Musculoskeletal injuries are a prevalent problem in horses, with injury to flexor tendons or the suspensory ligament accounting for up to 46% of all limb injuries in performance horses and racehorses.1–3 Of these injured structures, the SDFT has a particularly high injury rate, with a prevalence of 8% to 43% in racing Thoroughbreds.4–6 Despite the high prevalence, injuries to tendons and ligaments remain a therapeutic challenge, with up to 67% of horses affected by tendinopathy of the SDFT sustaining reinjury.5,7,8 Recovery requires a balance between rest and controlled exercise to improve the mechanical properties of the healing tissue and minimize risk of rei njur y.9,10
Serial ultrasonographic examination has commonly been used to assess tendon injury and to monitor healing, and the ultrasonographic appearance of a tendon has been the basis for instituting rehabilitation programs.9,11 Injury is detected by disruption of the ultrasonographically normal anatomy as observed by changes in fiber pattern, shape, cross-sectional area, and pixel intensity.9,11–13 During rehabilitation, small changes in the B-mode ultrasonographic appearance are associated with large changes in biomechanical strength. Consequently, it is difficult to determine when activity can be increased while avoiding reinjury.14
Elastography and acoustoelastography are noninvasive ultrasonographic methods for evaluation of tendon properties that offer the potential for clinical application in horses with tendon injury.15,16 Elastography can be used to evaluate tendon tissue by comparing ultrasonographic echoes obtained before and after compression with the ultrasound transducer in a transverse plane.15 However, tendons are physiologically loaded in tension, which may limit the applicability of elastography in evaluating these anatomic structures. In addition, this technique works best when increments of applied strain are smaller than the naturally occurring deformation of equine tendons.17,18
Acoustoelastography is a novel ultrasound-based technique that relates changes in echogenicity observed during deformation of a tendon from an unloaded to loaded state to the mechanical properties of the tissue.18,19 Thus, changes in pixel intensity as a tendon is stretched can be related to strain and stiffness of the tissue. Specifically, acoustoelastography can be used to determine the stiffness gradient by estimation of the PI, which represents the rate of change in stiffness in relation to the change in strain. Acoustoelastography can be used to accurately estimate strain and stiffness within porcine flexor tendons in vitro,18 and protocols developed for in vivo use in dogs20 and horses16 suggest that this technique is feasible and repeatable for use in measuring stiffness gradients in clinically normal tendons in these species.
It is important that a patient be adequately restrained during an ultrasonographic examination. Sedation is often required to make patients more cooperative.16,21 Detomidine and butorphanol or xylazine and butorphanol are the drug combinations most commonly used by equine veterinarians in North America for sedation of standing horses.22 Several studies23–28 have been conducted to assess the muscle relaxant properties of these drugs. The α2-adrenergic receptor agonists administered alone or in combination with butorphanol cause muscle relaxation evident as lowering of the head and drooping of the eyelids and lower lip.23,24,26,28–31
The use of sedation during ultrasonographic examination may cause relaxation of the superficial digital flexor muscle and may influence assessment of SDFT biomechanical properties. Because acoustoelastography is used to assess the stiffness gradient of a tendon, sedation during ultrasonographic examination might influence the acoustoelastography PI.
The purpose of the study reported here was to assess the effects of sedation with a combination of detomidine and butorphanol on the acoustoelastography PI of SDFTs in clinically normal horses. Our hypothesis was that the PI in clinically normal equine SDFTs would not be influenced by sedation.
Materials and Methods
Horses
Twenty-seven client-owned clinically normal horses of various ages and breeds were included in the study. Horses were defined as clinically normal on the basis of owner history and results of an orthopedic physical examination and a lameness evaluation. Examinations were performed by 1 investigator (DDG). Lack of tendon abnormalities was confirmed during ultrasonographic examination. Owner consent was obtained for inclusion of each horse in the study. All animal protocols were approved by the University of Wisconsin School of Veterinary Medicine Institutional Animal Care and Use Committee.
