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

https://www.thapca.org/

hyperlink

N=467/820

57%

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