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Bellander, Bo Michael

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Bellander, Bo Michael

  1. 1. Complementactivation and Traumatic Brain Injury<br />Bo-Michael Bellander MD., Ph.D.<br />Dept of Neuroscience, <br />Section for Neurosurgery<br />Karolinska hospital Solna<br />Stockholm, Sweden<br />National Neurotrauma Society<br />Fort Lauderdale <br />2011-07-11<br />
  2. 2. Primarybraininjury<br />The result of the mechanicalforces<br />on the braintissue at impact<br />neurons<br />Secondary insults<br />Hypoxia, Hypotension, Hypercarbia, Hypocarbia, Fever, Hyperglycemia, Hypoglycemia, Hyponatremia,<br />Intracranial hypertension, Hematoma, Oedema, Hyperemia, Vascular spasm, Seizures<br />endotelial<br />cells<br />glial cells<br />Biochemicalprocesses<br />ExcitatoryAminoacids, Free radical formation, Apoptosis, Auto-phagia, Mitochondrialdysfunction, Coagulation disorder, <br />Inflammation<br />Secondarybraindamage<br />
  3. 3. Classical<br />Pathway<br />Alternative<br />Pathway<br />Thecomplementcascade<br />The Lectin<br />pathway<br />Factor P<br />Factor B<br />C3<br />MASP<br />MBL<br />C1q<br />Factor D<br />C3 convertase<br />C2<br />Opsonization<br />C4a<br />C4<br />C3a<br />C5a<br />C5 convertase<br />Recruitment of<br />macrophages<br />C6<br />C8<br />C7<br />C5b9<br />C9<br />C9<br />C9<br />Cytolysis<br />
  4. 4. Complement and diseases<br />Alzheimers disease(Moss and Albert 1988)<br />Multiple Sclerosis(Tegla et al 2009)<br />MyastheniaGravis (Liszewski and Atkinson 1998)<br />Vasculitis(Peerschke et al 2010)<br />Bacterialmeningitis(Stahel and Barnum 1997)<br />AcuteMyocardialinfarction(Bjerre et al 2008)<br />Thrombosis and stroke (Davies et al 1992; Peerschke et al 2010)<br />Subarachnoidhemorrhage(Kasuya et al 1989; Lindsberg et al 1996)<br />Encephalitis<br />ICH (Hua et al 2000)<br />
  5. 5. Classical<br />Pathway<br />Alternative<br />Pathway<br />The Lectin<br />pathway<br />Factor B, D, P<br />C1q<br />C4b2a<br />C3bBb<br />C1-inh<br />C3<br />Factor H<br />CD59<br />DAF<br />MBP/CD46<br />sCR1<br />C3 convertase<br />C2<br />Factor I<br />Opsonisation<br />C4<br />C4b2a3b<br />C3bBb3b<br />C4-bp<br />Factor I<br />Anti-C5mAb<br />C5 convertase<br />Recruitment of <br />macrophages<br />Complement<br />regulation<br />C6<br />HRF<br />C8<br />C7<br />C5b9<br />C9<br />C9<br />C9<br />clusterin<br />Cytolysis<br />
  6. 6. Is the complement system activatedfollowingtraumaticbraininjury?<br />
  7. 7. Material & Methods<br />21 male <br />Spr-Dawley rats<br />+ 4 controls<br />Craniotomy <br />3 mm posterior to bregma <br />and 2 mm lateral to the midline<br />Stereotactic, standardized <br />drop device technique<br />modified from Feeney<br />Bellander et al, JNS 1996<br />Sacrified 2, 4, 7 and 14 dpi<br />
  8. 8. Tight junction protein<br />Absence of thightjunction – IR in the borderzone<br />indicatingdisruption of BBB 4 dpi<br />B. Immunoreactivity for thightjunction protein in intactbrain<br />Bellander et al, JNS 1996<br />
