Lasers Med Sci DOI 10.1007/s10103-014-1555-y
ORIGINAL ARTICLE
The effects of a minimally invasive laser needle system on complete Freund’s adjuvant-induced arthritis Heesung Kang & Taeyoon Son & Aeju Lee & Inchan Youn & Dong Hyun Seo & Han Sung Kim & Byungjo Jung
Received: 16 September 2013 / Accepted: 20 February 2014 # Springer-Verlag London 2014
Abstract The present study aimed to investigate the effects of a minimally invasive laser needle system (MILNS) on the acute progression of arthritis. Previous studies showed controversial clinical results regarding the effects of low-level laser therapy on arthritis, with the outcomes depending upon stimulation parameters such as laser wavelength and dosage. Based on the positive effects of MILNS on osteoporotic mice, we hypothesized that MILNS could potentially suppress the progression of arthritis owing to its biostimulation effects. Eight C57BL/6 mice with complete Freund’s adjuvant (CFA)-induced arthritis were used as acute progression arthritis models and divided into the laser and control groups (n=4 each). In the laser group, after minimally invasive laser stimulation, laser speckle contrast images (LSCIs) were obtained every 6 h for a total of 108 h. The LSCIs in the control group were obtained without laser stimulation. The effects of MILNS on the acute progression of arthritis were indirectly evaluated by calculating the paw area and the average laser speckle index (LSI) at the arthritis-induced area. Moreover, the macrophage population was estimated in the arthritisinduced area. Compared to the control group, the laser group showed (1) lower relative variations of the paw area, (2) lower average LSI in the arthritis-induced area, and (3) lower macrophage population in the arthritis-induced area. These results H. Kang : T. Son : D. H. Seo : H. S. Kim : B. Jung (*) Department of Biomedical Engineering, Yonsei University, 1 Yonseidae-gil, Wonju, Gangwon-do 220-710, South Korea e-mail: [email protected] A. Lee Department of Laboratory Medicine, College of Medicine, Korea University, Seoul 136-705, South Korea A. Lee : I. Youn Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-Dong, Seongbuk-gu, Seoul 136-791, South Korea
indicate that MILNS may suppress the acute progression of CFA-induced arthritis in mice and may thus be used as a potential treatment modality of arthritis in clinics. Keywords Arthritis . Low-level laser therapy . Laser speckle contrast image . Minimally invasive
Introduction Because of its biostimulative effect, low-level laser therapy (LLLT) has been widely used in various fields for orthopedic conditions such as osteoporosis, muscle injuries, and arthritis [1, 2]. Arthritis, a representative orthopedic disease, is a chronic joint disorder that originates from conditions involving inflammation of the musculoskeletal system, such as synovitis [3, 4]. Numerous studies have indicated the positive clinical performance of LLLT as a treatment modality for arthritis and the improvement of the skeletal joint movement such as hand grip strength and morning stiffness following this treatment [5]. However, several research papers regarding the clinical performance and treatment efficacy of LLLT in arthritis showed conflicting and controversial results because the mechanism of LLLT is still not clearly defined. Moreover, the clinical studies used differing therapeutic parameters such as laser wavelength and laser energy dosage, which makes direct comparisons of results difficult [2, 4, 6–8]. Most of the light irradiated on the skin is scattered and then reflected. Therefore, only a small amount of light is transmitted and delivered to the deep target tissue. In their study evaluating laser irradiation with respect to laser penetration, Nakano et al. reported that the penetration of the Ga-Al-As laser decreased to 33.3 and 8.3 % in rat skin and in rat skin as well as the gastrocnemius muscle, respectively [9]. In addition, Ninomiya et al. reported that only 15.9 % of laser light penetrated the skin surrounding rat femurs [10].
Lasers Med Sci
Laser scattering in tissue is always an inherent and potential problem in LLLT. In a previous study, a minimally invasive laser needle system (MILNS) was developed to address the problem of light scattering and to increase the therapeutic efficacy of LLLT [11]. The positive therapeutic outcome from the use of MILNS was confirmed through the prevention of further bone loss in osteoporotic mice [11–13]. The MILNS has the ability to deliver the laser light to the deep target tissue without any energy loss. Therefore, direct stimulation on biological cells may be achieved, thereby enhancing the treatment efficacy of the LLLT. Furthermore, when using the MILNS, ultraviolet and visible light which have a stronger light scattering and higher energy as compared to near-infrared (NIR) light can be used for deep target tissue because penetration depth does not need to be considered. In previous publications, most studies evaluated the treatment efficacy of LLLT in arthritis by assessing patient movement with a scoring system such as the pain score and grip strength [2, 4, 8]. A few papers revealed the histological observation of the effect of laser stimulation in an arthritic animal model. Alves et al. reported that LLLT modulates the inflammatory process and has a positive effect on histological events in a collagen-induced arthritis (CIA) rat model. They observed the improvement of histological features such as necrosis, hyperemia, and medullary hemorrhage as a result of the use of LLLT [3]. Zhang et al. observed that low-level laser irradiation (LLLI) significantly decreased CCL2 mRNA levels, which may be one of the mechanisms involved in LLLI-mediated reduction of rheumatoid arthritis (RA) inflammation in the CIA rat model [14]. In the present study, C57BL/6 mice with complete Freund’s adjuvant (CFA)-induced arthritis were used as acute progression arthritis models. For the suppression of the inflammatory response, MILNS was used as the treatment modality. The treatment efficacy was evaluated using laser speckle contrast images (LSCIs) and histological analysis.
