Control of Burn Wound Infection by Methylene Blue-Mediated Photodynamic Treatment With Light-Emitting Diode Array Illumination in Rats.

BACKGROUND AND OBJECTIVES: Control of burn wound infection is difficult due to the increase in drug-resistant bacteria and deteriorated immune responses. In this study, we examined the usefulness of methylene blue (MB)-mediated antimicrobial photodynamic therapy (aPDT) with illumination by a light-emitting diode (LED) array for controlling invasive infections from the wound to inside the body for rats with an extended deep burn infected with Pseudomonas aeruginosa. STUDY DESIGN/MATERIALS AND METHODS: An MB solution with the addition of ethanol, ethylene-diamine-tetra-acetic acid disodium salt, and dimethyl sulfoxide was used as a photosensitizer (PS). An extended deep burn was made on the dorsal skin in rats and the wounds were infected with P. aeruginosa. The rats were divided into three groups: control (no treatment; n = 14), PS mixture application alone (PS alone group; n = 10), and aPDT group (n = 14). For aPDT, after the PS mixture was applied onto the surface of infected wounds, the wounds were illuminated with a 665-nm LED array at an intensity of 45 mW/cm2 three times per treatment, with an illumination duration of 20 minutes and an interval of 10 minutes. The treatment was repeated each day for 7 consecutive days (day 0-day 6). Bacterial numbers on the wound surface and the weights and survival rates of the animals were evaluated daily. At the endpoints, bacterial numbers in the liver and blood were counted. Since the PS mixture showed high dark toxicity against P. aeruginosa in vitro, the influence of the PS mixture application onto healthy skin was also examined in vivo. RESULTS: Even in the aPDT group, rapid bacterial regrowth was observed on the wound surface after each day's treatment, but the geometric mean values of the bacterial numbers before and after each aPDT were considerably lower than those in the control group. Application of the PS mixture alone showed a clear bactericidal effect only at day 0, which is attributable to the formation of biofilms after day 1. Rats in the aPDT group showed a smaller weight loss, a higher ratio of no bacterial migration at the endpoints, and significantly higher survival rates than those in the other two groups. Effects of repeated application of the PS mixture onto healthy skin were not evident. CONCLUSIONS: Application of MB-mediated aPDT with illumination by a high-intensity LED array daily for seven consecutive days was effective for suppressing invasive infection from the wound to inside the body in rats with an extensive deep burn infected with P. aeruginosa, resulting in significant improvement of their survival. Lasers Surg. Med. © 2021 Wiley Periodicals LLC.

Meningeal Damage and Interface Astroglial Scarring in the Rat Brain Exposed to a Laser-Induced Shock Wave(s).

In the past decade, signature clinical neuropathology of blast-induced traumatic brain injury has been under intense debate, but interface astroglial scarring (IAS) seems to be convincing. In this study, we examined whether IAS could be replicated in the rat brain exposed to a laser-induced shock wave(s) (LISW[s]), a tool that can produce a pure shock wave (primary mechanism) without dynamic pressure (tertiary mechanism). Under certain conditions, we observed astroglial scarring in the subpial glial plate (SGP), gray-white matter junctions (GM-WM), ventricular wall (VW), and regions surrounding cortical blood vessels, accurately reproducing clinical IAS. We also observed shock wave impulse-dependent meningeal damage (dural microhemorrhage) in vivo by transcranial near-infrared (NIR) reflectance imaging. Importantly, there were significant correlations between the degree of dural microhemorrhage and the extent of astroglial scarring more than 7 days post-exposure, suggesting an association of meningeal damage with astroglial scarring. The results demonstrated that the primary mechanism alone caused the IAS and meningeal damage, both of which are attributable to acoustic impedance mismatching at multi-layered tissue boundaries. The time course of glial fibrillary acidic protein (GFAP) immunoreactivity depended not only on the LISW conditions but also on the regions. In the SGP, significant increases in GFAP immunoreactivity were observed at 3 days post-exposure, whereas in the GM-WM and VW, GFAP immunoreactivity was not significantly increased before 28 days post-exposure, suggesting different pathological mechanisms. With the high-impulse single exposure or the multiple exposure (low impulse), fibrotic reaction or fibrotic scar formation was observed, in addition to astroglial scarring, in the cortical surface region. Although there are some limitations, this seems to be the first report on the shock-wave-induced IAS rodent model. The model may be useful to explore potential therapeutic approaches for IAS.

