1. PORPHYRIN-BASED IN VIVO MITOCHONDRIAL RESPIROMETRY Floor Harms, Sander Bodmer, Robert Jan Stolker, Bert Mik Department of Anesthesiology, Laboratory of Experimental Anesthesiology, ErasmusMC – University Medical Center Rotterdam, the Netherlands

2. Venous return Intravascular volume Pump function / valves Heart rate / rythm Vasomotor tone Vascular resistance Lung function Oxygen saturation Hemoglobin levels Vasomotor tone Regional distribution Shunting Oxygenation from a clinical perspective Oxygen content Cardiac output Regional blood flow Blood pressure

3. Meet Sara! Happy and Healthy

4. Sara = Septic

5. Treatment of sepsis Antibiotics Fluid resuscitation Mechanical ventilation

6. After Resuscitation

7. Mitochondrial Dysfunction Mitochondrial dysfunction in sepsis is a well known phenomenon called : Cytopathic Hypoxia  ↓ function of the mitochondrial respiratory chain Resulting in: ↓ production of adenosine triphosphate (ATP) ↓ cell metabolism.

8. Mitochondrial Dysfunction  The current knowledge of mitochondrial dysfunction is based on: -Swelling and disruption of normal mitochondrial architecture -↑ Tissue oxygen tension -↓ Respiratory rates of isolated mitochondria

9. Classical respirometry: isolated mitochondria / cells

10. ‘In vivo’ data about the mitochondrial oxygen metabolism is NOT is available. Due to the lack of an adequate in vivo measurement method Mitochondrial dysfunction “in vivo” Voeding Zuurstof W ater CO 2 Energie

11. Towards in vivo respirometry

12. ‘In vivo’ monitoring of mitochondrial function ‘In vivo’ measurement of the mitochondrial oxygen tension (mitoPO2) by the Protoporphyrin IX Triplet state lifetime measurement (PpIX-TSLM).

13. Mitochondrial oxygen tension measurements by Protoporphyrin IX (PpIX) Triplet state lifetime measurement E.G. Mik, 2006. Nat Methods

14. Mitochondrial localization of PpIX E.G. Mik, 2006. Nat Methods

15. PpIX shows delayed fluorescence E.G. Mik, 2006. Nat Methods Prompt fluorescence PpIX Excitation 405 nm High PO 2 Low PO 2

16. Oxygen-dependent delayed fluorescence 1  M PpIX in 4% BSA Temp 37˚C, pH = 7.4 Exitatie: 510 nm Emissie: 600-700 nm E.G. Mik, 2006. Nat Methods

17. Oxygen-dependent delayed fluorescence O2O2 collision relaxation no light red light green light QUENCHING DEL. FLUORESCENCE

18. “The old” setup

19. “New” in vivo setup Lens Mono- chromator PMT Integrator reset Pulsed tunable laser Fiber Tissue

20. Fiber based method

21. In vivo delayed fluorescence Rectus abdominis muscleSkin paw 4 hrs after intravenous ALA injection

22. Isolated organs EG Mik et al., Biophys J 95: 3977-90 (2008). EG Mik et al., J Mol Cell Cardiol 46: 943-51 (2009). Rat liverRat heart

23. Rat liver in vivo EG Mik et al., Biophys J 95: 3977-90 (2008).

24. Aim of my PhD project Towards clinical monitoring of mitochondrial function in sepsis

25. Towards clinical monitoring of mitochondrial function 1.Non invasive measurements on the skin. 2.Validation of PpIX TSLM for mitoPO2 measurements in the skin. 3.In vivo respirometry in the skin. 4.In vivo respirometry in sepsis. 5.Mitochondrial respirometry on the skin of a human volunteer.

26. Non invasive measurements on the skin. Applied on Harms FA, et al. (2011) J. Biophotonics.

27. Towards clinical monitoring of mitochondrial function 1.Non invasive measurements on the skin. 2.Validation of PpIX TSLM for mitoPO2 measurements in the skin 3.In vivo respirometry in the skin. 4.In vivo respirometry in sepsis. 5.Mitochondrial respirometry on the skin of a human volunteer.

28. Validation of PpIX TSLM for mitoPO2 measurements in the skin Harms FA, et al. (2012) Optics Letters Normal tissueNonrespiring tissue Tissue hypoxia during 100% nitrogen ventilation

29. Towards clinical monitoring of mitochondrial function 1.Non invasive measurements on the skin. 2.Validation of PpIX TSLM for mitoPO2 measurements in the skin 3.In vivo respirometry in the skin. 4.In vivo respirometry in sepsis. 5.Mitochondrial respirometry on the skin of a human volunteer.

30. In vivo respirometry in the skin

32. Example measurement

33. In vivo respirometry in the skin Tested in 8 anesthetized and mechanical ventilated wistar rats

34. The reliable monitor is developed!!!

35. Co-workers Bert Mik, MD PhD Head of laboratory ErasmusMC Sander Bodmer, MD PNVs technology NVA Harold Raat, PhD Blood substitutes Baxter, ErasmusMC Marit van Velzen Pletysmography ErasmusMC Jaqueline Voorbeijtel Biotechnician ErasmusMC Patricia Specht Biotechnician ErasmusMC Tanja Johannes, MD PhD Experimental sepsis ErasmusMC Sjoerd Niehof, PhD Thermography ErasmusMC Sander v.d. Heuvel, MD Beademing en IAH ErasmusMC

36. Questions