1. Jastrow Canlas BSc. Biotechnology (Medicine) Biological Science Flinders University Hons. Medical Biotech Pain and Pulmonary Neurobiology Laboratory Anatomy and Histology Department Flinders Medical Centre

2. Overview Title Introduction Methods Results Conclusion Significance

3. Title Condenses the paper’s content in a few words; Captures the readers’ attention; Differentiates the paper from other papers of the same subject area.

4. Why is my pee yellow? Researches into the Colouring- Matters of Human Urine, with an Account of the Separation of Urobilin MacMunn, A. Serotonin receptor 1A–modulated phosphorylation of glycine receptor α3 controls respiration Manzke, T., Niebert, M., Koch, U. R., Caley, A., Vogelgesang, S. & Hülsmann, S. What controls my breathing? Role of sphingosine kinase 2 and sphingosine 1-phosphate in murine spinal cord glial cells during chronic inflammation Canlas, J. What is the role of this substrate in pain signalling?

5. Introduction Role of sphingosine kinase 2 and sphingosine 1- phosphate in murine spinal cord glial cells during chronic inflammation Sphk2S1P generates enzyme substrate Type of pain Cells from the spinal cord

6. What is pain? An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage (IASP, 2012). Acutee.g. pin prick; dissipates quickly. Inflammatorye.g. sunburn; subsides when healed. Neuropathice.g. nerve injury; persistent.

8. OW!

9. NeurogliaGeneric cell diagram

10. Enzyme Y Y Y T precursor Hello! Hi! Huh? Representation of a cell -substrate another cell

11. Why S1P? Interaction of S1P with S1P receptor (S1PR) induced pain (Mair et al., 2011). Deletion of S1PR1 reduced inflammation-induced hypersensitivity. Deletion of S1PR3 attenuated spontaneous pain behaviour (Camprubi-Robles et al., 2013) PAIN Y S1PR S1P- Ow!

12. Why Sphk2? Sphk phosphorylates sphingosine into S1P (Pitson et al., 2011). Our lab has shown, that out of the two Sphk isoforms, Sphk2 is the predominant isoform expressed in the murine spinal cord (Fig. 1). Figure 1. Relative mRNA-expression levels as mean normalized expression (MNE) for the two sphingosine kinase isoform, Sphk1 and Sphk2, at different levels of the mouse spinal cord. Sphk2 is significantly higher expressed compared to the Sphk1 isoform in thoracic and lumbosacral spinal cord levels (n = 5, one-way ANOVA, p < 0.05).

13. How would you elucidate the role of Sphk2 in spinal glial cells during inflammation?

14. Sphk2-KO animal model Ethics: experimental procedure conducted on animals were approved by the Animal Welfare Committee. Animal model: Double isoform knock-outs is embryonic lethal; however, single knock-out, i.e. Sphk2, are viable.

15. Inflammation model Pain model: Inflammation induced by intraplantar injection of Complete Freund’s Adjuvant (CFA) to the hindpaw.

16. Behavioural studies: mechanosensitivity measurement using von Frey filaments

17. Figure 2. Ipsilateral 50% paw withdrawal threshold (g) in response to no treatment (0; baseline) and CFA-induced peripheral inflammation 4, 48, 120 and 168 hrs post injection. Solid circle data points represent C57BL/6 and clear square data points represents Sphk2 -/-, presented as mean ± SEM (n = 5 and n = 8). Results: 50% withdrawal threshold of inflamed left hindpaw of WT and Sphk2-KO

18. Results: 50% withdrawal threshold of uninjured right hindpaw of WT and Sphk2-KO WT Sphk2-KO Figure 3. This data shows that Sphk2 deficient mice develop bilateral hypersensitivity (mirror image-pain) in responses to peripheral inflammation, indicating a possible role of Sphk2 in the inhibition of communication between ipsi- and contralateral spinal cord.

19. What is happening in the level of the spinal cord during the spread of pain?

20. Spinal cord dissection g Lumbosacral spinal cord was dissected and sliced into 1 um thick sections in preparation for immunohistochemistry.

21. Immunohistochemistry

22. Astrocytes labelled with GFAP Microglia labelled with IBA-1

23. GFAP-immunoreactive astrocyte and IBA-1-immunoreactive microglia in the dorsal horn after 7 days of peripheral inflammation. Sphk2-KO WT

24. Conclusion Bilateral pain hypersensitivity in Sphk2-KO mice was accompanied by the absence of the usual increase in microglia activation. This suggests that Sphk2 is required for glia activation, which are responsible for the modulation of pain.

25. Sphk2 Pain signal microglia activation Y Inhibit astrocytes Generates S1P

26. ???? Pain signal microglia activation Y Astrocytes activation ? Spread of pain!

27. Summary We wanted to know the role of Sphk2 and S1P in pain. We generated Sphk2-KO mice And induced inflammation We measured behavioural response to mechanical stimulation and looked at glial cell reactivity at the level of the dorsal horn spinal cord. We showed Sphk2-KO developed mirror image pain and it was accompanied by the absence of spinal microglia activation. We conclude that Sphk2 is required for microglia activation and the activation of microglia is required for the supress of astrocytes, which is responsible for the spread of pain.