  9. 9. OX42 positive cells – <br />Activatedmicroglialcells/Macrofages 7 dpi<br />Microglial cells/ macrophages<br />Bellander et al, JNS 1996<br />
  10. 10. C3mRNA<br />Local synthesis of <br />Complement C3<br />Bellander et al, JNS 1996<br />In situ hybridization<br />
  11. 11. OX42<br />Anti-C3<br />Microglia vs C3<br />Co-localizationbetween OX42 positive activatedmicroglialcells/macrofages and anti-C3, at 4 dpi<br />Bellander et al, JNS 1996<br />
  12. 12. Clusterin-mRNA<br />Local synthesis of <br />clusterin<br />In situ Clusterin<br />Bellander et al, JNS 1996<br />
  13. 13. Clusterin-mRNA<br />Bar = 10 µm<br />Bellander et al, JNS 1996<br />
  14. 14. Neurons<br />in the border zone double stained with anti-MAP5 and anti-Clusterin<br />Anti-Clusterin<br />Anti-MAP5<br />Bellander et al, JNS 1996<br />
  15. 15. Astrocytes<br />in the border zone double stained with anti-GFAP and anti-Clusterin<br />Anti-GFAP<br />Anti-Clusterin<br />Bellander et al, JNS 1996<br />
  16. 16. Conclusions I<br />Microglialcells/macrophages are activated in the borderzone of contusions<br />Localsynthesis of the complement protein C3 in the borderzone<br />Microglialcells/macrophagesco-localizes with complementC3<br />Neurons and astrocytesin the vicinity of contusionsco-localizes with the complement regulator clusterin<br />The balancebetweencomplement proteins and regulators might be a challenge for cells in the borderzone of contusions<br />
  17. 17. Is the complement system activatedin the human brainfollowingtraumat?icbraininjury?<br />
  18. 18. Complement and human TBI<br />Patients: 16 suffering from cerebral contusions<br />Age: 12-76 years (mean 50)<br />Gender: 13 males, 3 females<br />GCS at admission: 3-13<br />Indication for craniotomywas ICP>25 mmHg and CPP< 60 mm Hg<br />Trauma-surgery: 2,5 – 82 hours<br />GOS: 1-5, 1 nk<br />
  19. 19. C1q<br />C3b<br />C3d<br />C5b9<br />Bar: 10µm<br />Bellander et al, JNT 2001<br />
  20. 20. In situ hybridization C3-mRNA<br />Bellander et al, JNT 2001<br />
  21. 21. C5b9 and NeuN<br />C5b9 and NeuN<br />Border zone<br />10 µm<br />50 µm<br />Clusterin and NeuN<br />Anti-Clusterin<br />Anti-Clusterin and NeuN<br />Bellander et al, JNT 2001<br />
  22. 22. Conclusions II<br />C is activated in the borderzone of human contusions<br />There is a localsyntesis of the complement protein C3 in the borderzone (in situhybridization for C3-mRNA)<br />Neurons in the vicinity of the contusion express the complement regulator clusterin<br />Neurons in the borderzone are challenged by the complementmembrane attack complex<br />Bellander et al. J. Neurotrauma, 18, 12, 2001<br />
  23. 23. Hypothesis III<br />Circulating blood is not a necessity for complement activation. The complement activation is an ”endogenous” process.<br />
  24. 24. Rat entorhinal-hippocampal slice cultures<br />96 slices from 12 days old S-D rats<br />300 µm viable cerebral slices<br />Cultivation for 14 days in 37°C prior to experiment<br />Injury by 2 mm steel ball<br />Slices fixed 1 to 8 dpi<br />Antibodies used<br />OX42 – macrophages/microglia<br />GFAP – astrocytes<br />ED1 – macrophages<br />C1q – complement<br />C5b9 - MAC<br />
  25. 25.
