Effect of photodynamic therapy on local muscle treatment in a rat muscle injury model: a controlled trial
© Inage et al.; licensee BioMed Central. 2015
Received: 21 October 2014
Accepted: 9 March 2015
Published: 18 April 2015
Muscle injury is common and is thought to account for 10%–50% of all sports-related injuries. The use of rest, ice, compression, and elevation is common in clinical practice, but many treatments over a long period are required to produce a therapeutic effect. We evaluated the utility of photodynamic therapy as a new treatment option for the acute stage of muscle injury.
Twenty 8-week-old Sprague-Dawley male rats underwent experimental injury of the right gastrocnemius muscle with a drop-mass method. After muscle injury was induced, a liposomally formulated indocyanine green derivative (7 mg/kg) near-infrared laser irradiation was performed at 18 h after injury. Local time-dependent changes in the treatment (n = 14) and no treatment (n = 14) groups were evaluated with in vivo imaging, histologic examination, and enzyme-linked immunosorbent assay methods.
In vivo imaging fluorescence values were significantly higher in the no treatment group, whereas interleukin-6 and tumor necrosis factor-α levels were significantly higher in the treatment group at 18 h after injury. Histologic examination results revealed that the treatment group had less bleeding and more degeneration repair processes than the no treatment group at 24 h and 1 week after muscle injury.
These findings suggest that photodynamic therapy promotes a tissue-repairing effect during the early stage of muscle injury.
Muscle injuries are common and reportedly account for 10%–50% of all sports-related injuries . These injuries occur when the muscle fibers are damaged by external pressure, often during contact sports such as football, rugby, and American football. Muscle injuries most commonly occur on the frontal surface of the thigh.
The so-called rest, ice, compression, and elevation (RICE) treatment is the most popular acute-phase treatment in clinical practice. However, previous magnetic resonance imaging (MRI)-based studies have shown that a large number of treatments over a long duration are required to produce a therapeutic effect . While echo assessments have shown that the pressure in a local hematoma is controlled, the appropriate treatment duration is unknown, and the acute period of treatment has not been sufficiently established . Importantly, inappropriate treatment in the early phase of muscle injury results in chronic pain in approximately 30% of patients . Therefore, appropriate treatment in the early phase is considered critical for the prevention of the transition to chronic pain. However, the treatment efficacy may depend on the severity of the injury.
Experiment 1: in vivo imaging
Experiment 2: enzyme-linked immunosorbent assay
Inflammatory mediators in the two groups were analyzed at 18 h after muscle injury. Resected gastrocnemius muscle samples were frozen in liquid nitrogen, pulverized or homogenized, and digested in a tissue lysis reagent. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 production at the muscle injury site were quantified using an enzyme-linked immunosorbent assay in accordance with the manufacturer’s protocols (R&D Systems, Minneapolis, MN, USA). Tissue protein levels were assayed using a kit in accordance with the manufacturer’s protocols (Bio-Rad, Hercules, CA, USA), and inflammatory mediator levels were normalized to tissue protein levels.
Experiment 3: histologic examination
The muscle injury sites (right side) of the two groups were dissected from the hind limbs under anesthesia with sodium pentobarbital (40 mg/kg, intraperitoneally) at 12, 18, 24 h and 1 week (nine rats; three rats per time period) after the muscle injury and perfused transcardially with 0.9% saline followed by 500 mL of 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). Each formalin-fixed tissue specimen was embedded in paraffin after dehydration for 14 h using an ascending series of ethanol in Tissue-Tek VIP (M1500; Sakura Finetek Japan Co. Ltd., Tokyo, Japan). Sections with a thickness of 4 μm were prepared from these paraffin blocks using a sliding microtome (LS113; Yamato-Kohki Industrial Co. Ltd., Saitama, Japan) and were stuck on a glass slide (#5116; Muto Pure Chemicals Co. Ltd., Tokyo, Japan). These sections were stained with Mayer’s hematoxylin (Muto Pure Chemicals Co. Ltd.) for 5 min after deparaffinization with xylene and ethanol. After washing with distilled water, these sections were dipped in 0.1% ammonium solution several times and washed again, and then washed using 100% ethanol. They were then stained with 1% eosin & phloxine-equivalent solution for 20 s. Finally, the sections were covered with mounting medium (Entellan- New; Merck KGaA, Darmstadt, Germany) after dehydration with a series of ethanol and xylene. The slides were observed under a microscope (BH20; Olympus Corporation, Tokyo, Japan) by a professional animal pathologist, and the degree of each finding was evaluated semiquantitatively. We evaluated the presence or absence of histologic degeneration, bleeding, and neutrophil recruitment in each slice. We also compared the histologic changes between the treatment and no treatment groups.
