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.

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.

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.

Model Calculations The tissue was modelled by the so called Krogh's cylinders, i.e. blood parallel vessels in a specific distance supply the tissue with oxygen. Oxygen transfer in the tissue takes place due to diffusion only which needs an decrease of oxygen concentration with increasing distance to the vessel. A oxygen diffusion rate of D = 2 10-5 cm /s was used. The rate equations for the population density in the levels of the photosensitizer molecule and in the oxygen molecule were build up. All transition shown in Fig. 1 have been considered with the marked rate constants. The system of coupled differential equations were solved numerically on a personal computer which needed computing time up to 36 hours.

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.

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/cm2, absorbed dose: 18,6J/cm2) 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.

Helicobacter pylori (HP), a gram-negative microaerophilic bacterium located in gastric mucosa, plays an important role in gastro carcinogenesis. Due to the increasing emergence of antibiotic resistance, photodynamic inactivation of bacteria presents a new approach to treat bacterial infections, like HP. In vitro experiments were performed to determine the irradiation conditions for a complete inactivation of HP with the photosensitizer Chlorin e6 (Ce6). The HP strain CCUG 38770 (Culture Collection, University of Gothenburg, Sweden) was routinely cultured under microaerophilic conditions, suspended in sodium chloride, incubated with Ce6 and irradiated briefly with red light of the appropriate wavelength of lambda = 660 nm. Series of measurements of different Ce6-concentrations (0.1 mu M - 100 mu M) were carried out, whereby the incubation time was kept constant at 1 min. The absorbed energy dose has been selected in varying the irradiation time (1 s - 300 s) and the power density (4.5 mW/cm(2), - 31 mW/cm(2)). Quantification of inactivation was performed by enumeration of the grown colonies. In addition, the accumulation of Ce6 in HP cells was studied more precisely by fluorescence spectroscopy. With a Ce6 concentration of 100 mu M and a power density of 9 mW/cm(2), a 6-log(10) reduction in the survival rate of HP was achieved within 30 seconds of irradiation. In conclusion the most relevant factor for the inactivation of HP is the exposure time of irradiation, followed by the concentration of Ce6 and the light intensity. Further studies with HP strains obtained from patient specimens are under current investigation.

The increasing prevalence of antibiotic resistance requires new approaches also for the treatment of infectious keratitis. Photodynamic Inactivation (PDI) using the photosensitizer (PS) Chlorin e6 (Ce6) was investigated as an alternative to antibiotic treatment. An in-vitro cornea model was established using porcine eyes. The uptake of Ce6 by bacteria and the diffusion of the PS in the individual layers of corneal tissue were investigated by fluorescence. After removal of the cornea’s epithelium Ce6-concentrations < 1 mM were sufficient to reach a penetration depth of 500 μm. Liquid cultures of microorganisms were irradiated using a specially constructed illumination chamber made of Spectralon(R) (reflectance: 99 %), which was equipped with high power light emitting diodes (λ = 670 nm). Clinical isolates of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) from keratitis patients were tested in liquid culture against different concentrations of Ce6 (1 - 512 μM) using 10 minutes irradiation (E = 18 J/cm2 ). This demonstrated that a complete inactivation of the pathogen strains were feasible whereby SA was slightly more susceptible than PA. 3909 mutants of the Keio collection of Escherichia coli (E.coli) were screened for potential resistance factors. The sensitive mutants can be grouped into three categories: transport mutants, mutants in lipopolysaccharide synthesis and mutants in the bacterial SOS-response. In conclusion PDI is seen as a promising therapy concept for infectious keratitis.

Photodynamic inactivation (PDI) of bacterial strains presents an attractive potential alternative to antibiotic therapies. Success is dependent on the effective accumulation in bacterial cells of photochemical substances called photosensitizers, which are usually porphyrins or their derivatives. The kinetics of porphyrin synthesis after treatment with the precursor ALA and the accumulation of the Chlorin e6 and the following illumination were studied. The goal was to estimate effectivity of the destructive power of these PS in vitro in respect of the physiological states of Mycobacteria. So the present results examine the cell destruction by PDI using ALA-induced Porphyrins and Chlorin e6 accumulated in Mycobacterium phlei and Mycobacterium smegmatis, which serve as models for the important pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis. We could show that both Mycobacterium after ALA and Chlorin e6 application were killed by illumination with light of about 662 nm. A reduction of about 97 % could be reached by using a light-dose of 70 mW/cm2.

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 protoporhyrin 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 protoporhyrin 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.

To detect bronchial carcinoma by autofluorescence, we measured in-vivo, in an in-vivo model, and in-vitro the spectra of tumor and normal tissue by a fiber-optic-spectrometer. The main difference between tumor and bronchial tissue is the intensity of the 505 nm main peak.

To evaluate the application of high power diode lasers for medical treatment, the interaction of a beam of up to 450 W (λ = 808 and λ = 940 nm) on lung tissue was studied.

The zebrafish embryo has been proposed as a ‘bridge model’ to study the effects of cigarette smoke on early development. Previous studies showed that exposure to total particulate matter (TPM) led to adverse effects in developing zebrafish, and suggested that the antioxidant and aryl hydrocarbon receptor (AHR) pathways play important roles. This study investigated the roles of these two pathways in mediating TPM toxicity. The study consisted of four experiments. In experiment I, zebrafish embryos were exposed from 6 h post fertilization (hpf) until 96 hpf to TPM0.5 and TPM1.0 (corresponding to 0.5 and 1.0 μg/mL equi-nicotine units) in the presence or absence of an antioxidant (N-acetyl cysteine/NAC) or a pro-oxidant (buthionine sulfoximine/BSO). In experiment II, TPM exposures were performed in embryos that were microinjected with nuclear factor erythroid 2-related factor 2 (Nrf2), AHR2, cytochrome P450 1A (CYP1A), or CYP1B1 morpholinos, and deformities were assessed. In experiment III, embryos were exposed to TPM, and embryos/larvae were collected at 24, 48, 72, and 96 hpf to assess several genes associated with the antioxidant and AHR pathways. Lastly, experiment IV assessed the activity and protein levels of CYP1A and CYP1B1 after exposure to TPM. We demonstrate that the incidence of TPM-induced deformities was generally not affected by NAC/BSO treatments or Nrf2 knockdown. In contrast, AHR2 knockdown reduced, while CYP1A or CYP1B1 knockdowns elevated the incidence of some deformities. Moreover, as shown by gene expression the AHR pathway, but not the antioxidant pathway, was induced in response to TPM exposure, providing further evidence for its importance in mediating TPM toxicity.

The quantification of the induced fluorescence in tumor tissue is important to design optical equipment for photodynamic diagnosis (PDD). The fluorescence intensities of Protoporphyrin IX (PpIX) solved in Dimethylsulfoxid and induced via application of Aminolevulinic Acid in cells, cultivated in the hen's eggs model, have been measured photometrically. With an optimized CCD-camera-systems fluorescent tumor areas were detected before and after photodynamic therapy (PDT). The ratio of dead cells was detected by staining with trypan blue after PDT. The measurements were carried out with various energy densities at the time of maximal PpIX-enrichement.