Rheo-Erythrocrine Dysfunction as a Biomarker for RIC Treatment in Acute Ischemic Stroke - Pilot, Single-center, Randomized, Patient-assessor Blinded, Sham-controlled Study
Phase: NA
Conditions: Stroke, Acute, Ischemic Stroke, Cerebrovascular Disorders, Central Nervous System Diseases
Sponsor: Grethe Andersen

ENOS stroke trial 
 
version 1.2 (October 9th 2019) 
 
 
 
Rheo-Erythrocrine dysfunction as a biomarker for 
RIC treatment in acute ischemic stroke 
RESEARCH PROTOCOL 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
2 
 
Project description 
This study aims to investigate whether Remote Ischemic Conditioning improves rheo-erythrocrine 
dysfunction in acute ischemic stroke 
 
 
 
 
 
 
Project acronym 
 
 
 
Project type  
 
 
 
Sponsor/Principal 
Investigator 
 
 
 
Investigator (US) 
 
 
 
Investigator (DK) 
 
 
 
Investigator (US) 
 
 
 
Study Coordinator & 
Investigator (DK) 
ENOS stroke trial 
Rheo-Erythrocrine dysfuNction as a biOmarker for RIC 
treatment in acute ischemic Stroke 
 
Pilot, randomized, patient-assessor blinded, sham-controlled 
study 
 
 
Grethe Andersen, Senior Consultant, MD, DMSc, Professor of 
Neurology, Department of Neurology & Danish Stroke Center, 
Aarhus University Hospital, DK-8000, Denmark 
 
 
David Hess, MD, Professor & Chairman, Department of 
Neurology, Georgia Medical College, Georgia, USA 
 
 
Rolf Ankerlund Blauenfeldt, MD, PhD-student, Department of 
Neurology & Danish Stroke Center, Aarhus University 
Hospital, DK-8000, Denmark 
 
Mohammad B. Khan, PhD, Research Scientist,  
(Research Faculty), Department of Neurology, Georgia 
Medical College, Georgia, USA 
 
Maria Kjølhede Sørensen, stud. med., Aarhus University, 
Denmark 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
3 
 
Abbreviations 
 
RIC 
Remote Ischemic Conditioning 
RBC 
Red Blood Cell 
NO 
Nitric Oxide 
-SNO 
S-nitrosylation 
EI 
Elongation Index 
DI 
Deformability Index 
mRS 
Modified Rankin Scale 
MRI 
Magnetic Resonance Imaging  
NIHSS 
National Institutes of Health Stroke Scale 
MRI 
Magnetic Resonance Imaging 
TIA 
Transient ischemic attack 
AE 
Adverse effects  
DSR 
Danish Stroke Registry 
CRF 
Case report form    
Stroke RIC 
Automatic Remote Ischemic Conditioning Device 
PVP 
Polyvinylpyrrolidone 
GPA 
Glycophorin A 
miRNA 
Micro ribonucleic acid 
EV 
Extracellular vesicles, also known as exosomes 
ELISA 
enzyme-linked immunosorbent assay 
MoCA 
Montreal Cognitive Assessment   
PASE  
Physical activity in the elderly 
SAE 
Serious Adverse Event  
SADE 
Serious Adverse Device Effect 
USADE 
Unanticipated Serious Adverse Device Effect  
ASADE 
Anticipated Serious Adverse Device Effect 
AIS  
Acute Ischemic Stroke 
ICH 
Intracerebral hemorrhage  
 
 
 
 
 
 
 
