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“In Theory, Theory and Practice are the same, but in Practice they’re different.” -Unknown “You can do combinations.” -John Connor Terminator II: Judgement.

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Presentation on theme: "“In Theory, Theory and Practice are the same, but in Practice they’re different.” -Unknown “You can do combinations.” -John Connor Terminator II: Judgement."— Presentation transcript:

1 “In Theory, Theory and Practice are the same, but in Practice they’re different.” -Unknown “You can do combinations.” -John Connor Terminator II: Judgement Day

2 CHILLING OUT WITH GROWTH FACTORS: NEW THERAPIES FOR CEREBRAL ISCHEMIA Jonathon M. Sullivan MD, PhD Associate Director Cerebral Resuscitation Laboratory Dept of Emergency Medicine Wayne State University

3 My Colleagues: Rita Kumar, PhD Thomas Sanderson PhD Anthony Lagina

4 “Tell ‘Em What You’re Gonna Tell ‘Em” There’s this thing called brain ischemia. We hate brain ischemia. We usually can’t fix it. Yet. It’s complicated. Hypothermia seems to help. Growth factors (including insulin) are promising. “You can do combinations.” Science stuff we’ve already done. Stuff we’re gonna do. Fade to black. Roll credits. Crowd goes wild.

5 Brain Ischemia is a Big Deal 700,000 suffer a focal ischemic stroke each year. –3d leading cause of death –Treatment options extremely limited: Supportive care Thrombolysis and other reperfusion strategies –Efficacious, not very effective (yet) –Risky: NNT is low, but so are NNH and NNK. –70,000 survive cardiac arrest each year Heart resuscitation: better. Cerebral resuscitation: not so much. Vast majority have serious brain damage Hypothermia the only proven neuroprotective strategy. –Still not universally employed –Effective in 20-25% of cases

6 Primum Non Nocere

7 Global vs. Focal Ischemia Focal Ischemic Stroke 8 m Global Ischemia at 14 days

8 Ischemia Cocks the Hammer, Reperfusion Pulls the Trigger

9 The Cerebral Ischemia Train Wreck

10 Single Drug Therapies Have Been Uniformly Disappointing A partial list: –Calcium Channel Blockers –Glutamate antagonists –Desferoxamine –Cyclooxygenase/lipoxygenase inhibitors –Steroids, lazaroids –NXY-059

11 Monotherapy Will Never Work Brain ischemia is the result of multiple, progressive, independently-lethal processes. Targeting one process (such as excitoxicity or free radicals) will do little more than alter the cell death phenotype.

12 Only One Therapy Has Been Cool Enough Hypothermia has been clinically demonstrated effective for transient global brain ischemia. Clinical data for focal ischemic stroke is still lacking. Laboratory data indicates likely effectiveness for focal ischemia

13 Hypothermia Isn’t Monotherapy Induces pro-survival signaling processes –Activation of Akt –Suppression of cytochrome c release and caspase activation Decreases free radical production Decreases lipolysis Alters glutamate receptor composition Other?

14 Single Agent Monotherapy Hypothermia is a case in point So are peptide growth factors.

15 GFs in Brain Ischemia: Historical Perspective Siemkowicz 1982: Increased glucose after global ischemia impedes CMRO2. Strong 1985: mitochondria from insulin-treated ischemic rats maintain respiratory control. Marsh 1986: hyperglycemia  cortical acidosis LeMay 1988: Insulin administration protects neurologic function in cerebral ischemia in rats. Voll and Auer 1988: Post-ischemic insulin decreases hippocampal and striatal neuronal death. Voll and Auer 1989: Makes rats smarter, too. Voll and Auer 1991: Insulin attenuates ischemic brain damage independent of its hypoglycemic effect.