paw of each mouse [15, 16]. Because the adjuvant elicited an acute response, minimally invasive laser stimulation was applied to the laser group 3 h after injection of the adjuvant. LSCIs were subsequently obtained every 6 h for a total of 108 h. Conversely, in the control group, no laser stimulation was performed and LSCIs were obtained. As a result, the laser group has taken totally 18 times of minimally invasive laser stimulations and both groups obtained totally 19 LSCIs. For the LSCI acquisition and minimally invasive laser stimulation, the mice were anesthetized with a mixture of isoflurane (2– 5 %) and oxygen (1–2 L/min). At the end of the experiment, all mice were euthanized by cervical dislocation for histological analysis. All animal experiments were performed in accordance with the guidelines and under the approval of the Institutional Animal Care and Use Committee of Yonsei University (YWC-130411-1). MILNS Figure 1 shows a schematic diagram of the MILNS used for the minimally invasive laser stimulation of the left hind paw of the mouse. A 660-nm diode laser (#ML101J27, ThorLabs, Newton, NJ, USA) on a laser diode mount (#TCLDM9, ThorLabs, Newton, NJ, USA) was used as a light source with a laser diode driver (#IP500, ThorLabs, Newton, NJ, USA) and a thermoelectric cooler (TEC) controller (#TCM1000T, ThorLabs, Newton, NJ, USA). The laser was collimated by using a collimation lens (#C230TME-B, ThorLabs, Newton, NJ, USA) and focused on an optical fiber (#AFS105/125Y-CUSTOM, ThorLabs, Newton, NJ, USA) with a fiber port (#PAF-X-11-PC-A, ThorLabs, Newton, NJ, USA). The end of the optical fiber was combined to a fine hollow needle (outer diameter, 300 μm; inner diameter, 130 μm) without a fiber jacket. The fine hollow needle, which delivered the laser at the center of the mouse’s left hind paw, was made of stainless steel that has anti-corrosion, heat-resistant, and nonmagnetic characteristics. The irradiated energy of the minimally invasive laser is 3 J with a 20-mW laser intensity for 150 s.
Materials and methods
Laser speckle imaging system
Animals and experimental design
Figure 2 shows a schematic diagram of the laser speckle imaging system. A diode laser (HL6512MG; 658 nm, 50 mW; Thorlabs, Newton, NJ, USA) is coupled into an optical fiber. A laser passes through a holographic diffuser for even illumination and is directed to a 1:1 beam splitter to eliminate shadowing in the sample. An image of the mouse’s left hind paw was captured with a charge-coupled device (CCD) camera (XC-HR57; Sony, Tokyo, Japan) and a macro zoom lens (MLM3X-MP; Computar, Commack, NY, USA). The exposure time was empirically determined based on preliminary experiments. The imaging areas were
Eight C57BL/6 mice (aged 14 weeks) were used as an animal experiment model of arthritis using an adjuvant. The mice were kept in a controlled standard condition (room temperature, 23±2 °C; humidity, 50±10 %), allowed to move freely, and fed a standard laboratory chow and water ad libitum. The mice were divided into two groups—namely the laser group (n=4) and the control group (n=4). To induce arthritis, 0.05 mL of complete Freund’s adjuvant (CFA) (#7001, Chondrex, Redmond, WA, USA) was injected in the left hind
Lasers Med Sci Fig. 1 Schematic diagram of the minimally invasive laser needle system
approximately 6×8 mm2, and the images were acquired at 30 Hz with a frame grabber (DOMINO IOTA; Euresys, Angleur, Belgium). The area and average laser speckle index (LSI) value of the left hind paw were calculated from the LSCIs. Analysis of LSCI In general, the key parameters from which the diagnosis of arthritis is derived are the degree of swelling and the presence of angiogenesis, which are correlated with inflammatory responses and reflect the progression stage of arthritis. The paw area was used to estimate the degree of swelling in twodimensional images using the LSCIs. The LSI computed from the LSCI was used to indirectly calculate the degree of angiogenesis. The paw area and LSI were obtained by calculating the total number of pixels and the average pixel value of the paw region on LSCI, respectively.
cut into slices with a thickness of 5 μm. For the purpose of assessing the macrophage population in the laser-stimulated area, immune-fluorescent anti-CD68 was used as a specific macrophage marker. In brief, tissue sections of the middle of the hind paw were washed with phosphate buffer solution (PBS; pH 7.4) and blocked in 5 % bovine serum albumin containing 0.1 % Triton X-100 for 30 min. The sections were then incubated in a cocktail of mouse anti-CD68 (1:1,000; Abcam) for 1 h at room temperature. After washing with 0.01 M PBS, the sections were further incubated for 30 min with a mixture of PE-conjugated goat anti-mouse IgG (1:5,000; Santa Cruz Biotechnology Inc., Santa Cruz), and fluorescence images were subsequently acquired by fluorescence microscopy (Olympus IX81, MetaMorph ver 7.5.3.0, NY, USA).