Fluorescence values at the muscle injury site were compared between the two groups at 12, 18, and 24 h after muscle injury using Student’s t test. Levels of inflammatory mediators at the muscle injury site were compared between the two groups at 18 h after the muscle injury using the Student’s t test. P values of <0.05 were considered statistically significant.
In vivo imaging
Enzyme-linked immunosorbent assay
In this study, we used in vivo fluorescence imaging to evaluate local fluorescent brightness after muscle injury and found that the fluorescence signal was significantly decreased in the treatment group compared to that in the no treatment group at 18 h after injury. According to a previous report, photodynamic therapy has a local hemostatic effect, such as neovascular vessel regression . Indeed, irradiation of LP-iDOPE causes the transformation of local triplet oxygen to singlet oxygen and free radicals; local cellular and tissue damage then occurs, and the local cells that line the blood vessels become damaged and block the blood vessel by creating a neovascular vessel . In actual clinical practice, this mechanism of action is widely used in the ophthalmologic field for age-associated macular degeneration . Our in vivo fluorescent findings are in line with this proposed mechanism of action.
The levels of two key cytotoxins reflect the inflammation and repair processes in muscle injury [9-11,17,18]. TNF-α is known not only as an irritable cytokine but also as an apoptosis-inducing factor (repair-starting signal) . In addition, previous reports have indicated that IL-6 plays a central role in inflammation and is involved in the repair and growth of the deep muscle . Thus, increases in irritable cytokine levels are important in the repair of injured tissue. We found that the levels of irritable cytokines significantly increased in the treatment group compared to those in the no treatment group at 18 h after injury and immediately after photodynamic therapy.
Additionally, histologic evaluations at 24 h after injury demonstrated low bleeding and enhanced degeneration repair processes in the treatment group. In addition, the repair reaction for degeneration tended to be very active even during the first week after muscle strain.
One limitation of this study is that we did not conduct objective tissue evaluation (i.e., grading for damage). In addition, we did not assess the maximum depth reached by the treatment method. Since these two criteria can be important for the clinical application of photodynamic therapy, we plan to examine them further in the future.
The LP-iDOPE used in this study was a liposomal preparation including ICG as a fluorescent dye. Although no reports have been published on the clinical use of this agent, the side effects are considered to be the same as those for ICG. ICG is a relatively safe trial drug with few side effects, but side effects (36/21,278 administration cases) were reported in 0.17% of cases in previous studies. The primary side effects reported were shock symptoms (0.02%: five cases), nausea/vomiting (0.08%: 16 cases), angialgia (0.04%: eight cases), and fever/heat sensations (0.02%: four cases) [19-21].
In conclusion, these findings suggest that photodynamic therapy promotes cytokine-mediated processes. We therefore suggest that photodynamic therapy elicits a tissue-repairing effect during the early stage of injury. Overall, these findings suggest that photodynamic therapy is a beneficial new treatment method for the acute period after muscle injury.
All procedures were performed at the Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University in Japan, 1-8-1 Inohana Chuo-ku, Chiba City, Chiba, Japan. 260-8670.
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