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Bellander, Bo Michael
1. Complementactivation and Traumatic Brain Injury Bo-Michael Bellander MD., Ph.D. Dept of Neuroscience, Section for Neurosurgery Karolinska hospital Solna Stockholm, Sweden National Neurotrauma Society Fort Lauderdale 2011-07-11
2. Primarybraininjury The result of the mechanicalforces on the braintissue at impact neurons Secondary insults Hypoxia, Hypotension, Hypercarbia, Hypocarbia, Fever, Hyperglycemia, Hypoglycemia, Hyponatremia, Intracranial hypertension, Hematoma, Oedema, Hyperemia, Vascular spasm, Seizures endotelial cells glial cells Biochemicalprocesses ExcitatoryAminoacids, Free radical formation, Apoptosis, Auto-phagia, Mitochondrialdysfunction, Coagulation disorder, Inflammation Secondarybraindamage
3. Classical Pathway Alternative Pathway Thecomplementcascade The Lectin pathway Factor P Factor B C3 MASP MBL C1q Factor D C3 convertase C2 Opsonization C4a C4 C3a C5a C5 convertase Recruitment of macrophages C6 C8 C7 C5b9 C9 C9 C9 Cytolysis
4. Complement and diseases Alzheimers disease(Moss and Albert 1988) Multiple Sclerosis(Tegla et al 2009) MyastheniaGravis (Liszewski and Atkinson 1998) Vasculitis(Peerschke et al 2010) Bacterialmeningitis(Stahel and Barnum 1997) AcuteMyocardialinfarction(Bjerre et al 2008) Thrombosis and stroke (Davies et al 1992; Peerschke et al 2010) Subarachnoidhemorrhage(Kasuya et al 1989; Lindsberg et al 1996) Encephalitis ICH (Hua et al 2000)
5. Classical Pathway Alternative Pathway The Lectin pathway Factor B, D, P C1q C4b2a C3bBb C1-inh C3 Factor H CD59 DAF MBP/CD46 sCR1 C3 convertase C2 Factor I Opsonisation C4 C4b2a3b C3bBb3b C4-bp Factor I Anti-C5mAb C5 convertase Recruitment of macrophages Complement regulation C6 HRF C8 C7 C5b9 C9 C9 C9 clusterin Cytolysis
7. Material & Methods 21 male Spr-Dawley rats + 4 controls Craniotomy 3 mm posterior to bregma and 2 mm lateral to the midline Stereotactic, standardized drop device technique modified from Feeney Bellander et al, JNS 1996 Sacrified 2, 4, 7 and 14 dpi
8. Tight junction protein Absence of thightjunction – IR in the borderzone indicatingdisruption of BBB 4 dpi B. Immunoreactivity for thightjunction protein in intactbrain Bellander et al, JNS 1996
11. OX42 Anti-C3 Microglia vs C3 Co-localizationbetween OX42 positive activatedmicroglialcells/macrofages and anti-C3, at 4 dpi Bellander et al, JNS 1996
14. Neurons in the border zone double stained with anti-MAP5 and anti-Clusterin Anti-Clusterin Anti-MAP5 Bellander et al, JNS 1996
15. Astrocytes in the border zone double stained with anti-GFAP and anti-Clusterin Anti-GFAP Anti-Clusterin Bellander et al, JNS 1996
16. Conclusions I Microglialcells/macrophages are activated in the borderzone of contusions Localsynthesis of the complement protein C3 in the borderzone Microglialcells/macrophagesco-localizes with complementC3 Neurons and astrocytesin the vicinity of contusionsco-localizes with the complement regulator clusterin The balancebetweencomplement proteins and regulators might be a challenge for cells in the borderzone of contusions
17. Is the complement system activatedin the human brainfollowingtraumat?icbraininjury?
18. Complement and human TBI Patients: 16 suffering from cerebral contusions Age: 12-76 years (mean 50) Gender: 13 males, 3 females GCS at admission: 3-13 Indication for craniotomywas ICP>25 mmHg and CPP< 60 mm Hg Trauma-surgery: 2,5 – 82 hours GOS: 1-5, 1 nk
19. C1q C3b C3d C5b9 Bar: 10µm Bellander et al, JNT 2001
21. C5b9 and NeuN C5b9 and NeuN Border zone 10 µm 50 µm Clusterin and NeuN Anti-Clusterin Anti-Clusterin and NeuN Bellander et al, JNT 2001
22. Conclusions II C is activated in the borderzone of human contusions There is a localsyntesis of the complement protein C3 in the borderzone (in situhybridization for C3-mRNA) Neurons in the vicinity of the contusion express the complement regulator clusterin Neurons in the borderzone are challenged by the complementmembrane attack complex Bellander et al. J. Neurotrauma, 18, 12, 2001
23. Hypothesis III Circulating blood is not a necessity for complement activation. The complement activation is an ”endogenous” process.
