First biocompatibility margins for optical stimulation at the eardrum via 532-nm laser pulses in a mouse model.

Hearing impairment affects ∼460 million people worldwide. Conservative therapies, such as hearing aids, bone conduction systems, and middle ear implants, do not always sufficiently compensate for this deficit. The optical stimulation is currently under investigation as an alternative stimulation strategy for the activation of the hearing system. To assess the biocompatibility margins of this emerging technology, we established a method applicable in whole-mount preparations of murine tympanic membranes (TM). We irradiated the TM of anesthetized mice with 532-nm laser pulses at an average power of 50, 89, 99, and 125 mW at two different locations of the TM and monitored the hearing function with auditory brainstem responses. Laser-power-dependent negative side effects to the TM were observed at power levels exceeding 89 mW. Although we did not find any significant negative effects of optical stimulation on the hearing function in these mice, based on the histology results further studies are necessary for optimization of the used parameters.

Frequency-specific activation of the peripheral auditory system using optoacoustic laser stimulation.

Hearing impairment is one of the most common sensory deficits in humans. Hearing aids are helpful to patients but can have poor sound quality or transmission due to insufficient output or acoustic feedback, such as for high frequencies. Implantable devices partially overcome these issues but require surgery with limited locations for device attachment. Here, we investigate a new optoacoustic approach to vibrate the hearing organ with laser stimulation to improve frequency bandwidth, not requiring attachment to specific vibratory structures, and potentially reduce acoustic feedback. We developed a laser pulse modulation strategy and simulated its response at the umbo (1-10 kHz) based on a convolution-based model. We achieved frequency-specific activation in which non-contact laser stimulation of the umbo, as well as within the middle ear at the round window and otic capsule, induced precise shifts in the maximal vibratory response of the umbo and neural activation within the inferior colliculus of guinea pigs, corresponding to the targeted, modelled and then stimulated frequency. There was also no acoustic feedback detected from laser stimulation with our experimental setup. These findings open up the potential for using a convolution-based optoacoustic approach as a new type of laser hearing aid or middle ear implant.

Bacterial keratitis: Photodynamic inactivation reduced experimental inflammation.

The successful treatment of bacterial keratitis remains an unsolved clinical problem. The current study aimed to establish a murine keratitis model and to investigate the effect of chlorin e6 (Ce6) and photodynamic inactivation (PDI) on corneal inflammation. The cornea of anesthetized mice was scratched and covered with a bacterial suspension of Pseudomonas aeruginosa. A paste containing Ce6 was applied to the cornea with subsequent exposure to specified light. Two days later the animals were sacrificed, and the globes were processed for light microscopy. Evaluation parameters were the maximal corneal thickness and the severity of the hypopyon. The maximal corneal thickness of 290±16 µm in the infected and untreated group was significantly reduced to 220±8 µm in the infected and treated group (P<0.05). In addition, the hypopyon was less severe in the infected and treated group. In conclusion, the present study indicates that PDI using Ce6 may be a potential approach to treat patients suffering with severe bacterial keratitis.

Chlorin e6 mediated photodynamic inactivation for multidrug resistant Pseudomonas aeruginosa keratitis in mice in vivo.

Following corneal epithelium scratches, mouse corneas were infected with the multidrug resistant (MDR) P. aeruginosa strain PA54. 24 hours later, 0% (for control group), 0.01%, 0.05% or 0.1% Chlorin e6 (Ce6), a second generation photosensitizer derived from chlorophyll, was combined with red light, for photodynamic inactivation (PDI). 1 hour or 2 days later, entire mouse eyes were enucleated and homogenized for counting colony forming units (CFU) of P. aeruginosa. For comparison, 0.1% Ce6 mediated PDI was started at 12 hours post infection, and 0.005% methylene blue mediated PDI 24 hours post infection. Clinical scores of corneal manifestation were recorded daily. Compared to the control, CFU 1 hour after PDI started 24 hours post infection in the 0.01% Ce6 and 0.05% Ce6 groups were significantly lower (more than one log10 reduction), the CFU 2 days post PDI higher in the 0.1% Ce6 group, clinical score lower in the 0.1% Ce6 group at 1 day post PDI. These findings suggest that PDI with Ce6 and red light has a transient efficacy in killing MDR-PA in vivo, and repetitive PDI treatments are required to fully resolve the infection. Before its clinical application, the paradoxical bacterial regrowth post PDI has to be further studied.