  26. 26. Bar = 50µm<br />Bellander et al. J. Neurotrauma, 2004<br />
  27. 27. Bar = 50µm<br />Bar = 10 µm<br />OX42-IR<br />Bellander et al. J. Neurotrauma, 2004<br />
  28. 28. Bar = 50µm<br />Bar = 10 µm<br />Bellander et al. J. Neurotrauma, 2004<br />
  29. 29. Bar = 50µm<br />Bar = 10 µm<br />Bellander et al. J. Neurotrauma, 2004<br />
  30. 30. Quantification<br />***<br />*<br />***<br />%<br />Bellander et al. J. Neurotrauma, 2004<br />
  31. 31. Results III<br />Activation of the complement cascade occurs in the absence of circulating blood cells and plasma components<br />Clusterin is upregulated as a result of the complement attack<br />C5b-9, the membrane attack complex – (MAC) is assembled despite absence of circulating blood cells and plasma components.<br />
  32. 32. Do pronouncedimmunologicalreactivityleadto <br />increasedcomplementactivation<br />and more extensive cellulardeath?<br />
  33. 33. Inbred rat strains:<br />><br />Dark Agoutirats (DA) <br />Piebald VirolGlaxo (PVG)<br />Antibodies:<br />OX42 (microglia/macrophages)<br />ED1 (macrophages)<br />GFAP (astrocytes)<br />Fluoro-Jade (neuronal degeneration)<br />In situ: C3 <br />Drop device technique <br />ad modum Feeney<br />PCR: C3<br />
  34. 34. OX42 – microglial cells/macrophages<br />Bellander et al. 2010,<br />DA - borderzone<br />DA - control<br />PVG - control<br />PVG - borderzone<br />
  35. 35. ED1 – macrophages<br />Bellander et al. 2010,<br />DA - borderzone<br />DA - control<br />PVG - control<br />PVG - borderzone<br />
  36. 36. Microglia/macrophages OX42<br />Bellander et al. 2010,<br />
  37. 37. In situ hybridization C3<br />Bellander et al. 2010,<br />DA - borderzone<br />DA - control<br />PVG - control<br />PVG - borderzone<br />
  38. 38. C3-mRNA in situ<br />C3-mRNA PCR<br />Bellander et al. 2010,<br />
  39. 39. Fluoro Jade – cellular degeneration<br />Bellander et al. 2010,<br />DA - borderzone<br />DA - control<br />PVG - control<br />PVG - borderzone<br />
  40. 40. Bellander et al. 2010,<br />
  41. 41. Results IV<br />There is a geneticinfluenceon inflammatoryresponsefollowing TBI<br />Complementactivation<br />correspondsto neuronalnecrosis<br />playsa role in the development of secondarybraindamage<br />
  42. 42. Does secondaryinsults influencecomplementactivation and the release of tissuedamage markers S100B and NSE?<br />
  43. 43. Patients<br />20 patients <br />Severe TBI (GCS 3-8)<br />18 males, 2 females, <br />Age 22-72 (mean 53)<br />ICP <br />Bellander et al, ActaNeurochir 2010<br />
  44. 44. Secondary insults at S.O.A.<br />Hypoxia <br />SaO2 < 85%<br />”insufficient breathing”<br />”cyanois”<br />Hypotension<br />SAP < 90 mmHg<br />Seizure<br />Bellander et al, ActaNeurochir 2010<br />
  45. 45. Secondary insults at S.O.A.<br />Of 20 patients, secondary insults verified in total<br />11 patients .<br />Bellander et al, ActaNeurochir 2010<br />
  46. 46. Secondary insults at NICU<br /> <br /> <br />Mild<br />Moderate<br />Severe<br />Intracranial hypertension<br />ICP (mm Hg)<br />20 - 30<br />30 – 40<br />> 40<br />Poor cerebral perfusion<br />CPP (mm Hg)<br />50 - 60<br />40 – 50<br />< 40<br />Hypotension<br />MAP (mmHg)<br />55 - 70<br />40 – 55<br />< 40<br />Hypotension<br />SAP (mmHg)<br />70 - 90<br />50 – 70<br />< 50<br />Hypertension<br />MAP (mmHg)<br />110 - 130<br />130 - 150<br />> 150<br />Hypertension<br />SAP (mmHg)<br />160 - 190<br />190 - 220<br />> 220<br />Hypoxia<br />SaO2 (%)<br />85 - 90<br />80 – 85<br />< 80<br />Bradycardia<br />bpm<br />40 - 50<br />30 – 40<br />< 30<br />Tachycardia<br />bpm<br />120 - 135<br />135 – 150<br />> 150<br />Pyrexia<br />C<br />38 - 39<br />39 – 40<br />> 40<br />Miller JD, Piper IR, Jones PA: (1995). Pathophysiology of headinjury. Neurotrauma. R. K. Narayan, Wilberger J:r, <br />J.E., Povlishock, J.T., McGraw-Hill: 61-69.<br />
  47. 47. BBB integrity(QA)<br />QuotientCsf-Albumin (mg/L)/B-Albumin (g/L)<br />Normal: < 7<br />Severedysfunction: > 20<br />Tibbling et al, J ClinLab Invest. 1977<br />
  48. 48.