Results Histological analysis After the sacrifice of eight C57BL/6 mice at 14 weeks, excised paw tissues (n=5) were dipped in Fixative/Decalcifier (Thermo Fisher Scientific) for 14 days, dehydrated with a graded ethanol, and embedded in paraffin. The paraffin blocks were
LSCI Figures 3 and 4 show results from the LSCIs of the control group and the laser group, respectively. Macroscopically, both groups show a decreasing trend of LSI as a function of time due to the characteristics of the adjuvant. However, compared to the laser group, the control group shows higher LSI across all measurement points.
Variation of the paw area
Fig. 2 Schematic diagram of the laser speckle imaging modality
Figure 5 shows the percentage variation of the paw area as a function of time. Up to two measurement points, both groups show similar percentage variation. However, after two measurement points, the laser group shows a lower percentage variation as a function of time when compared to the control group. Data are expressed as mean±standard error of measurement (SEM).
Lasers Med Sci
0 (before adjuvant
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Fig. 3 Representative laser speckle contrast image of the control group as a function of time
Variation of the average LSI
Histological analysis
Figure 6 shows the percentage variation of average LSI on the paw area as a function of time. Similar to that noted for paw area variation, both groups show a marked difference in LSI after two measurement points. The control group when compared to the laser group shows greater percentage variation in LSI as a function of time. Data are expressed as mean±SEM.
Macrophage population is used as a marker of inflammation in arthritis because macrophages are known to be exclusively generated in inflamed tissue. Therefore, to investigate the effect of minimally invasive laser stimulation, tissue sections of the left hind paw of both groups were examined using antiCD68 as a macrophage cell surface marker. As noted in Fig. 7,
Lasers Med Sci
0 (before adjuvant
1 (3 hours after
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Fig. 4 Representative laser speckle contrast image of the laser group as a function of time
the control group shows a stronger red fluorescence signal of the CD68 when compared to the laser group.
Discussion Arthritis is an autoimmune inflammatory disease that affects an individual’s activities of daily living due to the destruction
of the joints [3, 4, 8]. The application of LLLT as a treatment modality for arthritis patients was reported in several clinical papers to have a positive outcome in terms of relieving pain and improving joint movement [1, 5]. However, LLLT has the potential problem of laser energy loss due to light scattering in the tissue. This, in turn, results in the decrease of its therapeutic efficacy due to the reduction of the intensity, which makes it difficult to achieve biostimulation of the target tissue. When
Lasers Med Sci Fig. 5 Variation of paw area in the control and laser groups as a function of time
Laser group Control group
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efficacy of LLLT at the target tissue, high-intensity NIR light is generally used for conditions requiring deep penetration with sufficient intensity. In the present study, we aimed to address the problem of light energy loss and to increase the overall therapeutic efficacy in the treatment of arthritis, by using MILNS, which employs a fine hollow needle for stimulation at deep target tissues without the associated light energy loss. As a CFA-induced arthritis mouse model, we used C57BL/ 6 mice with a site-induced inflammatory response and acute
Fig. 6 Variation of laser speckle index in the control and laser groups as a function of time
Percentage variation of average LSI on paw area (%)
the appropriate laser energy density is reached, LLLT has been shown to be effective in the treatment of tissue disorders by activating a series of reactions leading to increased cellular metabolism, with the transformation of the absorbed light energy into cellular energy [1]. According to the Arndt-Schulz law, the response of biostimulation can either be an acceleration or an inhibition of physiologic activity depending on the energy dosage of stimulation [17]. Therefore, in order to increase the therapeutic
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Lasers Med Sci Fig. 7 Representative fluorescence images of the center of the left hind limb paw in the a control and b laser groups
progression of the condition. Due to the acute progression in this model, LSCIs of the laser and control groups were first obtained 3 h after the injection of adjuvant and subsequently after every 6 h for a total of 108 h. The LSCI is mainly used to measure blood flow perfusion in biological tissue [18]. During the progression of arthritis with synovial membrane inflammation, modulation of inflammatory cells from the vascular endothelium increases the vascular permeability, thus resulting in vasodilatation with edema [3]. Such transitions that are captured in LSCI can be quantified by LSI in order to determine the severity of the inflammatory response. In particular, a higher LSI reflects a more severe progression of arthritis. The results of the present study showed that CFAinduced arthritis progressed severely with higher LSI as a function of time in the control group (Fig. 3). However, the progression of arthritis in the laser group was suppressed, as indicated by a lower LSI (Fig. 4). MILNS may influence the function of cells in the immune system such as T, B, and NK lymphocytes in the arthritis-induced area. Alves et al. reported that LLLT has a positive effect in rheumatoid arthritis (RA) due to the decrease of the vascular permeability associated with infiltration of leukocytes and influence on the maintenance of synovial fluid lubricant composition [3]. Moreover, the inflammatory modulation by MILNS was confirmed through the variation of the paw area. The laser group showed a markedly smaller variation of the paw area when compared to the control group (Fig. 5). In biological tissue, when inflammation is induced, macrophages—because of their role as phagocytic cells—become activated and will interact with lymphocytes to facilitate antibody production. With the activation of macrophages, the synovial tissue releases cytokines and chemokines to promote chronic inflammation, such as in cases of arthritic synovial tissue [19]. The population of activated macrophages induced by the adjuvant was examined by using an anti-CD68 as a macrophage marker. As noted in Fig. 7, when a red fluorescence signal was used for the detection of macrophages, marked differences between the two groups were noted in the macrophage population. As compared to the control group, the laser group showed a lower fluorescence signal,
