Hypothermia Plus: New NeuroProtective Strategies with Stem Cells
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1 Hypothermia Plus: New NeuroProtective Strategies with Stem Cells The BEST of IPOKRaTES: an Update in Neonatology Leuven, Belgium, September 17-20, 2014
2 Clinical Background Perinatal Hypoxia-Ischemia (Birth Asphyxia): Incidence: 4-9 per 1000 live births High mortality and morbidity (1 per 1000 live births) Clinical Outcome - Motor deficits (cerebral palsy) - Epilepsy - Mental retardation/ learning disabilities - Visual and Auditive problems - Behavioral problems
3 Treatment strategies Birth asphyxia Reperfusion/reoxygenation damage intervention 0h 6h 12h 24h days
4 Hypothermia Selective Head cooling Whole Body cooling Neuroprotective after birth asphyxia
5 Edwards et al, British Medical Journal 2010 Meta-analysis hypothermia studies in full-term asphyxiated infants?
6 Treatment strategies Birth asphyxia X Reperfusion/reoxygenation damage intervention 0h 6h 12h 24h days hypothermia + pharmacotherapeutical intervention(s)
7 Time profile of destructive pathways induced by reoxygenation/reperfusion Superoxide FR formation ph FREE-Iron Pro-radical formation (NPBI) > OH FR Inflammation/cytokines / inos/apoptosis Ca 2+ glutamate Hypo- Xanthine nnos >NO > ONOO- Downregulation trophic factors Fetal hypoxiaischemia 30 min 3h 6h 12h 24h days Birth
8 Hypoxia-induced Free Radical Formation Dirnagel U et al, J Cereb Blood flow Metab 1995
9 Ion-channels - Phenobarbital - Ketamin - Amiloride - Noble gas-inhalation - Magnesium Anti-oxidants - Vitamin C/E - Allopurinol - Deferoxamine - Selective NOS-inhib - N-acetylcystein - Erythropoietin (EPO) - Lazaroids - Edaravone Anti-inflammation - IL-1RA - IL-4 - IL-10 - Melatonin - Erythropoietin (EPO) Trophic factors - Erythropoietin (EPO) - Growth factors - Thyroxin-derivates Others - Thiorphan - Bumetamide - 17-B-estradiol - (Stem) Cell therapy
10 Noble Gas Xenon Advantage - highly neuroprotective Inhibition of NMDA-receptor Xenon
11 P7 Rats: Hypoxia-Ischemia and Xenon * vs NT p<0.05 vs HT p<0.05 * * Hobbs et al, Stroke 2008
12 Current Clinical Studies Two clinical trials Bristol, CoolXenon2: 18 hours of 50% Xenon London TOBYXe: 24 hours of 30% Xenon
13 Advantage Disadvantages Noble Gas Xenon - highly neuroprotective - Very costly/fixed annual production - Anesthetic - Complex Ventilation set-up - Influences Background pattern aeeg-up
14 Xenon Ventilation Set-up Adapted from Thoresen et al RSC 2010
15 Noble gas Argon: An alternative? Advantages - Easy ventilation set-up - Cheap - No anesthetic - highly neuroprotective?* * Zhuang et al, Crit Care Med 2012
16 Neuroprotective properties Cell cultures of dissociated neurons (Jawad et al 2009) Organotypic hippocampal slice cultures (Loetscher 2009) Rodent/piglet models (Ryang et al 2011, Zhuang et al 2012)
17 Zhuang et al, Crit Care Med 2012 P7 Rats after global Hypoxia-Ischemia (Argon) * vs controls p<0.05
18 Neuroprotection by argon ventilation after perinatal asphyxia A safety study in newborn piglets T. Alderliesten, R. Neijzen, C, Rademaker, C. Nijboer, R. Marges, F. van Bel, F. Groenendaal Alderliesten et al, submitted
19 Conclusions Argon ventilation did not affect - Heart rate - Blood pressure - Cerebral oxygenation - aeeg No increased apoptosis after Argon
20 Clinical Limitations of Noble gas Ventilation If need for 100% Oxygen (PPHN) If need for NO-inhalation (PPHN) 0
21 ALLOPURINOL