  49. 49. Results C5b9secondary insults at S.O.A.<br />Bellander et al, ActaNeurochir 2010<br />
  50. 50. Results QAsecondary insults at S.O.A.<br />Bellander et al, ActaNeurochir 2010<br />
  51. 51. Resultssecondary insults at S.O.A.<br />Bellander et al, ActaNeurochir 2010<br />
  52. 52. C5b9csf vs BBB dysfunction (QA)<br />Stahel et al. JNT 18,8,773-781, 2001<br />Bellander et al, 2010<br />
  53. 53. C5b9 concentrations following isolated TBI (11 humans)<br />Is C5b9 also originating from the blood?<br />Nekludov et al, J Neurotrauma. 2007 Jan;24(1):174-80.<br />
  54. 54. Results: Secondary insults at NICU<br />Secondary insults in the NICU <br />Complementactivation<br />Disturbed BBB integrity<br />Release of biomarker S100B in csf<br />
  55. 55. Resultssecondary insults at NICU<br />
  56. 56. Results V:<br /><ul><li>Secondary insults at S.O.A. -> </li></ul>- csf-C5b9 ,<br />- disturbedBBB integrity<br /><ul><li>Severesecondary insults at NICU -> - csf-C5b9 ,
  57. 57. disturbedBBB integrity and
  58. 58. csf-S100B </li></li></ul><li>Classical<br />Pathway<br />Alternative<br />Pathway<br />The Lectin<br />pathway<br />Factor B, D, P<br />C1q<br />C4b2a<br />C3bBb<br />C1-inh<br />C3<br />Factor H<br />CD59<br />DAF<br />MBP/CD46<br />sCR1<br />C3 convertase<br />C2<br />Factor I<br />Opsonisation<br />C4<br />C4b2a3b<br />C3bBb3b<br />C4-bp<br />Factor I<br />Anti-C5mAb<br />C5 convertase<br />Recruitment of <br />macrophages<br />Complement<br />regulation<br />C6<br />HRF<br />C8<br />C7<br />C5b9<br />C9<br />C9<br />C9<br />clusterin<br />Cytolysis<br />
  59. 59. Complement receptor type 1 sCR1 – Animal studies<br />Pretreatment<br />ReducedAMI volume in dogs (Weisman et al 1990)<br />Reducedreperfusioninjury in ischemicgut (Hill et al 1992)<br />Reduced ARDS (Mulligan et al 1992)<br />Reducedleucocyteinfiltration following experimental TBI (Kaczorowski et al, 1995)<br />
  60. 60. sCR1 – spray treatment of cerebral contusion in rat<br />Bellander, Thesis<br />sCR1 treatment<br />Placebo<br />Ox42 vs sCR1<br />Ox42 vs NaCl<br />C9 vs sCR1<br />C9 vs NaCl<br />
  61. 61. Complement receptor type 1 sCR1 – Human studies<br />ARDS – Phase 1 (Zimmerman 2002)<br />Dose-dependentcomplementreduction<br />No unexpectedtoxicities<br />Inconclusiveendpoint<br />Lung transplant (Zamora et al 1999 [abstract])<br />Earlierextubation<br />Thermalinjury<br />Inconclusiveendpoint(Ryan 1995, [abstract])<br />Cardiacsurgery and cardiopulmonaryby-pass<br />Inconclusiveendpoint (Death, AMI, IABP or length of intubation) (Lazar et al 2004)<br />
