1. Sepsis, Immunity and Memory Tomás Huerta Mentors: Betty Diamond, Bruce T. Volpe Affiliations: Feinstein Medical Research Institute, Weill Medical College of Cornell University Tomás Huerta Mentors: Betty Diamond, Bruce T. Volpe Affiliations: Feinstein Medical Research Institute, Weill Medical College of Cornell University Title slide

2. What is Sepsis?  Baby is attacked by aggressive bacteria  Infection triggers a mix of:  pro-inflammatory responses  anti-inflammatory responses  Sepsis happens when there is too much pro-inflammatory responses  If unchecked  rapid death Introduction/Background

3. What is Sepsis? Introduction/Background

4. How big of a problem is Sepsis?  Serious problem in all emergency rooms in hospitals  Very frequent in babies and people over 70 years old  Common in already sick people (diabetic, heart attack, etc.)  In USA: 751,000 cases per year  215,000 deaths (29%)  In the world (estimate): 10-100 million cases  28-50% death Introduction/Background

5. Sepsis occurs in Stages ① “systemic inflammatory response syndrome” (pro-inflammatory response) ② severe sepsis ③ septic shock ④ multiple organ failure  Introduction/Background

6. What causes Sepsis?  Exaggerated inflammatory response of the MACROPHAGES of the “innate” immune system to microbes (bacteria, virus)  INFECTION triggers inflammation: Introduction/Background

7. Immune System  Innate immunity: always present (ready to attack); many pathogenic microbes have evolved to resist innate immunity  Adaptive immunity: stimulated by exposure to microbe; more potent Introduction/Background Macrophages are phagocytes

8. The main players in Sepsis Introduction/Background

9. The main players in Sepsis Introduction/Background innate IS Cell death nervous system adaptiv e IS microbe s

10. Order of events in Sepsis Introduction/Background microbial infection DEATH of immune cells – hypo-inflammation hyper-inflammatory response (macrophages) CYTOKINE STORM (too much cytokines)

11. Therapy for Sepsis Introduction/Background microbial infection DEATH of immune cells – hypo-inflammation hyper-inflammatory response (macrophages) CYTOKINE STORM (too much cytokines) Anti-inflammatory drugs Anti-apoptotic drugs Anti-microbial drugs

12. Post-Sepsis and Cognition  Up to 70% of post-sepsis survivors have problems with cognition:  memory loss (amnesia)  attention deficit  lack of executive control  anxiety disorder  It is not known why the cognitive deficits happen Introduction/Background

13. High Cytokines enter the Brain Cytokines in the brain may alter the well-being of the CNS Introduction/Background During the cytokine storm, elevated cytokines enter the brain

14. High Cytokines and Memory Loss The Problem cytokines accumulate in brain areas that encode memory high level of cytokines cause memory loss elevated cytokines enter the brain

15. How to Study Memory? The Problem  Use “animal model”  mouse  Study the HIPPOCAMPUS (brain region that encodes memory)  Study SYNAPTIC PLASTICITY (basic cellular process for memory)

16. Memory Centers in the Brain hippocampus parahippocampal / perirhinal cortex The Problem

17. Stating The Problem The Problem It is likely that the high level of cytokines that enter the brain during sepsis cause an impairment of synaptic plasticity, the cellular basis for memory

18. Our Hypothesis Hypothesis 2) We propose that hippocampal slices prepared from mice that had suffered sepsis (a few weeks before) will show impaired synaptic plasticity 1) We propose that adding high levels of cytokines to hippocampal slices will cause an impairment of synaptic plasticity

19. Methods  Extract hippocampal slices from the brain of a mouse (male, 2-4 weeks of age)  Keep slices alive (up to 24 hours) by incubating them in special medium  Place hippocampal slice in the recording chamber  Record excitatory post-synaptic potentials  Induce SYNAPTIC PLASTICITY

20. Excitatory Post-Synaptic Potential Stim 1 Rec DG CA1 Stim 2 SUB CA3 Methods

21. Stim 1 Stim 2 Rec

22. Synaptic potential AFTER synaptic plasticity Synaptic potential before synaptic plasticity Methods

23. Experimental Plan Methods Aim 1: high levels of cytokines will impair synaptic plasticity  Study 3 groups of slices = 15 slices per group  Experimental Group #1 (“High Cytokine” Group): synaptic plasticity in the presence of high cytokines (~100 micromolar)  Experimental Group #2 (“Low Cytokine” Group): synaptic plasticity in the presence of low cytokines (~10 micromolar)  Control Group (“No Cytokine” Group): Try to induce synaptic plasticity in the absence of cytokines  Statistical Analysis: compare the results from each experimental group against the control group, by using Student’s t test. A probability of less then 0.05 will be considered significant

24. Experimental Plan Methods Aim 2: post-sepsis slices will have impaired synaptic plasticity  Study 2 groups of mice:  Post-Sepsis Group: Try to induce synaptic plasticity in slices from mice that underwent sepsis  No-Sepsis Control: Try to induce synaptic plasticity in in slices from mice that did not suffer sepsis  Sample Number:  Study 3 mice per group, 5 slices per mouse, for a total of 15 slices per group  Statistical Analysis: compare whether the results from both groups are significantly different by using Student’s t test. A probability of less then 0.05 will be considered significant

25. Expected Results Results Aim 1: high levels of cytokines will impair synaptic plasticity  Experimental Group #1 (“High Cytokine” Group): synaptic plasticity will be absent or highly reduced (significantly lower than control group)  Experimental Group #2 (“Low Cytokine” Group): synaptic plasticity will be marginal or, perhaps, unaffected  Control Group (“No Cytokine” Group): synaptic plasticity will be strong

26. Expected Results Results  Post-Sepsis Group: synaptic plasticity will be absent or highly reduced (significantly lower than control group)  No Sepsis Group: synaptic plasticity will be strong Aim 2: post-sepsis slices will have impaired synaptic plasticity

27. Significance of this research Discussion  It would be useful to know whether synaptic plasticity is abolished by high levels of cytokines (applied directly to the brain)  It would be useful to know whether synaptic plasticity is absent in the post-sepsis brain  Therapies can be designed to stop the cytokines from entering the brain and affecting synaptic plasticity  Therapies can be designed to recover the deficient synaptic plasticity in post-sepsis patients that exhibit memory problems

28. References Citation slide  Tracey K.J. (2002) The inflammatory reflex. Nature 420: 853-859.  Tracey K.J. (2005) “Fatal sequence: The killer within”. Washington DC: Dana Press.  Murphy KM., Travers P. and Walport M. (2007) “Janeway’s Immunology”. New York: Garland Science.  Diamond B., et al (2009) Losing your nerves? Maybe it’s the antibodies. Nature Reviews Immunology May 5. [Epub ahead of print]