16 Growth Factors are Good. High dose insulin (12U/kg) decreases lesion volume and cell death in diabetic rats (focal model). Rizk, Rafols and Dunbar, 2006 Topical IGF-1 reduces infarct volume and caspase-3 staining after focal ischemia in rats. Abe et al 2001. Patients with lower IGF-1 and IGFBP-3 levels are at higher risk of stroke. Johnson et al 2005; Bondanelli et al 2006, Denti et al 2004. Expression of GFs and GFRs is enhanced in resistant neurons after ischemia, but reduced in SVNs. Hwang et al, 2004.

17 How Do GFs Work in Cerebral Ischemia? Short answer: nobody really knows. Single mechanism unlikely. Two places to look for answers: –Mechanisms of GF effect –Mechanisms of ischemic damage

18 Growth Factor Signaling Systems: The Obligatory Oversimplified Cartoon

19 NGF


21 GF Signaling Systems: Summary GF Metabolic Effects Genetic Responses Translational Control Survival Signaling

22 GlucoseBulk of data says no. Excitotoxicity incr GABA, decreased glutamate release, altered GlutR Oxidative Stress ? changes in SOD, Glutathione No strong in vivo data Calpain ProteolysisNo good data Genetic Modulation Lots of in vitro data, surprisingly little in vivo data. Mitochondrial and ER stress, Protein Synth, Apoptosis Coming right up. Potential Salutary Effects of GFs in Ischemia:

23 GFs in Ischemia:Translational Control Mechanisms

24 eIF2-α Kinases: The Usual Suspects HRI PKR GCN-2 PERK I want a lawyer.

25 ER Stress and Translational Control

26 eIF2α(P) Localizes to SVNs During Early Reperfusion DeGracia, Sullivan, Neumar, Alousi, Hikade, Pittman, White, Rafols, Krause.

27 Salutary Effects of GFs in Ischemia: Translational Control Sullivan, Alousi, Hickade, Bahu, Rafols, Krause, White

28 GFs in ER Stress Effect of insulin on eIF2α(P) is highly compelling, however…. Virtually no other published data in vivo. Some in vitro data that GFs are salutary in setting of ER stress Watch Dr. Kumar.

29 Salutary Effects of GFs in Ischemia: Programmed Cell Death

30 Apoptosis: For Real? Focal Ischemic Neuronal Death Pro-ApoptosisAnti-Apoptosis DNA laddering observed in penumbra within a few hours Necrotic phenotypes predominate in the core. Transcription of caspase 3 observed in vulnerable neurons Caspase inhibitors convert “apoptotic death phenotypes to “necrotic” phenotypes. Activated caspase 3 seen by 1-3 h of reperfusion Caspase 3 inhibitors decrease caspase cleavage products, reduce tissue damage, improve outcome in rodent stroke models.

31 Apoptosis: For Real? Global Ischemic Neuronal Death Pro-ApoptosisAnti-Apoptosis TUNEL staining observed in both animals and humans. Apoptotic bodies not observed by most investigators. Caspase inhibitors protect against neuronal death. “Necrosis” morph typical in primate neurons, even w. caspase activation. Activated caspase 3 seen as early as 1 hr of reperfusion. Little evidence of chromatin condens’n at light level (mixed results from EM). Caspase 3 mRNA appears early in SVNs. PARP breaks down between 24 and 72 hours. BAX and BclXs expressed in vulnerable CA1 neurons.

32 Manichean Biology EITHEROR

33 Intrinsic Apoptotic Bad-ness

34 Intrinsic Apoptosis and Bcl-2 Family Proteins: the Movie

35 GF-Mediated Survival Aktion TRANSLATION

36 So Perfect, It Had to Be Wrong PI3K INSULIN Akt BadpBadSequestered PS Survival

37 Insulin Maintains Hippocampal Architecture and Function Sanderson, Murariu, Kumar, Owen, Krause, Sullivan 2008

38 Insulin-Akt Dose-Response Sanderson, Murariu, Kumar, Owen, Krause, Sullivan 2008

39 Insulin Induces Akt Phosphorylation Through PI3K Sanderson, Murariu, Kumar, Owen, Krause, Sullivan 2008

40 p-Akt in CA1 Hippocampus ShamR30 R30 + Ins 20U/kg R30 + Ins 20U/kg + WM Sanderson, Murariu, Kumar, Owen, Krause, Sullivan 2008

