Low-level laser therapy uses light waves with a high degree of spatial and temporal coherence to activate chromophores within the treated area. This photobiostimulation upregulates production of ATP, nitric oxide, and reactive oxygen species within cells, alters gene transcription, and leads to an increase in cell proliferation, cellular motility, and growth factor production. The biochemical mechanism of action of LLLT has yet to be fully elucidated but appears to be dependent on the wavelength of light used for tissue penetration and molecular stimulation as well as irradiance or total light dose.1–7 Low-level laser therapy has been widely studied in human medicine, where its beneficial effects include decreasing inflammation associated with mucositis and arthritis,8–14 managing joint and spinal pain,15–17 increasing neurologic function and recovery,18,19 and promoting healing of chronic wounds and tendinopathies.5,6,20,21
Despite increasing evidence of the benefits of LLLT in a wide variety of medical applications in human patients, there is a paucity of literature evaluating the use of LLLT in veterinary patients. In dogs, the use of LLLT is associated with improvement in PVFs of pelvic limbs (as measured with a pressure mat system) following TPLO,22 shortened duration to ambulation following surgical correction of intervertebral disk herniation,23 and improvement in hair regrowth for patients with noninflammatory alopecia.24 In an experimental study,25 LLLT was positively associated with osteogenesis and fibrogenesis in dogs that underwent midpalatal expansion, as evidenced by an increase in suture reorganization during and after the repair. Conversely, open wound healing did not differ significantly between dogs that did and did not undergo LLLT,26 and wound healing for both groups of dogs in that study26 was slower than that for a historical control group, which suggested that LLLT might have a detrimental systemic effect on wound healing. However, each of those experimental studies25,26 used varying LLLT protocols, with differing light doses, intensities, and wavelengths, which makes direct comparisons challenging.
Cranial cruciate ligament rupture is the most common cause of pelvic limb lameness in dogs and frequently affects middle-aged and older large-breed dogs.27 Clinical signs associated with CCLR include pelvic limb lameness and effusion, instability, and signs of pain during hyperextension of the stifle joint. Numerous surgical procedures have been developed to stabilize the affected stifle joint of dogs with CCLR, with TPLO currently being the most common such procedure performed. Although the TPLO procedure is generally well tolerated by dogs, potential complications associated with the procedure include surgical site infection, delayed union of osteotomy fragments, osteomyelitis, fibular or tibial fracture, implant complications, hemorrhage, and angular limb deformities.28
The purpose of the study reported here was to evaluate the effect of LLLT on select markers of synovial inflammation and cartilage degradation and signs of pain, function, bone healing, and osteoarthritis following TPLO in dogs with spontaneous unilateral CCLR. We hypothesized that use of LLLT would decrease signs of pain, improve use of the affected limb, enhance the environment of the affected joint, and facilitate postoperative bone healing. The null hypothesis was that measured outcomes would not differ significantly between dogs that did and did not undergo LLLT after TPLO.
Materials and Methods
Animals
All study procedures were reviewed and approved by the Washington State University Institutional Animal Care and Use Committee, and informed consent was obtained from the owners of all dogs prior to study enrollment. Dogs with a spontaneous unilateral CCLR and a stable contralateral stifle joint that were evaluated and treated at the Washington State University Veterinary Teaching Hospital were considered for study enrollment. To be included in the study, dogs had to be ≥ 1 year old and weigh ≥ 15 kg. Sexually intact and neutered dogs were eligible for study inclusion, as were dogs that had a stabilization procedure performed on the stifle joint of the contralateral limb > 6 months prior to hospital admission. Dogs enrolled in the study also had to be healthy aside from the CCLR. Sexually intact male dogs used for breeding purposes, sexually intact female dogs that were pregnant or lactating, and dogs with a history of seizures or that were fractious or otherwise uncooperative were excluded from the study. Dogs that had received short-acting systemic corticosteroids within 14 days or long-acting corticosteroids within 30 days prior to examination and those with a known sensitivity to opioids or NSAIDs were also excluded from the study.
Study enrollment
Dogs were enrolled in the study 2 weeks prior to TPLO. During the initial evaluation, each patient underwent complete physical and orthopedic examinations, which included lameness and pain evaluations, the acquisition of orthogonal radiographs of the affected stifle joint, and a CBC, serum biochemical profile, and urinalysis. Baseline values were obtained by force plate analysis and cytologic and biochemical analysis of synovial fluid obtained by synoviocentesis of the affected stifle joint. Each dog was instrumented with a 3-axis accelerometer,a which was to be worn continuously for 2 weeks before the TPLO was performed for the acquisition of additional baseline measurements.
TPLO
Two weeks after study enrollment, each dog was brought back to the veterinary teaching hospital for TPLO surgery (time 0). Each dog was premedicated with hydromorphone and dexmedetomidine. Anesthesia was induced with propofol with or without midazolam and maintained with either isoflurane or sevoflurane in 100% oxygen. Anesthetic dosages and protocols varied among dogs and were dictated by individual patient needs and the preferences of the attending anesthetists. Once anesthetized, each dog received an epidural injection of preservative-free morphine before initiation of surgery. The affected stifle joint was evaluated arthroscopically to determine the extent of damage to the cranial cruciate ligament and menisci. A partial meniscectomy was performed for dogs with meniscal tears; meniscal releases were not performed. Then a TPLO was performed as previously described.29 Following surgery, each dog underwent a standardized rehabilitation program, which included passive range-of-motion exercises and gradually increasing controlled activity over the 8-week postoperative observation period. Each dog was also instrumented with a 3-axis accelerometera for collection of postoperative data.
Treatment group assignment and treatment application
The study was a randomized controlled trial. Prior to surgery, a random number generatorb was used to randomly assign dogs to either an LLLT group or a control group. Dogs in the LLLT group were treated with a dual-probe class 2 laserc with four 5-mW diodes with a wavelength of 635 nm while hospitalized and a customized class 2 laserd with one 5-mW diode with a wavelength of 635 nm after hospital discharge. Dogs in the control group were treated with the same laser units, in which the 5-mW diodes were replaced with red LED lightbulbs. The treatment application protocol was the same for dogs in both the LLLT and control groups. Each treatment consisted of the application of the laser at the TPLO incision site on the medial aspect of the stifle joint and the L6-7 nerve root region on the side ipsilateral to the affected limb. For each dog, the assigned treatment was administered for 5 minutes immediately before and after surgery and again at 6, 12, 24, 36, 48, 60, 72, 84, and 96 hours after surgery. Following the last in-hospital treatment at 96 hours after surgery, each dog was discharged and sent home, where owners were instructed to administer the assigned treatment for 3 minutes every other day for 4 weeks. For the LLLT group, the calculated radiant exposure was 2.25 J/cm2 during each in-hospital treatment and 1.5 J/cm2 during each at-home treatment. The people who administered the in-hospital treatments and dog owners remained unware of (ie, were blinded to) the treatment group assignment for all dogs throughout the duration of the study.
