I, or muscle blood flow, resulting in a further decrease of PMo2 by 68% and worse metabolic acidosis. Hemoglobin glutamer-200 infusion caused systemic and pulmonary vasoconstriction, however, without deterioration of cardiac output, Do2I, muscle blood flow, or PMo2 despite lack of oxygen content increase. Blood transfusion restored most variables. CONCLUSIONS: Low-volume crystalloid or hemoglobin glutamer-200 resuscitation posthemorrhage may improve (but not restore) macro- and microvascular functions and tissue oxygenation, while arginine vasopressin infusion may only improve blood pressures and result in lower overall systemic perfusion compared with low-volume saline or hemoglobin glutamer-200 treatment and worsening of anaerobic conditions in skeletal muscle.

Image fusion of computed tomographic and magnetic resonance images for the development of a three-dimensional musculoskeletal model of the equine forelimb.

Biomechanical models that compute the lengths and forces of muscle-tendon units are broadly applicable to the study of factors that promote injury and the planning and effects of orthopedic surgical procedures in equine athletes. A three-dimensional (3D) generic musculoskeletal model of the equine forelimb comprised of bony segment, muscle-tendon, and ligament information, was developed based on high-resolution computed tomographic (CT) and T1-weighted magnetic resonance (MR) images from an isolated forelimb of a Thoroughbred racehorse. Image fusion was achieved through coregistration of CT and MR images with an image analysis program (Analyze) by adjustment of the relative position and orientation of fiducial markers visible in both modalities until the mutual information between the images was maximized. 3D surfaces of the bones and origin/insertion sites, centroid paths and volumes of the muscle-tendon and ligamentous structures were obtained from the multimodal (CT/MR) images using semiautomated and manual segmentation combined with sagittal and transverse color-cryosection anatomic images obtained from three other cadaveric equine forelimbs. Once bony and soft-tissue structures were reconstructed in the same coordinate system, data were imported to a software package for interactive musculoskeletal modeling (SIMM). The combination of integrated CT and MR acquisitions and anatomical images provided an accurate and efficient means of generating a 3D model of the musculoskeletal structures of an average-sized equine adult horse.

Musculoskeletal modeling and dynamic simulation of the thoroughbred equine forelimb during stance phase of the gallop.

Because thoroughbred racehorses have a high incidence of forelimb musculoskeletal injuries, a model was desired to screen potential risk factors for injuries. This paper describes the development of a musculoskeletal model of the thoroughbred forelimb and a dynamic simulation of the motion of the distal segments during the stance phase of high-speed (18 m/s) gallop. The musculoskeletal model is comprised of segment, joint, muscle-tendon, and ligament information. The dynamic simulation incorporates a proximal forward-driving force, a distal ground reaction force model, muscle activations, and initial positions and velocities. A simulation of the gallop after transection of an accessory ligament demonstrated increased soft tissue strains in the remaining support structures of the distal forelimb. These data were consistent with those previously reported from in vitro experimental data and supported usefulness of the model for the study of distal forelimb soft tissue mechanics during the stance phase of the gallop.

Passive and active mechanical properties of the superficial and deep digital flexor muscles in the forelimbs of anesthetized Thoroughbred horses.

The superficial (SDF) and deep digital flexor (DDF) muscles are critical for equine forelimb locomotion. Knowledge of their mechanical properties will enhance our understanding of limb biomechanics. Muscle contractile properties derived from architectural-based algorithms may overestimate real forces and underestimate shortening capacity because of simplistic assumptions regarding muscle architecture. Therefore, passive and active (=total - passive) force-length properties of the SDF and DDF muscles were measured directly in vivo. Muscles from the right forelimbs of four Thoroughbred horses were evaluated during general anesthesia. Limbs were fixed to an external frame with the muscle attached to a linear actuator and load cell. Each muscle was stretched from an unloaded state to a range of prefixed lengths, then stimulated while held at that length. The total force did not exceed 4000 N, the limit for the clamping device. The SDF and DDF muscles produced 716+/-192 and 1577+/-203 N maximum active isometric force (F(max)), had ascending force-length ranges (R(asc)) of 5.1+/-0.2 and 9.1+/-0.4 cm, and had passive stiffnesses of 1186+/-104 and 1132+/-51 N/cm, respectively. The values measured for F(max) were much smaller than predicted based on conservative estimates of muscle specific tension and muscle physiological cross-sectional area. R(asc) were much larger than predicted based on muscle fiber length estimates. These data suggest that accurate prediction of the active mechanical behavior of architecturally complex muscles such as the equine DDF and SDF requires more sophisticated algorithms.

Determination of muscle architecture and fiber characteristics of the superficial and deep digital flexor muscles in the forelimbs of adult horses.