Photodynamic inactivation of multidrug-resistant Staphylococcus aureus by chlorin e6 and red light (λ=670nm).

Multidrug-resistant Staphylococcus aureus (MDR-SA) are a frequent cause of antibiotic treatment refractory bacterial corneal infections. Photodynamic therapy (PDT) is being discussed as a putative treatment option to cure this type of bacterial infection. Here we tested the in vitro susceptibility of a set of 12 clinically derived MDR-SA isolates with differing genetic backgrounds and antibiotic resistance profiles against photodynamic inactivation (PDI) by the porphyrin chlorin e6 (Ce6) and red light (λ=670nm). All tested clinical isolates displayed a 5-log10 reduction in viable cells by Ce6 and red light, when cells were preincubated with the photosensitizer at concentrations ≥128µM for 30min in the dark, and a subsequent irradiation with light at λ=670nm (power density: 31mW/cm(2), absorbed dose: 18,6J/cm(2)) was applied. Similarly, cells of the laboratory strain Newman required the same Ce6 pre-incubation and light dose for a 5-log10 reduction in cell viability. Inactivation of crtM in strain Newman, which interferes with pigment production in S. aureus, rendered the mutant more susceptible to this PDT procedure, indicating that the level of resistance of S. aureus to this therapy form is affected by ability of the pathogen to produce the carotenoid pigment staphyloxanthin. Incubation of freshly explanted porcine corneas with a 0.5% Ce6 gel demonstrated that the photosensitizer can diffuse into and accumulate within the stroma of the cornea in concentrations found to be sufficient to yield a 5-log10 reduction of the S. aureus cell pool in vitro. These data suggest that PDI with Ce6 and red light might be a promising new option for the treatment of MDR-SA induced corneal infections.

Pharmacokinetics of the photosensitizers aminolevulinic acid and aminolevulinic acid hexylester in oro-facial tumors embedded in the chorioallantois membrane of a hen's egg.

BACKGROUND: Oral squamous-cell carcinoma is a frequent form of cancer in the head and neck region. The survival rate is poor. Therapy success is highly dependent on the stage of cancer development at which diagnosis is made. The disease is mostly diagnosed at a late stage. Photodynamic diagnosis is a new tool for screening examinations. This technique calls for reliable photosensitizers, such as aminolevulinic acid (ALA) and aminolevulinic acid hexylester (h-ALA). ALA and h-ALA are the source material for the synthesis of protoporphyrin IX in tumor cells. Protoporphyrin IX has a high detection rate for tumor tissue within a reasonable period of time. METHODS: Tumor specimens were harvested from oral carcinomas and basaliomas of the face. The vital cells of the specimens and the human tumor cell line (CLS- 354) were cultured in a 90% RPMI and 10% fetal bovine serum medium. A constant number of 50,000 cells from each specimen and the cell line were transferred to an in vivo model on the hen's egg model. The grown specimens were tested for tumor fluorescence with ALA and h-ALA. The intensity of tumor fluorescence during the following 24 hours was measured spectroscopically as the degree of concentration of protoporphyrin IX within the cells. RESULTS: All tumors showed higher protoporphyrin IX enrichment and fluorescence, compared to healthy tissue. Using h-ALA, the peak concentration of protoporphyrin IX was achieved 20%-25% more quickly with 3- or 6-mM solutions than with ALA. The highest contrast between tumorous and healthy tissue achieved owing to fluorescence was 1:11 using h-ALA, compared to 1:5 using ALA with the peak concentrations of protoporphyrin IX. CONCLUSIONS: Using h-ALA, the peak concentration of protoporphyrin IX, compared to ALA, is achieved 20% percent more quickly and with twice as much contrast between tumorous and healthy tissue (1:11 compared and 1:5, respectively). This facilitates a faster, better discrimination between tumorous and healthy tissue.