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Acute morphological sequelae of photodynamic therapy with 5-aminolevulinic acid in the C6 spheroid model

  • Pitr Zelenkov
  • Reinhold Baumgartner
  • Karl Bise
  • Michael Heide
  • Richard Meier
  • Susanne Stocker
  • Ronald Sroka
  • Roland Goldbrunner
  • Walter StummerEmail author
Laboratory Investigation

Abstract

Objective

Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) may represent a treatment option for malignant brain tumors. We used a three-dimensional cell culture system, the C6 glioma spheroid model, to study acute effects of PDT and how they might be influenced by treatment conditions.

Methods

Spheroids were incubated for 4 h in 100 μg/ml ALA in 5% CO2 in room air or 95% O2 with subsequent irradiation using a diode laser (λ = 635 nm, 40 mW/cm2, total fluence 25 J/cm2). Control groups were “laser only”, “ALA only”, and “no drug no light”. Annexin V-FITC, a marker used for detection of apoptosis, propidium iodide (PI), a marker for necrotic cells and H 33342, a chromatin stain, were used for morphological characterization of PDT effects by confocal laser scanning and fluorescence microscopy. Hematoxylin–eosin staining and TdT-FragEL (TUNEL) assay were used on cryosections. Growth kinetics were followed for 8 days after PDT.

Results

PDT after incubation in 5% CO2 provided incomplete cell death and growth delay in spheroids of >350 μm diameter. However, complete cell death and growth arrest occurred in smaller spheroids (<350 μm). Incubation in 95% O2 with subsequent PDT resulted in complete cell death and growth arrest regardless of spheroid size. In incompletely damaged spheroids viable cells were restricted to spheroid centers. The rate of cell death in all control groups was negligible. Cell death was accompanied by annexin/PI costaining, but there was also evidence for annexin V-FITC staining without PI uptake.

Conclusions

PDT of experimental glioma results in rapid and significant cell death that could be verified as acute necrosis immediately after irradiation. This effect depended on O2 concentration and spheroid size.

Keywords

Malignant glioma 5-ALA photodynamic therapy C6-Spheroids 

Notes

Acknowledgments

This work was supported by Deutsche Krebshilfe e.V. Projekt-Nr. 70–2864. We gratefully acknowledge advice from Jorg Cristian Tonn, Prof., MD, Department of Neurosurgery, Herbert Stepp, Ph.D., Tobias Beck, Dipl. phys., Laser Research Laboratory, Ludwig-Maximilians University, Munich, Germany.

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Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Pitr Zelenkov
    • 1
    • 2
  • Reinhold Baumgartner
    • 2
  • Karl Bise
    • 3
  • Michael Heide
    • 2
  • Richard Meier
    • 2
  • Susanne Stocker
    • 2
  • Ronald Sroka
    • 2
  • Roland Goldbrunner
    • 1
  • Walter Stummer
    • 4
    Email author
  1. 1.Department of NeurosurgeryLudwig-Maximilians UniversityMunichGermany
  2. 2.Laser Research LaboratoryLudwig-Maximilians UniversityMunichGermany
  3. 3.Institute for NeuropathologyLudwig-Maximilians UniversityMunichGermany
  4. 4.Department of NeurosurgeryHeinrich-Heine UniversityDüsseldorfGermany

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