The Laser-Research Laboratory at the LIFE Center of the University Clinic Munich

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Medical Laser Application 24 (2009) 87–94 www.elsevier.de/mla

NEWS FROM THE LASER INSTITUTES

The Laser Research Laboratory at the LIFE Center of the University Clinic Munich

History and structure of the LIFE Center

Scientific director: Dr. Reinhold Baumgartner Contact:

Marchioninistr. 23 D-81377 Munich, Germany Phone: +49 89 7095 4882 Fax: +49 89 7095 4864 E-mail: [email protected] Internet: http:// laser.klinikum.uni-muenchen.de

Staff:

Physicists: 4 Engineers: 1 Post doctorates: 2 Ph.D. students: 5 MD students: 4 Assistants: 3 Total: 19

1615-1615/$ - see front matter doi:10.1016/j.mla.2009.02.048

Laser medicine in Munich dates back to 1970, when the first research groups settled in the former GSF, now the Helmholtz-Center, Munich, Neuherberg. They were headed by Prof. Hofstetter (Urology), Prof. Kiefhaber (Gastroenterology) and Prof. Leheta (Neurosurgery) and scientifically assisted by the Institute of Applied Optics (Prof. Waidelich). Increasing innovative laser applications soon required companies that had set their focus on clinical demands. Manufacturers of medical lasers and fiber optic components started to settle especially around Munich. To enable the technology transfer from the companies to the patients to be realized quickly and efficiently, the Laser Research Laboratory (LFL) was built on the Campus of the Klinikum of the University Munich, Großhadern, then constituting a research unit of the Clinic of Urology (headed by Prof. Hofstetter). In October 1995 the Research Pavilion took its present function as a well-equipped optical laboratory. The LIFE Center was established together with the Laboratory for Tumorimmunology, LTI (headed by Prof. Zimmermann). The concept of the LIFE Center is comprehensive tumor therapy (LIFE being an acronym for ‘‘Laser- und Immunologie-Forschungs-Einrichtungen’’). While scientists at the LFL focus their research on endoscope-based methods to detect and destroy the solid parts of the tumor, lasers and photodynamic procedures, the LTI staff tries to develop effective therapeutic strategies against single disseminated malignant cells causing tumor metastasis. The LIFE Center became an independent institution in April 2004. During the past few years, existing contacts to industrial as well as clinical partners have been continuously intensified and new ones established (see foundation of Transluminal Endoscopic Association Munich (T.E.A.M.)).

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Competence profile

TumorVision

In the Laser Research Laboratory the main research topics are:

LFL is investigating new fluorescent markers for malignant tumors. The project partners have developed new fluorescent probes (smart probes) for the staining of two different enzymes: (1) transketolase-like 1 (TKTL1) is a promising prognostic marker, (2) DNaseX may indicate early malignant changes. LFL has had to develop a highly sensitive fluorescence spectroscopic device to detect the marker fluorescence from native tissue samples. The device is based on fiberoptic excitation and detection and designed in such a way to also allow for endoscopic measurements. Therefore, the fluorochrome concentrations have to be measured quantitatively within scattering and absorbing media. Ray tracing and Monte Carlo simulations, as well as phantom measurements, have been carried out. Finally, a set-up has been constructed based on a multifiber applicator, switching fluorescence excitation and detection of emission spectrum with white-light illumination and detection of remission spectra (Fig. 1). LEDs and a laser diode have been used as light sources. Corrected ‘‘intrinsic’’ fluorescence spectra are calculated from acquired fluorescence and remission spectra, Phantom measurements have shown a good correction for varying absorption, whereas scattering could be compensated for less efficiently. Sensitivity was in the 1 nmol-region, which should be sufficient to detect physiological levels. Support: BMBF – Biophotonik III.

  

Development and clinical evaluation of techniques for ‘‘in-vivo optical pathology’’ Photodynamic therapy (PDT) with the focus on light application techniques and light dosimetry Evaluation of medical lasers for the treatment of soft and hard tissues.