Owners were required to keep a treatment journal and complete a CBPI30 at weekly intervals after surgery. The CBPI contained 4 questions that assessed signs of pain on a scale from 0 (no pain) to 10 (extreme pain), 6 questions that assessed the effects of pain on patient function on a scale from 0 (does not interfere) to 10 (completely interferes), and 1 question that assessed the patient's overall quality of life on a scale from 0 (poor) to 5 (excellent).
The same surgical team rechecked all dogs at 2, 4, and 8 weeks after surgery. During each recheck appointment, accelerometer data were downloaded, and complete physical and orthopedic examinations were performed, which included evaluation of lameness and signs of pain by use of the modified Glasgow Composite Pain Scale.31 Briefly, lameness, joint mobility, pain on palpation, contralateral limb lift, and composite well-being were each assessed on a scale from 0 (clinically normal) to 4 (worst possible; Supplementary Appendix S1, available at avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.8.893). Each dog also underwent force plate analysis, and orthogonal radiographs were obtained of and synoviocentesis was performed on the affected stifle joint during each recheck appointment.
Force plate analysis
Ground reaction force measurements were obtained by trotting dogs across 3 in-series force platese,f embedded in the center of a 14-m runway. For each dog, velocity was maintained between 1.8 and 2.0 m/s, and acceleration was maintained at ± 0.5 m/s2. Data were recorded by acquisition softwareg interfaced with a compatible desktop computer. Ground reaction force measurements during the vertical (z-axis; PVF and VI) and craniocaudal (y-axis; peak braking force, braking impulse, force, and propulsion impulse) components of the swing phase and stance phase were calculated for the affected pelvic limb. Each measurement was normalized to the body weight of the patient and expressed as a percentage.
Synovial fluid analysis
Markers of inflammation and cartilage degradation were quantified in synovial fluid samples obtained during synoviocentesis by use of validated commercially available assay kits. Synovial fluid PGE2,h TNF-α,i and IL-6j concentrations and MMP-3k activities were determined by ELISAs. The nitric oxide concentrationl was quantified on the basis of nitrate and nitrite concentrations. The sGAG concentration was quantified by use of Alcian blue dye supplied in an assay kit,m and soluble collagen was quantified by use of Sirius red dye supplied in another assay kit.n All assays were performed in accordance with the manufacturers' recommendations. All markers were determined in duplicate for each sample, and the mean was calculated and used for analysis purposes.
Radiographic assessment
Orthogonal radiographs of the affected stifle joint were obtained before and at 2, 4, and 8 weeks after surgery. All radiographs were assessed by a board-certified veterinary radiologist (RLT), who was blinded to the treatment group assignment of each dog. The radiologist used 3 modified radiographic evaluation instruments32–34 to evaluate extent of osteotomy healing, osteoarthritis, and soft tissue inflammation or reaction within the affected joints. Healing (callus formation and stage of osseous union) of the osteotomy line created during the TPLO was assessed on a scale from 0 to 4, where 0 = complete healing and 4 = no evidence of healing. The extent of osteoarthritis in the affected stifle joint was assessed on the basis of the margins of the tibial condyle, intercondylar eminence, femoral condyles and sesamoids, femoral epicondyle, and patella on a scale from 0 to 3, where 0 = no evidence of osteoarthritis and 3 = severe osteoarthritis. The extent of soft tissue inflammation was assessed on the basis of joint effusion and thickening of the joint capsule, patellar tendon, and other lateral and medial soft tissues on a scale from 0 to 3, where 0 = no inflammation and 3 = severe inflammation.
Statistical analysis
The data distributions for all continuous variables were assessed for normality by means of the Shapiro-Wilk test. Parametric variables were compared between the 2 treatment groups by use of repeated-measures ANOVA. Each ANOVA model included fixed effects for treatment group and time and a random effect for dog nested within treatment. The Akaike information criterion was used to determine the appropriate variance-covariance matrix for each model. Least squares mean differences were used to evaluate the respective associations between each outcome variable of interest and treatment group and time, and results were reported as the mean ± SD. The data for nonparametric variables were transformed, and comparisons between the treatment groups were performed with the Wilcoxon rank sum test. Results were reported as the median (IQR). All analyses were performed with commercially available software,o,p and values of P < 0.05 were considered significant.
Results
Dogs
Twelve dogs (6 spayed females and 6 neutered males) were enrolled in the study, of which 6 were assigned to the LLLT group and 6 were assigned to the control group. There were 4 mixed-breed dogs, 3 Labrador Retrievers, and 1 each of Australian Shepherd, English Mastiff, German Shepherd Dog, Golden Retriever, and Saint Bernard. Seven dogs had a darkcolored hair coat, and 5 dogs had a light-colored hair coat. The study population had a median age of 6 years (range, 2 to 10 years) and mean body weight of 35.9 kg (range, 17.1 to 60.5 kg). Neither age nor weight differed significantly between the 2 treatment groups. Body weight did not change significantly (P = 0.09) for any patient during the observation period.
Functionality
Mean accelerometric activity did not differ significantly between the LLLT and control groups at any of the measured time points (Table 1); however, there was a significant (P < 0.01) association between accelerometric activity and time. For both treatment groups, mean acceleration decreased between baseline (0 week) and 2 weeks after TPLO, then gradually increased between 2 and 8 weeks after TPLO. None of the goniometric variables differed significantly between the 2 treatment groups or over time within a treatment group except flexion of the affected stifle joint at baseline. The mean ± SD baseline flexion angle of the affected stifle joint for the LLLT group (35 ± 9°) was significantly (P = 0.03) greater than that for the control group (45 ± 5°; ie, the smaller the flexion angle, the greater the amount of flexion). All ground reaction forces measured varied significantly within treatment groups over time and between the 2 treatment groups at various times. For example, the mean ± SD baseline PVF for the LLLT group (53 ± 16%BW) was significantly (P = 0.04) greater than that for the control group (42 ± 8%BW). At 2 weeks after TPLO, all ground reaction forces for the LLLT group were significantly lower than the corresponding ground reaction forces for the control group. At 4 weeks after TPLO, all ground reaction forces for the LLLT group remained significantly lower than the corresponding ground reaction forces for the control group except PVF, which did not differ significantly (P = 0.14) between the 2 groups. However, by 8 weeks after TPLO, none of the ground reaction forces differed significantly between the LLLT and control groups.
