Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Molecular Biomedical Sciences
Non Technical Summary
Soft tissue musculoskeletal injuries in the horse are common and costly problems to the equine industry. Historically, tendons were thought to be largely avascular and the role of angiogenesis was considered minimal. However, vascularization of these structures is now known to play an important role in health, pathology and healing with histological studies showing alterations in the vasculature during pathologic states and extensive neovascularization during healing.5 Imaging studies investigating the vascularity of tendon pathology in vivo are limited in veterinary medicine, particularly using ultrasound. Regenerative therapies, specifically platelet-rich plasma (PRP), are commonly used to enhance healing1,2,3 and have been shown to improve clinical outcome in the horse by increasing neovascularization in the early stages of healing.4 Recently, low level laser therapy has been advocated to enhance healing of tendons,5 however there is little documentation describing its efficacy in horses.6 Though becoming commonplace, the combination of these two therapies effect on neovascularization has not been investigated and whether the two methods act synergistically or not is unknown.In our hospital, horses treated with both PRP and low level laser have exhibited pronounced neovascularization at the site of injury when evaluated with Doppler ultrasound. However, given the lack of research, the health and integrity of these structures is not known. The specific aim of this study is to document the quantity and distribution of neovascularization throughout the healing process of experimentally induced lesions of the equine superficial digital flexor tendon (SDFT) treated with PRP alone, PRP in combination with low level laser therapy and laser therapy alone. Our hypothesis is that tendons treated with both PRP and low level laser therapy will exhibit increased neovascularization compared to tendons treated with PRP or laser alone and that this neovascularization will correlate with improved tendon structure and healing.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The purpose of this study is to characterize the pattern of neovascularization detectable with ultrasound in experimentally induced lesions of the equine SDFT treated with PRP compared to PRP combined with low level laser and laser therapy alone throughout healing. The normal appearance of the vascularity of these conditions needs to be understood as imaging of the vasculature of tendon injuries in horses adds incredible value to the assessment of disease and enhances the understanding of pathophysiology and healing. Additionally, given the similarities between the equine SDFT and Achilles tendon, information may be extrapolated for use in humans
Project Methods
Animals: Nine horses donated to NCSU-CVM for terminal research or being culled from the NCSU-CVM reproductive herd will be included in the study. Each horse will be evaluated for lameness by a board-certified orthopedic surgeon. The forelimbs will be clipped and prepared in standard fashion and grey scale ultrasound examinations of the forelimb SDFTs will be performed by a board-certified radiologist using a MyLab 70 ultrasound system (Biosound Easote Inc. Indianapolis, IN) with a 6-18MHz linear probe. Only limbs without evidence of lameness or pathology diagnosed with ultrasound will be included in the study. Baseline grey scale examinations of normal SDFTs will consist of images obtained in longitudinal and transverse orientations every 2cm distal to the accessory carpal bone. Cross sectional area will be measured in transverse images. Elastrographic evaluations will be performed to estimate stiffness, as an additional determinate of health,24,25 throughout the study. Doppler evaluation will be performed using the same ultrasound system with the horse standing as well as while the limb is held in a non-weight bearing position. Each Doppler evaluation will consist of obtaining images in longitudinal and transverse orientation. Images from the Doppler examination will be recorded as still frames and as cine video clips for real-time evaluation. Lesion induction: Collagenase-induced lesions will be created in the tensile region of the SDFT of both forelimbs, using filter sterilized bacterial collagenase type I (Sigma, St. Louis, MO) under sedation with detomidine (Dormosedan, Zoetis, Florham Park, NJ) and local anesthesia using mepivicaine (Carbocaine V, Pfizer Inc. New York, NY).26 Using a recently developed technique27 to more accurately control the size of the tendon lesions, a physical defect will be created by longitudinally placing a curved 16 gauge 8.89cm Weiss Epidural needle with a Tuohy tip (BD, Franklin Lakes, NJ) under ultrasonographic guidance from the palmar surface of the limb. The