Experimental procedures
Hair was clipped on the palmar aspect of both thoracic limbs of each horse from the proximal aspect of the metacarpus to the level of the metacarpophalangeal joint. The skin was washed with water to remove any dirt in preparation for an ultrasonographic examination. To establish that no B-mode ultrasonographic abnormalities were evident, longitudinal and transverse images of the right and left thoracic limb SDFTs at locations 5, 10, 15, and 20 cm distal to the accessory carpal bone were obtained with a linear transducer by use of a portable ultrasound machine.a A standardized frequency setting of 12 MHz was maintained during imaging. Focal zone position was set at the level of the SDFT, and gain was subjectively maintained between 56% and 60% to provide the optimal image for each examination. When no abnormalities were detected in the SDFTs, 3 cineloop video recordings were obtained in longitudinal orientation at each examination location as the tendon was loaded by shifting the horse from a baseline square stance to an increased weight-bearing stance and then back to a baseline square stance. This was accomplished by having an assistant lift the contralateral thoracic limb into a non–weight-bearing position with the carpus flexed such that additional weight was shifted onto the limb being scanned and then placing the contralateral thoracic limb back down on the ground. This maneuver was performed as smoothly as possible. After image acquisition was completed, each horse was sedated by IV administration of a combination of detomidine hydrochlorideb (0.01 mg/kg) and butorphanol tartratec (0.01 mg/kg). When signs of complete sedation (eg, lowering of the head) were evident, ultrasonographic examination was repeated. All ultrasonographic examinations were performed by the same investigator (DDG).
Data analysis
Uncompressed audio-video interleave format files for each of the cineloop video recordings were used for PI analyses, which were performed by use of analysis softwared by 1 investigator (SLD). A region of interest consistent in size and location that was approximately the central half of the tendon section was selected for each cineloop. To maintain this same region of interest throughout the video recording, a region-based optical flow tracking technique was used to estimate the movement of speckles between consecutive frames.18 Changes in relative pixel intensity and location over time were analyzed via acoustoelastography to estimate the PI for each pixel.
To quantify stiffness gradients, PI values for each cineloop were calculated by use of the stiffness gradients for each pixel within a 50 × 300-pixel area located centrally within the region of interest. A mean PI value was calculated.
Statistical analysis
Summary data were reported as mean ± SD. Analysis was performed by use of commercial software.e The PI data did not have a normal distribution, as determined by use of the Shapiro-Wilk W test. Therefore, data were logarithmically transformed for analysis. A repeated-measures ANOVA was used to determine the effect of limb (left and right), SDFT examination location, and sedation on the PI (limb, examination location, and sedation were analyzed as repeated measures). The contribution of each repeated measure to data variance was calculated. Results were considered significant at P < 0.05.
Results
Of the 27 horses included in the study, 13 were geldings and 14 were mares. Horses ranged from 5 to 31 years of age (mean ± SD, 14.7 ± 5.86 years). Breeds of horses included Thoroughbred (n = 11), Quarter Horse (5), Paint (5), American Saddlebred (1), Oldenburg (1), Missouri Fox Trotter (1), Arabian crossbred (1), and Thoroughbred crossbred (1); breed was not recorded for 1 horse.
Mean PIs for the SDFT at locations 5, 10, 15, and 20 cm distal to the accessory carpal bone were summarized (Table 1). Horse, sedation, and examination location had significant effects on the PI. Overall, the mean ± SD PI after sedation (0.0581 ± 0.0111) was significantly (P < 0.001) lower than the PI before sedation (0.0667 ± 0.1250). The PI at 15 cm distal to the accessory carpal bone was significantly lower than the PI at 5 cm (P < 0.001) and 10 cm (P = 0.003) distal to the accessory carpal bone, and the PI at 20 cm was significantly (P = 0.028) lower than the PI at 5 cm distal to the accessory carpal bone. The PI was not significantly (P = 0.06) affected by limb (right vs left). In addition, there was not a significant interaction between sedation and limb (P = 0.54) or between sedation and location (P = 0.31). Overall, the magnitude of variance effects for the PI (from largest to smallest) was horse (0.407; 95% CI, 0.387 to 0.516), sedation (0.187; 95% CI, 0.122 to 0.252), examination location (0.064; 95% CI, 0.021 to 0.110), and limb (0.009; 95% CI, 0 to 0.036).