Mean ± SD values for select acceleration, goniometric, and ground reaction force variables, and modified Glasgow composite pain scores for adult dogs with unilateral CCLR before (baseline) and at 2, 4, and 8 weeks after TPLO for stifle joint stabilization that did (LLLT group; n = 6) and did not (control group; 6) receive LLLT.
| Week after TPLO | |||||
|---|---|---|---|---|---|
| Variable | Group | Baseline | 2 | 4 | 8 |
| Accelerometric activity (mean activity counts/d) | LLLT | 134 ± 126 | 75 ± 23 | 93 ± 35 | 98 ± 31 |
| Control | 128 ± 66 | 75 ± 41 | 105 ± 38 | 119 ± 46 | |
| Goniometric variables | |||||
| Extension of affected stifle joint (°) | LLLT | 146 ± 13 | 144 ± 16 | 144 ± 8 | 141 ± 6 |
| Control | 145 ± 12 | 145 ± 7 | 148 ± 8 | 148 ± 15 | |
| Flexion of affected stifle joint (°) | |||||
| LLLT | 35 ± 9* | 41 ± 8 | 46 ± 10 | 43 ± 8 | |
| Control | 45 ± 5 | 44 ± 7 | 44 ± 6 | 39 ± 4 | |
| Ground reaction forces | |||||
| PVF (%BW) | LLLT | 53 ± 16* | 10 ± 11* | 29 ± 18 | 52 ± 11 |
| Control | 42 ± 8 | 26 ± 12 | 38 ± 10 | 48 ± 7 | |
| Peak VI (%BW) | LLLT | 6.4 ± 1.3 | 1.2 ± 1.3* | 3.7 ± 2.2* | 6.4 ± 1.5 |
| Control | 6.2 ± 0.6 | 4.6 ± 2.5 | 6.8 ± 3.1 | 8.0 ± 1.8 | |
| Stance phase (%BW) | LLLT | 211 ± 24 | 102 ± 117* | 181 ± 91* | 220 ± 31 |
| Control | 237 ± 54 | 274 ± 119 | 283 ± 106 | 247 ± 49 | |
| Y-axis braking force (%BW) | LLLT | −2.7 ± 1.5 | −0.6 ± 0.7* | −2.0 ± 1.6* | −3.5 ± 1.7 |
| Control | −2.2 ± 0.5 | −2.5 ± 1.1 | −3.6 ± 2.2 | −2.7 ± 1.3 | |
| Y-axis braking impulse (%BW) | LLLT | −0.08 ± 0.06 | −0.02 ± 0.02* | −0.08 ± 0.09* | −0.13 ± 0.09 |
| Control | −0.10 ± 0.05 | −0.12 ± 0.08 | −0.22 ± 0.24 | −0.13 ± 0.08 | |
| Y-axis propelling force (%BW) | LLLT | 6.9 ± 1.9 | 1.6 ± 1.9* | 3.0 ± 2.5* | 5.4 ± 1.5 |
| Control | 6.4 ± 1.6 | 3.7 ± 19 | 5.6 ± 1.5 | 6.4 ± 1.0 | |
| Y-axis propelling impulse (%BW) | LLLT | 0.56 ± 0.14 | 0.15 ± 0.17* | 0.25 ± 0.21* | 0.44 ± 0.13 |
| Control | 0.54 ± 0.18 | 0.38 ± 0.25 | 0.60 ± 0.30 | 0.63 ± 0.13 | |
| Modified Glasgow Composite Pain scores | |||||
| Lameness | LLLT | 3.2 ± 1.2 | 2.8 ± 1.0 | 2.0 ± 1.6 | 0.3 ± 0.5 |
| Control | 2.6 ± 1.5 | 2.3 ± 1.2 | 1.7 ± 1.0 | 0.7 ± 0.5 | |
| Joint mobility | LLLT | 0.7 ± 0.5 | 1.0 ± 0.0 | 1.2 ± 0.8 | 0.0 ± 0.0 |
| Control | 1.2 ± 0.4 | 0.8 ± 0.8 | 1.0 ± 0.9 | 0.3 ± 0.8 | |
| Signs of pain on palpation | LLLT | 1.7 ± 0.5 | 0.8 ± 0.8 | 0.5 ± 0.8 | 0.0 ± 0.0 |
| Control | 2.0 ± 0.7 | 0.3 ± 0.5 | 0.3 ± 0.5 | 0.0 ± 0.0 | |
| Willingness to have contralateral pelvic limb lifted | LLLT | 2.2 ± 0.8 | 2.2 ± 0.4* | 1.8 ± 0.8 | 0.3 ± 0.5 |
| Control | 2.6 ± 0.9 | 1.3 ± 0.5 | 1.3 ± 0.5 | 0.7 ± 0.8 | |
| Composite well-being | LLLT | 2.5 ± 0.8 | 2.2 ± 0.8 | 1.7 ± 0.8 | 0.3 ± 0.5 |
| Control | 2.4 ± 0.5 | 1.8 ± 0.8 | 1.5 ± 0.5 | 0.8 ± 0.8 | |
Dogs in the LLLT group were treated with a dual-probe class 2 laser with four 5-mW diodes with a wavelength of 635 nm while hospitalized and a class 2 laser with one 5-mW diode with a wavelength of 635 nm after hospital discharge. Dogs in the control group were treated with the same laser units in which the 5-mW diodes were replaced with red LED lightbulbs. The treatment application protocol was the same for all dogs in both the LLLT and control groups. Each treatment consisted of the application of the laser at the TPLO incision site on the medial aspect of the stifle joint and the L6-7 nerve root region on the side ipsilateral to the affected limb. For each dog, the assigned treatment was administered for 5 minutes immediately before and after surgery and again at 6, 12, 24, 26, 48, 60, 72, 84, and 96 hours after surgery. Following the last in-hospital treatment at 96 hours after surgery, each dog was discharged and sent home, where owners were instructed to administer the assigned treatment for 3 minutes every other day for 4 weeks. For the LLLT group, the calculated radiant exposure was 2.25 J/cm2 during each in-hospital treatment and 1.5 J/cm2 during each at-home treatment. Dogs were instrumented with an accelerometer continuously for 2 weeks before (baseline) TPLO and following hospital discharge to collect acceleration data. The same surgical team rechecked all dogs at 2, 4, and 8 weeks after TPLO. During each recheck appointment, accelerometer data were downloaded, and complete physical and orthopedic examinations were performed, which included evaluation of lameness and signs of pain by use of the modified Glasgow Composite Pain Scale.31 Briefly, lameness, joint mobility, pain on palpation, contralateral limb lift, and composite well-being were each assessed on a scale from 0 (clinically normal) to 4 (worst possible). Each dog also underwent force plate analysis to measure ground reaction forces.
