Ventricular CSF proteomics in patients with acute brain injury: Protocol of the observational trial

C. Santacruz^1-2, 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

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 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:

 Sample preparation

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.%).

Hypoxemia events before and 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.

References

1.     McMullan JT, William A. Knight WA, Clark JF, Beyette FR, Pancioli A. Time-critical neurological emergencies: the unfulfilled role for point-of-care testing. Int J Emerg Med. 2010 Jun; 3(2): 127–131.

2.     Mirski MA, Cherylee W. J. Chang, Cowan R. Impact of a Neuroscience Intensive Care Unit on Neurosurgical Patient Outcomes and Cost of Care. Evidence-Based Support for an Intensivist-Directed Specialty ICU Model of Care. Journal of Neurosurgical Anesthesiology. Vol. 13, No. 2, pp. 83–92.

3.     Hinson HE, Rowell S, Schreiber M. Clinical evidence of inflammation driving secondary brain injury: a systematic review. J Trauma Acute Care Surg. 2015 Jan;78(1):184-91.

4.     Al-Khindi T, Macdonald RL, Schweizer TA. Cognitive and Functional Outcome After Aneurysmal Subarachnoid Hemorrhage. Stroke 2010; 41: e519-e536.

5.     Chiu D, Peterson L, Elkind MS, Rosand J, Gerber LM, Silverstein MD; Glycine Antagonist in Neuroprotection Americas Trial Investigators. Comparison of outcomes after intracerebral hemorrhage and ischemic stroke. J Stroke Cerebrovasc Dis. 2010 May;19(3):225-9.

6.     Oddo M, Rossetti AO. Predicting neurological outcome after cardiac arrest. Curr Opin Crit Care. 2011 Jun;17(3):254-9.

7.     Cooper DJ, Rosenfeld JV, Murray L, et al. Decompressive craniectomy in diffuse traumatic brain injury. N Engl J Med 2011; 364:1493 – 1502.

8.     Prabhakar H, Singh GP, Anand V, Kalaivani M. Mannitol versus hypertonic saline for brain relaxation in patients undergoing craniotomy. Cochrane Database Syst Rev. 2014 Jul 16;7:CD010026.

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The importance of network collaboration for the development of proteomic research during critical illness

Background
Proteins are the building blocks of biological systems. Recently, more groups are performing descriptive proteomic analysis in patients with acute kidney injury (AKI), sepsis, ARDS and acute brain injury. The main focus of research has commonly been the identification of an early biomarker that with alert the clinician on the development of organ failure. The work of Supavekin et al. and Mishra et al. lead to the identification of NGAL as an early biomarker related to AKI. Chang et al describes the qualitative analysis of protein expression from patients with ARDS. In patients with acute brain injury (ABI), several groups have described many biomarkers that have been related to functional outcome. However, the notion that a single biomarker is responsible for the entire pathophysiologic phenomena is naive. Also, even if the biomarker has a good profile to detect end-organ injury and is cost-effective, no available treatment that would reduce mortality or improve neurological outcome can be offered at the moment.

The importance of network collaboration
Proteomic analysis of serum and other fluids from critically ill patients is an emerging field. However,  samples from patients in the ICU are not often easy to obtain, due to ethical issues, and costs are very high. Also, comparison among different groups research on the same topic is often difficult because of different techniques use to identify the targeted proteins (SELDI-TOF-MS, DIGE,etc), and further methods to validate the results such as ELISA testing, Westerblot, among others. Moreover, the introduction of new technologies like the AB Sciex 5600 Triple TOF with MS/MS Swath spectra quantification allows for more in-deep analysis of the data. Identification of an interactome that orchestrates inflammatory, apoptotic and ischemic pathways may lead to performing better experiments and to develop pharmacologic strategies.  
Sharing this knowledge is of key importance. Collaboration and communication between different groups working on the same project would save time and money. Free and easy to use web-based analytical tools would help develop the field and attract new researchers to join-in the search for novel biomarkers or potential pharmacologic interventions. Only with the collaboration from the industry, the private sector and academic groups, can a network of professionals dedicated to advance the field of proteomic research during critical illness be made.