41 Bad Data Sanderson and Sullivan: our very own White Whale. NEVER saw a change in Bad phosphorylation or translocation. p112 Bad p136 Bad Total Bad

42 Looking Downstream: Cytochrome c SHAM (Non-Isch) 8 min Isch + 240 min Rep 240 min Rep + Insulin 2U/kg

43 From Insulin to Cytochrome c INSULIN PI3K Akt Bcl-Xl-Bax Binding Mitochondrial Bax Pores Cytochrome c Release

44 Insulin Restores Bcl-XL-Bax Binding Sanderson, Kumar, Sullivan, Krause 2008

45 Insulin Suppresses Mitochondrial Bax Localization Sanderson, Kumar, Sullivan, Krause 2008

46 Insulin Suppresses Release of Cytochrome c Sanderson, Kumar, Sullivan, Krause 2008

47 Insulin Suppresses Release of Cytochrome c Sanderson, Kumar, Sullivan, Krause 2008

48 The Potential for GF Treatment in Cerebral Ischemia Insulin and IGF-1 are already in clinical use. Insulin is inexpensive, familiar to clinicians, and easily administered. Potential limitations: –20U/kg? Are you out of your mind? –Hypoglycemia, hypokalemia –Fluid shifts, cerebral edema

49 You Can Do Combinations Combination therapy for stroke/brain ischemia has been identified as a priority by the NIH. Early targeting of mulitple pathways far more likely to be successful than singly targeted therapy. –By “early,” we mean “upon reperfusion.” Preclinical evaluation using STAIR criteria.

50 Building on the Foundation THERAPEUTIC HYPOTHERMIA Anti-ROSGFs Protease Inhibition Other AGGRESSIVE REPERFUSION

51 The Potential for GF Treatment in Cerebral Ischemia: Combined Therapy

52 CVRI-Supported Research “Combination Therapy for Focal Ischemic Stroke.” –J. Sullivan, A. Lagina, R. Kumar, J. Li –hypothermia + insulin for focal ischemic stroke –MCAO model of focal ischemia –Guided by STAIR recommendations –Dose-response for hypothermia and insulin –Affect on biochemical targets Insulin: Akt, cytochrome c release Hypothermia: free radicals (4-HNE, MDA), Akt, cyto c release –Short- and Long-term outcomes: Histological Neurobehavioral Similar study for Global Brain Ischemia (Lagina, Sullivan, Sanderson) funded by Emergency Medicine Foundation (IGF-1 + Hypothermia)

53 “Combination Therapy for Global Brain Ischemia” Lagina, Sullivan; Emergency Medicine Foundation Preclinical investigation of hypothermia + insulin for global ischemia 2v-OH model of global ischemia Dose-response for hypothermia and insulin Affect on biochemical targets –Insulin: Akt, cytochrome c release –Hypothermia: free radicals, Akt Short- and Long-term outcomes: –Histological –Neurobehavioral

54 “Tell ‘Em What You Told ‘Em” Brain ischemia is a Big Deal, and current treatment strategies are limited. Single drug therapy will never work. Hypothermia and growth factors are both neuroprotective and act on multiple pathophysiological processes Combination therapy CVRI-supported preclinical work underway in our laboratory

55 Special Thanks: Tom Sanderson, PhD Rita Kumar PhD Anthony Lagina MD Karin Przyklenk PhD Mike Deogracia Suzanne White MD, FACEP Joseph Dunbar PhD Jose Rafols PhD James Rillema PhD Gary S. Krause MD, MS Work shown here supported by: AHA PDFG 0415380Z (Sanderson) Emerg Med Found CDG (Kumar) NIH KO8 NS-02008 (Sullivan) NIH R01 NS4919 (Sullivan) Emerg Med Found CDG (Sullivan) NIH R01 NS33196 (Krause)

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