Value differs significantly (P < 0.05) from the corresponding value for the control group.
Pain assessments
When signs of pain were assessed by the surgical team, who used the modified Glasgow composite scoring system, the mean ± SD score did not differ significantly between the LLLT and control groups for any variable assessed at any time except for contralateral limb lift at 2 weeks after surgery. At that time, dogs in the LLLT group were significantly (P = 0.04) less willing to allow the contralateral pelvic limb to be lifted than were dogs in the control group (Table 1). When signs of pain were assessed by dog owners, who used the CBPI, the mean ± SD overall score did not differ between the LLLT and control groups at any time, but significant differences were detected between the 2 groups for various aspects of the CBPI at various times throughout the observation period (Table 2). In general, when significant differences were detected between the 2 groups, the dogs in the control group had lower pain scores and improved function, compared with the dogs in the LLLT group.
Mean ± SD CBPI scores for the dogs of Table 1 as assigned by their owners.
| Week after TPLO | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| CBPI question | Group | Baseline | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Worst pain | LLLT | 6.3 ± 1.8 | 7.7 ± 0.5* | 5.2 ± 1.5 | 4.2 ± 2.1 | 4.2 ± 2.4* | 4.0 ± 2.9* | 2.7 ± 1.9 | 2.0 ± 1.9 | 1.8 ± 1.5 |
| Control | 5.8 ± 2.2 | 5.2 ± 2.6 | 4.7 ± 3.3 | 2.3 ± 1.4 | 1.8 ± 1.6 | 1.2 ± 0.8 | 0.8 ± 0.8 | 0.7 ± 0.5 | 0.2 ± 0.5 | |
| Least pain | LLLT | 2.8 ± 1.2 | 4.2 ± 1.2* | 5.0 ± 2.1* | 3.5 ± 3.0* | 2.0 ± 1.3* | 1.7 ± 1.5 | 1.0 ± 0.9 | 0.7 ± 0.8 | 0.5 ± 0.6 |
| Control | 3.0 ± 0.8 | 2.0 ± 2.1 | 1.5 ± 0.8 | 1.0 ± 0.6 | 0.5 ± 0.6 | 0.3 ± 0.5 | 0.3 ± 0.5 | 0.3 ± 0.5 | 0.2 ± 0.5 | |
| Average pain | LLLT | 4.5 ± 4.1 | 5.7 ± 1.0* | 5.2 ± 1.5* | 3.3 ± 1.9 | 3.0 ± 1.8* | 2.5 ± 1.6* | 1.7 ± 1.4 | 1.3 ± 1.4 | 1.0 ± 1.2 |
| Control | 4.5 ± 1.3 | 3.2 ± 2.0 | 2.7 ± 1.6 | 2.0 ± 1.1 | 1.0 ± 1.6 | 0.5 ± 0.6 | 0.5 ± 0.6 | 0.3 ± 0.5 | 0.2 ± 0.5 | |
| Current pain | LLLT | 4.3 ± 1.3 | 4.8 ± 1.6* | 4.7 ± 1.4* | 3.2 ± 1.8* | 2.5 ± 1.8 | 2.3 ± 1.5* | 1.2 ± 1.2 | 1.2 ± 1.5 | 1.0 ± 1.2 |
| Control | 4.1 ± 2.2 | 3.2 ± 1.9 | 1.8 ± 1.7 | 1.3 ± 1.4 | 1.0 ± 1.6 | 0.7 ± 0.8 | 0.3 ± 0.5 | 0.3 ± 0.5 | 0.2 ± 0.5 | |
| General activity | LLLT | 5.8 ± 1.9 | 5.7 ± 2.2 | 5.2 ± 1.7 | 3.2 ± 1.2 | 2.7 ± 1.6 | 2.3 ± 1.5 | 1.3 ± 1.0 | 1.3 ± 1.4 | 0.8 ± 1.0 |
| Control | 5.3 ± 3.5 | 5.7 ± 3.1 | 5.2 ± 2.3 | 2.7 ± 2.0 | 2.0 ± 1.8 | 2.0 ± 1.6 | 1.2 ± 1.2 | 1.0 ± 0.9 | 0.6 ± 0.9 | |
| Enjoyment of life | LLLT | 5.3 ± 2.3 | 5.2 ± 2.8 | 4.8 ± 2.6 | 2.8 ± 1.5 | 2.5 ± 1.5 | 2.2 ± 1.5 | 1.7 ± 1.8 | 0.8 ± 1.2 | 0.8 ± 1.0 |
| Control | 5.1 ± 2.5 | 3.8 ± 2.8 | 3.3 ± 1.4 | 2.3 ± 2.3 | 1.8 ± 1.5 | 1.2 ± 0.8 | 0.8 ± 0.8 | 0.7 ± 0.8 | 0.2 ± 0.5 | |
| Ability to rise | LLLT | 5.0 ± 1.9 | 5.2 ± 1.5 | 5.0 ± 1.7 | 3.3 ± 1.4 | 2.8 ± 1.5 | 2.5 ± 1.4 | 1.5 ± 1.1 | 1.3 ± 1.2 | 1.5 ± 1.3 |
| Control | 5.0 ± 1.7 | 4.5 ± 3.2 | 4.2 ± 1.7 | 1.7 ± 1.4 | 1.7 ± 1.2 | 1.2 ± 1.2 | 1.0 ± 1.1 | 0.8 ± 0.8 | 0.6 ± 0.9 | |
| Ability to walk | LLLT | 4.1 ± 1.7 | 5.2 ± 2.3 | 5.2 ± 2.1 | 3.0 ± 1.3 | 2.0 ± 1.8 | 1.5 ± 1.5 | 0.8 ± 0.8 | 0.7 ± 1.0 | 0.3 ± 0.5 |
| Control | 5.1 ± 1.4 | 5.3 ± 2.8 | 3.2 ± 1.2 | 2.0 ± 1.1 | 1.2 ± 0.8 | 1.0 ± 0.9 | 0.8 ± 0.8 | 0.7 ± 0.5 | 0.4 ± 0.6 | |
| Ability to run | LLLT | 6.7 ± 2.3 | 8.7 ± 1.4 | 8.0 ± 1.4 | 5.0 ± 2.2 | 4.3 ± 2.7 | 3.3 ± 2.2 | 2.7 ± 2.1 | 1.0 ± 1.1 | 0.8 ± 1.0 |
| Control | 6.6 ± 2.9 | 7.2 ± 3.6 | 7.0 ± 2.1 | 4.3 ± 4.2 | 4.4 ± 3.4 | 3.2 ± 3.5 | 2.5 ± 3.3 | 2.5 ± 3.3 | 2.4 ± 0.6 | |