Conclusion
Proteomic analysis of bodily fluids reveals the pathophysiologic picture of the tissue during acute illness. Development of experiments and computational analysis by individual groups should be available to others in order to generalize results and reduce costs.




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Comprehensive summary

#ISICEM-BRACE Meeting: Brain Critical Care and Emergencies.

In this opportunity, we heard from world-wide experts the latest concepts on neurocritical care management. Faculty members included a multi-disciplinary team conformed by intensivists and neurologists. Key topics on the management of several acute neurological emergencies were discussed. Here, a brief summary of the highlights from the BRACE meeting.

Cerebral blood flow and oxygenation 
Mauro ODDO (Lausanne, Switzerland)

"The most important aspect is the balance between flow and CMRO2".

Vasospasm is a multifactorial event that involves the dysfunction of molecular pathways activated during the acute phase. Dysfunctional pathways included the nitric oxide pathway (impaired blood flow stability) and prostacyclin (impaired vasodilation). Other abnormalities include microvascular dysfunction,  micro-thrombotic vascular occlusions and cortical spreading depolarizations.

Is cerebral micro dialysis helpful?
Raimund HELBOK (Innsbruck, Austria)
"The primary objective after brain injury is the prevent secondary insults".

Cerebral micro dialysis (MD) is still considered an experimental technique and no guidelines are available to use MD in clinical practice. However, it is becoming more evident that regional monitoring of cerebral tissue may be associated with better outcomes. Indeed, PbO2 may not detect as many as 50% of ischemic episodes.

How systemic hemodynamic influence brain function?

Daniel deBacker (Brussels, Belgium)
"The alteration of brain microcirculation during sepsis affects cerebral blood flow"

During sepsis, leucocyte and platelets adhesion the endothelia induce brain microcirculatory dysfunction. These microcirculatory derangements lead to cerebral blood flow alteration and reduction on tissue oxygen tension (PbO2).

Anemia and RBC transfusion
Fabio Taccone (Brussels, Belgium)
"How low can you go?"

The decision to transfuse is related to the severity of the disease. Anemia is associated with poor outcome if associated with low PbO2. RBCT may improve tissue and brain tissue oxygenation. However, liberal RBCT strategy is associated with increase rate of secondary complications such as ARDS, infectious, and microcirculatory alterations.

Fever control
Paul Vespa (LA,USA)
"The brain is 1 degree warmer than the rest of the body"
Fever is very common among patients with brain injuries. Fever is associated with worsening neurological outcome. However, fever can be effectively controlled and intravascular cooling devices seem to be better than surface cooling devices.

ICP crisis
Nino Stocchetti (Milan, Italy)
"What matters is the consequence of increase ICP and not the actual number"

If ICP increases from 8 to 18 but with associated reduce awareness, mydriasis or hemiparesis then treatment must be initiated immediately. However, if transient increase in ICP is not associated with any neurological signs, than immediate treatment is probably not necessary. 








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Comprehensive summary

#ISICEM-Course on Metabolic and Nutritional Issues 

in the ICU.

Day1

Today was the first day of the advance metabolic and nutritional ICU course, were world-wide experts debated on critical issues of daily ICU management. Authors come from different countries such as Belgium, France, Sweden, Netherlands, Germany and Switzerland.
Here we present a comprehensive summary of todays key messages from each speaker.




Metabolic response to stress. 
Dr. Jean-Charles Preiser (Brussels, Belgium)
"Homeostasis is needed for life"
During critical illness, circulating levels of orixigenic hormones (gherlin), are reduced. Also,  circulating levels of anorexigenic levels (PYY), are elevated.

The benefits and risks of insulin resistance.
Dr. Peter Soeter (Maastricht, Netherlands)
"The brain does not rely exclusively on glucose"
In critically ill patients, a target blood blood glucose level of 10 mmol (180 mg/dL). Lower levels increase the risk of hypoglycemia and decreases the host response to injury. With 50% of energy requirements covered by glucose, this risk is diminished in host response promoted.

