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Neuroprotection
Jamie Hutchison, MD
Professor of Paediatrics, University of Toronto
CCCF
Caring for the Brain Injured Patient - I
November, 2012
Primary and secondary brain injury
timeline
OutcomePrimary Injury Secondary injury
Prevention of cell death
Today’s Grand Rounds:
• Global Cerebral Ischemia and Cardiac Arrest
• Traumatic Brain Injury
Molecular mediators of secondary brain
injury
Time
Brain
Injury
Heat shock protein
Cytokines, Adhesion molecules
Apoptotic proteins
Reactive oxygen species
Excitatory amino acids
PCr/ATP energy failure
Therapeutic
window
Sham 2VO
forebrain
hindbrain
no
perfusion
MICe – Dermot Doherty and John Sled, SickKids
Perfusion CT - Global forebrain ischemia
Laser doppler – Cerebral blood flow
2VO Normothermic (37 C)
0
20
40
60
80
100
120
0 20 40 60 80 100 120 140
Time (min)
%rC
BF
Ischemia
2VO Hypothermic (32 C)
0
20
40
60
80
100
120
0 20 40 60 80 100 120 140
Time (min)
% r
CB
F
Ischemia
Jeffrey Shih, MSc, Boston University
Interleukin (IL)-1β is expressed in Neurons
Following 2VO ischemia
Hippocampus (CA1)
IL-1β Neuronal - N Merged
Ryan Salewski, MSc, Institute for Medical Science, University of Toronto
Hippocampus (CA1)
macrophage/microglia (Iba-1)
Sham
Normothermia
2VO
Hypothermia
2VO
Nguyen A, MSc , Institute for Medical Science, University of Toronto
Hypothermia protects against cell death
post ischemia
Detection of degenerating neurons with FluoroJade B in the hippocampus (CA-1) at 72 h
Normothermia
shamNormothermia
2VO
Hypothermia
2VO
Nguyen A, MSc , Institute for Medical Science, University of Toronto
Theoretical paradigm
Hypothermia
_
Cardiac
Arrest
The Use of Hypothermia Therapy after Pediatric
Cardio-Respiratory Arrest - a 5 Centre International
Observational Study
Conclusions: No Benefit of Hypothermia therapy
Equipoise
D. Doherty, C. Parshuram, R. Farrell, D. Bohn, A. Hoskote, M. Tucci, A.
Joffe, K. Choong, J. Hutchison for the Canadian Critical Care Trials Group
Circulation 2009;119:1492-1500.
Hypothermia for Cardiac Arrest in
Pediatrics (HypCAP) – Pilot study
J. Hutchison, A-M. Guerguerian, C. Parshuram, B. Bissonette, Igor
Luginbuehl , R. Sananes, A. Hoskote, J. Beca, R. Gaiteiro, H. Frndova, S.
Hussain, J. Lacroix
HypCAP
R
Eligibility:
- Coma post-CA
Informed consent Te = 33-34 °C (48 h.)
Te = 36.5-37.5 °C (48 h.)
•N = 38
•Masked follow-up 3, 6 and 12 months
- Functional and Neuropsychological tests
Outcomes
Characteristic
N (%)Hypothermia
Group
(N=19)
Normothermi
a Group
(N=19)
P-value
PCPC 4-6 at 6
months 13 (68) 10 (53) 0.60
Mortality
(28 days) 12 (63) 8 (42) 0.19
No significant increase in adverse effects
PCPC = Pediatric Cerebral Performance Category score
Summary
Hypothermia therapy in cardiac arrest and
global cerebral ischemia
• Hypothermia therapy in global cerebral
ischemia
– Potent anti-inflammatory mechanisms
• Hypothermia therapy in cardiac arrest in
children – HypCAP pilot complete
– It is feasible and likely safe
• THAPCA – funded by NHLBI-NIH – study
in progress
Proposed parallel studies of molecular mechanisms
of global cerebral ischemia in mice and cardiac
arrest in infants and children
Sham 2VO
forebrain
hindbrain
no
perfusion
Peripheral
Blood
Immunology
Neurological
Functional
Outcomes
Death –
Brain Molecular
Expression and
Cell Death
Intravital
Microscopy
Time
Traumatic Brain Injury (TBI)
How do we prevent cell death in
our patients?
• Cerebral physiology-based therapy
– We manage ABCs with great care!
– We monitor for and control intracranial
hypertension
– We treat HERNIATION as a
neurosurgical/medical emergency
– We tried treating molecular mechanisms of
cell death with early institution of hypothermia
therapy – didn’t work!
