The effectiveness of LLLT for the treatment of musculoskeletal conditions such as tendon injuries or osteoarthritis is not universally accepted, as studies1,2 have failed to unequivocally show clinical benefit. Reported positive effects include decreased inflammation,3 decreased pain perception,4,5 improvement of delayed wound healing,6 and improvement in healing of injured deeper tissues such as tendons.2 The cellular and molecular mechanisms underlying these effects have not been established. However, it is thought that absorption of light energy by cellular components triggers a chain of chemical reactions, resulting in altered cellular metabolism—a process termed photobiostimulation or photobiomodulation.7,8 For example, LLLT increased ATP production and oxygen consumption in cells cultured in vitro,9,10 which may be explained by absorption of laser light (wavelength, 600 to 1,000 nm) by cytochrome c,11,12 a component of the mitochondrial electron transport chain. Further, absorption of light energy by cytochrome b and flavoproteins of the cell membrane-bound nicotinamide adenine dinucleotide phosphate (reduced form [NADPH]) oxidase complex13 has been suggested to induce subtle changes in cells’ redox potential by promoting the production of reactive oxygen species,13–15 resulting in altered gene expression.16
Although in vitro investigations in other species have suggested possible benefits of LLLT in the treatment of tendon injuries, such as promotion of fibroblast proliferation,17 increased decorin and type I collagen gene expression in tenofibrocytes,17 and stimulation of tenocyte migration,18 LLLT failed to improve the histologic appearance of experimentally induced tendon lacerations in horses.19 Also, LLLT did not appear to affect clinical outcome in racehorses with superficial digital flexor tendon injuries.20 These negative results may be attributable to energy doses (termed fluence [measured in J/cm2]) of LLLT that were inappropriate. The relationship between laser fluence and biological response appears to be biphasic, with low fluence associated with biostimulatory effects and high fluence associated with no or negative effects, emphasizing the importance of appropriate fluence selection in LLLT.21 Alternatively, it is possible that LLLT is ineffective in equids because of unknown species-specific factors.
When attempting to affect deeper tissues with LLLT, translation of effective in vitro fluence to the in vivo setting is hampered by loss of laser energy via reflection, absorption, and scattering by the skin. Preparation of skin and hair has been shown to influence low-level laser energy penetration through horse limbs, with clipping of hair followed by cleaning the skin with alcohol allowing greatest energy penetration through the tissues.22 However, it is unknown how much laser energy is transmitted through equine skin. In the only previous study22 to investigate the penetration of laser light through equine tissues, measurements were made with the laser beam penetrating the entire distal portion (from lateral to medial) of the equine limbs. Thus, the objective of the study reported here was to measure penetration efficiency of low-level laser energy through equine skin and to determine what fraction of delivered laser energy is absorbed by the superficial and deep digital flexor tendons of horses. We hypothesized that, similarly to what has been shown previously in vivo, skin preparation influences penetration of laser energy through skin ex vivo. We further hypothesized that hair color, skin color, and skin thickness influence penetration of laser energy through equine skin ex vivo.
Materials and Methods
Laser unit and measurement setup
A class IV therapeutic lasera with wavelength capabilities of 800 and 970 nm was used at its minimum beam diameter (12 mm) and at a constant distance (9.0 cm) from a photodetector. Results of preliminary experiments indicated that the distance between the photodetector and the laser did not influence energy penetration efficiencies. The photodetector was a silicon PIN photodiodeb with an active region of 9.7 × 9.7 mm, reverse biased by a 9.3-V battery in series with a 100-Ω load resistor. To assure a sample area smaller than the active region of the photodetector, a 5-mm-diameter aperture in a 0.5-mm-thick aluminum plate was centered over the photodetector. To protect the detector from contamination by the tissue samples, a film of plastic wrapc was placed over the aluminum plate, and tissue samples were situated directly over the aperture onto the plastic wrap.
The optical signal was quantified by measuring the voltage produced across the load resistor by the detector photocurrent. The voltage across the load resistor was connected to the 1ΜΩ input of a direct current-coupled 100-MHz oscilloscope.d With a time sweep of 500 μs/major division and a reduced bandwidth of 20 MHz, and by continuously averaging 16 sweeps to reduce the noise level, each measurement was recorded on 3 occasions and a mean value calculated. The signal level for the 100% laser signal with no tissue sample but with clean plastic wrapc and aperture in place was maintained < 8 V to assure detector linearity at all signal levels.
Collection of tissue samples
Tissue samples were collected from a randomly chosen forelimb of each of 19 horses euthanized by means of an IV overdose (≥ 1 mL/4.5 kg of body weight) of pentobarbital and phenytoine for reasons unrelated to the investigation. Breeds included American Quarter Horse (n = 11), Thoroughbred (4), warmblood (3), and Miniature horse (1). All horses were ≥ 2 years of age. One sample per horse of grossly healthy-appearing skin with normal hair coat and superficial and deep digital flexor tendons was collected en bloc from the middle to distal third of the metacarpal region (minimum proximal to distal length, approx 7 cm), dissected to separate skin and both tendons, and frozen at −20°C. All samples (n = 19) were allowed to thaw to room temperature (approx 21°C) before measurements were obtained.
