Ventricular CSF proteomics in patients
with acute brain injury: Protocol of the observational trial
C. Santacruz1-2, N. Sadeghi, G. Cabrera, D. Communi, M. Bruneau, J.L. Vincent, J. Creteur, S. Brimioulle.
1.Hopital Erasme,
Brussels, Belgium.
2. Fundación
CardioInfantil, Bogotá, Colombia.
Introduction
After acute brain injuries,
intra-thecal proteins related to brain inflammation, apoptosis and oxidative
stress induce migration and production of quimiotatic factors that will
ultimately lead to blood-brain barrier (BBB) dysfunction and edema formation
(1). After experimental ischemic brain injury, the expression of
neuro-protective proteins extracted from the ipsilateral hippocampus differs
when compared against sham controls (2). In a rat model of TBI, expression of
proteins related to apoptosis during the first week after lateral fluid
percussion injury (LFPI) was part of a temporal pattern of neuro-degeneration
and programmed cell death (3). In
patients with TBI, alteration in vCSF proteins related to cholesterol
metabolism, apoptosis and neuroendocrine system differ when compared to control
patients (4-6). In patients with sSAH, changes in vCSF proteins related to
oxidative stress, apoptosis and micro-vascular reactivity were related to worst
neurological outcome and increase risk of vasospasm (7-8).
Characterization of these
proteins would help understand the complex pathophysiology of neuronal injury
after ANE and identify potential candidates for biomarkers of impaired clinical
and long-term neurological outcome.
Hypothesis
We hypothesized that alteration
in vCSF protein concentration from patients with ABI would be related to worst
3-month neurological outcome as measured by the Glasgow Outcome Scale (GOS)(9).
Objectives
Our objective is to compare the effects of
abnormal protein expression in CSF of patients with ABI on the 3-month GOS
score, head CT-scan hemorrhage and peri-hemorrhagic edema volume (in mL)
evolution during ICU stay and ICU mortality.
Materials
and methods
We
will collect CSF from patients with ANI that have an
ICP catheter (intra-ventricular) in place inserted by the Neurosurgery
Department and are hospitalized in the ICU at Erasme Hospital. The study is already approved by the hospital
Ethical Committee
1.
Inclusion criteria
- ABI (traumatic
brain injury, subarachnoid hemorrhage, acute ischemic stroke, intra-parenchymal
hemorrhage, etc.) with an ICP catheter.
-
Glasgow coma scale (GCS) ≤ 8 at admission in the ICU.
- Less
than 24 hours from ICP catheter placement.
2.
Exclusion criteria
-
Bacterial, viral or fungal meningitis.
-
Bacterial, viral or fungal ventriculitis.
-
Brain tumor resection.
-
Imminent death.
3. CSF
sampling and proteomic analysis:
CSF (3 to 4 mL) samples from
patients with ABI en ICP monitoring will be collected from the ventriculostomy
catheter at admission and then every 24 hours for 5 consecutive days.
Samples will be centrifuged,
frozen and stored at -80°C until the time of analysis. 250µl of CerebroSpinal
Fluid will be concentrated on Amicon 3KDa (Millipore). The concentrated volume
will be diluted in 100µl of 25mM NH4HCO3. Protein samples will be then reduced
with 10 mM DL-dithiothreitol during 30min at 56°C and alkylated with 55 mM
iodoacetamide during 20min at room temperature. After, proteins digestion
overnight with 2µg of trypsin (Promega, Belgium) at 37°C, formic acid will be
added to 1% (v/v) and peptides will be purified using StageTips C18
(ThermoFischer Scientific) according to the manufacturer's instructions. The
sample will be evaporated to dryness in a vacuum centrifuge and resuspended in
15µl of 5% ACN/0.1% HCOOH. 5µl will be injected in DIA acquisition modes.
DIA mass spectrometry
DIA mass spectra will be acquired
using an AB Sciex 5600 Triple TOF mass spectrometer (AB Sciex, Concord, Canada)
interfaced to an Eksigent NanoLC Ultra 2D HPLC System (Eksignet, Dublin, CA).
Peptides will be injected and concentrated on a trapping column (Waters
Symmetry C18 NanoAcquity 2G v/v, 20mm x 180µm, 5µm) with a loading solvent (5%
CAN / 0.1% HCOOH). After 10min, peptides will be separated on a separation
column (Waters Acquity UPLC HSS T3, 250mm x 75µm, 1.8µm) using a two steps
acetonitrile gradient (5-25% ACN / 0.1% HCOOH in 60min then 25%-60% CAN / 0.1%
HCOOH in 40min) and will be sprayed online in the mass spectrometer. MS1
spectra will be collected in the range 400-1200 m/z for 250ms. The 20 most
intense precursors with charge state 2-4 will be selected for fragmentation,
and MS2 spectra will be collected in the range 100-2000 m/z for 100ms;
precursor ions will be excluded for reselection for 12s.
Swath MS/MS data
Targeted data extraction of the
MS/MS spectra generated by data independent acquisition (DIA) method of all
proteins from ABI patients and controls will be used as previously described
(20). The DIA approach uses 32 cycles to iterate through precursor ion windows
from 400-426 Da to 1175-1201 Da and at each step acquire a complete,
multiplexed fragment ion spectrum of all precursors present in that window.
After 32 fragmentations, the cycle is restarted and the first window (400-426
Da) is fragmented again, delivering complete "snapshots" of all
fragments of a specific window every 3.2 seconds.