Monroe- Kellie Doctrine
Rogers (1996) Textbook of Pediatric Intensive Care p. 646
The brain has the
ability to control its
blood supply to match
its metabolic
requirements
Chemical or metabolic
by products of cerebral
metabolism can alter
blood vessel caliber
and behavior
Blood: Cerebral Blood Flow
Laffey & Kavanagh,
New Engl J Med 2002
Bregma
Impounder
MC
Steel rod
CaudalRostral
Ventral
Dorsal
Mouse Model – Weight-drop TBI
Hutchison, et al. J Neurotrauma 2001
Mikrogianakis, et al. J Neurotrauma 2007
Brain
Protein
(NAIP)
Neuronal apoptosis inhibitory protein
(NAIP) at 24 hours following 2 hits
1st Hit
2nd Hit
Brain cell death at 24 hours following 2 hits
Dead
Brain
Cells
1st Hit
2nd Hit
Mikrogianakis, et al., J Neurotrauma 2007
Effect of hypothermia therapy in rodent
models of traumatic brain injury (TBI)
• Reduces cerebral edema, blood brain
barrier permeability, inflammation, cerebral
atrophy, contusion size, necrotic and
apoptotic cell death, axonal injury and
mortality and improves neurobehavior
scores following TBI in rodents
Hypothermia Paediatric Head Injury
Trial (HyP-HIT)• Randomized controlled trial of 24 hours of
hypothermia therapy in Paediatric patients with Severe TBI
• 17 centres in Canada, UK and France
• Trial coordinated by the Chalmers Group and Epidemiology Group (CHEO and University of Ottawa) on behalf of the Canadian Critical Care Trials Group
• Funded by the Canadian Institutes of Health Research and other granting agencies
HyP-HIT cerebral perfusion pressure (CPP)
Hutchison et al. Dev Neuroscience 2010
HyP-HIT Hypotension
• There was a significant association between the number of hypotensive or low cerebral perfusion pressure events and unfavorable outcome.
Hutchison et al. Dev Neuroscience 2010
What more can we do for our patients
in the future to develop and test novel
therapies?
• Find the ideal neuroprotective therapy
– Models of brain injury
• TBI ? + multi-system trauma
– Study mechanisms of cell death
• Eg. Apoptosis
Neuroprotection• Novel neuroprotective agents in TBI
• Multiple negative trials and trials in
progress– NMDA and AMPA receptor antagonists
– Necrosis and apoptosis inhibitors
– Immunophilin ligands eg. Cyclosporin A
– Ovarian hormones - Progesterone
– Erythropoetin
– Steroids - CRASH Lancet 2005 – Harm
– Nimodipine
– Magnesium sulfate, tirilizad and selfotel
Review: McConeghy KW, et al. CNS Drugs 2012
Bregma
Impounder
MC
Steel rod
CaudalRostral
Ventral
Dorsal
Mouse Model – Weight-drop TBI
Hutchison, et al. J Neurotrauma 2001
Mikrogianakis, et al. J Neurotrauma 2007
TAT- NAIP Construct
BIR
BIR = Baculovirus Inhibitor of apoptosis protein Repeat
GFP TATBIRBIR NOD LRR
Model of cell death in rodent neurons
Cleaved PARP Positive cells 16 hours of etoposide
Hoechst Cleaved PARP
100 X 100 X
Cleaved PARP following 16 hours of
etoposide
0
5
10
15
20
25
30
DM
SO
+ E
top
osid
e A
lon
e
TA
T-G
FP
-BIR
123xt
Eto
po
sid
e
Perc
en
tag
e o
f C
leaved
PA
RP
Po
sit
ive C
ells
Percentage of NG108 Cells Incubated for 90 minutes with PBS alone or TAT-GFP-Bir123xt Positive for Cleaved PARP in a 40x Field of Magnification following 16 hours of DMSO or Etoposide Exposure
DM
SO
Alo
ne
*
Negative
Control
Etoposide
+ Control
Etoposide
+ TAT-NAIP
Cell
Death
Recombinant TAT-GFP-NAIP enters brain cells rapidly (3-
4h.) following injection into the peritoneum of mice
E F
GFP Hoechst
PBS
TAT-GFP-NAIP
Effect of recombinant TAT- NAIP on area of
contusion post-TBI
0
5
10
15
20
% Damage
GFP-BIR123xt TAT-GFP-BIR123xt
*
Percent
Area of
Damage
TBI
+ TAT – NAIP
TBI
+ NAIP
(no TAT)
Specific Biomarkers May Be Responsive To Neuroprotective
Therapy
Oligodendrocyte:
Myelin basic protein
Astrocyte: S100B
Glial fibrillary acid protein
Basement
membrane
Endothelial-selectin
Soluble adhesion molecules
Endothelial cell
Molecular Biomarkers
Microglia:
Cytokines
Chemokines
Adherent leukocyte:
Cytokines
Chemokines
Neuron:
Neuron-specific enolase
Phosphorylated neurofilament H protein
Blood-brain
barrier
Lipids:
Eicosanoids
Leukotrienes
Sphingolipids
Oxidized
lipids
Conclusions – Neuroprotection in TBI
• Prevent secondary brain injury by preventing
cerebral hypoxic-ischemia
• Hypothermia therapy holds promise in cardiac
arrest but not in TBI in children
• Novel anti-inflammatory and anti-apoptotic
therapies hold promise
• Combination therapies or therapeutics may be
successful
• Brain specific biomarkers hold promise as
surrogate endpoints for pilot randomized
controlled trials of novel neuroprotective agents
Acknowledgements– HypCAP, HyP-HIT and Biomarkers in TBI Clinical study
coordinators and investigators
• Rose Gaiteiro, Roxanne Ward, Judy Van Huyse
• Anne-Marie Guerguerian
Laboratory:
– Technicians:• Rachel Shaye, Phil Griffen, Julia Lockwood
– Graduate students and post-Docs:• Angelo Mikrogianakis, Natalie Gendron, Arsalan Haqqani, Dermot Doherty,
Milly Lo, Ian Sutcliffe, Jeffery Shih, Ryan Salewski, Vera Nenadovic, Anh
Nguyen
– Collaborators:• Danica Stanimirovic, Alex MacKenzie, Rand Askalan, Cynthia Hawkins,
Sheena Josselyn, Paul Frankland, Martin Post, Mike Salter