Skin samples
Tissue thickness of shaved skin was determined in the area of laser application with a dial caliper.f Perceived colors of hair and skin were recorded separately. For data analysis involving measurements with hair present, samples were categorized into 3 groups on the basis of hair color (light, medium, or dark). For data analysis involving measurements of clipped or shaved skin, samples were categorized into 3 groups on the basis of subjectively perceived skin pigmentation (light, medium, or dark).
To ascertain that subjectively perceived skin color was consistent with more objective measures of skin pigmentation and to provide a continuous independent variable for skin pigmentation, the percentage of scattered light at the 750-nm wavelength was measured for all shaved skin samples. For these measurements, the wavelength of 750 nm was selected because it is the visible wavelength23 that is closest to the wavelengths of the therapeutic laser used in this study. The percentage of scattered light was determined by directing a white-light quartz halogen microscope illuminatorg onto the skin from near-normal incidence and a distance of 10 cm. The scattered light spectrum from 400 to 800 nm was collected at near-normal incidence with a 250-µm-diameter optical fiber and miniature spectrometer.h The 100% reference scattering spectrum was measured by use of a sheet of bright white paperi instead of a skin sample. Subsequently, skin samples were ordered (highest to lowest) on the basis of the percentage of scattered light at the 750-nm wavelength (Table 1). It was apparent from this ordering that grouping based on subjectively perceived skin color was adequate to reflect measured skin pigmentation at a wavelength of 750 nm. The percentage of scattered light at a wavelength of 750 nm for white-colored skin samples (group designated as light-colored skin; n = 4) was 66.0% to 48.9%. For dark brown–, gray-, and all dark gray–colored skin samples but 1 (group designated as medium-colored skin; n = 9), the percentage of scattered light at a wavelength of 750 nm was 25.0% to 9.6%. For 1 dark gray– and the black-colored skin samples (group designated as dark-colored skin; n = 6), the percentage of scattered light at a wavelength of 750 nm was 4.7% to 3.2%.
Characteristics of 19 equine skin samples collected from 1 metacarpal area of each of 19 equine cadavers and used to determine skin penetration of laser energy at 3 different wavelength settings (800 nm, 970 nm, and both 800 and 970 nm emitted simultaneously).
| Percentage of scattered light at 750 nm for each sample | Perceived skin color | Assigned skin color group | Perceived hair color | Assigned hair color group | Skin thickness (mm) |
|---|---|---|---|---|---|
| 66.0 | White | Light | White | Light | 2.6 |
| 64.2 | White | Light | White | Light | 1.6 |
| 52.1 | White | Light | White | Light | 2.8 |
| 48.9 | White | Light | Palomino | Light | 2.7 |
| 24.9 | Gray | Medium | Dark chestnut | Medium | 2.5 |
| 23.1 | Gray | Medium | Gray | Medium | 3.5 |
| 21.1 | Dark brown | Medium | Chestnut-sorrel | Medium | 2.4 |
| 18.2 | Gray | Medium | Brown | Medium | 1.6 |
| 12.6 | Gray | Medium | Palomino | Medium | 2.7 |
| 12.3 | Gray | Medium | Light brown | Medium | 1.5 |
| 12.1 | Dark gray | Medium | Light gray | Light | 1.9 |
| 9.7 | Dark gray | Medium | Mixed light and dark brown | Medium | 1.7 |
| 9.6 | Gray | Medium | Mixed black, gray, and white | Medium | 1.5 |
| 4.7 | Dark gray | Dark | Black with single cream and brown-colored hairs | Dark | 1.8 |
| 4.6 | Black | Dark | Black | Dark | 1.6 |
| 4.1 | Black | Dark | Black | Dark | 2.5 |
| 3.9 | Black | Dark | Black | Dark | 2.0 |
| 3.6 | Black | Dark | Black | Dark | 1.9 |
| 3.2 | Black | Dark | Black | Dark | 2.4 |
Grossly healthy-appearing skin with normal hair coat and superficial and deep digital flexor tendons were collected en bloc from the middle to distal third of the metacarpal region and then dissected to separate skin and both tendons. Subjectively perceived hair and skin colors of samples were recorded, and then the percentage of scattered light at the 750-nm wavelength was measured for skin samples after shaving and cleaning with water. Those values were ordered (highest to lowest) and confirmed that sample grouping based on subjectively perceived skin color was adequate to reflect measured skin pigmentation at a wavelength of 750 nm.
