Investigation of brain tissue oxygenation, cytochrome-c-oxidase … · 2018-05-03 · Investigation...
Transcript of Investigation of brain tissue oxygenation, cytochrome-c-oxidase … · 2018-05-03 · Investigation...
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)
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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
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0.1
0.2
0.3
0.4
0.5
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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
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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]