| Ability to climb up | LLLT | 4.8 ± 2.5 | 6.2 ± 3.4 | 5.8 ± 3.0 | 3.2 ± 1.1 | 3.0 ± 1.4 | 2.2 ± 1.1 | 1.8 ± 0.5 | 1.0 ± 0.9 | 1.3 ± 0.6 |
| Control | 5.6 ± 2.3 | 5.0 ± 3.0 | 4.4 ± 1.3 | 3.0 ± 1.7 | 3.0 ± 2.6 | 2.2 ± 3.1 | 1.8 ± 3.1 | 1.8 ± 3.1 | 1.8 ± 3.5 | |
| Overall impression of well-being | LLLT | 2.4 ± 0.8 | 2.2 ± 0.4 | 2.7 ± 0.8 | 3.0 ± 0.6 | 3.2 ± 0.8 | 3.3 ± 0.5 | 3.7 ± 0.5 | 4.0 ± 0.6 | 4.0 ± 0.0 |
| Control | 2.6 ± 0.5 | 3.0 ± 0.9 | 3.3 ± 0.8 | 3.8 ± 0.8 | 4.0 ± 0.9 | 4.2 ± 0.8 | 4.2 ± 0.8 | 4.2 ± 0.8 | 4.0 ± 1.2 | |
The CBPI contained 4 questions that assessed signs of pain on a scale from 0 (no pain) to 10 (extreme pain), 6 questions that assessed the effects of pain on patient function on a scale from 0 (does not interfere) to 10 (completely interferes), and 1 question that assessed the patient's overall quality of life on a scale from 0 (poor) to 5 (excellent).
See Table 1 for remainder of key.
Synovial fluid and radiographic assessments
None of the markers of synovial inflammation and cartilage degradation assessed differed significantly between the LLLT and control groups at any time (Table 3). Likewise, none of the radiographic variables assessed differed between the 2 groups at any time except for the extent of soft tissue inflammation at 8 weeks after TPLO. The median soft tissue inflammation score at 8 weeks after TPLO for the LLLT group (3.0) was significantly (P = 0.04) greater (ie, worse) than that for the control group (2.0; Table 4).
Median (IQR) concentrations and activities for markers of inflammation and cartilage degradation in synovial fluid samples obtained from the affected stifle joints of the dogs of Table 1.
| Week after TPLO | |||||
|---|---|---|---|---|---|
| Variable | Group | Baseline | 2 | 4 | 8 |
| PGE2 (pg/mL) | LLLT | 2,642 (2,512–2,768) | 2,774 (2,577–2,862) | 2,531 (2,329–3,039) | 2,185 (1,852–2,770) |
| Control | 1,880 (1,462–2,657) | 2,187 (1,094–2,544) | 1,809 (418–2,236) | 1,840 (430–1,198) | |
| MMP-3 (ng/mL) | LLLT | 3.00 (2.97–3.03) | 2.98 (2.98–2.99) | 2.98 (2.96–3.00) | 2.98 (2.97–3.00) |
| Control | 3.03 (2.97–3.23) | 3.07 (3.03–4.29) | 3.36 (3.00–5.02) | 3.03 (2.98–3.91) | |
| IL-6 (pg/mL) | LLLT | 10.6 (10.1–12.8) | 12.2 (9.0–13.3) | 12.5 (11.4–17.2) | 9.8 (4.0–18.1) |
| Control | 7.4 (6.9–12.8) | 7.4 (4.5–18.9) | 14.9 (6.0–25.0) | 14.4 (8.0–104.3) | |
| Total nitrogen (μM) | LLLT | 19.7 (19.0–40.3) | 25.9 (24.0–26.8) | 24.1 (15.1–36.5) | 16.3 (15.2–26.5) |
| Control | 32.8 (28.3–33.5) | 60.7 (44.3–72.3) | 17.5 (17.1–33.9) | 28.6 (14.2–36.0) | |
| Nitrite (μM) | LLLT | 3.78 (2.35–4.04) | 16.90 (11.05–44.17) | 1.96 (0.68–9.17) | 1.77 (0.18–3.65) |
| Control | 5.21 (4.43–12.49) | 11.70 (7.68–35.40) | 9.88 (0.79–24.30) | 1.57 (0.66–9.88) | |
| Nitrate (μM) | LLLT | 15.90 (13.58–16.66) | 3.77 (−17.35–8.97) | 11.63 (10.04–23.00) | 13.95 (11.14–16.13) |
| Control | 19.63 (9.45–27.46) | 16.26 (8.94–48.29) | 17.34 (1.01–33.07) | 11.00 (4.36–35.30) | |
| sGAG (μg/mL) | LLLT | 56.4 (51.5–76.8) | 113.0 (75.2–114.9) | 63.1 (52.8–76.1) | 54.0 (43.4–67.6) |
| Control | 73.9 (71.2–77.3) | 72.0 (71.8–78.2) | 50.6 (25.5–53.1) | 48.9 (45.7–50.2) | |
| Soluble collagen (μg/mL) | LLLT | 2,102 (1,469–3,521) | 4,553 (2,957–7,672) | 4,473 (4,083–5,248) | 2,770 (2,401–2,887) |
| Control | 2,142 (913–5,606) | 2,507 (649–5,805) | 3,846 (2,819–11,693) | 2,074 (1,463–2,911) | |
| TNF-α (pg/mL) | LLLT | 1.85 (1.78–2.93) | 2.93 (1.08–3.24) | 1.78 (1.08–2.07) | 1.78 (1.18–2.32) |
| Control | 2.48 (1.71–7.10) | 1.31 (0.66–1.97) | 0.85 (0.68–1.79) | 2.55 (1.16–8.09) | |
None of the concentrations or activities differed significantly (P < 0.05) between the 2 groups at any time.