Techniques for ‘‘in-vivo optical pathology’’ Introduction In order to assess diseased tissue without taking biopsies, it is necessary to localize these regions precisely, preferentially via endoscopes. This can be performed by fluorescence detection using fluorescing molecules, which accumulate selectively in malignant regions. In addition, it is necessary to have information about the degree of infiltration (staging) and the biological properties of the cells (grading). While optical coherence tomography (OCT) is able to deliver information about disturbed tissue layers, endomicroscopy permits for tissue imaging with high resolution in the mm-range. For fluorescence staining, 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) is the fluochrome of preference. 5-ALA was firstly used in Munich for endoscopic visualization of bladder tumors in the early 1990s. Following instillation of a 5-ALA solution and detection with a special lamp equipped with optical filters for color contrast imaging (Karl Storz GmbH, Germany), a significant improvement could be shown in detecting the carcinoma in situ. Motivated by this data, clinical multicenter trials were performed, which resulted in a European approval of Hexvixs (an ester derivative of 5-ALA) in 2005. Similarly successful was the systemic application of 5-ALA to patients suffering from malignant gliomas in the brain. First clinical experience with a specially equipped Zeiss operation microscope was obtained in Munich in 2004, followed by a multicenter trial in Germany. This study led to the approval of 5-ALA for fluorescence-assisted surgical removal of malignant gliomas in 2008. A lamp-based detection system for the visualization of lung tumors and malignancies in the oral cavity has been developed in parallel in cooperation with Karl Storz GmbH, Germany. It highlights diseased tissue by using autofluorescence detection exclusively. The technique received FDA approval in 2002.

FemtoSCOPE The FemtoSCOPE project aims to develop an innovative diagnostic system for use in patients suspected of having cancer. It consists of a flexible high-resolution two-photon endoscope in combination with in vivo suitable adopted contrast agents (Fig. 2). Using this endoscope, benign or malign changes in hollow organs can be diagnosed to the single cell level. Since laboratory preparation and analyses of histological samples currently constitute a large portion of the workload, this system may in the future significantly improve the pathological classification of diseases without the need for biopsies taken invasively. For this project, it is intended to focus on superficial bladder cancer. Until now, only very few clinically applicable fluorochromes which might provide a good morphological or functional contrast on a cellular level have been identified and characterized. In vitro cell as well as pre-clinical tissue examinations must be performed to identify and optimize the most suitable of these candidates. For the generation of the necessary short laser pulses, a compact high-performance femtosecond (fs)-laser source is used, providing a high repetition rate for fast imaging, which may also reduce

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the overall necessary radiation exposure to a minimum. Furthermore, this source is designed to be tunable over a relevant wavelength range for the sequential excitation of different fluorophores leading to highly contrasted images. Support: DFG Cluster of Excellence ‘‘Munich-Centre for Advanced Photonics (MAP)’’ (see also: http:// www.map.uni-muenchen.de).

3D-TissueScreen

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Fig. 1. (A) Device for fiber-based fluorescence spectroscopy of fluorochromes in turbid media. The pictures inset at the left bottom corner show the top view of device during red-excited fluorescence detection (left) and white-light remission detection (right). (B) Performance of the algorithm to correct for scattering and absorption: the spectrum of the clear solution is depicted as the black solid line. In turbid samples, the same fluorochrome concentration results in the colored solid lines, which are transformed to the dashed lines by the algorithm.

LFL is a microscope manufacturer sub-contractor and manufacturer of fs lasers in this project. The laser and specific components of an innovative microscope (laser beam scanner, detectors) were tested for their suitability as main components of a device for twophoton microendoscopy. Pulse distortions, produced in the fibers of an imaging light guide have been measured and will be minimized by appropriate prechirping. The laser beam scanner enables image repetition rates of a few Hertz. A gradient index (GRIN) lens-based distal objective with a high numerical aperture was also constructed and compared with a standard microscope objective and another lens-based microobjective. The current set-up enables highly resolved imaging both in the one-photon confocal mode and in the two-photon mode. To obtain two-photon images from low fluorescent tissue, the overall sensitivity has to be improved. Further steps will be taken to reduce linear and nonlinear pulse distortion and the optimization of the set-up. Two-photon microendoscopy has the potential to significantly improve clinical intraoperative diagnostics by providing information about the degree of malignancy (grading) of small tissue areas; for the first time it may be possible to resolve cellular and tissue

In-vivo diagnosis of cancer cell structures (Courtesy of Dr David Becker)

Injection of adopted contrast agents

Short-pluse laser source Beam manipulation and lateral scanning

Applicator system in-vivo therapy monitoring

Detector Axial scanning unit Data analysis database and expert system

Lumen for contrast agent

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in-vivo analysis of inflammatory deseases

Fig. 2. Basic set-up of a highly resolving in vivo diagnosis system referring to the MAP-approach.