22 Superoxide Formation upon reoxygenation Severe Fetal Hypoxia Hypoxanthine + Hypoxanthine ALLOPURINOL O2 Birth Xanthine Oxidase Superoxide!! Xanthine + Uric acid x x (XO)
23 Allopurinol and Superoxide after Ischemia Superoxide anion radical Prod. Takeru Ono et al, Brain Research 2009
24 TimeProfile of ROS/Superoxide Formation Re-oxygenation ph FREE-Iron Ca 2+ glutamate Superoxide-prod Free-iron (Fenton Reaction) Fetal Hypoxia 30 min 3h 6h 12h 24h days Treatment during Reperfusion-reoxygenation (Maternal Therapy)
25 Clinical Randomized Controlled Trial during fetal hypoxia : Multicenter RCT with 500 mg Allopurinol or placebo iv to mother: n= 111/111 Results: - No Adverse effects - Currently analysis of results
26 Postnatal Allopurinol Treatment versus Control Favorable vs adverse outcome * Infants with severe perinatal asphyxia excluded Study ALLO (n) CONT (n) Risk Ratio Weight Risk Ratio Van Bel 1998* 3/9 4/8 17.8% 0.67 [0.21, 2.11] Benders, 2006* 1/6 5/7 19.4% 0.23 [0.04, 1.48] Gunes, /28 15/ % 0.73 [0.41, 1.30] Total 95% (CI) % 0.62% [0.38, 1.02] Favors allopurinol Favors control Adapted from Chaudhari, McGuire; Database of Systemic Reviews 2008, issue 2
27 Outcome at (Pre)school age: WPPSI-III/WISC-III-NL Kaandorp et al, Arch dis Child Fet 2011
28 Erythropoietin (EPO)
29 Neuroprotective properties (endo-/exogeneous EPO) Anti-oxidative (incl. NO-prod-inhibition) Anti-inflammatory (EPO-anti-inflamm-cytokine) Anti-apoptotic Stimulates neurogenesis and angiogenesis
30 Evidence for Neuroprotection EPO There is a fair amount of evidence from Experimental studies in different species (incl.primates) and from Human adult and neonatal studies (Zhu et al!) that rh-epo exert neuroprotection and may be a suitable (additional) candidate for neuroprotection after perinatal asphyxia. Van der Kooy et al, Brain Res Rev, 2008
31 EPO and Outcome after Birth Asphyxia (I) Convulsions (%) (n) * <0.05 * * (n=15) (n=15) * * Adapted from Elmahdy H et al, Pediatrics 2010
32 EPO and Long-term Outcome after Birth Asphyxia Zhu et al, Pediatrics 2010
33 Other EPO studies (ClinicalTrials.gov) Neonatal Erythropoietin in Asphyxiated Term Newborns (NEAT, n=24) Darbe Administration in Newborns Undergoing Cooling for Encephalopathy (DANCE) n=24 Efficacy of Erythropoietin to Improve Survival and Neurological Outcome in Hypoxic Ischemic Encephalopathy (Neurepo) n=330
34 Melatonin
35 Pro-Inflammatory Pathway Perinatal Asphyxia Melatonin Glial Cell (Apoptotic) Pre-OD death Pro-Inflammatory Cytokines IL-1β, IL-6, IL-8, TNFα, Interferon-γ
36 Experimental and Clinical Evidence of Neuroprotection of Melatonin Several experimental fetal/neonatal studies in different species, using a wide range of doses, mostly pointing to white matter protection Four studies in (preterm) neonates during different clinical conditions. Up to now, no adverse effects reported
37 Clinical Evidence of Neuroprotection of Melatonin after Perinatal HI Free radical (MDA) and NOS Prod (Nitr) at 24 h of age ** <0.05 * * (n= 10) (n= 10) (n= 10) Adapted from Gitto et al, Pineal Res 2009
38 Other Melatonin studies (ClinicalTrials.gov) Melatonin to Prevent Brain Injury in Unborn Growth Restricted Babies n=12 Melatonin As A Novel Neuroprotectant In Preterm Infants- Dosage Study (MIND) n=24
39 Cell Therapy
40 Stem cells Pluripotent stem cell Committed stem cell Hematopoietic stem cell Neural stem cell HI Mesenchymal stem cell Differentiated cell Lymphocyte Erythrocyte Granulocyte Platelets Neuron Astrocyte Oligodendrocyte Cardiomyocyte Fibroblast Chondrocyte Adipocyte Osteocyte