which indicates that minimally invasive laser stimulation is effective in the suppression of inflammatory reaction on arthritis progression. To our knowledge, it may be the first study employing the MILNS for arthritis treatment. The MILNS was employed as a minimally invasive stimulation method to improve the therapeutic efficacy of LLLT in arthritis, and its therapeutic efficacy was evaluated by analyzing the LSCI and histology. The laser group when compared to the control group resulted in better therapeutic efficacy by presenting remarkable lower macrophage population which is the fundamental parameter directly related to the suppression of inflammatory response and decreasing paw area and LSI. Although the therapeutic and diagnostic methods for arthritis are effective in the animal model due to the shallow animal skin compared to the human skin, this study may have some limitations in clinical application: (1) the strength and size of the laser needle may have to be reconsidered depending on the region of interest; (2) the lower quality of LSCI which may be caused by the shallow penetration depth of the laser in the human skin has to be considered and may be solved by integrating an optical tissue clearing method into the imaging modality to reduce tissue turbidity. In previous LLLT studies, infrared light was used to increase the laser penetration depth of deep target tissues such as bone [10, 20]. However, in the present study, MILNS minimized the energy loss problem incurred in the use of LLLT and addressed the limitation of the wavelengths available for the penetration depth. As a means of localized treatment, MILNS may suppress the acute inflammatory response and increase treatment efficacy by modulating the inflammatory response and the activity of the inflammatory cells. Nonsteroidal anti-inflammatory drugs (NSAIDs) are generally used for the systemic treatment of arthritis [21]. However, further studies are expected to show that MILNS may have a synergic effect with NSAIDs in a systemic-developed arthritis animal model. Furthermore, each cell in biological tissue has a different cellular reaction to stimulation conditions such as wavelength, energy density, and pulse duration. Although the present study shows positive effects in the suppression of acute
Lasers Med Sci
arthritis progression, various stimulation conditions have yet to be investigated in order to further enhance the therapeutic efficacy.
Conclusion In the present study, MILNS has been shown to be therapeutically efficacious in suppressing the acute progression of CFA-induced arthritis. However, further studies are needed to investigate the effects of different therapeutic parameters such as wavelength, dosage, and the number of sessions needed to achieve the most effective therapeutic parameter settings. Moreover, the therapeutic mechanism of MILNS in arthritis has to be investigated. We believe that MILNS can potentially be used as a treatment modality of arthritis in the clinical setting. Acknowledgements This research was supported by the Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2010-00757).
References 1. Basford JR (1995) Low intensity laser therapy: still not an established clinical tool. Lasers Surg Med 16(4):331–342 2. Johannsen F, Hauschild B, Remvig L, Johnsen V, Petersen M, Bieler T (1994) Low energy laser therapy in rheumatoid arthritis. Scand J Rheumatol 23(3):145–147 3. Alves AC, de Carvalho PT, Parente M, Xavier M, Frigo L, Aimbire F, Leal EC Jr, Albertini R (2013) Low-level laser therapy in different stages of rheumatoid arthritis: a histological study. Lasers Med Sci 28(2):529–536. doi:10.1007/s10103-012-1102-7 4. Goats GC, Hunter JA, Flett E, Stirling A (1996) Low intensity laser and phototherapy for rheumatoid arthritis. Physiotherapy 82(5):311– 320 5. Brosseau L, Robinson V, Wells G, Debie R, Gam A, Harman K, Morin M, Shea B, Tugwell P (2005) Low level laser therapy (classes I, II and III) for treating rheumatoid arthritis. Cochrane Database Syst Rev 4, CD002049. doi:10.1002/14651858.CD002049.pub2 6. Heussler JK, Hinchey G, Margiotta E, Quinn R, Butler P, Martin J, Sturgess AD (1993) A double blind randomised trial of low power laser treatment in rheumatoid arthritis. Ann Rheum Dis 52(10):703–706 7. Bliddal H, Hellesen C, Ditlevsen P, Asselberghs J, Lyager L (1987) Soft-laser therapy of rheumatoid arthritis. Scand J Rheumatol 16(4): 225–228