 
 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
4 
Contents 
1. Background ............................................................................................................................................................ 6 
2. Project thesis ......................................................................................................................................................... 6 
Hypothesis ..................................................................................................................................................................... 6 
Primary endpoint ........................................................................................................................................................... 6 
Secondary endpoints ..................................................................................................................................................... 7 
3. 
Methods ........................................................................................................................................................... 7 
Design ............................................................................................................................................................................ 7 
Patient selection ............................................................................................................................................................ 7 
Informed consent .......................................................................................................................................................... 8 
Method of blinding ........................................................................................................................................................ 9 
Discontinuation of study participation .......................................................................................................................... 9 
Time schedule ................................................................................................................................................................ 9 
4. Study procedures and assessment .......................................................................................................................... 9 
Blood samples ............................................................................................................................................................. 10 
Intervention (Remote Ischemic Conditioning) ............................................................................................................. 11 
RBC – deformability - Ectacytometry ........................................................................................................................... 11 
Erythrocrine dysfunction - Flow Cytometry ................................................................................................................. 12 
MicroRNA analysis ...................................................................................................................................................... 12 
Extracellular vesicle analysis ....................................................................................................................................... 12 
Use of routine collected blood samples ........................................................................................................................ 13 
Montreal Cognitive Assessment (MoCA) ..................................................................................................................... 13 
PASE - physical activity in the elderly .......................................................................................................................... 14 
Remote Ischemic Conditioning questionnaire ............................................................................................................. 14 
5. Statistical analysis ................................................................................................................................................ 14 
Primary endpoint analysis ........................................................................................................................................... 14 
6. Ethical considerations .......................................................................................................................................... 14 
Advantages and disadvantages of participating in the study ..................................................................................... 15 
7. Assessment of safety ............................................................................................................................................ 15 
Emergency unblinding procedure ................................................................................................................................ 15 
Adverse events ............................................................................................................................................................ 15 
Adverse event (AE) ....................................................................................................................................................... 16 
Serious Adverse Event (SAE) ......................................................................................................................................... 16 
Device Deficiency ......................................................................................................................................................... 16 
Adverse Device Effect ................................................................................................................................................... 16 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
5 
Serious Adverse Device Effect (SADE) ........................................................................................................................... 16 
Unanticipated Serious Adverse Device Effect (USADE) ................................................................................................. 16 
Anticipated SADE (ASADE) ........................................................................................................................................... 17 
Event report and causality assessment ........................................................................................................................ 17 
8. Data handling and record keeping ......................................................................................................................... 20 
9. Further documentation ........................................................................................................................................ 20 
10. Economics ......................................................................................................................................................... 21 
11. Publication ......................................................................................................................................................... 22 
12. Source data access and monitoring ..................................................................................................................... 22 
13. Appendix ........................................................................................................................................................... 23 
Appendix 1: Baseline data and treatment characteristics ........................................................................................... 23 
Appendix 2: Study flowchart ....................................................................................................................................... 24 
Appendix 3: Montreal Cognitive Assessment .............................................................................................................. 25 
Appendix 4: RIC questionnaire (Danish) ...................................................................................................................... 26 
14. References ......................................................................................................................................................... 28 
 
 
 
 
 
 
 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
6 
1. Background 
Stroke is a leading cause of death and disability worldwide(1, 2). Of all strokes, 85% are ischemic 
strokes caused by a thrombus or an embolus. The additional 15% are caused by hemorrhage(3). 
Currently the only approved treatments for ischemic strokes are thrombolysis given within 4.5 
hours and thrombectomy performed within 6 hours of symptom onset – in some cases up to 24 
hours(4-7).  The majority of stroke patients are not however eligible for acute reperfusion therapy, 
mainly due to time constrains and late presentation. Novel neuroprotective strategies available for 
all stroke patients are thus urgently needed. 
 
Remote Ischemic Conditioning (RIC) is a simple intervention in which transient ischemia is induced 
in an extremity by repetitive inflation-deflation of a blood pressure cuff. It remains uncertain 
exactly how the protective effect of RIC is transmitted and communicated between the extremity 
and the brain. Both humoral, immunological and neuronal pathways seem to be involved (4, 8-10). 
Treatment with RIC and has proven to be a safe, feasible and low-cost treatment in clinical 
settings(9-12).   
 
Biomarkers of the RIC treatment is a new area of stroke research and are important to establish in 
order to assess and predict responders of the conditioning treatment. Rheo-erythrocrine 
dysfunction of the Red Blood Cell (RBC) is a novel biomarker in both ischemic strokes in general 
and on the effect of RIC. Red Blood Cells with a diameter of 6-8 μm must be highly deformable in 
order to deliver oxygen to brain tissue by travelling through micro vessels with a diameter of just 
2-3 μm. RBC’s can carry nitric oxide as NO2−/s-nitrosylated proteins. These proteins improve RBC 
deformability and induce hypoxic vasodilation thereby improving passage through the 
microvasculature(13-18). RBC’s also express Erythrocyte Nitric Oxide Synthase 3, which regulate 
the rheo-erythrocrine function. Erythrocyte Nitric Oxide Synthase 3 is activated by shear stress 
and provide an extra source of NO for hypoxic vasodilation(19-22). Preliminary data have shown 
that experimental stroke on mice seems to cause a rheo-erythrocrine dysfunction of the RBC’s 
leading to a loss of deformability. The RBC’s become rigid, which can lead to occlusion of micro 
vessels in the brain and further ischemic damage. Loss of deformability can be measured as a 
reduced Elongation Index (EI) by ektacytometry and may be attenuated by RIC(23).  
2. Project thesis  
Hypothesis 
RIC improves rheo-erythrocrine dysfunction in acute ischemic stroke as compared to sham RIC. 
 
