Investigation of brain tissue oxygenation,

cytochrome-c-oxidase and intracellular

metabolites during perinatal cerebral hypoxia-

ischaemia in piglets

aDept. of Medical Physics and Bioengineering, University College London, U.K. bMedical Physics and Bioengineering, UCLH NHS Foundation Trust, U.K. cInstitute for Women’s Health, University College London, U.K. dInstitute of Neurology, University College London, U.K. Multimodal Spectroscopy Group: http://www.ucl.ac.uk/medphys/research/borl/nirs/mms

I. Tachtsidisa, A. Bainbridgeb, E. Bȁra, K. Broad, S. Faulknerc, D.

Priceb, E. Powellc, D. Thomasd, E Cadyb, N. Robertsonc, X. Golayd

Introduction

Perinatal hypoxic-ischaemic brain

injury remains an important cause of

neurologic disability accounting for 15

to 28% of children with cerebral palsy.

Himmelmann et al. Acta Pediatrics 2005; 1:e382

The precise haemodynamic and metabolic events in the early

aftermath following perinatal hypoxia-ischaemia (HI) and how they

relate to subsequent injury severity may provide vital clues to

injury pathways and neuroprotective strategies.

Introduction-Energy Metabolism- NIRS & MRS

Probe By:

1H MRS

31P MRS

NIRS

Glucose Lactate Pyruvate Glycolisis Absence of O2

Presence of

O2

Acetyl - CoA

TCA Cycle

NADH Electron Transport Chain

Cytochrome-c-Oxidase (CCO)

ATP Synthase

ADP + Pi ATP

Energy Buffering: Creatine Kinase

PCr + ADP + H+ ATP

Energy Utilisation

ATP ADP + Pi

Introduction (1H MRS & NIRS)

BASELINE HI RECOVERY

BASELINE HI RECOVERY

Introduction (1H MRS & NIRS)

Scale bar: 2.5mm in A-C&E-G and 0.25mm in D&H

To investigate brain haemodynamic and metabolic changes

during transient HI and recovery using broadband Near-

Infrared Spectroscopy (NIRS) and phosphorus (31P)

magnetic resonance spectroscopy (MRS) in a neonatal

preclinical model.

Aim (Measurements)

Explore the relationship between the NIRS measured changes in

the oxidation state of cytochrome-c-oxidase (oxCCO) and the 31P

MRS measurement (ATP, PCr, Pi).

Look for evidence of early mitochondrial impairment following HI.

Methods (Broadband NIRS) We utilize the UCLn algorithm and fit the changes in the attenuation spectra between 780nm and 900nm to resolve:

-Oxidation cytocrome-c-oxidase: D[oxCCO]

-Oxy-haemoglobin:D[HbO2]

-Deoxy- haemoglobin:D[HHb]

Haemoglobin-difference

D[HbDiff]=D [HbO2]-[DHHb]

Total-haemoglobin

D[HbT]=D [HbO2]+D [HHb]

The optical pathlength was measured continuously at the 840-nm water absorption feature by fitting the second differential of the attenuation spectra to the second differential of water between 800 and 880nm

•A simplified scheme of the flow of electrons through cytochrome c

oxidase and the relevant absorbing wavelengths for each centre.

•NIRS utilizes the specific

extinction spectra of the

oxidised minus reduced

difference spectra of

cytochrome c oxidase to

monitor changes in the

redox state.

560/695nm 605nm 655/780nm 830nm

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

700 750 800 850 900 950

Oxidised CCO

Reduced CCO

Ox-Red CCO

Sp

ecif

ic E

xti

ncti

on

Co

eff

icie

nt

(OD

/cm

/mM

)

Wavelengths (nm)

Methods (broadband NIRS)

Cooper and Springett

Phil.Trans.R.Soc.London B 1997

Nucleotide triphosphate (NTP): has three peaks (α-, β- and γ-NTP) one for each

phosphorous atom in the molecule.

The NTP peaks are the total peaks from ATP (adenosine triphosphate), GTP (guanosine

triphosphate), CTP (cytidine triphosphate) and UTP (uridine triphosphate), where ATP is

by far the one with the highest concentration.

Methods (31P MRS) • 7cm x 5cm Tx/Rx elliptical surface coil

placed on top of head

• Pulse and acquire sequence:

-TR = 10s, NA = 6

Phosphocreatine (PCr) in neurons can

be considered as a reserve of energy in the

cell.

Inorganic phosphate (Pi) is an end

product of energy utilization.

Exchangeable Phosphate Pool (epp) is the sum of Pi+PCr+3NTP

Methods (Protocol) •Piglets (aged < 24 hr) were anaesthetised and physiologically monitored

with intensive life support (SA instruments, New York, USA).

-Combined NIRS and 31P MRS – 22 animals studied

Results (Systemic Data)

0

50

100

150

200

250

-10 0 10 20 30 40 50 60 70 80

MB

P (

mm

Hg

) S

aO

2 (

%)

HR

(b

ea

ts/m

in)

Time (min)

HR

MBP

SaO2

During HI:

Arterial saturation drops (SaO2~50%)

Heart Rate increases.

Mean Blood Pressure increases initially but drops during HI.