8. Meireles SM, Jones A, Jennings F, Suda AL, Parizotto NA, Natour J (2010) Assessment of the effectiveness of low-level laser therapy on the hands of patients with rheumatoid arthritis: a randomized doubleblind controlled trial. Clin Rheumatol 29(5):501–509. doi:10.1007/ s10067-009-1347-0 9. Nakano J, Kataoka H, Sakamoto J, Origuchi T, Okita M, Yoshimura T (2009) Low-level laser irradiation promotes the recovery of atrophied gastrocnemius skeletal muscle in rats. Exp Physiol 94(9): 1005–1015. doi:10.1113/expphysiol.2009.047738 10. Ninomiya T, Hosoya A, Nakamura H, Sano K, Nishisaka T, Ozawa H (2007) Increase of bone volume by a nanosecond pulsed laser irradiation is caused by a decreased osteoclast number and an activated osteoblasts. Bone 40(1):140–148. doi:10.1016/j.bone. 2006.07.026 11. Kang H, Ko CY, Ryu Y, Seo DH, Kim HS, Jung B (2012) Development of a minimally invasive laser needle system: effects on cortical bone of osteoporotic mice. Lasers Med Sci 27(5):965– 969. doi:10.1007/s10103-011-1014-y 12. Ko CY, Kang H, Ryu Y, Jung B, Kim H, Jeong D, Shin HI, Lim D, Kim HS (2013) The effects of minimally invasive laser needle system on suppression of trabecular bone loss induced by skeletal unloading. Lasers Med Sci. doi:10.1007/s10103-013-1265-x 13. Ko CY, Kang H, Seo DH, Jung B, Schreiber J, Kim HS (2013) Low-level laser therapy using the minimally invasive laser needle system on osteoporotic bone in ovariectomized mice. Med Eng Phys 35(7):1015–1019. doi:10.1016/j.medengphy. 2012.10.002 14. Zhang L, Zhao J, Kuboyama N, Abiko Y (2011) Low-level laser irradiation treatment reduces CCL2 expression in rat rheumatoid synovia via a chemokine signaling pathway. Lasers Med Sci 26(5): 707–717. doi:10.1007/s10103-011-0917-y 15. Parvathy SS, Masocha W (2013) Gait analysis of C57BL/6 mice with complete Freund’s adjuvant-induced arthritis using the CatWalk system. BMC Musculoskelet Disord 14:14. doi:10.1186/1471-2474-1414 16. Kamala T (2007) Hock immunization: a humane alternative to mouse footpad injections. J Immunol Methods 328(1–2):204–214. doi:10. 1016/j.jim.2007.08.004 17. Sommer AP, Pinheiro AL, Mester AR, Franke RP, Whelan HT (2001) Biostimulatory windows in low-intensity laser activation: lasers, scanners, and NASA’s light-emitting diode array system. J Clin Laser Med Surg 19(1):29–33. doi:10.1089/ 104454701750066910 18. Yuan S, Devor A, Boas DA, Dunn AK (2005) Determination of optimal exposure time for imaging of blood flow changes with laser speckle contrast imaging. Appl Optics 44(10):1823–1830 19. Ma Y, Pope RM (2005) The role of macrophages in rheumatoid arthritis. Curr Pharm Des 11(5):569–580 20. Pires-Oliveira DA, Oliveira RF, Amadei SU, Pacheco-Soares C, Rocha RF (2010) Laser 904 nm action on bone repair in rats with osteoporosis. Osteoporos Int 21(12):2109–2114. doi:10.1007/ s00198-010-1183-8 21. Berard A, Solomon DH, Avorn J (2000) Patterns of drug use in rheumatoid arthritis. J Rheumatol 27(7):1648–1655
ORIGINAL ARTICLE
The effects of a minimally invasive laser needle system on complete Freund’s adjuvant-induced arthritis Heesung Kang & Taeyoon Son & Aeju Lee & Inchan Youn & Dong Hyun Seo & Han Sung Kim & Byungjo Jung
Received: 16 September 2013 / Accepted: 20 February 2014 # Springer-Verlag London 2014
Abstract The present study aimed to investigate the effects of a minimally invasive laser needle system (MILNS) on the acute progression of arthritis. Previous studies showed controversial clinical results regarding the effects of low-level laser therapy on arthritis, with the outcomes depending upon stimulation parameters such as laser wavelength and dosage. Based on the positive effects of MILNS on osteoporotic mice, we hypothesized that MILNS could potentially suppress the progression of arthritis owing to its biostimulation effects. Eight C57BL/6 mice with complete Freund’s adjuvant (CFA)-induced arthritis were used as acute progression arthritis models and divided into the laser and control groups (n=4 each). In the laser group, after minimally invasive laser stimulation, laser speckle contrast images (LSCIs) were obtained every 6 h for a total of 108 h. The LSCIs in the control group were obtained without laser stimulation. The effects of MILNS on the acute progression of arthritis were indirectly evaluated by calculating the paw area and the average laser speckle index (LSI) at the arthritis-induced area. Moreover, the macrophage population was estimated in the arthritisinduced area. Compared to the control group, the laser group showed (1) lower relative variations of the paw area, (2) lower average LSI in the arthritis-induced area, and (3) lower macrophage population in the arthritis-induced area. These results H. Kang : T. Son : D. H. Seo : H. S. Kim : B. Jung (*) Department of Biomedical Engineering, Yonsei University, 1 Yonseidae-gil, Wonju, Gangwon-do 220-710, South Korea e-mail: [email protected] A. Lee Department of Laboratory Medicine, College of Medicine, Korea University, Seoul 136-705, South Korea A. Lee : I. Youn Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-Dong, Seongbuk-gu, Seoul 136-791, South Korea
indicate that MILNS may suppress the acute progression of CFA-induced arthritis in mice and may thus be used as a potential treatment modality of arthritis in clinics. Keywords Arthritis . Low-level laser therapy . Laser speckle contrast image . Minimally invasive
Introduction Because of its biostimulative effect, low-level laser therapy (LLLT) has been widely used in various fields for orthopedic conditions such as osteoporosis, muscle injuries, and arthritis [1, 2]. Arthritis, a representative orthopedic disease, is a chronic joint disorder that originates from conditions involving inflammation of the musculoskeletal system, such as synovitis [3, 4]. Numerous studies have indicated the positive clinical performance of LLLT as a treatment modality for arthritis and the improvement of the skeletal joint movement such as hand grip strength and morning stiffness following this treatment [5]. However, several research papers regarding the clinical performance and treatment efficacy of LLLT in arthritis showed conflicting and controversial results because the mechanism of LLLT is still not clearly defined. Moreover, the clinical studies used differing therapeutic parameters such as laser wavelength and laser energy dosage, which makes direct comparisons of results difficult [2, 4, 6–8]. Most of the light irradiated on the skin is scattered and then reflected. Therefore, only a small amount of light is transmitted and delivered to the deep target tissue. In their study evaluating laser irradiation with respect to laser penetration, Nakano et al. reported that the penetration of the Ga-Al-As laser decreased to 33.3 and 8.3 % in rat skin and in rat skin as well as the gastrocnemius muscle, respectively [9]. In addition, Ninomiya et al. reported that only 15.9 % of laser light penetrated the skin surrounding rat femurs [10].
