We investigated the oxygenated hemoglobin concentration (CHbO), deoxygenated hemoglobin concentration (CHbR), methemoglobin concentration (CmetHb), total hemoglobin concentration (CHbT), tissue oxygen saturation (StO2) and methemoglobin saturation (StMet) in rat burn wound model using a diffuse reflectance spectral imaging (DRSI) method. Superficial dermal burns (SDBs), deep dermal burns (DDBs), and deep burns (DBs) were induced in rat dorsal skin by exposing the skin of approximately 20% of the total body surface area (4×10 cm2) to water maintained at 70, 78, and 98 °C for 10 second, respectively, using a Walker–Mason template. A set of spectral images for dorsal regions were acquired pre-burn and immediately post burn under anesthesia. Subsequent measurements were acquired at 24, 48 and 72 hours after injury. We found that the time courses of CHbO, CHbR, CmetHb, CHbT, StO2 and StMet have different trends depending on the depth of burn wounds. The results in this study demonstrated the feasibility of our proposed DRSI method for in vivo monitoring of hemoglobin derivatives in rat burn wound model to differentiate the burn depth groups in rats.
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.
Pseudomonas (P.) aeruginosa is a typical Gram-negative bacterium to cause burn wound infection, which is hard to control since P. aeruginosa efficiently forms biofilms and easily acquires drug resistance, resulting in septicemia. Antimicrobial photodynamic therapy (aPDT) is a new approach for controlling such burn wound infection, and we showed a certain effectiveness of methylene blue (MB)-mediated aPDT with LED array illumination for a rat model with an extended, full-thickness burn infected with P. aeruginosa. However, rapid bacterial regrowth was observed even after consecutive daily aPDT, indicating efficient bacterial invasion into the tissue. Thus, it is crucial to evaluate the aPDT efficacy in a depthresolved manner. In this study, we analyzed depth distributions of bacteria for the rat treated with saline (control), rat treated with a photosensitizer mixture alone (PS alone) and rat treated with a PS mixture plus light (aPDT) based on Gramstained tissue sections for the same rat model as that described above. For the control rat, large number of bacteria were observed on the tissue surface and in the wide depth range of the skin. For the PS alone rat, many bacteria were observed on the tissue surface and in the epidermal region, but the number of bacteria was limited in the deeper region of the skin, indicating a PS mixture dark toxicity. The aPDT showed drastic decreases in the number of bacteria both on the tissue surface and in the subsurface region; however, there were still certain numbers of bacteria in the deeper region of the skin.
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