needle will be advanced starting from 12cm distal to the accessory carpal bone (ACB), through the skin and subcutaneous tissues to the center of the SDF tendon and then advanced distally until the tip is 18cm distal to the ACB. A dual injector syringe adapter (Duploject, Baxter Healthcare, Westlake Village, CA) will be used to delivered 1300 units of collagenase in 500 units of bovine thrombin (MP Biomedical, Solon, OH) combined during injection with autogenous cryoprecipitated equine fibrinogen, for a total injected volume of 1ml over a distance of 2cm. As the needle is withdrawn, the final 3.5cm of the physical defect will be injected with thrombin (without collagenase) combined with fibrinogen.27 Horses will be administered phenylbutazone (4.4mg/kg bwt PO BID) (Phenylbutazone, Zoetis, Florham Park, NJ) for pain control.Platelet-rich plasma: Each horse will serve as its own control. The treatment of the forelimbs will be randomly assigned in each horse as follows: one forelimb treated with PRP alone and the other treated with PRP combined with laser in three horses, one forelimb treated with laser alone and the other treated with PRP combined with laser in three horses, one forelimb treated with laser alone and one forelimb treated with PRP alone in three horses. Six days post-collagenase injection (t=0), selected SDFT lesions will be injected under ultrasonographic guidance with 3mL of leukocyte-reduced PRP using the commercially available ACP kit according to the manufacturer's directions (Arthrex, Inc., Naples, FL). This leukocyte-reduced PRP kit was specifically chosen as it has been extensively demonstrated in the literature that leukocyte-reduced PRP enhances tendon healing compared to leukocyte-rich preparations.28,29Rehabilitation and monitoring: Horses will be confined to stalls with twice daily hand-walking for 30 days after the PRP is administered at the NCSU LAR unit. After 30 days, horses will be housed in small paddocks at the Equine Health Center at Southern Pines for the duration of the study, an additional 90 days. Laser therapy will be performed on assigned limbs using MR4 ACTIVet portable laser (Multiradiance Medical, Solon, OH) following the manufacturer's recommended protocol, which consists of twice daily treatments of 10min at 1000Hz, 5min at 3000Hz and 2min at 5000Hz for the first 14 days, followed by daily treatments of 5min at 250Hz, followed by 5min of 50Hz, then 3min of 1000Hz and 5min of 250Hz for one week. The latter protocol will be repeated every other day for the following week.Ultrasound examinations of each forelimb SDFT will be performed using the same ultrasound system by the same board-certified radiologist once weekly for the first 8 weeks and then every other week for the duration of the study. Clinical evaluation will be performed prior to each ultrasound and will consist of palpation to detect evidence of heat or swelling, response to palpation and evidence of lameness. Grey scale ultrasound examinations, Doppler evaluation and elastographic examinations will be performed as described above.Histology: Upon completion of the study, horses will be euthanized with intravenous pentobarbital euthanasia solution (CIII Beuthanasia-D, Schering Plough Animal Health, Union, NJ). Samples of the SDFTs evaluated will be obtained and analyzed histologically by a board certified pathologist at NCSU with special staining for endothelial markers as well as by the PIs for a series of tendon health parameters as previous reported by Schnabel et al.26 The quantity of endothelium will be compared with power Doppler images.Image analysis: Images will be stored in a DICOM format and evaluated in a dedicated DICOM viewer (eFilm, Merge Healthcare, Chicago, IL). Quality of healing will be assessed in the grey scale ultrasound images by analysis of fiber pattern organization, quantification of cross sectional area, and quantification of the stiffness as estimated using elastography.30 Blood vessels detected with power Doppler will be quantified in still images saved in .bitmap format using in house developed software by manually drawing a region of interest over each structure within the color Doppler box. The program will calculate the number of blue and red pixels within the region of interest and blood volume will be estimated proportionate to the number of these pixels.Statistical analysis: Numerical data will be analyzed using paired t-tests to compare treatments performed on different limbs of the same horses. Ordinal data (scores) will be analyzed using Wilcoxon signed rank tests to compare treatments performed on different limbs of the same horses. To compare the three treatment groups to each other, numerical data will be analyzed using ANOVA blocked by horse and ordinal data analyzed by Kruskal-Wallis. For all tests, JMP software (SAS, Cary, NC) will be used and significance set at P < 0.05.