Lasers Med Sci
Laser scattering in tissue is always an inherent and potential problem in LLLT. In a previous study, a minimally invasive laser needle system (MILNS) was developed to address the problem of light scattering and to increase the therapeutic efficacy of LLLT [11]. The positive therapeutic outcome from the use of MILNS was confirmed through the prevention of further bone loss in osteoporotic mice [11–13]. The MILNS has the ability to deliver the laser light to the deep target tissue without any energy loss. Therefore, direct stimulation on biological cells may be achieved, thereby enhancing the treatment efficacy of the LLLT. Furthermore, when using the MILNS, ultraviolet and visible light which have a stronger light scattering and higher energy as compared to near-infrared (NIR) light can be used for deep target tissue because penetration depth does not need to be considered. In previous publications, most studies evaluated the treatment efficacy of LLLT in arthritis by assessing patient movement with a scoring system such as the pain score and grip strength [2, 4, 8]. A few papers revealed the histological observation of the effect of laser stimulation in an arthritic animal model. Alves et al. reported that LLLT modulates the inflammatory process and has a positive effect on histological events in a collagen-induced arthritis (CIA) rat model. They observed the improvement of histological features such as necrosis, hyperemia, and medullary hemorrhage as a result of the use of LLLT [3]. Zhang et al. observed that low-level laser irradiation (LLLI) significantly decreased CCL2 mRNA levels, which may be one of the mechanisms involved in LLLI-mediated reduction of rheumatoid arthritis (RA) inflammation in the CIA rat model [14]. In the present study, C57BL/6 mice with complete Freund’s adjuvant (CFA)-induced arthritis were used as acute progression arthritis models. For the suppression of the inflammatory response, MILNS was used as the treatment modality. The treatment efficacy was evaluated using laser speckle contrast images (LSCIs) and histological analysis.
paw of each mouse [15, 16]. Because the adjuvant elicited an acute response, minimally invasive laser stimulation was applied to the laser group 3 h after injection of the adjuvant. LSCIs were subsequently obtained every 6 h for a total of 108 h. Conversely, in the control group, no laser stimulation was performed and LSCIs were obtained. As a result, the laser group has taken totally 18 times of minimally invasive laser stimulations and both groups obtained totally 19 LSCIs. For the LSCI acquisition and minimally invasive laser stimulation, the mice were anesthetized with a mixture of isoflurane (2– 5 %) and oxygen (1–2 L/min). At the end of the experiment, all mice were euthanized by cervical dislocation for histological analysis. All animal experiments were performed in accordance with the guidelines and under the approval of the Institutional Animal Care and Use Committee of Yonsei University (YWC-130411-1). MILNS Figure 1 shows a schematic diagram of the MILNS used for the minimally invasive laser stimulation of the left hind paw of the mouse. A 660-nm diode laser (#ML101J27, ThorLabs, Newton, NJ, USA) on a laser diode mount (#TCLDM9, ThorLabs, Newton, NJ, USA) was used as a light source with a laser diode driver (#IP500, ThorLabs, Newton, NJ, USA) and a thermoelectric cooler (TEC) controller (#TCM1000T, ThorLabs, Newton, NJ, USA). The laser was collimated by using a collimation lens (#C230TME-B, ThorLabs, Newton, NJ, USA) and focused on an optical fiber (#AFS105/125Y-CUSTOM, ThorLabs, Newton, NJ, USA) with a fiber port (#PAF-X-11-PC-A, ThorLabs, Newton, NJ, USA). The end of the optical fiber was combined to a fine hollow needle (outer diameter, 300 μm; inner diameter, 130 μm) without a fiber jacket. The fine hollow needle, which delivered the laser at the center of the mouse’s left hind paw, was made of stainless steel that has anti-corrosion, heat-resistant, and nonmagnetic characteristics. The irradiated energy of the minimally invasive laser is 3 J with a 20-mW laser intensity for 150 s.