Primary endpoint 
Rheo-erythrocrine dysfunction in RBC’s (deformability and Erythrocyte Nitric Oxide Synthase 3) 
will serve as a biomarker of the conditioning response and predictor of the clinical outcome in 
stroke patients. 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
7 
Secondary endpoints 
 
• RBC deformability presentation across stroke subtypes 
• RBC deformability in relation to infarct size/stroke severity 
• RBC erythrocrine dysfunction presentation across stroke subtypes 
• RBC erythrocrine dysfunction in relation to infarct size/stroke severity 
• Difference in 7 days cognitive impairment between treatment groups. 
• RBC erythrocrine dysfunction and deformability as a marker for difference in 7 days 
cognitive impairment  
• Circulating microRNA and/or extracellular vesicle profile of RIC-induced neuroprotection 
• Circulating microRNA and/or extracellular vesicle profile as a marker for difference in 7 
days cognitive impairment  
• Circulating microRNA and/or extracellular vesicle profile as a marker for RBC erythrocrine 
dysfunction and deformability 
3. Methods 
Design 
Pilot, randomized, patient-assessor blinded, sham-controlled study on stroke patients admitted to 
the Stroke Unit at Aarhus University Hospital.  
 
Patient selection 
The study population is selected according to the criteria in table 1: 
 
 
Table 1. Inclusion and exclusion criteria for stroke patients and controls  
Stroke patients Inclusion/Exclusion criteria 
Inclusion criteria 
Exclusion criteria 
Onset to randomization < 48h 
 
Prior stroke, dementia or other known neurological condition 
 
Age 18-80 
Pregnancy* 
Independent in daily living (mRS 0-2) Contraindications to MRI 
Legal competent 
Investigators discretion 
Ambulatory  
Known upper extremity peripheral arterial stenosis         
Documented ischemic stroke on 
baseline MRI 
Diabetes 
 
 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
8 
*Women of child-bearing age will be asked about their use of safe birth control methods (contraceptive pill, intrauterine devices 
both hormonal and non-hormonal, hormonal implants, hormonal depot injection and transdermal hormonal patch). A safe birth 
control method shall be used throughout the study period.  A pregnancy test will be performed (ruling out pregnancy) on women of 
child-bearing age before study participation is possible.  
 
Informed consent 
Patients who fulfill the study criteria will be invited to participate in the study. It will be 
endeavored to create a calm and safe environment for this conversation either in the patient 
room or in an interview room at the ward. We will prioritize the presence of nearest relatives at 
the time of study information and inclusion. Furthermore, we will inform the patient on the right 
to 24-hour consideration time before entering the study. All information will be given orally as 
well as in writing. A patient can only participate in the study if he/she has signed the consent 
forms (“samtykkeerklæring og fuldmatserklæring”) 
 
A patient can withdraw from the study at any time. Withdrawal can also be due to the principal 
investigator’s discretion. In case the patient cannot be reached by telephone, every effort will be 
made to contact the patient or to document the outcome regarding new vascular events via 
registries/electronic health records. If the patient withdraws from the study, the date and the 
reason for the patient’s withdrawal will be recorded. The patient is encouraged to provide 
information about his or her reason(s) for withdrawal and any experienced adverse effects (AE) 
during the study.  
 
The study will follow the course of the flow chart (please refer to appendix 2). A total of 45 
patients will be included in the study: 15 will receive stroke-RIC, 15 will receive sham-RIC and 15 
will constitute the control group. The initial RIC treatment regime will be applied as soon as 
possible and less than 48 hours after stroke onset. If patients are discharged from the Stroke Unit 
before day 7, they will continue the RIC treatment at home according to written instructions. On 
day 7 patients will return to the Stroke Unit for further tests (Table 2 – study procedures) and 
return the RIC device. The control group will consist of patients with putative stroke/TIA’s ending 
up with a non-vascular diagnosis upon examination in the outpatient clinic or at the stroke unit. In 
addition to the study procedures all patients will receive standard treatment. 
 