Mean ± SD value of the PI of the SDFT of each thoracic limb derived by use of acoustoelastography in 27 clinically normal horses before and after horses were sedated with detomidine and butorphanol.
| Â | Left thoracic limb | Right thoracic limb | ||
|---|---|---|---|---|
| Location | Before sedation | After sedation | Before sedation | After sedation |
| 5 cm | 0.0683 ± 0.0152 | 0.0582 ± 0.0107 | 0.0707 ± 0.0119 | 0.0646 ± 0.0114 |
| 10 cm | 0.0659 ± 0.0116 | 0.0591 ± 0.0107 | 0.0710 ± 0.0118 | 0.0577 ± 0.0097 |
| 15 cm* | 0.0614 ± 0.0107 | 0.0564 ± 0.0127 | 0.0627 ± 0.0111 | 0.0556 ± 0.0100 |
| 20 cm†| 0.0665 ± 0.0124 | 0.0566 ± 0.0112 | 0.0673 ± 0.0150 | 0.0569 ± 0.0122 |
| Overall‡ | 0.0655 ± 0.0125 | 0.0576 ± 0.0113 | 0.0679 ± 0.0124 | 0.0587 ± 0.0108 |
Location is the distance distal to the accessory carpal bone.
Values for this location differed significantly from the values for 5 cm (P < 0.001) and 10 cm (P = 0.003).
Values for this location differed significantly (P = 0.028) from the values for 5 cm.
The mean value for both thoracic limbs after sedation was significantly (P < 0.001) lower than the mean value for both thoracic limbs before sedation.
Discussion
Results of the study reported here indicated that sedation with detomidine and butorphanol caused a decrease in the stiffness gradient or PI of thoracic limb SDFTs in clinically normal horses and did not support our a priori hypothesis. Muscle relaxation may have explained this observation. Stimulation of α2-adrenoceptors in the CNS decreases the discharge rate of central and peripheral neurons, which results in sedation, analgesia, and muscle relaxation.32 The α2-adrenergic receptor agonists induce excellent relaxation of the muscles of the head, neck, and ears, which is followed by drooping of the head, ears, lips, and eyelids. In standing horses, the effects of α2-adrenoceptor agonists can also contribute to a degree of ataxia. Such effects seen in horses correlate well with the degree of sedation and are accepted as a method to assess the depth of sedation.26,31,32 When α2-adrenoceptor agonists are combined with opioids, the opioids are thought to contribute to CNS effects of the α2-adrenoceptor agonists. Synergistic and additive interactions increase muscle relaxation, which results in deeper and more prolonged sedation and analgesia. Such effects can be achieved with reduced dosages of each drug, compared with dosages required when either drug is administered alone.28,32 Therefore, once the horses of the present study were sedated, they may have responded to loading of a thoracic limb with less muscle contraction and consequently less strain to the tendon and smaller changes in tendon stiffness, which led to lower PI values.
These findings contradict previous thoughts about the degree of influence that the superficial digital flexor muscle fibers have on the degree of strain applied to the SDFT. It has been suggested33,34 that the effect of contraction of the digital flexor muscle on the muscle-tendon unit length is minimal and that lengthening and shortening of muscle fibers contribute very little to the overall length of the SDFT. The digital flexor muscle fibers are extremely short and change in length by only 1 to 2 mm.34 As a result, changes in length are almost entirely attributable to stretching of the spring-like tendons via changes in the angle of the metacarpophalangeal joint and not to changes in the length of the muscle.33–35 In addition, the accessory ligament of the SDFT links the tendon distal to the muscle belly to the caudal surface of the distal aspect of the radius, which limits changes in muscle length.34 This would have been more consistent with our hypothesis that sedation would not have an effect on the PI. However, it is conceivable that small changes in muscle activity may alter the PI and that use of acoustoelastography may potentially detect subtle changes in tendon stiffness.
The clinical importance of the lower PI found after sedation is unknown; nevertheless, the use of sedation is important because it makes horses more indifferent to environmental stimulation and physical manipulation required for acquisition of an accurate acoustoelastographic image. In a recent study,20 investigators tested an in vivo protocol for measurement of stiffness gradients in the gastrocnemius tendons of anesthetized dogs. They concluded that acoustoelastography is feasible and repeatable for use in measuring stiffness gradients in clinically normal anesthetized dogs. Furthermore, investigators of another study16 found that acoustoelastography is also feasible and repeatable for evaluation of the SDFTs of clinically normal horses. However, although all horses in that study16 were sedated in a manner similar to the anesthetic regimen used for the present study, the effect of sedation on the PI was not evaluated.