Materials and methods
Laser speckle imaging system
Animals and experimental design
Figure 2 shows a schematic diagram of the laser speckle imaging system. A diode laser (HL6512MG; 658 nm, 50 mW; Thorlabs, Newton, NJ, USA) is coupled into an optical fiber. A laser passes through a holographic diffuser for even illumination and is directed to a 1:1 beam splitter to eliminate shadowing in the sample. An image of the mouse’s left hind paw was captured with a charge-coupled device (CCD) camera (XC-HR57; Sony, Tokyo, Japan) and a macro zoom lens (MLM3X-MP; Computar, Commack, NY, USA). The exposure time was empirically determined based on preliminary experiments. The imaging areas were
Eight C57BL/6 mice (aged 14 weeks) were used as an animal experiment model of arthritis using an adjuvant. The mice were kept in a controlled standard condition (room temperature, 23±2 °C; humidity, 50±10 %), allowed to move freely, and fed a standard laboratory chow and water ad libitum. The mice were divided into two groups—namely the laser group (n=4) and the control group (n=4). To induce arthritis, 0.05 mL of complete Freund’s adjuvant (CFA) (#7001, Chondrex, Redmond, WA, USA) was injected in the left hind
Lasers Med Sci Fig. 1 Schematic diagram of the minimally invasive laser needle system
approximately 6×8 mm2, and the images were acquired at 30 Hz with a frame grabber (DOMINO IOTA; Euresys, Angleur, Belgium). The area and average laser speckle index (LSI) value of the left hind paw were calculated from the LSCIs. Analysis of LSCI In general, the key parameters from which the diagnosis of arthritis is derived are the degree of swelling and the presence of angiogenesis, which are correlated with inflammatory responses and reflect the progression stage of arthritis. The paw area was used to estimate the degree of swelling in twodimensional images using the LSCIs. The LSI computed from the LSCI was used to indirectly calculate the degree of angiogenesis. The paw area and LSI were obtained by calculating the total number of pixels and the average pixel value of the paw region on LSCI, respectively.
cut into slices with a thickness of 5 μm. For the purpose of assessing the macrophage population in the laser-stimulated area, immune-fluorescent anti-CD68 was used as a specific macrophage marker. In brief, tissue sections of the middle of the hind paw were washed with phosphate buffer solution (PBS; pH 7.4) and blocked in 5 % bovine serum albumin containing 0.1 % Triton X-100 for 30 min. The sections were then incubated in a cocktail of mouse anti-CD68 (1:1,000; Abcam) for 1 h at room temperature. After washing with 0.01 M PBS, the sections were further incubated for 30 min with a mixture of PE-conjugated goat anti-mouse IgG (1:5,000; Santa Cruz Biotechnology Inc., Santa Cruz), and fluorescence images were subsequently acquired by fluorescence microscopy (Olympus IX81, MetaMorph ver 7.5.3.0, NY, USA).
Results Histological analysis After the sacrifice of eight C57BL/6 mice at 14 weeks, excised paw tissues (n=5) were dipped in Fixative/Decalcifier (Thermo Fisher Scientific) for 14 days, dehydrated with a graded ethanol, and embedded in paraffin. The paraffin blocks were
LSCI Figures 3 and 4 show results from the LSCIs of the control group and the laser group, respectively. Macroscopically, both groups show a decreasing trend of LSI as a function of time due to the characteristics of the adjuvant. However, compared to the laser group, the control group shows higher LSI across all measurement points.
Variation of the paw area
Fig. 2 Schematic diagram of the laser speckle imaging modality
Figure 5 shows the percentage variation of the paw area as a function of time. Up to two measurement points, both groups show similar percentage variation. However, after two measurement points, the laser group shows a lower percentage variation as a function of time when compared to the control group. Data are expressed as mean±standard error of measurement (SEM).
Lasers Med Sci
0 (before adjuvant
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Fig. 3 Representative laser speckle contrast image of the control group as a function of time
Variation of the average LSI
Histological analysis
Figure 6 shows the percentage variation of average LSI on the paw area as a function of time. Similar to that noted for paw area variation, both groups show a marked difference in LSI after two measurement points. The control group when compared to the laser group shows greater percentage variation in LSI as a function of time. Data are expressed as mean±SEM.
Macrophage population is used as a marker of inflammation in arthritis because macrophages are known to be exclusively generated in inflamed tissue. Therefore, to investigate the effect of minimally invasive laser stimulation, tissue sections of the left hind paw of both groups were examined using antiCD68 as a macrophage cell surface marker. As noted in Fig. 7,
Lasers Med Sci
0 (before adjuvant
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Fig. 4 Representative laser speckle contrast image of the laser group as a function of time
the control group shows a stronger red fluorescence signal of the CD68 when compared to the laser group.