Protein catabolism.
Dr. Jan Wernerman (Stockholm, Sweden).
" Techniques to asses protein metabolism have inherent limitations".
During critical illness, protein catabolism is unevenly distributed in between organs. This means that protein stores  could not be use to maintain homeostasis. However, measurement of proteins metabolism during critical illness is related to measurement biases.











Anabolic resistance
Dr. Jean-Paul Thissen (Brussels, Belgium)
"We should try to increase anabolic stimulus and decrease the resistance threshold"
Anabolic resistance was defined as the inability of skeletal muscle to respond to anabolic stimuli by stimulating protein synthesis and inhibiting proteolysis. There are several factors associated with increase  anabolic resistance such as age, inflammation, cancer, splachnic sequestration and micro-vascular alterations.

Autophagy
Dr. Michael Casaer ( Leuven, Belgium)
" Suppression of Autophagy was associated with organ failure"
Unfortunately, results from a animal studies depicting the role of autophagy have been un-conclusive. More data is needed to understand the role of this highly preserved pathway during critical illness.

Gastrointestinal dysfunction
Alain-Michel Dive (Yvoir, Belgium)
"Patients in the ICU have abnormal inter digestive migratory motor complex function and are not able to start a normal feeding pattern".
Patients that are mechanically ventilated have abnormal retrograde duodenal bolus propagation with almost zero activity at the gastric level. This increase the risk of ileus and broncho-inhalation.

Management of gastric residual volumes
Dr. Jean Reignier (La Roche Sur Yon, France)
"The myth of gastric volume"
Gastric residual volume monitoring should be removed from the standard of care of critically ill patients receiving mechanical ventilation and early enteral nutrition.

Measurements of energy expenditure
Dr. Elisabth de Waele (Brussels, Belgium)
Formulas for estimating the caloric need of critically ill patients are not reliable. Theses formulas may over-estimate the caloric need and thus increase the risk of over-feeding.
However, indirect calorimetry can be performed in only 50% of patients in the ICU, and technical issues make this technique also difficult to perform.

Protein turn-over
Dr. Jan Wernerman (Stockholm, Sweeden)
" The use of protein turnover measurements has the potential to guide protein feeding to critically ill patients"

Assessment of body composition
Dr. Claude Pichard (Geneva, Switzerland)
"It is almost impossible to measure body composition in ICU patients".
Assessment of body composition by CT-scan or MRI could correlate with nutritional risk and outcome. It could reflect the efficiency of nutrition support and thus allow to tailor treatment to patient´s characteristics.








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The era of meta-data in health and during critical illness.

A new personalized approach to critical illness is necessary. Evidence base medicine compares heterogeneous populations and so far has brought few (if any) available treatments fro critically ill patients. This approach lacks individualization of treatment. This kind of "signature medicine" could only come from the analysis of genomic, proteomic and metabolomic data from large population-based set of healthy individuals and compare it to specific patterns present during the disease state. During critical illness, identification of specific patterns of activation could increase the chance of therapeutic success by inhibiting or stimulating different parts of the interactome. More over, integration of hemodynamic, respiratory, and/or metabolic data, will allow multiple variables to be analyzed. This requieres a significant challenge for the medical community. From governmental policies to allow massive collection of blood samples from a given population, to researchers, clinical physicians, laboratory, protein data-banks and the general population. After the collection of millions of samples from healthy controls and from every disease state, we could have a population-based profile of homeostasis and track the deviation from this pattern of individual patients. Knowledge of every component involved in the dis-regulation of homeostasis would allow for specific treatments in each case. We could even tailored drug exposure to reduce secondary effects and toxicity according to models done with bio-informatics. Only after identifying if the individual pattern of each patients as a possible responder, then the best "evidence based" therapy should be initiated.
I do not address to the multiple possibilities that could develop from this hypothesis, since interaction and individuality are now at the center of human evolution.


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