Controls Inclusion/Exclusion criteria 
Inclusion criteria 
Exclusion criteria 
Independent in daily living (mRS 0-2) 
Ambulatory 
Prior stroke, dementia or other known neurological 
condition 
 
Age 18-80 
Pregnancy* 
Legal competent 
Contraindications to MRI 
Non vascular diagnosis (e.g. epilepsy, migraine etc.) 
Investigators discretion 
 
Known upper extremity peripheral arterial stenosis        
 
Diabetes 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
9 
Method of blinding 
Patients and the assessors of endpoints are blinded. No information regarding randomization 
status will be recorded in the patient record.  
 
Discontinuation of study participation 
A patient can withdraw from the study at any time. Patients can be withdrawn from the study at 
the principal investigator’s discretion. If the patient withdraws from the study, the date and the 
reason for the patient’s withdrawal will be recorded. The patient is encouraged to provide 
information about his or her reason(s) for withdrawal and any experienced adverse effects (AE) 
during the study.   
 
Time schedule 
The study is scheduled to start 1st of February 2020 and end upon 31st of January 2021. The 
inclusion period is scheduled to 5 months. 
4. Study procedures and assessment 
Baseline data (demographics) and process indicators are collected from the Danish Stroke Registry 
(DSR). Additional information regarding medical history, treatment characteristics, use of 
medication, clinical and physiological data during the hospitalization will be obtained from the 
electronic health records. This includes information about clinical examinations, scan results and 
biochemical results. Data are recorded in an electronic case report form (CRF) at discharge from 
the stroke unit. For a complete list of registered data, see Appendix 1. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
10 
Table 2. Study procedures for patients with acute ischemic stroke.  
                                                      
Enrollment 
      Pre-
allocation 
Post-allocation 
Follow-
up 
Time points 
0h 
+2h* 
Day 0-7 
(8AM and PM) 
+7days 
Informed consent 
X 
 
 
 
 
 
Randomisation 
 
X 
 
 
 
 
Intervention 
 
Remote Ischemic Conditioning 
(RIC/Sham) 
 
 
X 
 
X                  X 
 
Blood samples 
 
Blood samples  
(2x3,0 mL EDTA + 2x3,5 mL citrate) 
 
X 
 
X 
 
X 
RBC deformability 
 
X 
 
X 
 
 
RBC eythrocrine dysfunction 
 
X 
 
X 
 
X 
Biobank samples (EVs and 
microRNA) 
 
X 
 
X 
 
X 
Clinical evaluation 
NIHSS 
PASE questionnaire 
 
X 
X 
 
 
 
X 
MoCA 
 
X 
 
 
 
X 
RIC questionnaire  
 
 
 
 
 
X 
End of study 
 
 
 
 
 
X 
RIC: Remote Ischemic Conditioning. NIHSS: National Institute of Health Stroke Scale. PASE: Physical Activity scale for 
the elderly. RBC: Red Blood Cells. EVs: Extracellular Vesicles. microRNA: Micro ribonucleic acid. For patients without a 
vascular diagnosis study participation will exclude RIC/Sham-RIC treatment and no RIC questionnaire. 
*2hours after completion of first RIC/Sham-RIC treatment 
 
Blood samples  
Study blood samples will be drawn at the ward in a total of 3 times (Table 2 - Study procedures). 
Each sample set will consist of 2x3,0 mL EDTA + 2x3,5 mL citrate. Ektacytometry and 
flowcytometry will be performed on fresh whole-blood samples, excess material (plasma) will be 
placed in a research biobank and used for nitrite and EV/microRNA analysis. Furthermore, we will 
use biochemical lab results taken as part of routine care at admission. 
 
Analysis of blood samples will be performed during the study and until 2 years after inclusion of 
the last patient (no later than 31st of January 2023). After the last analysis and no later than 31st of 
January 2023 the blood samples will be destroyed.  
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
11 
If routine blood samples are taken, we will endeavor to include the baseline study blood samples 
in these in order to minimalize discomfort for the patient. If, however, the routine blood samples 
cannot be drawn within less than 2 hours after inclusion, the study personnel will draw them. The 
blood samples will be safely stored at Aarhus University Hospital. No samples or biological 
material will be sent abroad. Research documents are stored for 15 years, after which they are 
destroyed  
 
Blood samples 
Accepted time frame 
Baseline samples 
< 2 hours from inclusion 
+2-hour samples  
1-3 hours from completed baseline RIC 
+7 days samples 
< 4 hours from completed 08 AM RIC 
 
Intervention (Remote Ischemic Conditioning) 
The investigational device used to induce RIC is an automatic Remote Ischemic Conditioning 
device (Stroke-RIC) – a blood pressure cuff for application on an upper arm. It is preprogrammed 
for 5 cycles of RIC. Each of these rounds consist of 5 minutes of inflation followed by 5 minutes of 
deflation. For the RIC device cuff pressure will be 200 mmHg unless the systolic blood pressure 
exceeds 175 mmHg in which case a pressure of 35mmHg will be added to the systolic pressure.  
 