Experimental conditions
Optical transmission measurements were made for continuous wave laser power of 1.0 W at 800 nm, at 980 nm, and then at 800 and 980 nm emitted simultaneously. Measurements at these wavelengths were performed without any tissue between the laser and the photodetector (100% reference laser energy) and then with skin with the natural hair cleaned with water to determine the influence of hair color on energy penetration through haired skin samples. Subsequently, to determine the effect of skin preparation on energy penetration, measurements of skin-only samples were repeated after clippingj the hair and cleaning the skin with water and then after shavingk the hair and cleaning the skin with water in the same location as previous measurements. Measurements from shaved-skin-only samples were then used to determine the effects of skin color, skin thickness, and laser energy wavelength on penetration of laser energy.
Laser energy absorption by digital flexor tendons
To calculate the percentages of laser energy absorbed by shaved skin, superficial digital flexor tendons, and deep digital flexor tendons, measurements were obtained from shaved skin, shaved skin with the superficial digital flexor tendon arranged in anatomically correct orientation, and shaved skin with the superficial and deep digital flexor tendons arranged in anatomically correct orientation. The respective values were expressed as a percentage of reference laser energy, which was measured without any tissue between laser and photodetector. For the purpose of these calculations (Appendix 1), it was assumed that all energy that did not penetrate tissue placed between the laser and the detector was absorbed by the tissue or tissues of interest.
Data analysis
Penetration of laser energy through tissues was determined as a percentage of the laser energy detected without any tissue between the laser and the photodetector at the same laser settings. Values are reported as mean ± SEM to provide an estimate of the precision of the sample mean in relation to the population mean. Statistical analysis to determine differences in laser energy penetration through equine skin under the various experimental conditions was performed by ANOVA and post hoc F-protected t tests.1 Association between tissue thickness and laser energy penetration was determined by multiple linear regression analysis,m,n with wavelength and percentage of scattered light at 750 nm in the model. Significance was set at a value of P < 0.05.
Results
When the beam was directed through skin-only samples prior to clipping or shaving, the subjectively determined hair color significantly influenced energy penetration at all 3 wavelength conditions (ie, continuous wave laser power of 1.0 W at 800 nm, at 980 nm, and then at 800 nm and 980 nm emitted simultaneously). Energy penetration through skin with light-color haired was higher, compared with skin samples with medium- or dark-colored hair (all P < 0.001). For skin samples with light-colored hair, mean ± SEM percentage laser energy penetration (compared with laser energy penetration in the absence of tissue) was 14.9 ± 3.6%, 11.3 ± 2.1%, and 13.3 ± 2.7% at wavelengths of 800 nm and 970 nm and at both wavelengths emitted simultaneously, respectively. For skin samples with medium-colored hair, mean percentage laser energy penetration was 0.3 ± 0.1%, 0.8 ± 0.2%, and 0.6 ± 0.2% at wavelengths of 800 nm and 970 nm and at both wavelengths emitted simultaneously, respectively. For skin samples with dark-colored hair, mean percentage laser energy penetration was 0.04 ± 0.1%, 0.6 ± 0.6%, and 0.1 ± 0.1% at wavelengths of 800 nm and 970 nm and at both wavelengths emitted simultaneously, respectively.
Skin preparation had a significant effect on laser energy penetration through equine skin samples (Figure 1). For skin samples with subjectively determined light- or medium-colored hair, penetration was increased after clipping or shaving of the hair, compared with the effect of only cleaning the hair, in all wavelength settings. However, for skin samples with subjectively determined dark-colored hair, this effect was detected at only the 970-nm wavelength setting.
Mean ± SEM laser energy penetration (expressed as a percentage of laser energy detected without any tissue between laser and photodetector) of equine skin samples with light-colored hair (n = 5; A), medium-colored hair (8; B), or dark-colored hair (6; C) at 3 different wavelength settings (800 nm, 970 nm, and both 800 and 970 nm emitted simultaneously). A class IV therapeutic laser with wavelength capabilities of 800 and 970 nm was used at its minimum beam diameter (12 mm) and at a constant distance (9.0 cm) from a photodetector. Measurements of laser energy penetrating skin from 1 metacarpal area of each of 19 equine cadavers were obtained first with hair present that was cleaned with water (light gray bars), then after hair was clipped and skin was cleaned with water (gray bars), and finally after hair was shaved and skin was cleaned with water (black bars). Repeated measurements were performed in the same location as previous measurements. *Value for samples with cleaned hair present is significantly (P < 0.001) less than the value for the same samples after clipping or shaving. †Value for samples with cleaned hair present is significantly (P < 0.01) less than the value for the same samples after clipping or shaving.
Citation: American Journal of Veterinary Research 77, 9; 10.2460/ajvr.77.9.991
For shaved skin samples, subjectively determined skin color significantly influenced laser energy penetration at all 3 wavelength settings (Figure 2). Laser energy penetration of light-colored skin was greater than that of medium- or dark-colored skin. Laser energy penetration of medium-colored skin was greater than that of dark-colored skin.