OBJECTIVE: To provide a quantitative description of the architecture of superficial digital flexor (SDF) and deep digital flexor (DDF) muscles in adult horses to predict muscle-tendon behavior and estimate muscle forces. SAMPLE POPULATION: 7 forelimb specimens from 7 adult Thoroughbreds. PROCEDURE: Muscle and tendon lengths and volumes were measured from 6 fixed forelimbs. After processing, fiber bundle and sarcomere lengths were measured. Optimal fascicle lengths and muscle length-to-fascicle length, muscle length-to-free tendon length, and fascicle length-to-tendon length ratios were calculated, as were tendon and muscle physiologic cross-sectional areas (PCSAs). Pennation angles were measured in 1 embalmed specimen. RESULTS: The SDF optimal fascicle lengths were uniformly short (mean +/- SD, 0.8 +/- 0.1 cm), whereas DDF lengths ranged from 0.9 +/- 0.2 cm to 10.8 +/- 1.6 cm. The DDF humeral head had 3 architectural subunits, each receiving a separate median nerve branch, suggestive of neuromuscular compartmentalization. Pennation angles were small (10 degrees to 25 degrees). The PCSAs of the SDF and DDF muscle were 234 +/- 51 cm2 and 259 +/- 30 cm2, with estimated forces of 4,982 +/- 1148 N and 5,520 +/- 544 N, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The SDF muscle appears to provide strong tendinous support with little muscle fascicular shortening and fatigue-resistance properties. The DDF muscle combines passive and dynamic functions with larger tension development and higher shortening velocities during digital motion. Architectural parameters are useful for estimation of forces and have implications for analysis of muscle-tendon function, surgical procedures involving muscle-tendon lengthening, and biomechanical modeling.

Sonographic diagnosis of a presumed ureteral tear in a horse.

A 14-year-old thoroughbred gelding was presented for the evaluation of acute abdominal pain. Rectally, there was a soft fluctuant painful swelling dorsal to the bladder and to the right of the midline. The creatinine concentration of the peritoneal fluid was 15 mg/dl. Transrectal ultrasonographic examination of the urinary tract revealed a large collection of fluid dissecting from the pelvic portion of the right ureter ventrally through the right side of the bladder wall and into the retroperitoneal space, and a thickened right ureter and bladder wall at the level of the trigone. Cystoscopically there was moderate hemorrhage within the wall of the bladder. Ultrasonography revealed air within the retroperitoneal fluid collection after ureteral catheterization, confirming the preliminary diagnosis of a tear in the right ureter. The gelding was treated medically. After 48 h of hospitalization, nuclear scintigraphy revealed normal clearance from both kidneys and no apparent leakage from the right ureter. The ureteral tear and urinoma were monitored using transrectal ultrasound until resolution. The horse was successfully returned to racing. This case establishes the value of diagngstic ultrasound in the diagnosis and monitoring of a traumatic ureteral tear in a horse.

An in vivo equine forelimb model for short-term recording of peak isometric force in the superficial and deep digital flexor muscles.

OBJECTIVE: To develop and test an experimental model for in vivo short-term recording of peak isometric forces of the digital flexor muscles in the forelimb of adult horses. STUDY DESIGN: In vivo experimental study. SAMPLE POPULATION: Four healthy, anesthetized, adult Thoroughbred horses (3 to 7 years old; 527 +/- 87 kg) METHODS: In dorsal recumbency, ulnar and median nerves were exposed and instrumented with insulated bipolar cuff stimulation electrodes for later connection to an electrical stimulator. In left lateral recumbency, a biplanar fixator was applied to the right humerus and a custom-made, rigid, aluminum frame connected to it, to allow loading of muscles distal to the fixator. Threaded transfixation pins through the radial and metacarpal condyles were clamped to the rigid frame so that the humerus, radius, ulna, and metacarpus were fixed in position. Each digital flexor muscle insertion tendon was transected just above the metacarpophalangeal joint, extracted from the carpal canal, and secured in a metal clamp positioned at the distal myotendinous (MT) junction. Distally, the clamp was connected in series to a load cell and a pneumatic actuator to record force and to maintain muscle length during nerve stimulation. A linear potentiometer was connected in parallel to the actuator to record MT junction position. Initial trials were conducted to identify median and ulnar nerve stimulation variables to achieve maximal muscle contraction. Isometric contractions were performed at different muscle lengths and peak forces registered during 3 seconds of supramaximal dual (ulnar and median) nerve stimulation. RESULTS: A stimulation voltage of 2.5 to 5.0 V at 50 Hz usually produced maximal force for both the superficial digital flexor (SDF) and deep digital flexor (DDF) muscles. Single ulnar and median nerve stimulation elicited force development not only in the DDF muscle but also in the SDF muscle. At voltages higher than 1 V, normalized force was greatest with combined median and ulnar nerve stimulation for both the DDF and SDF muscles; however, normalized force was greater for median nerve stimulation than ulnar nerve stimulation in the DDF muscle, and the opposite relationship was observed for the SDF muscle. Final recording of dual supramaximal nerve stimulation of SDF and DDF muscles resulted in peak isometric forces of 716 +/- 192 N and 1,577 +/- 203 N, respectively. CONCLUSIONS: The instrumentation technique and experimental protocol enabled recording of peak isometric forces in the SDF and DDF muscles of anesthetized adult horses. CLINICAL RELEVANCE: Studies using this model will improve knowledge of SDF and DDF muscle mechanics with insight to functional implications of the complex architecture of these muscles. Knowledge of the dynamic performance of the SDF and DDF muscles would also be useful for the development of new treatment strategies for flexor deformities and tendon injuries in horses.