Patients allocated to the sham-RIC group will go through the same program as the intervention 
group, except the sham-RIC device cuff pressure will be only 20 mmHg during inflation. 
 
The devices are designed and developed in corporation with Aarhus University, The faculty of 
Biomedicine technologies, 8200, Aarhus N, Denmark, Seagull Aps, 4160, Herlufmagle, Denmark 
and the department of Neurology, Aarhus University Hospital, 8000-DK, Aarhus C. 
 
RIC/sham-RIC protocol 
Accepted time frame 
Baseline RIC 
< 2 hours from inclusion 
RIC twice daily for 7 days – 08.00 PM and AM 
 
06.00 to 10.00 PM and 06.00 to 10.00 AM 
 
RBC – deformability - Ectacytometry 
The rheological properties of RBCs are quantified using a Deformability or Elongation Index (DI or 
EI). A higher EI at the optimum viscosity (300 Osmolality) indicates highly deformable RBCs 
indicative of better microcirculation, while a lower EI indicates rigid RBC’s. Briefly, 6 uL of 
heparinized or EDTA treated fresh blood is mixed with 600-uL of polyvinylpyrrolidone (PVP) 
solution (300 Osm) and transferred to a disposable kit. The kit is placed inside the laser-assisted 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
12 
ectacytometer for automated read out, data and image collection as per the vendors instructions. 
In a similar approach aggregation and shear stress will be measured using 0,5 mL of blood.  
With the current knowledge on RBC deformability it is impossible to predict a risk of developing 
specific diseases in the future.  
 
Erythrocrine dysfunction - Flow Cytometry 
Analytical flow cytometry for Erythrocyte Nitric Oxide Synthase 3 phosphorylation 
(pNOS3Ser1177) and s-nitrosylation (–SNO) in RBCs: Red blood cells are separated from 50-uL of 
freshly fixed blood samples, using a cocktail of monoclonal antibodies to RBC-specific markers 
(Glycophorin A (GPA)) and Hemoglobin. To assess the functional features, after fixation and 
permeabilization, RBCs are incubated with antibodies conjugated to fluorochrome either directly 
or through secondary antibodies to s-nitrosocysteine (–SNO), pNOS3Ser1177 and AMPKα1. A 
titration study is currently ongoing to determine the optimal antibody concentration.  Next, RBC 
samples are run through a flow cytometer (Cytoflex S, Beckman Coulter, Colorado, USA), and data 
is collected using CellQuest software to process for FC analysis. With the current knowledge on the 
Erythrocrine dysfunction it is impossible to predict a risk of developing specific diseases in the 
future.  
 
MicroRNA analysis 
MicroRNAs will be identified with Illumina next-generation sequencing using the TruSeq Small RNA 
Sample Preparation kit (Illumina) which allows for the addition of unique barcode sequences to 
each sample. Such barcoding allows pooling and simultaneous sequencing of multiple samples in a 
single-sequencing run on the Illumina NextSeq500 sequencer thereby significantly reducing the 
cost of sequencing. Pooling of multiple samples will generate adequate data amounts for detailed 
miRNA profiling, yet at limited costs. Data generated from the NextSeq500 sequencer will be 
filtered based on sequence quality as part of our established bioinformatics pipeline. This will also 
include matching the filtered data to annotated RNA databases such as miRNA sequences from 
miRBase (mirbase.org). The output from our bioinformatic pipeline will be quantitative miRNA 
expression levels for each sample, which will form the basis for a miRNA differential analysis 
where miRNAs with statistically significant expression changes will be found.  
 
The microRNA (and extracellular vesicles) analysis is not a mapping of the genome. The microRNA 
profile is a snapshot of microRNA activity at the specific time of the blood sampling. Levels of 
microRNA are changing constantly and with the current knowledge on the topic it is impossible to 
predict a risk of developing specific diseases in the future. Cryotubes not used to for 
microRNA/exosome analysis will be destroyed.  
 