Mean ± SEM laser energy penetration of the equine skin samples in Figure 1 classified on the basis of skin color (light-colored skin [n = 4; light-gray bars], medium-colored skin [9; gray bars], or dark-colored skin [6; black bars]) at 3 different wavelength settings (800 nm, 970 nm, and both 800 and 970 nm emitted simultaneously). First, subjectively perceived skin color (white, gray, dark gray, dark brown, or black) of samples was recorded, and then the percentage of scattered light at the 750-nm wavelength was measured for samples after shaving and cleaning with water. The values were ordered (highest to lowest) and confirmed that sample grouping based on subjectively perceived skin color was adequate to reflect measured skin pigmentation at a wavelength of 750 nm. The group designated as the light-colored skin sample group included samples with skin color perceived as white. The group designated as the medium-colored skin sample group included samples with skin perceived as dark brown and gray. The group designated as the dark-colored skin sample group included 1 sample perceived as dark gray and the samples perceived as black. *,†,‡Within a given wavelength setting, different superscript symbols indicate significant (all P < 0.001) differences between the means. See Figure 1 for key.
Citation: American Journal of Veterinary Research 77, 9; 10.2460/ajvr.77.9.991
Wavelength setting (800 nm, 980 nm, or 800 and 980 nm emitted simultaneously) had a significant effect on laser energy penetration in the shaved skin samples (Table 2). For shaved light-colored skin samples, laser energy penetration at a wavelength of 800 nm was more efficient than that achieved at a wavelength of 970 nm. For shaved medium- and dark-colored skin samples, laser energy penetration at a wavelength of 970 nm was more efficient than that achieved at a wavelength of 800 nm. With the combined wavelength setting, the efficiency of laser energy penetration was intermediate for shaved skin samples of all colors.
Mean ± SEM laser energy penetration (expressed as a percentage of laser energy detected without any tissue between laser and photodetector) at 3 different wavelength settings (800 nm, 970 nm, and both 800 and 970 nm emitted simultaneously) through shaved equine skin samples in Table 1 that were classified as light-, medium-, or dark-colored skin.
| Skin color | Wavelength (nm) | Mean percentage laser energy penetration | P value |
|---|---|---|---|
| Light (n = 4) | 800 | 25.1 ± 2.1a | < 0.001 |
| 970 | 19.1 ± 1.7b | ||
| 800 and 970 combined | 21.9 ± 1.6c | ||
| Medium (n = 9) | 800 | 5.9 ± 0.8a | < 0.001 |
| 970 | 9.0 ± 1.3b | ||
| 800 and 970 combined | 7.5 ± 1.0c | ||
| Dark (n = 6) | 800 | 1.2 ± 0.4a | 0.006 |
| 970 | 3.5 ± 0.9b | ||
| 800 and 970 combined | 2.4 ± 0.7a,b |
A class IV therapeutic laser with wavelength capabilities of 800 and 970 nm was used at its minimum beam diameter (12 mm) and at a constant distance (9.0 cm) from a photodetector.
Within a given skin color group, values with different letters are significantly different.
See Table 1 for key.
Results of the multiple linear regression analysis indicated that skin thickness, as well as skin pigmentation measured as a percentage of scattered light at 750 nm, had a significant effect on laser energy penetration through shaved skin samples (adjusted R2 = 0.87; P < 0.001; multiple regression equation: y = 8.6 – 3.5x1 + 0.38x2, with x1 being skin thickness in millimeters and x2 being the percentage of scattered light at 750 nm). As skin thickness increased, laser energy penetration decreased (P < 0.001). Furthermore, the greater the percentage of scattered light at 750 nm, the greater the laser energy penetration (P < 0.001).
Laser energy penetration was determined for samples of shaved skin, shaved skin with the superficial digital flexor tendon, and shaved skin with the superficial and deep digital flexor tendons, arranged in anatomically correct fashion. Laser energy absorption by skin, the superficial digital flexor tendon, and the deep digital flexor tendon was calculated (Table 2)
Discussion
Low-level laser therapy has been shown to positively affect tendon-derived fibroblasts of some species in vitro. The positive effects include cell activities thought to be necessary for tendon healing, such as increased cell proliferation,17,24 cell migration,18 and expression of tendon matrix proteins.17,18 These effects were found to occur at a relatively narrow range of low fluence, with 2 J/cm2 possibly having the greatest effects. Translation of effective in vitro fluence to an in vivo setting requires adjustment for losses of laser energy via reflection, absorption, and scattering by tissues overlying the injured tendon. In the present study, the influence of subjectively assessed hair color, skin pigmentation, skin preparation, and wavelength on laser energy penetration through equine skin was investigated. The intent was that the study data would provide guidance for equine practitioners with regard to adjustment of laser energy outputs for individual patients to achieve delivery of appropriate in vivo energy fluence for augmentation of healing of flexor tendon injuries in horses. The data obtained in the present study suggested that without laser energy output adjustment based on perceived equine skin color and skin thickness, it is unlikely that effective energy fluences are delivered to injured tendons.