Discussion Arthritis is an autoimmune inflammatory disease that affects an individual’s activities of daily living due to the destruction
of the joints [3, 4, 8]. The application of LLLT as a treatment modality for arthritis patients was reported in several clinical papers to have a positive outcome in terms of relieving pain and improving joint movement [1, 5]. However, LLLT has the potential problem of laser energy loss due to light scattering in the tissue. This, in turn, results in the decrease of its therapeutic efficacy due to the reduction of the intensity, which makes it difficult to achieve biostimulation of the target tissue. When
Lasers Med Sci Fig. 5 Variation of paw area in the control and laser groups as a function of time
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efficacy of LLLT at the target tissue, high-intensity NIR light is generally used for conditions requiring deep penetration with sufficient intensity. In the present study, we aimed to address the problem of light energy loss and to increase the overall therapeutic efficacy in the treatment of arthritis, by using MILNS, which employs a fine hollow needle for stimulation at deep target tissues without the associated light energy loss. As a CFA-induced arthritis mouse model, we used C57BL/ 6 mice with a site-induced inflammatory response and acute
Fig. 6 Variation of laser speckle index in the control and laser groups as a function of time
Percentage variation of average LSI on paw area (%)
the appropriate laser energy density is reached, LLLT has been shown to be effective in the treatment of tissue disorders by activating a series of reactions leading to increased cellular metabolism, with the transformation of the absorbed light energy into cellular energy [1]. According to the Arndt-Schulz law, the response of biostimulation can either be an acceleration or an inhibition of physiologic activity depending on the energy dosage of stimulation [17]. Therefore, in order to increase the therapeutic
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Lasers Med Sci Fig. 7 Representative fluorescence images of the center of the left hind limb paw in the a control and b laser groups
progression of the condition. Due to the acute progression in this model, LSCIs of the laser and control groups were first obtained 3 h after the injection of adjuvant and subsequently after every 6 h for a total of 108 h. The LSCI is mainly used to measure blood flow perfusion in biological tissue [18]. During the progression of arthritis with synovial membrane inflammation, modulation of inflammatory cells from the vascular endothelium increases the vascular permeability, thus resulting in vasodilatation with edema [3]. Such transitions that are captured in LSCI can be quantified by LSI in order to determine the severity of the inflammatory response. In particular, a higher LSI reflects a more severe progression of arthritis. The results of the present study showed that CFAinduced arthritis progressed severely with higher LSI as a function of time in the control group (Fig. 3). However, the progression of arthritis in the laser group was suppressed, as indicated by a lower LSI (Fig. 4). MILNS may influence the function of cells in the immune system such as T, B, and NK lymphocytes in the arthritis-induced area. Alves et al. reported that LLLT has a positive effect in rheumatoid arthritis (RA) due to the decrease of the vascular permeability associated with infiltration of leukocytes and influence on the maintenance of synovial fluid lubricant composition [3]. Moreover, the inflammatory modulation by MILNS was confirmed through the variation of the paw area. The laser group showed a markedly smaller variation of the paw area when compared to the control group (Fig. 5). In biological tissue, when inflammation is induced, macrophages—because of their role as phagocytic cells—become activated and will interact with lymphocytes to facilitate antibody production. With the activation of macrophages, the synovial tissue releases cytokines and chemokines to promote chronic inflammation, such as in cases of arthritic synovial tissue [19]. The population of activated macrophages induced by the adjuvant was examined by using an anti-CD68 as a macrophage marker. As noted in Fig. 7, when a red fluorescence signal was used for the detection of macrophages, marked differences between the two groups were noted in the macrophage population. As compared to the control group, the laser group showed a lower fluorescence signal,
which indicates that minimally invasive laser stimulation is effective in the suppression of inflammatory reaction on arthritis progression. To our knowledge, it may be the first study employing the MILNS for arthritis treatment. The MILNS was employed as a minimally invasive stimulation method to improve the therapeutic efficacy of LLLT in arthritis, and its therapeutic efficacy was evaluated by analyzing the LSCI and histology. The laser group when compared to the control group resulted in better therapeutic efficacy by presenting remarkable lower macrophage population which is the fundamental parameter directly related to the suppression of inflammatory response and decreasing paw area and LSI. Although the therapeutic and diagnostic methods for arthritis are effective in the animal model due to the shallow animal skin compared to the human skin, this study may have some limitations in clinical application: (1) the strength and size of the laser needle may have to be reconsidered depending on the region of interest; (2) the lower quality of LSCI which may be caused by the shallow penetration depth of the laser in the human skin has to be considered and may be solved by integrating an optical tissue clearing method into the imaging modality to reduce tissue turbidity. In previous LLLT studies, infrared light was used to increase the laser penetration depth of deep target tissues such as bone [10, 20]. However, in the present study, MILNS minimized the energy loss problem incurred in the use of LLLT and addressed the limitation of the wavelengths available for the penetration depth. As a means of localized treatment, MILNS may suppress the acute inflammatory response and increase treatment efficacy by modulating the inflammatory response and the activity of the inflammatory cells. Nonsteroidal anti-inflammatory drugs (NSAIDs) are generally used for the systemic treatment of arthritis [21]. However, further studies are expected to show that MILNS may have a synergic effect with NSAIDs in a systemic-developed arthritis animal model. Furthermore, each cell in biological tissue has a different cellular reaction to stimulation conditions such as wavelength, energy density, and pulse duration. Although the present study shows positive effects in the suppression of acute
Lasers Med Sci
arthritis progression, various stimulation conditions have yet to be investigated in order to further enhance the therapeutic efficacy.
Conclusion In the present study, MILNS has been shown to be therapeutically efficacious in suppressing the acute progression of CFA-induced arthritis. However, further studies are needed to investigate the effects of different therapeutic parameters such as wavelength, dosage, and the number of sessions needed to achieve the most effective therapeutic parameter settings. Moreover, the therapeutic mechanism of MILNS in arthritis has to be investigated. We believe that MILNS can potentially be used as a treatment modality of arthritis in the clinical setting. Acknowledgements This research was supported by the Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2010-00757).
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