24. Rat entorhinal-hippocampal slice cultures 96 slices from 12 days old S-D rats 300 µm viable cerebral slices Cultivation for 14 days in 37°C prior to experiment Injury by 2 mm steel ball Slices fixed 1 to 8 dpi Antibodies used OX42 – macrophages/microglia GFAP – astrocytes ED1 – macrophages C1q – complement C5b9 - MAC
25.
26. Bar = 50µm Bellander et al. J. Neurotrauma, 2004
27. Bar = 50µm Bar = 10 µm OX42-IR Bellander et al. J. Neurotrauma, 2004
28. Bar = 50µm Bar = 10 µm Bellander et al. J. Neurotrauma, 2004
29. Bar = 50µm Bar = 10 µm Bellander et al. J. Neurotrauma, 2004
31. Results III Activation of the complement cascade occurs in the absence of circulating blood cells and plasma components Clusterin is upregulated as a result of the complement attack C5b-9, the membrane attack complex – (MAC) is assembled despite absence of circulating blood cells and plasma components.
41. Results IV There is a geneticinfluenceon inflammatoryresponsefollowing TBI Complementactivation correspondsto neuronalnecrosis playsa role in the development of secondarybraindamage
52. C5b9csf vs BBB dysfunction (QA) Stahel et al. JNT 18,8,773-781, 2001 Bellander et al, 2010
53. C5b9 concentrations following isolated TBI (11 humans) Is C5b9 also originating from the blood? Nekludov et al, J Neurotrauma. 2007 Jan;24(1):174-80.
54. Results: Secondary insults at NICU Secondary insults in the NICU Complementactivation Disturbed BBB integrity Release of biomarker S100B in csf
59. Complement receptor type 1 sCR1 – Animal studies Pretreatment ReducedAMI volume in dogs (Weisman et al 1990) Reducedreperfusioninjury in ischemicgut (Hill et al 1992) Reduced ARDS (Mulligan et al 1992) Reducedleucocyteinfiltration following experimental TBI (Kaczorowski et al, 1995)
60. sCR1 – spray treatment of cerebral contusion in rat Bellander, Thesis sCR1 treatment Placebo Ox42 vs sCR1 Ox42 vs NaCl C9 vs sCR1 C9 vs NaCl
61. Complement receptor type 1 sCR1 – Human studies ARDS – Phase 1 (Zimmerman 2002) Dose-dependentcomplementreduction No unexpectedtoxicities Inconclusiveendpoint Lung transplant (Zamora et al 1999 [abstract]) Earlierextubation Thermalinjury Inconclusiveendpoint(Ryan 1995, [abstract]) Cardiacsurgery and cardiopulmonaryby-pass Inconclusiveendpoint (Death, AMI, IABP or length of intubation) (Lazar et al 2004)
62. Classical Pathway Alternative Pathway The Lectin pathway Factor B, D, P C1q C4b2a C3bBb C1-inh C3 Factor H CD59 DAF MBP/CD46 sCR1 C3 convertase C2 Factor I Opsonisation C4 C4b2a3b C3bBb3b C4-bp Factor I Anti-C5mAb C5 convertase Recruitment of macrophages Complement regulation C6 HRF C8 C7 C5b9 C9 C9 C9 clusterin Cytolysis
63.
64. Anti-C5ab – Human studies Human studies CABG and AMI – Phase 3 Improved 90 dayssurvival(Granger 2003) Mortality benefit for high-risk surgical patients (Smith et al 2010).
65. Classical Pathway Alternative Pathway The Lectin pathway Factor B, D, P C1q C4b2a C3bBb C1-inh C3 Factor H CD59 MBP/CD46 sCR1 DAF C3 convertase C2 Factor I Opsonisation C4 C4b2a3b C3bBb3b C4-bp Factor I Anti-C5mAb C5 convertase Recruitment of macrophages Complement regulation C6 HRF C8 C7 C5b9 C9 C9 C9 clusterin Cytolysis
66. Crry – mice analogue for DAF TransgenicmiceoverexpressingCrry Improvedneurologicaloutcomeup to 30 dpi Less BBB dysfunction Systemictreatment with recombinantCrry Improvedneurologicaloutcomeup to 7 dpi Preserved neurons in CA3/CA4 Upregulation of candidateneuroprotective genes Bcl-2, C1-inh, CD55, CD59 Rancan et al, J CerebBlood Flow Metab 2003 Leinhase et al. ExpNeurol, 2006
67. Transgenic C3 -/- deficientmice Cryogenicbraininjury decreasedneutrophilextravasation decreasedinjurysize Intracerebralinjection of C3 increasedneutrophilextravasation Sewell et al, J Neuroimmunology 2004
69. Conclusions Complement is activated in TBI Complementregulationmightimproveoutcome Time for a RCT in human TBI?
70. Collaborators Mikael Svensson Olov Bendel Hamid Ghatan Lars-Olof Hansson Iver Langmoen Olle Lidman Per Mattsson Britt Meijer RumiMerzoug Marcus Ohlsson Ingvar Olafsson Fredrik Piehl Mårten Risling ElhamRostami Sim Singhrao PernilleSkejö Gabriel von Euler Hans von Holst Michael Wanecek