41 Mesenchymal SC: Proposed Action Oligodendrocyte FGF2 BDNF VEGF TNFα IL1 NGF Mouse MSCs + Hypoxia Astrocyte Microglia Neuron
42 Treatment with Mesenchymal Stem Cells after Neonatal Hypoxic Ischemic Brain Injury
43 Neon HI-model Rice-Vanucci : - 9-day-old mice - Ligation Ri-carotid artery - Recovery > 60 min - Subsequent Hypoxia 10%- 45 mins
44 Results: Lesion volume Neuronal (MAP-2) loss White matter (MBP) loss Van Velthoven et al, J Neurosci 2010
45 Route of Administration Intracerebral Injection Intravenous Injection Nasal Instillation
46 Nasal administration of MSC Both nostrils, 21 days after HI Motor function MAP-2 MBP Van Velthoven et al, Pediatr Res 2010
47 Long-term Effectivity & Safety after Neonatal MSC Treatment (I) Long lasting improvement Increase malignancies local/systemic Immunogenic derailment
48 Long-term Effectivity & Safety after Neonatal MSC Treatment (II) Investigated at age 14 Month in 13/17/17 Sh-Veh-MSC mice resp. Cognition: MSC mice not different from Sham mice No evidence for neoplasia in brain or nasal turbinates No difference in neoplasmata in other organs between groups No immunogenic effects after MSC treatment
49 Application in Man Currently: - Autologous Cord Blood Transfusion (s) for Perinatal asphyxia / Stroke?
50 Current Studies with Autologous Stem Cells Autologous Stem Cells in Newborns With Oxygen Deprivation (Mexico, n=20) Characterization of the Cord Blood Stem Cell in Situation of Neonatal Asphyxia (NEOCORD) (Marseille, France, n=5+5) Cord Blood for Neonatal Hypoxic-ischemic Encephalopathy (Duke, USA, n=25)
51 Application in Man In (near) future: - Autologous Cord Blood Transfusion (s) for Perinatal asphyxia / Stroke? On the Long-term: - Allogeneic (cultured) MSC s?
52 Human Study with Allogenic Stem Cells 20 neonates with Perinatal arterial-ischemic Stroke (PAIS) treated with MSCs compared to 20 PAIS historic controls
53 Manufacturing MSCs for clinical Trial Quality control analysis: - Sterility - Phenotyping - Function (induction of neuroregeneration) Preparation of the intranasal delivery system
54 Optimal intervention strategy, a Dream? XO-formation ph FREE-Iron Ca 2+ glutamate Fetal Hypoxia free (pro) radical formation cytokines / inos nnos trophic factors Therapeutic Window anti-apoptotic 30 min 3h 6h 12h 24h days Maternal allopurinol* Hypothermia Noble gas Vent/Melatonin? * Gender effect? rhepo* MSCs
55 Perinatal Center UMCU Floris Groenendaal Manon Benders Linda de Vries Thomas Alderliesten Joepe Kaandorp Jan Derks Carin Rademaker NIDOD Lab UMCU Cindy van Velthoven Cora Nijboer Michael van der Kooij Vanessa Donega Hilde Bonestroo Annemieke Kavelaars Cobi Heijnen
56 Thank you for your attention Questions?
57 Reviews J Pediatr 2012 Ann Neurol 2012 J Pediatr 2012
58
59 Clinical Evidence MgSO4 after Perinatal Asphyxia Neonataldeathcumulative Studyname Cumulativestatistics Cumulativeoddsratio(95%CI) Lower Upper Mg Point limit limit Z-Value p-value group Control Ichiba et al, / 17 1 / 16 Groenendaal et al, / 25 7 / 30 Kashaba et al, / / 54 Bath et al, / / MetaAnalysis Evaluation copy Control MgSO4
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1 Perinatal Center, Wilhelmina 2 Department of Neonatology, Leiden University Medical Center, Leiden, The Netherlands 3 Department of Neonatology, Beatrix Children s Hospital, University Medical Center
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