Depth distributions of bacteria for the Pseudomonas aeruginosa-infected burn wounds treated by methylene blue-mediated photodynamic therapy in rats: effects of additives to photosensitizer.

SIGNIFICANCE: Pseudomonas(P.) aeruginosa, a common cause of infection in burns, acquires antibiotic resistance easily and forms biofilms efficiently. Thus, it is difficult to control P. aeruginosa infection in burn wounds, which causes lethal septicemia. Antimicrobial photodynamic therapy (aPDT) is attractive as a new strategy to treat burn wound infections with drug-resistant bacteria. AIM: We examined the efficacy of methylene blue (MB)-mediated aPDT with various additives in a tissue depth-resolved manner to find conditions that minimize the bacterial invasion. APPROACH: We applied MB-mediated aPDT with LED array illumination to an extensive, full-thickness burn infected with P. aeruginosa in rats for three consecutive days (days 0, 1, and 2). On day 2, the depth distributions of bacteria were assessed based on the histological analysis using Gram staining. We examined how the addition of ethylenediaminetetraacetic acid (EDTA), ethanol, and dimethyl sulfoxide (DMSO) affected the efficacy of aPDT. RESULTS: Pure MB-mediated aPDT significantly reduced the numbers of bacteria with biofilms on the wound surface and in the epidermis compared with those for the control tissue (saline only). However, there were many bacteria in the deeper region of the tissue. In contrast, MB/EDTA/ethanol/DMSO-mediated aPDT minimized the numbers of bacteria in the broad depth region of the tissue. Still, a limited number of bacteria was observed in the subcutaneous tissue. CONCLUSIONS: The depthwise analysis of bacteria demonstrated the efficacy of the MB-mediated aPDT with the addition of EDTA, ethanol, and DMSO in controlling burn wound infections. However, further improvement of the therapy is needed to suppress bacterial migration into the deep tissue completely.

Combined Addition of Ethanol and Ethylenediaminetetraacetic Acid Enhances Antibacterial and Antibiofilm Effects in Methylene Blue-Mediated Photodynamic Treatment against Pseudomonas aeruginosa In Vitro.

Antimicrobial photodynamic treatment (aPDT) for infection with drug-resistant bacteria has received much attention. For P. aeruginosa, however, efficient formation of biofilms and the nature of Gram-negative bacteria often limit the efficacy of aPDT. In this study, we investigated the effects of ethanol and ethylenediaminetetraacetic acid (EDTA) as additives on bacterial viability, biofilm biomass, and structures of bacteria and biofilms in methylene blue (MB)-mediated aPDT in vitro. Matured P. aeruginosa biofilms were incubated with 32-µm MB solutions with different concentrations of additives and then illuminated with 665-nm light from an LED array. The combined addition of 10% ethanol and 10 mm EDTA to MB resulted in significantly greater bactericidal effects than those of MB alone and of MB with 10% ethanol or 10 mm EDTA. Crystal violet assays showed significant reductions in biofilm biomass by aPDT with addition of both ethanol and EDTA compared to that in the case of aPDT with MB alone. Scanning electron microscopy showed broken bacterial cells and reduction in the cell density and amount of biofilm under those conditions. Ethanol addition alone did not improve aPDT efficacy. Reduced amount of biofilm by EDTA addition would have improved the transportation of MB and ethanol to bacteria.