Nociception Analysis of Mice Lacking the EP1 and EP2 Receptor

1. Nociception Analysis of Mice Lacking the EP1 and EP2 Receptor Martin F. Quiroga, BS 2nd Year Master’s Student in Biomedical Sciences, Barry University, Vanderbilt Bridges Student Ronald G. Wiley, MD, Ph.D Neurology Service, Experimental Neurology VA Medical Center, Vanderbilt University

2. Introductive Terminology • Pain –Unpleasant sensation associated with actual or potential tissue damage • Nociception – The appreciation or transmission of pain • Hyperalgesia –increased sensitivity to pain stimuli • Allodynia –painful reaction to non-noxious stimuli • Prostaglandins – class of lipids present in tissues and bodily fluids involved in many processes including pain, inflammation, kidney function, labor, and gastro-duodenal protection • PGE2 (dinoprostone) –Prosta-5,13-dien-1-oic acid • PGE2Rs (EP1-EP4) – G-Protein receptors for PGE

3. Can be caused by burns, cramps, injuries since these tissues will heal (ideally) Plays an important role in self-survivability The principle message used to halt the continued transmission of noxious stimuli Background InformationTypes of Pain Acute Pain • Temporary and caused by tissue damage • Disappears as tissue heals • Can last a few seconds to many months

4. Chronic Pain Continuing even after tissues have healed Can last from days to years Can be due to disease, trauma, surgery, and stress. Does not have a clinically significant purpose Can even slow down recovery in some cases In the U.S. 1/3 of the population will require medical therapy for a recurring chronic pain. Background InformationTypes of Pain

5. Background Information cont.How is pain transmitted and how many types of nerve fibers are involved? • Nociceptors receive external stimuli • Information is sent through peripheral nerve fibers • Then through the dorsal horns of the spinal cord and up to the thalamus and the cerebral cortex of the brain. • There are 3 nerve fibers types.

6. Types of Nerve FibersA and C Fibers Note: A delta (1) fibers are also referred to as A beta

7. Role of Prostaglandins in Pain • Prostaglandins mediate inflammation, a nonspecific response of the immune system to damaged cells • It is characterized by redness, pain, heat, and swelling • Prostaglandin E2 (PGE2) is the form of prostaglandin most associated with inflammation • PGE2 prolongs pain and may also stimulate emigration of phagocytes through capillary wall. • Studies have shown that PGE2 is involved in the CNS, not only PNS • Intrathecal injections directly into the dorsal horns have shown changes in inflammatory response

8. Cyclooxygenase PathwayProstaglandin Biosynthesis Arachidonic Acid COX Pathway Prostaglandin E2

9. Types of COXConstitutive – always presentInducible – produced when needed

10. NSAIDs and COX2 Inhibitors NSAIDs • Work by blocking the COX enzyme • Both COX1 and COX2 are blocked • Only blocking COX2 is desirable • Blocking of COX1 leads to serious side effects such as gastric ulcers • Even worse, the analgesic effects of NSAIDS can mask the pain of the ulcer

11. NSAIDs and COX2 Inhibitors COX2 Inhibitors • Are much more specific for the inducible form of COX • Eliminate PGs involved in inflammation more effectively • The enzyme is not blocked irreversibly so the effects wear off quicker • Higher doses are safer • Still acts on COX1, however, making it less safe and effective than desired

12. Types of Test Used to Measure Pain Three main Test • Hot Plate –This test consist of placing the mice on a hot surface, and measuring the latency for the first lick or guard, measuring thermal pain. • Tail Flick – This measures the spinal reflex of the tail by subjecting it to a beam of light that heats up very rapidly. • Acetic Acid –This is a measure of visceral pain, by an erratic movement known as a writhe. To induce writhing the mice received an intraperitoneal injection of acetic acid.

13. Methods and EquipmentTail Flick

14. Methods and EquipmentHot Plate

15. Methods and EquipmentAcetic Acid

16. EP2 Tail Flick ResultsBaseline &Hyperalgesia

17. EP1 Tail Flick ResultsBaseline

18. EP2 Hot Plate Baseline • No difference between wild type and EP2 knockouts EP2 Hot Plate

19. EP1 Hot Plate Graph p value= 0.3408931323 (47-53)0 p value= 0.01219763

20. EP1 Hot Plate Test for Hyperalgesia

21. EP2 and EP1 Acetic Acid

22. Comparing EP1 to EP2 Acetic Acid test P-value= 0.007135

23. What does this mean? • EP2 is involved in visceral pain, but not thermal pain • EP1 is involved in thermal pain, but not visceral pain (CNS Mediated). • Different transducers involved • Double knockouts are needed • Agonist/Antagonist can be used

24. References • Vane JR; Bakhle YS; Botting RM Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol, 1998, 38:, 97-120 • Cryer B; Feldman M Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am J Med, 1998 May, 104:5, 413-21 • Bjorkman DJ The effect of aspirin and nonsteroidal anti-inflammatory drugs on prostaglandins. Am J Med, 1998 Jul 27, 105:1B, 8S-12S • Kawamori T; Rao CV; Seibert K; Reddy BS Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res, 1998 Feb, 58:3, 409-12 • Lipsky PE; Isakson PC Outcome of specific COX-2 inhibition in rheumatoid arthritis. J Rheumatol, 1997 Jul, 24 Suppl 49:, 9-14 • Wu KK. Biochemical pharmacology of nonsteroidal anti-inflammatory drugs Biochemical Pharmacology. 1998; 55(5):543-7.

25. Bridges Program Ronald G. Wiley, MD, Ph.D Roger Chalkley, Ph.D Dr. Lou DeFelice, Ph.D Barry University Gerhild R. Packert, Ph.D Flona Redway, Ph.D Sr. John Karen Frei, O.P., Ph.D Acknowledgements