Similar to findings in a previous study22 in horses, penetration of laser energy through skin samples was significantly less when the hair was not clipped or shaved, although there was no difference between the effect of clipping and shaving in the present study. In the presence of natural hair, only white or cream-colored hair allowed laser energy penetration through equine skin, whereas gray, brown, chestnut, or black hair blocked > 99% of the laser energy penetration. Thus, the data obtained in the present study supported previous recommendations to clip or shave and then clean the skin over the area to be treated via LLLT.22 After clipping or shaving the skin, penetration of laser energy through equine skin was significantly affected on the basis of the subjectively perceived skin color; laser energy penetration was greatest for nonpigmented skin, followed by that for moderately pigmented skin and then black-pigmented skin. This was not surprising, given that skin color is influenced by the type and amount of melanin within the skin and light of a wavelength of 800 or 970 nm is absorbed by melanin.21
In the present study, skin thickness was significantly associated with laser energy penetration through equine skin samples, but the effect was small, compared with the effect of skin pigmentation. This may be a result of the minor variability in thickness of the skin samples used (thickness range, 1.5 to 3.5 mm). Correction of laser energy settings on the basis of skin thickness would theoretically be possible by ultrasonographically measuring skin thickness at the region requiring treatment.25 However, correction of laser energy settings on the basis of skin thickness is apparently much less important than corrections based on skin pigmentation.
It has been stated that longer-wavelength laser light penetrates deeper into tissues, compared with the effect of shorter-wavelength laser light,8 but in the present study, this was dependent on skin pigmentation of the equine skin samples. In skin samples with moderate or dark pigmentation, laser energy penetration was greater at a wavelength of 970 nm than at a wavelength of 800 nm. However, laser energy penetration in nonpigmented skin samples was greater at a wavelength of 800 nm than at a wavelength of 970 nm. A possible explanation is that more laser energy was absorbed by melanin in the skin at the 800-nm wavelength than at the 970-nm wavelength and that more laser energy was absorbed by water at the 970-nm wavelength than at the 800-nm wavelength.8 In nonpigmented skin, the major chromophore may be water because of the lack of melanin, which would result in better skin penetration by laser energy at the 800-nm wavelength, compared with the effects of longer wavelengths.
In our opinion, the best estimate for a clinically effective laser fluence delivered to tendon fibroblasts in the area of a tendon lesion in vivo is the fluence that has been shown to improve cellular functions in vitro. After receiving a laser fluence of 2.0 to 2.5 J/cm2 (660 nm, 50 mW, and continuous mode), rat Achilles tenocytes in monolayer culture had increased cell proliferation24 and cell migration.18 Furthermore, administration of 1 to 3 J/cm2 (820 and 635 nm, 40 mW, and 50-Hz pulse frequency) was associated with increased gene expression of decorin and collagen type I in porcine Achilles tendon fibroblasts.17 Cell proliferation was enhanced at a fluence of 2.00 to 2.16 J/cm2, but not at higher fluences.17,26 On the basis of these findings, a fluence of 2 J/cm2 may be the most appropriate fluence in vitro to stimulate cellular functions that possibly benefit tendon healing. With as little as 1.9% of emitted laser energy penetrating shaved, black-pigmented equine skin in the present study, the question arises whether it is feasible to deliver this energy fluence to injured flexor tendons within a reasonable amount of time. When attempting to treat the superficial digital flexor tendon in a horse with black-pigmented skin, the prediction based on the data obtained in the present study is that use of a laser beam at a wavelength of 800 nm would most likely result in only 1.9% of the laser energy penetrating the skin (Table 2). Also, in general, 1.8% of the laser energy is absorbed by the superficial digital flexor tendon (Table 3). Thus, with the laser set at a 1-W power output and a spot diameter of 1.2 cm, it would take 10 minutes and 24 seconds of treatment time to deliver a fluence of 2 J/cm2 to a tendon lesion that was determined to be 1 cm in width and 5 cm in length (Appendix 2). However, even the assumption that 1.8% of laser energy is absorbed by the superficial digital flexor tendon is probably an overestimation because additional energy is likely to be reflected or scattered by the tendon. This energy loss is very difficult, if not impossible, to assess27 and subsequently has not been taken into account in the example calculation.