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morphology in vivo in sub-micron resolution, even without drug-enhanced contrast. Support: BMBF – Biophotonik III.

Fluorescence-imaging using indocyanine green (ICG) ICG is a clinically approved infrared (IR) fluorochrome with a very short half-life in the circulation. It is used for liver function diagnostics and in ophthalmology. New possible indications are being explored in cooperation with the endoscope manufacturer (Fig. 3). One of the aims is to display the invisible infrared fluorescence on a monitor in such a way that an overlay with a simultaneously recorded standard white-light image will become possible. Image processing algorithms should extract valuable information from the recorded fluorescence and white-light video. In a clinical study comprising 25 patients, the perfusion of free composite flaps transplanted into the oral cavity was studied. In two cases, a malperfused flap could be identified by its significantly lower ICG fluorescence compared to the surrounding tissue. The reason was arterial kinking that occurred following anastomosis and, which could be successfully corrected by surgery. Support: Industrial partners.

Clinical investigations with endoscopic OCT The US company Imalux is hitherto the only manufacturer of a forward-looking, endoscopically applicable flexible OCT probe that is licensed for in vivo use. LFL coordinated the first clinical trials performed in Western Europe. OCT images show, similar to ultrasound images, a cross-sectional view of the tissue displaying the layered structure of the epithelium, connective tissue and muscle to a depth of approx. 2 mm (Fig. 4). First clinical investigations were performed within the framework of fluorescence cystoscopy. The aim was to test OCT for its ability to detect

flat malignant lesions, and especially to discriminate between inflammatory and malignant lesions. The fluorescence diagnosis served as localizing suspicious bladder wall areas. Currently, investigations are being performed in gynecology (cervix dysplasia) and ENT (premalignant lesions of the upper aerodigestive tract). In all disciplines, OCT clearly delineates the layered structure of normal tissue. Also, invasive tumors reproducibly show a complete loss of layered patterns in the OCT image. In order to judge the performance of OCT to correctly classify early malignant changes, more experience must be gained and technological improvements in resolution, scanning speed and field of view also appear to be necessary. Support: Industrial partners.

Photodynamic therapy White light PDT in the bladder Due to the proven high sensitivity of 5-ALA-induced PpIX for bladder tumors by means of fluorescence techniques, in addition to the well-known photosensitizing capability of PpIX, a method has been developed for integral PDT of bladder tumors. It consists of a whitelight source (Karl Storz GmbH, Germany) and a special flexible light catheter (OptiMed). A phase-I clinical trial shows that the method of so-called white light PDT is safe and clinically reliable.

Interstitial PDT in neurosurgery A further topic is PDT with 5-ALA in neurosurgery. A clinical pilot study and a phase-I/II study have been already finished for stereotactic interstitial therapy of glioblastoma. The irradiation has been performed using 3–6 nearly parallel fiber devices emitting a homogeneous light distribution over the last 3 or 4 distal centimeters.

Fig. 3. ICG-fluorescence images of free-flap reconstruction of the upper aerodigestive tract. Almost no signal is detected prior to ICG administration (left). After bolus injection ICG fluorescence first becomes visible in the tissue surrounding the free flap (middle) until finally both flap and surrounding tissue seem to exhibit almost equal fluorescence (right).

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Fig. 4. OCT image of normal urinary bladder wall shows dark urothelial layer, clearly delineated against bright connective tissue and muscle layer, being dark with bright horizontal structures (left). This example of a carcinoma in situ is characterized by a loss of delineation between urothelial and connective tissue, whereas the muscle layer appears to be uninvolved (middle). OCT image of invasive bladder cancer is diffuse, lacks horizontal structures almost completely (right).