-6-5.5-5-4.5-4-3.5-3-2.5-2-1.5-1-0.500.51

-60

-55

-50

-45

-40

-35

-30

-25

-20

-15

-10

-5

0

5

10

-10 0 10 20 30 40 50 60 70 80 90 100 110

Δ[o

xC

CO

] (m

M)

Δ[H

bD

iff]

(μ

M)

HbDiff

oxCCO

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0

0.05

0.1

0.15

0.2

0.25

0.3

-10 0 10 20 30 40 50 60 70 80 90 100 110

PC

r/e

pp

& P

i/e

pp

NT

P/e

pp

Time (minutes)

NTP/epp

PCr/epp

Pi/epp

HI

LWP 175

Results (31P MRS & NIRS)

-6-5.5-5-4.5-4-3.5-3-2.5-2-1.5-1-0.500.51

-60

-55

-50

-45

-40

-35

-30

-25

-20

-15

-10

-5

0

5

10

-10 0 10 20 30 40 50 60 70 80 90 100 110

Δ[o

xC

CO

] (m

M)

Δ[H

bD

iff]

(μ

M)

HbDiff

oxCCO

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0

0.05

0.1

0.15

0.2

0.25

0.3

-10 0 10 20 30 40 50 60 70 80 90 100 110

PC

r/e

pp

& P

i/e

pp

NT

P/e

pp

Time (minutes)

NTP/epp

PCr/epp

Pi/epp

HI

LWP 183

Recovery Fraction & Outcome

Estimate the % of the signal recovery to baseline following HI.

Baseline: 10minutes mean before HI

HI: nadir value during HI (before titration)

Recovery: 10minutes mean between 50-60minutes from the start of HI

Good (n=13) vs Poor (n=9) Outcome group

Subjects all terminated prior to 48Hrs due to poor recovery and

physiology.

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

-10 0 10 20 30 40 50 60 70 80

D[o

xC

CO

] m

M

Time (min)

oxCCO

0

0.05

0.1

0.15

0.2

0.25

0.3

-10 0 10 20 30 40 50 60 70 80

NT

P/e

pp

Time (min)

NTP/epp

Metabolic Recovery Fraction (NIRS vs MRS)

[oxCCO] Recovery Fraction

97%

NTP/epp Recovery Fraction

85%

LWP 210 [Good Outcome]

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

-10 0 10 20 30 40 50 60 70 80

D[o

xC

CO

] m

M

Time (min)

oxCCO

0

0.05

0.1

0.15

0.2

0.25

0.3

-10 0 10 20 30 40 50 60 70 80

NT

P/e

pp

Time (min)

NTP/epp

Metabolic Recovery Fraction (NIRS vs MRS)

[oxCCO] Recovery Fraction

14%

LWP 185 [Poor Outcome]

NTP/epp Recovery Fraction

27%

R² = 0.72

0

10

20

30

40

50

60

70

80

90

100

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

NT

P/e

pp

Re

co

ve

ry (

%)

oxCCO Recovery (%)

All (n=22)

R² = 0.72

0

10

20

30

40

50

60

70

80

90

100

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

NT

P/e

pp

Re

co

ve

ry (

%)

oxCCO Recovery (%)

Good Outcome (n=13)Poor Outcome (n=9)All (n=22)

Recovery Fraction (oxCCO vs NTP/epp)

R² = 0.46

0

10

20

30

40

50

60

70

80

90

100

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

NT

P/e

pp

Re

co

ve

ry (

%)

HbDiff Recovery (%)

All (n=22)

Linear (All (n=22))

Recovery Fraction (HbDiff vs NTP/epp)

0

10

20

30

40

50

60

70

80

90

100

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

NT

P/e

pp

Rec

ove

ry (

%)

HbDiff Recovery (%)

Poor Outcome (n=9)

Good Outcome (n=13)

Recovery

Fraction Pcr/Pi Pcr/epp Pi/epp NTP/epp

oxCCO 0.72 0.78 -0.78 0.85

HbDiff 0.69 0.71 -0.68 0.68

Results Correlation

The NIRS oxCCO % recovery show the highest correlation with

the metabolic MRS % recovery markers and in particular

NTP/epp.

The NIRS oxygenation HbDiff % recovery also show very good

correlations.

The NIRS brain blood volume HbT % recovery signal did not

correlate with the MRS metabolic markers.

Discussion

•During transient HI, CCO becomes reduced due to oxygen

depletion; ATP levels are initially preserved by the creatine

kinase reaction leading to PCr decline whereas energy

utilisation without oxidative phosphorylation leads to

increased Pi.

•Poor NTP/epp (ATP) and ∆[oxCCO] recovery is an

indicator of metabolic insufficiency, mitochondrial

dysfunction and cell death.

Conclusions

•Our novel multimodal spectroscopy methodology

described here suggests that is possible to monitor in-vivo

the metabolic events during and following HI.

• The cytochrome-c-oxidase NIRS signal is a sensitive

indicator of metabolic insufficiency which can be used as a

early marker of injury severity.

•This signal is a useful marker of brain energy metabolism

and can be applied continuously at the cot site.

Acknowledgements

• Piglet group

–Stuart Faulkner, Csilla Andorka, Elizabeth Powell, Lucy Lecky-

Thompson, Nikki Robertson

• Brain histology

–Patricia Refinetti, Gennadij Raivich

• MR Physics

–Alan Bainbridge, Ern Cady, David Price, Xavier Golay, David

Thomas, Madgalena Sokolska

Dr Ilias Tachtsidis is supported by a Wellcome Trust fellowship.

• Multimodal Spectroscopy Group

-Tracy Moroz, Tharindi Hapuarachchi [modelling]

-Aaron Taylor, Tingting Zhou, Gemma Bale, Esther Baer

[measurements]

-Tushaar Madaan, Na (Tina) Yu [signal processing]

-Luke Dunne [instrumentation development]