Mean ± SEM laser energy absorption (expressed as the percentage of the total laser energy detected without any tissue between the laser and the photodetector) at 3 different wavelength settings (800 nm, 970 nm, and both 800 and 970 nm emitted simultaneously) in samples of shaved skin, superficial digital flexor tendons, and deep digital flexor tendons collected en bloc from 1 metacarpal area of each of the 19 equine cadavers in Table 1 that were classified as having light-, medium-, or dark-colored skin.
| Skin color group | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Light (n = 4) | Medium (n = 9) | Dark (n = 6) | |||||||
| Tissue | 800 nm | 970 nm | 800 and 970 nm | 800 nm | 970 nm | 800 and 970 nm | 800 nm | 800 and 970 nm | 970 nm |
| Skin | 74.9 ± 2.1 | 80.9 ± 1.7 | 78.1 ± 1.6 | 94.1 ± 0.8 | 91.0 ± 1.3 | 92.5 ± 1.0 | 98.8 ± 0.4 | 96.5 ± 0.9 | 97.6 ± 0.7 |
| Superficial digital flexor tendon | 20.7 ± 2.7 | 16 ± 1.7 | 18.2 ± 1.9 | 5.2 ± 0.7 | 7.9 ± 1.1 | 6.6 ± 0.8 | 1.2 ± 0.4 | 3.3 ± 0.9 | 2.3 ± 0.6 |
| Deep digital flexor tendon | 4.1 ± 1.7 | 2.8 ± 1.4 | 3.4 ± 1.5 | 0.7 ± 0.2 | 1.1 ± 0.3 | 0.9 ± 0.2 | 0.1 ± 0.0 | 0.2 ± 0.0 | 0.1 ± 0.0 |
To calculate the percentages of laser energy absorbed by shaved skin, superficial digital flexor tendons, and deep digital flexor tendons, measurements were obtained from shaved skin, shaved skin with the superficial digital flexor tendon, and shaved skin with the superficial and deep digital flexor tendons, arranged in anatomically correct fashion. The respective values were expressed as a percentage of reference laser energy, which was measured without any tissue between laser and photodetector.
See Tables 1 and 2 for key.
When attempting to treat the deep digital flexor tendon, it may be theoretically beneficial to avoid having to penetrate skin and superficial digital flexor tendon by directing the beam toward the lesion in the lateral or medial plane, as opposed to the sagittal plane, from the palmar or plantar aspect because only approximately 1% of laser energy (at the 800-nm wavelength) penetrated black-pigmented skin and the superficial digital flexor tendon in the present study. However, we did not attempt to determine and thus do not know how much laser energy would be absorbed by healthy tendons when treatment is directed in this fashion.
A limitation of the present study was that we used cadaver tissues with lack of blood flow. Hemoglobin is a chromophore, and it is conceivable that perfused equine skin attenuates the laser beam to a greater extent than does nonperfused skin, resulting in target tissues that receive even less laser energy than what was calculated in this study. Also, absorption of laser energy by damaged areas of tendon, such as core lesions, may be different from findings for the normal tendons used in the present study. Furthermore, it is unknown how much laser energy is absorbed by tenocytes located at different depths within the tendon. This illustrates the difficulty of translating biostimulatory effects of LLLT in monolayer cultures to 3-D tissues. It is also unknown whether tenocytes surrounded by tendon matrix would have the same biostimulatory responses as tenocytes in monolayer culture. Additionally, inflammatory mediators that are found in tendon lesions28 may also alter tenocytes’ responses to LLLT. Finally, it is possible that LLLT is ineffective in influencing tendon healing in equids because of species-specific factors, such as biomechanics or wound healing.
The data obtained in the present study have clearly suggested that to deliver laser output equivalent to that which provides effective laser energy fluences in vitro, adjustments to laser energy output must be made depending on the preparation of the skin and the subjectively determined skin color. Hair color influenced penetration of laser energy through equine skin at wavelengths of 800 and 970 nm. Only hair of light color (white or cream colored) allowed penetration of sufficient laser energy to treat an area without clipping or shaving the overlying skin and without excessively long treatment times. As has been shown previously, penetration of laser energy through equine skin was improved by clipping or shaving of the hair, followed by cleaning of the skin. Laser energy penetration of equine skin was dependent on wavelength. Penetration of nonpigmented skin was better with a wavelength of 800 nm, compared with the effect of a wavelength of 970 nm, and penetration of pigmented skin was better with a wavelength of 970 nm, compared with the effect of a wavelength of 800 nm. Laser energy penetration decreased with increasing skin thickness, but it was influenced to a much greater extent by skin pigmentation: the darker the skin pigmentation, the less laser energy penetration of the skin. Thus, we accept our hypotheses that hair color, skin preparation, skin color, and skin thickness influence penetration of laser energy through equine skin ex vivo. We also conclude that laser energy of 2 J/cm2 can be delivered to equine superficial digital flexor tendons, even in horses with black-pigmented skin, although adjustments must be made to assure adequate duration of treatment. In our opinion, delivery of that fluence to the deep digital flexor tendon or the suspensory ligament appears to be impossible if the laser beam is directed through skin and the superficial digital flexor tendon.