The dosimetry planning is based on a coarse measurement of the mean optical brain tissue properties in vivo in combination with calculations about the expected light and temperature distributions. For the improvement of dosimetry we plan to determine the optical properties and their special and patient-specific variations via measurement of light transmission between movable emitters and detectors in adjacent fiber canals. It is also intended to measure the spatial distribution of concentration and bleaching of the photosensitizer. The aim is a patient-specific, controlled dosimetry, which enables identification and compensation of local undesired light dose or sensitizer concentration during treatment. These investigations are planned during a multicentric phase-II study, which was recently applied for.

PDT-induced immune responses Photodynamic therapy preferentially induces destruction of malignant tissue by light-mediated reactions of photosensitizing molecules accumulating in diseased cells. The primary effect is due to the generation of singlet oxygen and subsequent destruction of tumor cells. On the other hand, systemic immune reactions were reported in mice following PDT with Photofrins. In clinical trials with interstitial PDT with 5-ALA-induced PpIX as sensitizer of malignant, diffuse infiltrating glioma recurrences (see interstitial PDT in neurosurgery), a significantly prolonged survival could be shown despite the poor prognosis of these patients. This indicates that systemic tumor immune reactions could also be triggered in humans. The aim of this project was to elucidate the mechanisms by which the tumor cells trigger immune responses upon sublethal PpIXmediated PDT using murine and human prostate- and glioma-cell lines. Genome wide transcription analyses demonstrated significant upregulation of genes encoding early response transcription factors (c-Fos, c-Jun) heat

shock proteins (especially HSP70) as well as cytokines and chemokines following low-dose PDT. The latter proteins could explain the known prominent influx of myeloid cells into PDT-treated tumors, resulting in systemic antitumor immune responses. Support: Deutsche Krebshilfe.

5-ALA-PDT of early childhood cancer Neuroblastoma and hepatoblastoma are cancers of the early childhood. The project investigates diagnostic and therapeutic potentials of 5-ALA-based fluorescence diagnosis of resection margins or post-surgical PDT in an early preclinical stage (Fig. 5). Cell culture and cellular spheroid experiments (cell lines of the abovementioned tumors and fibroblasts) were performed to study the selectivity of photosensitizer accumulation, pharmacokinetics and cell viability. Pharmacokinetics and the extent of tumor necrosis following PDT were also studied in animal experiments (nude rat). Tumor selectivity was observed in all experimental models both for the recorded fluorescence intensity as well as for the therapeutic efficiency. Therefore, a continuation project aiming at a clinical trial appears desirable and is to be recommended. Support: DFG.

Optical tissue clearing for improving acne-PDT Blue light irradiation is known to be an efficient therapy for acne vulgaris. In order to increase the light dose to the sebaceous glands and hair follicles, the light penetration might be enhanced by tissue clearing agents. Different methods to measure the agent-induced change of optical tissue properties developed are: (1) simple measurement of reflectance and transmission on tissue slices, (2) optical coherence tomography, (3) analyzing the distribution of light transmitting a semitransparent mirror in contact with a tissue cross-section perpendicular to the surface during a surface illumination, and

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Fig. 5. Hepatoblastoma cells were implanted into the peritoneum of nude rats (left). Fluorescence laparoscopy reveals highly selective PpIX accumulation after i.p. injection of 5-ALA (middle). PpIX fluorescence peaks at approx. 2 h after injection (right).

Fig. 6. Corresponding cross-sections of OCT prior to laser intervention (left), post laser intervention (middle) and HE-section showing the local effect of perforation without any circumferential thermal damage.

(4) probing the light distribution in tissue by thin interstitial fibers. Using such a method, different agent formulas will be compared quantitatively in order to find the most effective one. Support: Bayerische Forschungsstiftung.