In vitro studies36,37 have confirmed homogenous stiffness along the SDFT in the metacarpal region of clinically normal horses. However, regardless of sedation, results for the study reported here indicated a lower PI at 15 cm distal to the accessory carpal bone, compared with the PI at 5 and 10 cm distal to the accessory carpal bone, and a lower PI at 20 cm distal to the accessory carpal bone, compared with the PI at 5 cm distal to the accessory carpal bone. Interestingly, these locations of the lower stiffness gradients coincided with the midmetacarpal region and immediately distal to that region, which is a region exposed to high tensional forces and is the area most commonly affected by tendinitis.9 The fact that there was a difference in the PI between locations emphasized that it is important to always compare results for the same locations and, if possible, to avoid making comparisons among locations in the same limb and between limbs.
Differences among individual horses accounted for the largest variance effect in the heterogeneous population of horses in the present study. This is consistent with the previously reported wide variation in mechanical properties of SDFTs for various individual horses.38 Although age and sex potentially could have influenced this variance effect, investigators of another study16 found no effect of these factors on the PI of clinically normal horses; however, a small sample size of overall younger horses was included in that study. Although an increase in SDFT stiffness with aging has been reported39 and could have accounted for individual differences in the present study, a recent investigation40 revealed that only the interfascicular matrix becomes stiffer, whereas the mechanical properties of the entire tendon are maintained with increasing age. In addition, differences in breed, level of exercise activity, and the manner by which individual horses load the SDFT may have contributed to the large variance effect of individual horses in the present study. If it is necessary to make comparisons among horses, horses of a similar breed and with a similar level of exercise activity ideally should be compared. The other variance effects, such as sedation, examination location, and thoracic limb, were much smaller in the present study, compared with the variance effect attributable to each individual horse. Therefore, the importance of sedation on the PI in the study reported here would appear to be small for a clinical setting. We recommend that drugs used for sedation and their respective doses should be consistent during assessment and reassessment of a patient to minimize variability in PI during acoustoelastography evaluation.
The present study had several limitations, one of which was the use of only 1 sedation regimen with a specific drug combination and dosages. Effects of α2-adrenergic receptor agonists are dose related up to a maximal point, after which additional increases of the dose only increase the duration of the effect.26 Whether administration of higher doses or other drug combinations would change the PI values is unknown. Sedative effects are evident within 2 minutes after IV administration of detomidine,24 and the effects last for 30 to 200 minutes, depending on the dose.24,26 In the present study, the total time for acquisition of acoustoelastographic images, including the images acquired before and after sedation, was approximately 1 h/horse. However, the images obtained after sedation were acquired within 30 minutes after drug administration, and our experiences indicate that acoustoelastographic examination may be completed in a shorter period. Additionally, differences in age, sex, breed, and exercise regimens may be factors that could influence the PI measured by use of acoustoelastographic and need to be evaluated in future studies. Horses included in the present study had physiologically normal SDFTs as determined on the basis of history, results of thorough orthopedic and lameness evaluations, and results of a complete ultrasonographic examination. However, histologic evaluation was not performed. Despite the exclusion of horses with obvious abnormalities, subclinical tendon injury could have been present in some of the horses in the present study.
The PI of the SDFTs measured by use of acoustoelastography was decreased after administration of detomidine and butorphanol to clinically normal horses. If sedation is required to enable an ultrasonographic examination for acoustoelastography, we recommend that the sedation regimen be consistent for the same horse for each examination in a series. Because the PI was similar between limbs, but differences existed between some locations, comparisons should be made at the same locations in the same limb and between limbs. Additional studies are needed to determine the utility of acoustoelastography for detecting tendon injury and monitoring tendon healing in clinically affected patients.
Acknowledgments
Ms. Dzierzak was supported by a grant from the Merck-Merial Summer Scholars Program.
Dr. Vanderby Jr. holds a patent associated with some aspects of this concept for ultrasonographic analysis.
Presented in abstract form at the 2016 American College of Veterinary Surgeons Surgery Summit, Seattle, October 2016.
ABBREVIATIONS
| CI | Confidence interval |
| PI | Pathology index |
| SDFT | Superficial digital flexor tendon |
Footnotes
GE LOGIQ e Vet, Sound, Carlsbad, Calif.
Dormosedan, Pfizer Animal Health, New York, NY.
Torbugesic, Fort Dodge Animal Health, New York, NY.
Echosoft, Echometrix LLC, Madison, Wis.
STATA, version 14.0, StataCorp LP, College Station, Tex.
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