Acknowledgments
Supported by the Department of Clinical Sciences, College of Veterinary Medicine, Oregon State University.
Presented in part in poster form at the 2014 American College of Veterinary Surgeons Surgery Summit, San Diego, Calif, October 2014.
ABBREVIATIONS
| LLLT | Low-level laser therapy |
Foonotes
K Series 1200, K-Laser, Franklin, Tenn.
FDS1010, Thorlabs, Newton, NJ.
Saran, SC Johnson, Racine, Wis.
TDS 220, Tektronix, Beaverton, Ore.
Beuthanasia-D Special, Merck Animal Health, Madison, NJ.
Series 505, Mitutoyo, Aurora, Ill.
L2, Leica, Buffalo Grove, Ill.
USB4000, Ocean Optics, Dunedin, Fla.
X-9, Boise White Paper, Boise, Ind.
40 blade, Oster, McMinnville, Tenn.
Disposable single-blade shaver, Bic, Clichy, France.
SAS, version 9.4, SAS Institute Inc, Cary, NC.
Daniel's XL Toolbox add in version 6.52 for Excel, Daniel Kraus, Würzburg, Germany.
Excel 2010, Microsoft, Redmond, Wash.
References
1. Ramey DW, Basford JR. Laser therapy in horses. Compend Contin Educ Pract Vet 2000; 22: 263–271.
2. Tumilty S, Munn J, McDonough S, et al. Low level laser treatment of tendinopathy: a systematic review with meta-analysis. Photomed Laser Surg 2010;28: 3–16.
3. Marcos RL, Leal ECP Jr, de Moura Messias F, et al. Infrared (810 nm) low-level laser therapy in rat Achilles tendinitis: a consistent alternative to drugs. Photochem Photobiol 2011; 87: 1447–1452.
4. Alfredo PP, Bjordal JM, Dreyer SH, et al. Efficacy of low level laser therapy associated with exercises in knee osteoarthritis: a randomized double-blind study. Clin Rehabil 2012; 26: 523–533.
5. Bjordal JM, Johnson MI, Lopes-Martins RA, et al. Short-term efficacy of physical interventions in osteoarthritic knee pain. A systematic review and meta-analysis of randomised placebo-controlled trials. BMC Musculoskelet Disord 2007; 8: 51.
6. Hawkins D, Houreld N, Abrahamse H. Low level laser therapy (LLLT) as an effective therapeutic modality for delayed wound healing. Ann N Y Acad Sci 2005; 1056: 486–493.
7. Vladimirov YA, Osipov AN, Klebanov GI. Photobiological principles of therapeutic applications of laser radiation. Biochemistry (Mosc) 2004; 69: 81–90.
8. Chung H, Dai T, Sharma SK, et al. The nuts and bolts of low-level laser (light) therapy. Ann Biomed Eng 2012; 40: 516–533.
9. Karu T, Pyatibrat L, Kalendo G. Irradiation with He-Ne laser increases ATP level in cells cultivated in vitro. J Photochem Photobiol B 1995; 27: 219–223.
10. Benedicenti S, Pepe IM, Angiero F, et al. Intracellular ATP level increases in lymphocytes irradiated with infrared laser light of wavelength 904 nm. Photomed Laser Surg 2008; 26: 451–453.
11. Passarella S, Casamassima E, Molinari S, et al. Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by helium-neon laser. FEBS Lett 1984; 175: 95–99.
12. Yu W, Naim JO, McGowan M, et al. Photomodulation of oxidative metabolism and electron chain enzymes in rat liver mitochondria. Photochem Photobiol 1997; 66: 866–871.
13. Lavi R, Shainberg A, Friedmann H, et al. Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells. J Biol Chem 2003; 278: 40917–40922.
14. Grossman N, Schneid N, Reuveni H, et al. 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species. Lasers Surg Med 1998; 22: 212–218.
15. Zhang J, Xing D, Gao X. Low-power laser irradiation activates Src tyrosine kinase through reactive oxygen species-mediated signaling pathway. J Cell Physiol 2008; 217: 518–528.
16. Lubart R, Eichler M, Lavi R, et al. Low-energy laser irradiation promotes cellular redox activity. Photomed Laser Surg 2005; 23: 3–9.
17. Chen CH, Tsai JL, Wang YH, et al. Low-level laser irradiation promotes cell proliferation and mRNA expression of type I collagen and decorin in porcine Achilles tendon fibroblasts in vitro. J Orthop Res 2009; 27: 646–650.
18. Tsai WC, Hsu CC, Pang JHS, et al. Low-level laser irradiation stimulates tenocyte migration with up-regulation of dynamin ii expression. PLoS ONE 2012; 7: e38235.
19. Kaneps AJ, Hultgren BD, Riebold TW, et al. Laser therapy in the horse: histopathologic response. Am J Vet Res 1984; 45: 581–582.