Laser for medical treatment Endoluminal vein treatment Endoluminal thermal procedures occluding incompetent saphenous veins for the treatment of varicose veins are becoming accepted alternatives for surgical high ligation and stripping. The aim of endoluminal thermal treatment of veins is to induce controlled damage to the vessel wall, resulting in an irreversible occlusion of the lumen. Following the description of such procedures, first trials report on the clinical efficacy of endovenous radiofrequency and laser therapy. Nevertheless, there is only limited data available on systematic experimental evaluation of these procedures. The purpose of this project was to develop a practicable model to allow standardized experimental evaluation and comparison of endovenous thermal occlusion procedures. The model consists of the subcutaneous foot vein from freshly

slaughtered cows. Using this model, primary and acute effects could be studied together with initial mechanisms on the vein vessel. In this study different energy sources (laser and radiofrequency generator), and different energy application parameters (velocity, fluence, fluence rate, temperature) were compared. The dependency of using bare fiber and cylindrical diffusors was investigated with respect to the induced effects on the vessel walls. Contraction of the vessels were measured and investigated macroscopically and microscopically, as well as by means of OCT (Fig. 6). As a result, an optimized treatment protocol was developed. Support: Industrial partners.

Laser-induced stone fragmentation Different clinically approved pulsed laser systems were compared with respect to their impact on stone management. The ablation threshold, the fragmentation rate and the sputtering rate of artificial and human calculi (urologic and salivary) depending on composition, were measured and different fragmentation rates were calculated in relation to the fluence. In the same manner, different equipment devices were tested. In order to underline the different primary laser-induced processes that lead to fragmentation, the generated

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Fig. 7. Nitinol basket wire after irradiation with Ho:YAG laser.

shockwave pressures of the different laser devices were measured. The ablation threshold value of infrared lasers was overstepped by the lowest laser setting independent of the repetition rate and fiber diameter. There was no difference in the fragmentation and sputtering rate between different IR lasers neither for stone phantoms, nor for clinical stones. On stone phantoms, visible (VIS) lasers showed a 20 times higher fragmentation rate and a 10 times higher sputtering rate compared to IR lasers. All kinds of clinical calculi could be destroyed by IR lasers contrary to VIS lasers. Equipment destruction could be induced by each laser as shown in Fig. 7. Investigations into phantom stone fragmentation are useful to compare clinical laser parameter settings but can partially be transferred to clinical stone fragmentation. IR lasers induced ablation and fragmentation in all the examined human calculi at the lowest energy settings in contrast to VIS lasers, which could not induce ablation in few of the stone composites. The VIS lasers are solely useful for laser-induced shock wave lithotripsy while the IR lasers are in use for other clinical applications (e.g. coagulation, ablation). Support: Industrial partners.

BetaMod – Modulation of wound healing by means of local b-irradiation Wound healing is a complex, dynamic process in which the proliferation of fibroblasts is preferentially responsible for inducing strictures or in the worst-case scenario, stenosis. In the follow-up such tissue alterations e.g. in cylindrical organs may decrease the functionality as well as the quality of life. Strictures occur especially in organs, such as the urethra, bile duct

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and tear duct and need further medical care. Within the very first days of wound healing, the switch for the development of the future is set. It has been shown that radiotherapy within this time, using local b-irradiation implants, results in a promising development of the process. It is proposed to apply low-dose radioactive b-radiation to prevent hyper-proliferation during scar formation. The focus of the BetaMod project aims to develop radioactive implants for such radiotherapeutic procedures and to investigate the modulated wound healing process. The precise and reproducible induction of strictures in animal models by means of laser irradiation is the first step of the BetaMod project. OCT techniques are used to investigate the feasibility of (1) control measurements of the exact position of the catheters within cylindrical organs, (2) evaluating the degree of encrustation on urologic catheters as well as (3) deriving tissue optical parameters from the OCT signal with the aim to differentiate between normal and diseased tissue, in correlation to histology. And finally, all kinds of endoscopy-based techniques of optical pathology will be used to study the process of scar and kelloid formation, as well as the process during and after b-irradiation. Support: Bayerische Forschungsstiftung.