Cytokine assay
The vCSF concentrations of
Apolipoprotein E (ApoE) will be measured in the vCSF from
ABI and control patients using a
commercial ELISA test (Quantikine® ELISA Human Apolipoprotein E/ApoE
Immunoassay, R&D Systems® Minneapolis, MN) according to the manufacturer’s
protocol.
4. Scanographic evaluation of head CT-scan hemorrhage and peri-hemorrhagic
edema volume (in mL)
CT Technique
Scans
will be performed with a 64-row multisection CT (Somaton Sensation 64; Siemens
Medical Systems, Forcheim, Germany). Patients
will be examined in supine position, and a lateral 26-cm scout view was first
obtained at 120 kVp and 35 mA. This is followed by a 12-cm-high helical CT
acquisition of the entire cranium, with a detector collimation of 32 x 0.6 mm,
rotation time of 0.5 seconds, pitch of 1, 100 kVp, and 200 effective milliamps,
resulting in a CT dose index of 18.95 mGy and a dose-length product of 300
mGy.cm.
Volume Measurements
The
intracranial hematoma (ICH) is defined by using the automated segmentation and
manual tracing tools in the segmentation module from the Analyze software
(LiveWire Software®, Carestream Health, Inc. Rochester, NY). Corrections will be applied when necessary
using a correction tool. This procedure will be reproduced for each section as
well as each separate target area as determined by the reader. Only areas of intraparenchymal
haemorrhage will be included in the ICH volume measurement. Areas of subarachnoid
and subdural hematomas will be excluded. After all areas were defined by the
reader, the analyze software calculated the segmental volume (cubic
millimetres) of each region of interest.
This segmental volume of each region of interest is the product of the
section thickness and its area. Finally, the total volume of the hematoma will
be automatically obtained by the summation of all segmental volumes for a given
lesion. Oedema volumes will be similarly
calculated by using the manual tracing tools in the segmentation module from
the same analyze software. When the oedema is surrounding the hematoma, a
volume corresponding to a conglomerate of hematoma and oedema will be obtained.
Oedema volume will be then calculated by subtracting the hematoma volume from
the conglomerate volume (Oedema Volume= Conglomerate Volume- Hematoma Volume).
Volumes in cubic millimeters will be converted to milliliters for purposes of
reporting final results.
5.
Clinical data:
Along
with proteomic analyses, several clinical data will be collected:
Age:
years
Sex: male/female (%)
Cause
of injury: traumatic, medical.
Time
to initial medical attention.
Pupil
status at first contact evaluation.
Glasgow
coma scale: overall score (median, interquartile range), motor score[j1]
(median, interquartile
range).
Hypotension
events during ICU stay (No.%).
APACHE
score (No.%).
GCS
score (No.%).
Complete
laboratory data during the study period (Ph, blood glucose, etc.)
IMPACT
score for patients with TBI.
Fisher
score for patients with SAH.
Marshall
class findings on computed tomography (No.%).
Hematoma
volume on head CT-scan in all patients.
6.
Outcomes
-
3-month neurological outcome as assessed by the Glasgow outcome scale.
-
Intra-parenchymal hematoma volume on head-CT scan.
-
Intra-cranial pressure (ICP).
-
ICU-mortality.
7.
Statistical analysis:
All statistical analysis wil be
performed using R language software v.3.0.1. and the
statistical tool Sciex and Peak Analyst
®software packages available with the AB Sciex 5600 Triple TOF mass
spectrometer (AB Sciex, Concord, Canada) . Heat maps will be used to represent
the level of expression of selected proteins and their relationships across all
comparable samples. Clusters of protein expression will be analyzed using
principal component analysis (PCA). Only proteins related to inflammation,
apoptosis and ischemia that are statistically different between groups and
between days would be included for analysis. Categorical and continuous data
will be compared using t-test or Mann-Whitney U test, as appropriated.
Regression models will be used to evaluate the relationship between specific
protein´s SWATH MS/MS data with 3-month GOS, ICU-mortality and changes (in mL)
in hemorrhage and peri-hemorrhagic edema volume during ICU stay. We will use clustering,
topology, Bayesian networks, Boolean/Fuzzy logic decision trees and
differential equations analysis of the data. Alfa error is set at 5%.
8.
Ethical Issues
The
study complies with the declaration of Helsinki and Good clinical practices
laws and with the bill to collect human biological material for human
scientific research purposes. Since samples are a surplus biological material
collected as part of every-day surveillance procedure in patients with a
intra-ventricular catheter, this study does not apply for the may 2004 law. So,
this trial is considered in the MCH category. An informed consent for use of
the data will be obtained from the patient or from the legal guardian.
Implications
of the study
The
complete characterization of the local environment during ABI could help
understand the pathophysiological pathways involved during neuro-critical
illness and identify a biomarker that is related to worst neurological and
clinial outcomes. Finally, limiting the expression of such biomarker by
pharmacological or surgical treatment could reduce the mortality and severe
neurological impairment related to ABI.
Limitations
Low
volume studies in neurocritical sciences are common and may lead to increase
false positive “discoveries”. High cost associated with proteomic studies may
limit the number of included patients, and thus reduce the power of the trial.
Only by effective collaboration between different research teams involved in ABI-neuroproteomic
can resources be optimized, methodological quality improve and more patient-related
questions be answer.
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