20. Marr CM, Love S, Boyd JS, et al. Factors affecting the clinical outcome of injuries to the superficial digital flexor tendon in National Hunt and point-to-point racehorses. Vet Rec 1993; 132: 476–479.
21. Huang YY, Chen AC, Carroll JD, et al. Biphasic dose response in low level light therapy. Dose Response 2009; 7: 358–383.
22. Ryan T, Smith R. An investigation into the depth of penetration of low level laser therapy through the equine tendon in vivo. Ir Vet J 2007; 60: 295–299.
23. Curtis H, Barnes NS. Photosynthesis, light, and life. In: Curtis H, Barnes NS, eds. Invitation to biology. 5th ed. New York: Worth Publishers, 1994; 161–175.
24. Tsai WC, Cheng JW, Chen JL, et al. Low-level laser irradiation stimulates tenocyte proliferation in association with increased NO synthesis and upregulation of PCNA and cyclins. Lasers Med Sci 2014; 29: 1377–1384.
25. Jain SM, Pandey K, Lahoti A, et al. Evaluation of skin and subcutaneous tissue thickness at insulin injection sites in Indian, insulin naive, type-2 diabetic adult population. Indian J Endocrinol Metab 2013; 17: 864–870.
26. Yu W, Naim JO, Lanzafame RJ. The effect of laser irradiation on the release of bFGF from 3T3 fibroblasts. Photochem Photobiol 1994; 59: 167–170.
27. Hutchison AM, Beard DJ, Bishop J, et al. An investigation of the transmission and attenuation of intense pulsed light on samples of human Achilles tendon and surrounding tissue. Lasers Surg Med 2012; 44: 397–405.
28. Hosaka Y, Kirisawa R, Yamamoto E, et al. Localization of cytokines in tendinocytes of the superficial digital flexor tendon in the horse. J Vet Med Sci 2002; 64: 945–947.
Appendix 1
Calculations used to determine absorption of laser energy by samples of shaved skin, SDFTs, and DDFTs obtained from the metacarpal areas of equine cadavers.
| Variable | Calculation |
|---|---|
| Value measured with no tissue between laser and detector | Etotal = 100% of emitted energy |
| Value measured with skin only between laser and detector | Tskin = % of energy transmitted through skin |
| Value measured with skin and SDFT between laser and detector | Tskin + SDFT = % of energy transmitted through skin and SDFT |
| Value measured with skin, SDFT, and DDFT between laser and detector | Tskin + SDFT + DDFT = % of energy transmitted through skin, SDFT, and DDFT |
| When only skin is between laser and detector | Etotal = (%)Askin (%) + Tskin (%) |
| When skin and SDFT are between laser and detector | Etotal = (%)Askin (%) + Asdft (%) + Tskin+SDFT (%) |
| When skin, SDFT, and DDFT are between laser and detector | Etotal = (%)Askin (%) + ASDFT (%) + ADDFT (%) + Tskin + SDFT + DDFT (%) |
| Energy absorbed by skin | Askin (%) = 100% – Tskin (%) |
| Energy absorbed by SDFT | ASDFT (%) = 100% – Askin (%) – Tskin + SDFT (%) |
| Energy absorbed by DDFT | ADDFT (%) = 100% – Askin (%) – ASDFT (%) – Tskin + SDFT + DDFT (%) |
DDFT = Deep digital flexor tendon. SDFT = Superficial digital flexor tendon.
E = emitted energy. A = absorbed energy. T = transmitted energy.
Appendix 2
Example calculation of treatment time with a therapeutic laser to provide LLLT in an SDFT in metacarpal area of a horse.
| Size of the tendon lesion (in the SDFT): 5 × 1 cm = 5 cm2 | |
| Attenuation of laser energy by black pigmented skin (through absorption and scattering): 98.2% | |
| Target fluence to be delivered to tendon lesion: 2 J/cm2 = 10 J/5 cm2 | |
| Laser settings: power output = 1 W; spot size diameter = 12 mm | |
| 1) | Calculation of energy emitted by laser: 1 W/1.13 cm2 = 0.89 W/cm2 = 0.89 J/s/cm2 |
| 2) | Calculation of actual fluence delivery rate to the SDFT: 0.89 J/s/cm2 × 1.8% = 0.01602 J/s/cm2 |
| 3) | Calculation of time necessary to deliver 2 J/cm2 to the SDFT: 2 J/cm2 / 0.01602 J/cm2 = 124.84 seconds |
| The treatment time required to deliver 2 J to 1 cm2 of an SDFT is 124.8 seconds. The treatment time required to deliver 2 J to 5 cm2 of an SDFT is 624.2 seconds. Thus, the treatment time required to deliver 10 J to 5 cm2 of SDFT is 10 minutes and 24 seconds. | |
SDFT = Superficial digital flexor tendon.