Interdisciplinary activities In November 2008, the Transluminal Endoscopic Association Munich (T.E.A.M.) was founded as an interdisciplinary association to develop and improve innovative endoscopic technologies at the Clinic of the University Munich. Up to now T.E.A.M. consisted of a total of 12 clinics and departments. Mostly all of them are familiar with techniques like fluorescence endoscopy, OCT and microendoscopy and consider improvements as a challenge for the future. To increase efficacy, interdisciplinary working groups have been established:







Natural Orifice Endoscopic Surgery (NOTES)/Natural Orifice Surgery (NOS): Surgery without scars as a future trend of laparoscopic surgery. Surgery is performed for example through the stomach, vagina, or colon. Optical endoscopic diagnosis: Non-invasive tissue diagnosis in hollow organs (bladder, lung, and esophagus) in real time and with high sensitivity for malignant alterations. For that purpose, fluorescence endoscopy is extended by OCT and microendoscopy. Functional implants: The aim is to develop active implants reactive to tissue. This is performed by controlled release of drugs or locally effective radioactivity (see BetaMod project).

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Recent publications [1]

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Hillemanns P, Wang X, Hertel H, Andikyan V, Hillemanns M, Stepp H, et al. Pharmacokinetics and selectivity of porphyrin synthesis after topical application of hexaminolevulinate in patients with cervical intraepithelial neoplasia. Am J Obstet Gynecol 2008;198(3):300.e1–7. Seitz M, Soljanik I, Stanislaus P, Sroka R, Stief C. Explosive gas formation during transurethral resection of the prostate (TURP). Eur J Med Res 2008;13(8):399–400. Seitz M, Sroka R. Re: Gunnar Wendt-Nordahl, Stephanie Huckele, Patrick Honeck, et al. 980-nm diode laser: A novel laser technology for vaporization of the prostate. Eur Urol 2007;52:1723–8. Eur Urol 2008;54(3):697; author reply 698. Stummer W, Beck T, Beyer W, Mehrkens JH, Obermeier A, Etminan N, et al. Long-sustaining response in a patient with non-resectable, distant recurrence of glioblastoma multiforme treated by interstitial photodynamic therapy using 5-ALA: case report. J Neurooncol 2008;87(1):103–9. Zaak D, Sroka R, Khoder W, Adam C, Tritschler S, Karl A, et al. Photodynamic diagnosis of prostate cancer using 5-aminolevulinic acid – first clinical experiences. Urology 2008;72(2):345–8. Beck TJ, Kreth FW, Beyer W, Mehrkens JH, Obermeier A, Stepp H, et al. Interstitial photodynamic therapy of nonresectable malignant glioma recurrences using 5-aminolevulinic acid induced protoporphyrin IX. Lasers Surg Med 2007;39(5):386–93. Beck TJ, Burkanas M, Bagdonas S, Krivickiene Z, Beyer W, Sroka R, et al. Two-photon photo-

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dynamic therapy of C6 cells by means of 5aminolevulinic acid induced protoporphyrin IX. J Photochem Photobiol B 2007;87(3):174–82. Hautmann H, Pichler JP, Stepp H, Baumgartner R, Gamarra F, Huber RM. In-vivo kinetics of inhaled 5-aminolevulinic acid-induced protoporphyrin IX fluorescence in bronchial tissue. Respir Res 2007;8:33. Meissner OA, Schmedt CG, Hunger K, Hetterich H, Sroka R, Rieber J, et al. Endovascular optical coherence tomography ex vivo: venous wall anatomy and tissue alterations after endovenous therapy. Eur Radiol 2007;17(9):2384–93. Schmedt CG, Meissner OA, Hunger K, Babaryka G, Ruppert V, Sadeghi-Azandaryani M, et al. Evaluation of endovenous radiofrequency ablation and laser therapy with endoluminal optical coherence tomography in an ex vivo model. J Vasc Surg 2007;45(5):1047–58. Sostak P, Theil D, Stepp H, Roeber S, Kretzschmar HA, Straube A. Detection of bone marrowderived cells expressing a neural phenotype in the human brain. J Neuropathol Exp Neurol 2007; 66(2):110–6. Wang XL, Wang HW, Huang Z, Stepp H, Baumgartner R, Dannecker C, et al. Study of protoporphyrin IX (PpIX) pharmacokinetics after topical application of 5-aminolevulinic acid in urethral condylomata acuminata. Photochem Photobiol 2007;83(5):1069–73. Zelenkov P, Baumgartner R, Bise K, Heide M, Meier R, Stocker S, et al. Acute morphological sequelae of photodynamic therapy with 5-aminolevulinic acid in the C6 spheroid model. J Neurooncol 2007;82(1):49–60.