See Table 1 for remainder of key.
Median (IQR) radiographic assessment scores for the dogs of Table 1.
| Week after TPLO | |||||
|---|---|---|---|---|---|
| Variable | Group | Baseline | 2 | 4 | 8 |
| Osteotomy healing | LLLT | 4.0 (4.0–4.0) | 3.5 (3.0–4.0) | 2.0 (2.0–2.8) | 1.0 (1.0–1.8) |
| Control | 4.0 (4.0–4.0) | 3.5 (3.0–4.0) | 2.0 (2.0–2.8) | 1.0 (1.0–1.0) | |
| Severity of osteoarthritis | LLLT | 2.0 (1.3–2.0) | 1.5 (1.0–2.0) | 2.0 (1.3–2.0) | 2.0 (1.3–2.0) |
| Control | 1.5 (1.0–2.0) | 1.5 (1.0–2.0) | 1.5 (1.0–2.0) | 2.0 (1.3–2.0) | |
| Soft tissue inflammation | LLLT | 2.0 (2.0–2.0) | 2.0 (2.0–2.8) | 2.5 (2.0–3.0) | 3.0 (2.3–3.0)* |
| Control | 2.0 (2.0–2.0) | 2.5 (2.0–3.0) | 2.0 (2.0–2.8) | 2.0 (2.0–2.0) | |
Orthogonal radiographs of the affected stifle joint were obtained before TPLO and during each recheck appointment. All radiographs were assessed by a board-certified veterinary radiologist, who was blinded to the treatment group assignment of each dog. Healing (eg, callus formation and stage of osseous union) of the osteotomy line (osteotomy healing) created during the TPLO was assessed on a scale from 0 to 4, where 0 = complete healing and 4 = no evidence of healing. The extent of osteoarthritis in the affected stifle joint was assessed on the basis of the margins of the tibial condyle, intercondylar eminence, femoral condyles and sesamoids, femoral epicondyle, and patella on a scale from 0 to 3, where 0 = no evidence of osteoarthritis and 3 = severe osteoarthritis. The extent of soft tissue inflammation was assessed on the basis of joint effusion and thickening of the joint capsule, patellar tendon, and other lateral and medial soft tissues on a scale from 0 to 3, where 0 = no inflammation and 3 = severe inflammation.
See Table 1 for remainder of key.
Discussion
In the present study, few significant differences were detected between dogs with unilateral CCLR that did (LLLT group) and did not (control group) receive LLLT after TPLO to stabilize the affected stifle joint. Function of the affected pelvic limb (as determined by force place analysis and measurement of ground reaction forces) at 2 and 4 weeks after surgery was poorer for dogs of the LLLT group, compared with that for the dogs in the control group. When signs of pain were assessed by the surgical team and use of the modified Glasgow composite pain scoring system,31 no significant differences were identified between the LLLT and control groups at any time during the observation period. However, when signs of pain were assessed by dog owners and use of the CBPI, significant differences between the 2 groups were occasionally observed, and in those instances, the dogs in the control group generally had lower pain scores or improved limb function, compared with dogs in the LLLT group. Therefore, we failed to reject (ie, accepted) our null hypothesis that the outcome variables assessed would not differ between dogs that did and did not receive LLLT following TPLO.
Accelerometry involves detection of motion by a gyroscope and is used to assess the general activity level of patients.35–39 In the present study, accelerometric activity was not significantly associated with treatment group but was significantly associated with time. In general, for dogs of both treatment groups, activity decreased from baseline (preoperative) levels immediately after surgery, then gradually increased during the remainder of the observation period. This was an expected finding for patients recovering from TPLO and provided evidence that the physical rehabilitation program prescribed for all dogs was beneficial. Use of accelerometry to monitor activity of canine patients in their home environments is fairly new, 37,38 and these findings provided credence that it is a sensitive tool for monitoring the activity of dogs.
Goniometry has been used in multiple studies40–48 to assess pelvic limb function of dogs with CCLR following surgical stabilization of the affected stifle joint and can detect functional deficits even after ground reaction forces have returned to within reference limits.49 A change in stifle joint flexion or extension > 10° is positively correlated with clinical lameness score.46 In the present study, although the mean ± SD extent of flexion for the affected stifle joint for dogs of the LLLT group (35 ± 9°) was significantly (P = 0.03) greater than that for dogs of the control group (45 ± 5°) at baseline, that difference was not clinically relevant. Cranial cruciate ligament rupture decreases the range of motion for the stifle joint.48 Surgical stabilization of CCLR-affected stifle joints generally improves their flexion and extension40,41,43,45,47; however, no significant changes in stifle joint flexion or extension were observed over time for the dogs of the present study. That might have been the result of insufficient power to detect significant changes owing to the small sample size or simply reflected the subjective nature of goniometric measurements.50
Force plate analysis is the gold standard for objective assessment of limb use and outcomes following surgical stabilization of cranial cruciate ligament–deficient stifle joints.40,41,49,51–58 In the present study, nearly all ground reaction force variables differed significantly between the LLLT and control groups at 2 and 4 weeks after surgery, and the actual measurements indicated that function of the affected limb for dogs of the control group was better than that for dogs of the LLLT group at those assessment times. This suggested that LLLT had a detrimental effect or the control treatment (red light) had a beneficial effect on return of limb function. Collectively, the significant differences in ground reaction force variables observed between the 2 treatment groups might have been representative of a clinically relevant difference in the healing process, but as healing and limb use progressed, that effect was no longer significant by 8 weeks after surgery. Although the ground reaction force and stance phase variables for the control group were, in general, significantly greater than those for the LLLT group at 2 and 4 weeks after surgery, the clinical relevance of those results should be interpreted cautiously because the clinical lameness and pain scores determined by use of the modified Glasgow pain scoring system did not differ between the 2 groups at 2 and 4 weeks after surgery.
Both the CBPI and modified Glasgow pain scoring system have been validated for evaluation of pain in dogs after surgery.30,31,52 In the present study, the mean pain scores as assigned by use of both the CBPI and modified Glasgow pain scoring system for the control group were often lower (ie, signs of pain were less severe), sometimes significantly, than the corresponding mean scores for the LLLT group during the first 5 weeks after surgery. As previously mentioned, those differences might have reflected a possible detrimental effect of the LLLT or beneficial effect associated with the control treatment.