  62. 62. Classical<br />Pathway<br />Alternative<br />Pathway<br />The Lectin<br />pathway<br />Factor B, D, P<br />C1q<br />C4b2a<br />C3bBb<br />C1-inh<br />C3<br />Factor H<br />CD59<br />DAF<br />MBP/CD46<br />sCR1<br />C3 convertase<br />C2<br />Factor I<br />Opsonisation<br />C4<br />C4b2a3b<br />C3bBb3b<br />C4-bp<br />Factor I<br />Anti-C5mAb<br />C5 convertase<br />Recruitment of <br />macrophages<br />Complement<br />regulation<br />C6<br />HRF<br />C8<br />C7<br />C5b9<br />C9<br />C9<br />C9<br />clusterin<br />Cytolysis<br />
  63. 63. C5a receptor antagonist – animal study<br /><ul><li>I.pinjection prior to cryogenicbraininjury</li></ul>- Decreasedneutrophilextravasation<br />I.v. 500 ug anti-C5a antibody in experimental sepsis (rat)<br />Restored BBB integrity<br />Sewell et al, J Neuroimmunology 2004<br />Flierlet al, Crit Care, 13:R12, 2009<br />
  64. 64. Anti-C5ab – Human studies<br />Human studies<br />CABG and AMI – Phase 3<br />Improved 90 dayssurvival(Granger 2003)<br />Mortality benefit for high-risk surgical patients (Smith et al 2010).<br />
  65. 65. Classical<br />Pathway<br />Alternative<br />Pathway<br />The Lectin<br />pathway<br />Factor B, D, P<br />C1q<br />C4b2a<br />C3bBb<br />C1-inh<br />C3<br />Factor H<br />CD59<br />MBP/CD46<br />sCR1<br />DAF<br />C3 convertase<br />C2<br />Factor I<br />Opsonisation<br />C4<br />C4b2a3b<br />C3bBb3b<br />C4-bp<br />Factor I<br />Anti-C5mAb<br />C5 convertase<br />Recruitment of <br />macrophages<br />Complement<br />regulation<br />C6<br />HRF<br />C8<br />C7<br />C5b9<br />C9<br />C9<br />C9<br />clusterin<br />Cytolysis<br />
  66. 66. Crry – mice analogue for DAF<br />TransgenicmiceoverexpressingCrry<br />Improvedneurologicaloutcomeup to 30 dpi<br />Less BBB dysfunction<br />Systemictreatment with recombinantCrry<br />Improvedneurologicaloutcomeup to 7 dpi<br />Preserved neurons in CA3/CA4<br />Upregulation of candidateneuroprotective genes Bcl-2, C1-inh, CD55, CD59<br />Rancan et al, J CerebBlood Flow Metab 2003<br />Leinhase et al. ExpNeurol, 2006<br />
  67. 67. Transgenic C3 -/- deficientmice<br />Cryogenicbraininjury<br />decreasedneutrophilextravasation<br />decreasedinjurysize<br />Intracerebralinjection of C3<br />increasedneutrophilextravasation<br />Sewell et al, J Neuroimmunology 2004<br />
  68. 68. Transgenic C5 -/- deficientmice<br />Cryogenicbraininjury<br />Decreasedneutrophilextravasation<br />Sewell et al, J Neuroimmunology 2004<br />
  69. 69. Conclusions<br />Complement is activated in TBI<br />Complementregulationmightimproveoutcome<br />Time for a RCT in human TBI?<br />
  70. 70. Collaborators<br />Mikael Svensson <br />Olov Bendel <br />Hamid Ghatan<br />Lars-Olof Hansson <br />Iver Langmoen<br />Olle Lidman<br />Per Mattsson<br />Britt Meijer <br />RumiMerzoug<br />Marcus Ohlsson<br />Ingvar Olafsson<br />Fredrik Piehl<br />Mårten Risling<br />ElhamRostami<br />Sim Singhrao<br />PernilleSkejö<br />Gabriel von Euler<br />Hans von Holst<br />Michael Wanecek<br />
  71. 71. Thank You!<br />

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