Pascale Vérant Laboratoire de Spectrométrie Physique. Grenoble.

1. A Method for Measuring Cerebral Blood Volume of Mouse using Multiphoton Laser Scanning Microscopy Pascale Vérant Laboratoire de Spectrométrie Physique. Grenoble.

2. Relation between Cerebral Blood Volume (CBV) and tumor growth process. Role of the angiogenesis (formation of new blood vessels). Effects of a microbeam radiotherapy on the cerebral microvasculature. Why measure cerebral blood volume ?

3. Optical methods : • Histology (ex vivo) • Two-photon microscopy • Depth of imaging : up to 1000 µm (Denk et al., Optics letters, 2003); 450 µm in our experiments. • Used to study vascular anatomy and changes in capillary blood flow (Chaigneau et al, PNAS, 2003, group S. Charpak), thrombotic stroke (Kleinfeld et al)… • In vivo measurement of the Cerebral Blood Volume without morphometric analysis of the blood vessels. How to measure cerebral blood volume ? • Medical imaging methods used in vivo : • Positron emission tomography • Magnetic Resonance Imaging (MRI) : • Non invasive • Limited resolution in space (mm 3) and in time ()  

4. Experimental procedure Swiss Nude Mice (4-6 weeks) • Injection of dyes by the femoral vein : • 200 µl 10% (w/vol)rhodamine labelled dextran 70 kDa • 10 µl 10% (w/vol)sodium fluorescein Ex vivo experiments : On excised brain (mice sacrified 3 min after the injection) In vivo experiments : Through a 3 mm diameter hole drilled in the skull, over the parietal cortex

5. Microscopy set-up Femtosecond Ti:Sapphire Laser (Excitation 800 nm) Bio-Rad scanhead MRC 1024 Neutral density filters Scan mirrors (x,y) Pinhole Internal PMTs Dichroic mirrors Olympus microscope BX50WI red Filter cube green External PMTs 20x water-immersion (NA=0.95) Xlum Plan Olympus Z-translation Bio-Rad interface mouse

6. Vessel with fluorescein Intensity in grey levels The excitation volume • Generation of two-photon fluorescence in a small volume around the focal point : the « two-photon point spread function »

7. Acquisition of images • Acquisition of planar scans of the fluorescent intensity at successive depths in the cortex. • steps in z of 1-5 µm • 0.9 s/image • Incident intensity manually increased so as to saturate of a few of the brightest pixels. • Observation of vessels as far as 450 µm below the dura (60% of the whole cortex) • Capillaries present for z > 50 µm 100 µm 100 µm Images acquired ex vivo from 50 to 300 µm (in steps of 2 µm) in left parietal cortex of a Nude mouse injected with 200 µl of a 100 mg.ml-1 rhodamine-dextran solution

8. The relative blood volume BV is : F(x,y,z)=1 if (x,y,z) is in a vessel else F(x,y,z)=0 with - - VB : observed volume We measure : k : efficiency of fluorescence generation and light collection C : dye concentration. : L : laser intensity. L2(x,y,z) = L02PSF(x,y,z) with assumption C=C0 kL02 kept constant We have then: with K = kL02C0 The blood volume is proportional tosum of thefluorescence intensities

9. vessel Numerical treatment on each images Increase of laser beam intensity The calibration How to keep kL02 constant when z varies? Normalization of image intensities

10. Histogram of the z-projection Blue : the full projection Red : the top left selection 100 µm 100 µm Z-projection of the 101 images acquired ex vivo from 0 to 300 µm (in steps of 3 µm) in left parietal cortex of a nude mouse injected with 200 µl of a 100 mg.ml-1 rhodamine-dextran solution Method sensitive to local variations in blood volume Estimation of CBV from experimental acquisitions

11. Averages out the heterogeneity of the blood fluorescence (presence of blood cells in plasma) • More tissue movement tolerated • Short acquisition time High resolution or large PSF ? • The advantages of a large PSF : • Resolution sufficient to be sensitive to local variations of CBV

12. A method to measure in vivo local and temporal variations of CBV Preliminary results Preliminary values of CBV in mouse cortex : between 2.5 % and 3 % in areas with only capillaries In vivo studies Use of cranial window to be in physiological conditions. => evolution of the CBV in the same mice.

13. 200 µm X-Ray beam profile tumor 1000 Gy Effects of unidirectional irradiation in the grey matter of a rat (histological section) Effects of irradiation on the cerebral blood volume of a mouse ? The microbeam radiotherapy Developed by Jean Laissue 200 µm 25 µm

14. 100 µm 100 µm Observation of the rhodamine-dextran (red PMT) Observation of the sodium fluorescein (green PMT) Evolution of CBV Study of vascular permeabilty Preliminary results on microbeam irradiation • Observation 24h post irradiation (1000 Gy) • Simultaneous injection of rhodamine-dextran and sodium fluorescein

15. Laboratoire de Spectrométrie physique. Grenoble • P. Verant • J. C. Vial • Acknowledgements : • Ligue contre le cancer • - Association pour la recherche sur le cancer • Programme interdisciplinaire CNRS-INSERM-CEA « IPA » • - Région Rhône-Alpes • Unité Mixte INSERM 594 – Université Joseph Fourier. Grenoble. • Neuro-imagerie fonctionnelle et métabolique • R. Serduc • J. Coles • R. Farion • C. Rémy • B. van der Sanden • The European Synchrotron Radiation Facility. Grenoble. • G. Le DUC