In both human and veterinary medicine, LLLT is a still-developing treatment modality with few robust guidelines established for best practices or standardized protocols for target tissues or disease processes.20,59–61 The medical literature contains descriptions of LLLT protocols that involve use of a wide variety of wavelengths, powers, and application frequencies and durations, which makes it difficult to compare results among studies. However, results of in vitro studies1,3,60,61 indicate that tissues have a biphasic dose response to LLLT, which follows the Arndt-Schulz curve. The Arndt-Schulz curve illustrates the effects of varying irradiance and treatment time on stimulatory or inhibitory biological responses. Although laser unit manufacturer–recommended settings are typically used for LLLT, protocols for specific tissues have not been established. It is possible that the LLLT protocol used for the dogs of the present study caused an inhibitory biological response, which was reflected by the increase in pain scores and diminished ground reaction forces relative to those at baseline observed during the immediate postoperative period. Results of some studies61,62 suggest red LED light, as was used for the control group of the present study, might have a beneficial effect on wound healing. The red LED lightbulbs used in the laser units for treatment application to the dogs of the control group emitted light with a lower wavelength that could not penetrate tissues or stimulate molecules as well as the light emitted by the 5-mW diodes used in the laser units for treatment application to the dogs of the LLLT group. It is possible that beneficial effects of the low-wavelength light emitted by the red LED lightbulbs on stifle joint healing outweighed those of the light provided by the 5-mW laser diodes. In other words, the light emitted from the red LED lightbulbs might have confounded our results. Investigators of similar future studies may want to consider the use of no light as a control treatment; however, that would prevent the individuals responsible for administering the treatments from being blinded to the treatment group assignment and potentially introduce bias or limit the feasibility of certain assessments.
Several synovial markers of inflammation and cartilage degradation have been assessed for humans and various veterinary species in both in vitro and in vivo studies,63–84 and the diagnostic sensitivity and specificity of those markers vary. For patients in the early stages of osteoarthritis, radiography is an insensitive method for detection of intra-articular molecular changes prior to the development of radiographically evident cartilage and bone abnormalities characteristic of the disease85; therefore, identification of biomarkers that are indicative of the early stages of osteoarthritis is desirable from a clinical standpoint. Concentrations of the inflammatory markers PGE2, IL-6, nitric oxide, and TNF-α63–71,75–78 and activities of cartilage degradation enzymes and products such as sGAG, soluble collagenase, and MMP-3 increase in the synovial fluid of joints affected by osteoarthritis.65,72,73,81 Attenuation of the synovial concentrations and activities of those markers of inflammation and cartilage degradation helps ameliorate osteoarthritis.63–65,68–71 That, in addition to the fact that LLLT has been suggested to have anti-inflammatory activity,86 was why we chose to measure those markers for the dogs of the present study. However, none of the biomarkers assessed differed significantly between the LLLT and control groups at any sampling point, which suggested that neither the LLLT nor the red LED light used for the control treatment had a significant effect on inflammation and cartilage degradation associated with osteoarthritis during the 2 months immediately following TPLO to stabilize the affected stifle joints of dogs with CCLR. Those findings appear to contradict the results of other in vivo studies,8–15 which suggest that LLLT has anti-inflammatory effects and modulates cartilage degradation in arthritic joints. Possible reasons for the apparent conflict between results of the present study and those of other studies8–15 include differing LLLT protocols and insufficient power owing to the small sample size of the present study.
The criteria outlined by the radiographic scoring systems32–34 used in the present study were designed to detect fairly progressive and marked bone and soft tissue changes. The dogs of the present study were evaluated for only 8 weeks after TPLO; therefore, it was not surprising that the radiographic scores did not differ significantly between the LLLT and control groups at any time except for the soft tissue inflammation score at 8 weeks after TPLO. At that time, the median soft tissue inflammation score for the LLLT group (3.0) was significantly (P = 0.04) greater than that for the control group (2.0), which suggested LLLT might have a proinflammatory effect. However, that was not substantiated by or consistent with clinical assessments or synovial fluid biomarker results. The radiographic scoring criteria included evaluation of the extent of joint effusion and thickening of the joint capsule, medial and lateral soft tissues, and patellar tendon. It was expected that radiographically evident inflammation of any of those structures would be positively correlated with inflammatory biomarker concentrations in synovial fluid. Radiographically evident bone and soft tissue changes characteristic of osteoarthritis have been described for dogs with CCLR following stifle joint stabilization in other studies40,87–89 but were not observed for the dogs of the present study. The lack of radiographic changes for the dogs of this study might have been a function of the short postoperative observation period or small sample size. Although those limiting factors must be considered during interpretation of the results of the present study, the collective lack of significant differences observed between the dogs of the LLLT and control groups suggested that the LLLT protocol used in this study did not have demonstrable anti-inflammatory and cartilage-sparing effects for the stifle joints of dogs with CCLR following TPLO when applied for 8 weeks after surgery.
To our knowledge, prior to the present study, there was only 1 other study22 in which the effects of LLLT were evaluated in dogs undergoing TPLO. In that study,22 27 dogs were randomly assigned to receive a single dose of LLLT (wavelength, 800 to 900 nm; power, 6 W; radiant exposure, 3.5 J/cm2) or sham treatment over the surgical site prior to TPLO. For each patient, clinical behavior, lameness, response to manipulations, force plate analysis, and radiographic evaluation of the affected joint were assessed before and at 24 hours and 2 and 8 weeks after surgery.22 The only significant finding in that study22 was that PVF of the affected limb at 8 weeks after TPLO was significantly greater for dogs in the LLLT group, compared with that for sham-treated dogs; none of the other variables evaluated, including VI, differed significantly between the 2 groups at any assessment time. In the present study, the mean PVF of the affected limb for the LLLT group was significantly lower than that for the control group at 2 and 4 weeks after surgery but did not differ significantly from that for the control group at 8 weeks after surgery. However, given the differences in timing, power, dose, wavelength, frequency, and duration of the LLLT protocols between that study22 and the present study, comparisons between the 2 studies may be inconsequential.90 Additionally, patient activity was not assessed in the other study,22 whereas it was assessed by means of accelerometry in the present study. Early return to function after surgery is beneficial to outcome, and lack of activity assessment for the dogs of that study22 may have obscured assessment of the effects of the LLLT owing to differences in the extent of movement and ambulation among individual dogs.89,91 Nonetheless, as in the present study, the number of dogs evaluated at 8 weeks after TPLO in the other study22 was small (n = 20); therefore, type II error was possible in both studies. Although both the present study and the other study22 were underpowered, the collective results of the 2 studies suggested that ground reaction forces as determined by force plate analysis may be the most reliable objective variables for assessment of limb function following LLLT.92–95 Our conclusions are in agreement with those of the investigators of the other study22; further investigation is necessary to assess whether LLLT has any beneficial effects for dogs undergoing TPLO and, if it does, to determine the optimal LLLT protocol.
In a study by Draper et al,23 LLLT decreased the duration to ambulation following surgery for dogs with thoracolumbar intervertebral disk disease that underwent hemilaminectomy (n = 17), compared with that for similar dogs that did not receive LLLT (18). However, in a study by Bennaim et al96 in which a similar LLLT protocol was used, LLLT appeared to have no effect on the duration of recovery for dogs with thoracolumbar intervertebral disk disease that underwent hemilaminectomy (n = 12), relative to that for dogs that received a control treatment that consisted of LED light (11). For the LLLT group of both studies,23,96 a laser unit with five 200-mW diodes with a wavelength of 810 nm was used to deliver approximately 2 to 8 J/cm2 to the target tissues for 1 minute daily for 5 days after surgery. However, the dogs of the Bennaim et al96 study received an additional LLLT treatment immediately after surgery, whereas the dogs in the Draper et al23 study did not. Moreover, physical rehabilitation was specifically controlled for both treatment groups in the Bennaim et al96 study but was not detailed in the Draper et al23 study. It is possible that the differences in the number of LLLT treatment applications, observation duration (> 15 days after surgery for the Draper et al23 study vs 10 days for the Bennaim et al96 study), and use of an LED control treatment in the Bennaim et al96 study contributed to the conflicting conclusions regarding the effects of LLLT between the 2 studies.23,96 Differences in LLLT protocols will affect tissue penetration and assessment of the overall effect of LLLT among studies. Given the apparent beneficial effects of the LLLT protocols used in the Rogatko et al22 and Draper et al23 studies, it appears that LLLT protocols that provide a higher dose of light with a greater wavelength for a shorter duration than the LLLT protocol used in the present study should be evaluated to assess potential beneficial deep tissue effects of that modality in veterinary patients.
The present study had multiple limitations. One was the lack of established LLLT protocols for veterinary patients to provide guidance for protocol development. Also, because of the randomized nature of the study, the SDs for the measured variables tended to be fairly large, which suggested there was a high risk for type II error. The small study population increased the possibility that outliers could overly influence estimates, although obvious outliers were not identified during any of the analyses. A study with a larger population than that of the present study would allow for control of the effects of patient body condition score, limb circumference, disease duration, partial or complete CCLR, meniscal injury, and concurrent subclinical disease on measured outcomes; increase statistical power; and decrease the risk for type II error.
Results of the present study suggested that LLLT had no beneficial effects on clinical signs of pain or pelvic limb function following TPLO for dogs with CCLR. In fact, variables associated with signs of pain were generally decreased (ie, indicative of less pain) and those associated with function of the affected limb were generally increased (ie, indicative of improved function) for control dogs that did not receive LLLT relative to those for dogs that received LLLT during the 4 weeks immediately following surgery. Those findings suggested that, for the dogs of this study, LLLT had detrimental effects or the control treatment (red LED light) had beneficial effects that exceeded those of the LLLT. Further studies with study populations greater than that evaluated in the present study are necessary to improve the power to detect significant differences, to assess whether LLLT is beneficial for dogs with CCLR following TPLO, and, if it is, to determine the optimal LLLT protocol.
Acknowledgments
Supported by Erchonia Laser Healthcare through the Washington State University Comparative Orthopedic Research Laboratory. The investigators did not receive any remuneration, and Erchonia Laser Healthcare was not involved in the study design; collection, analysis, and interpretation of data; writing of the manuscript; or decision to submit the manuscript for publication. The authors had full access to all study data and take complete responsibility for the integrity of the data and accuracy of the data analysis.
Presented as a Mark Bloomberg Resident Research Award abstract at the Veterinary Orthopedic Society Conference, Breckenridge, Colo, March 2014.
The authors thank Kelly Nansen, MS; Nicole Kuhn; Tyler Hudacek; and Ben Spall, DVM, MS, for technical assistance and Joe Hauptman, DVM, MS, for assistance with statistical analyses.
ABREVIATIONS
| %BW | Percentage of body weight |
| CBPI | Canine Brief Pain Inventory |
| CCLR | Cranial cruciate ligament rupture |
| IL-6 | Interleukin-6 |
| IQR | Interquartile (25th to 75th percentile) range |
| LED | Light-emitting diode |
| LLLT | Low-level laser therapy |
| MMP-3 | Metalloproteinase-3 |
| PGE2 | Prostaglandin E2 |
| PVF | Peak vertical force |
| sGAG | Sulfated glycosaminoglycan |
| TNF-α | Tumor necrosis factor α |
| TPLO | Tibial plateau leveling osteotomy |
| VI | Vertical impulse |
Footnotes
Respironics Inc, Murrysville, Pa.
Research Randomizer, version 4.0, Geoffrey C. Urbaniak and Scott Plous, Middletown, Conn. Available at: www.randomizer.org. Accessed Jan 30, 2012.
3LT PL5000, Erchonia Lasers Ltd, Wallingford, England.
THL, Erchonia Lasers Ltd, Wallingford, England.
OR6-6, Advanced Medical Technology Inc, Watertown, Mass.
OR6-7, Advanced Medical Technology Inc, Watertown, Mass.
Acquire, Sharon Software Inc, DeWitt, Mich.
PGE2 ELISA kit, Cayman Chemical Co, Ann Arbor, Mich.
TNF-α (human) ELISA kit, Cayman Chemical Co, Ann Arbor, Mich.
IL-6 (human) ELISA kit, Cayman Chemical Co, Ann Arbor, Mich.
Human MMP-3 ELISA, RayBiotech Inc, Norcross, Ga.
Nitric Oxide Quantitation Kit, Active Motif Inc, Carlsbad, Calif.
Kamiya Biomedical Co, Seattle, Wash.
Sircol Soluble Collagen Assay, Biocolor Ltd, Newtownabbey, Northern Ireland.
Excel, version 15, Microsoft Corp, Redmond, Wash.
SAS Analytics, version 9.4, SAS Institute Inc, Cary, NC.
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