Extracellular vesicle analysis 
Extracellular vesicles (EVs, also known as exosomes) will be isolated from plasma samples before 
characterization of surface markers and content. EVs are isolated by a number of different 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
13 
techniques, ultra-centrifugation, precipitation, size exclusion chromatography among others. 
While protein characterization will be done using classical molecular biological techniques such as 
ELISA and Western blots in addition to array techniques, EV-array, that utilizes a panel of 
antibodies directed against known EV surface markers. These analyses might be supported by 
proteomic analysis of all proteins as well as post-translational modifications such as 
phosphorylations and glycosylations. To broaden the feasibility of finding stroke type specific EV 
surface markers, we will utilize recombinant antibody library techniques to find novel disease 
binders with the potential of diagnosing stroke types in blood samples.  
 
In addition, the nucleic acid (DNA and RNA including miRNA) content of EVs will be analyzed using 
next generation sequencing (NGS), qRT-PCR, and other nucleic acid detection techniques. Both the 
protein data and the nucleic acid data will be subjected to bioinformatic analysis using different 
pipelines and analysis tools depending on the dataset and the purpose of the analysis. A number 
of validated published databases will be used for annotation and comparison. EVs do not contain 
genomic DNA and the analysis are therefore not a mapping of the subject’s genome. EV secretion 
and nucleic acid content in these are thought to change rapidly in the body due to external and 
internal signals and are considered a snapshot at the specific time of blood sampling. With the 
current knowledge on the topic it is impossible to predict a risk of developing specific diseases in 
the future.  
 
Use of routine collected blood samples 
The following biochemical entities will be used (lab results performed as a part of standard 
operating procedure): C-reactive protein, Glucose, HBA1c, Potassium, Natrium, Calcium, Albumin, 
Creatinine, eGFR (estimated Glomerular Filtration Rate), Total WBC (White Blood Cell 
count/Leukocytes), Hemoglobin, Erythrocytes, Erythrocytes (Volume Fraction, EVF), Erythrocytes 
distribution widths (RDW), Erythrocyte median cell volume (MCV), Hemoglobin concentration 
(MCHC and MCH), Reticulocyte count, Platelet count, Iron, Transferring, Ferritin, International 
Normalized Ratio (INR), Activated Partial Tromboplastin Time (APTT), Cholesterol (Total, HDL, LDL, 
Triglyceride). 
 
Montreal Cognitive Assessment (MoCA)  
The Montreal Cognitive Assessment (MoCA) is a brief screening tool originally designed to identify 
mild cognitive impairment. MoCA is a 1-page (healthcare administered), 30-point test, 
administrable in ≈10 minutes, which evaluates different domains: visuospatial abilities, executive 
functions, short-term memory recall, attention, concentration, working memory, language, and 
orientation to time and space (appendix 4). 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
14 
PASE - physical activity in the elderly 
Participants will be asked to complete the physical activity in the elderly (PASE) questionnaire 
during the acute hospital admission. PASE is a 12-item questionnaire, which quantifies the amount 
of PA over a 7-day period. The PASE questionnaire was developed with the purpose of assessing 
the level of PA in middle-aged and elderly individuals. The PASE score is calculated by taking the 
average number of hours spent on an activity (sports, occupational activity, household activities, 
and leisure time activities) per day over a 7-day period multiplied by an activity coefficient. Item 
scores are added to reveal the PASE score. The score may range from zero to more than 400.  
 
Remote Ischemic Conditioning questionnaire 
In the RIC questionnaire the study participants are asked to reveal the level of discomfort (if any) 
experienced during the investigational treatment and whether they knew which treatment they 
received (Sham/Sham-RIC) despite the blinded study design. Control subjects will not be 
interviewed with the RIC questionnaire. The questionnaire is administered by study personnel. 
(Appendix 4) 
5. Statistical analysis 
Sample size estimation is based on yet unpublished data on elongation index in experimental 
stroke models. The maximum difference in IE between stroke and non-stroke (experimental 
models) was observed between 16 and 48 hours from stroke onset (IE of 0,43 and 0,58 at 
maximum deformability, 300Osm). Thus, a sample size of 12 stroke patients and 12 patients 
without stroke will be required to detect a deference of 0,15 on the elongation index with a power 
90% (significance level 5%). To account for dropouts (estimated to maximum 3 per arm) and the 
additional study arm of RIC treated stroke patients a total of 45 patients will be included - 15 
stroke patients (RIC), 15 stroke patients treated with sham-RIC and 15 patients (non-stroke 
controls). The maximum number of included patients will be 45.  
 
Primary endpoint analysis 
Differences in RBC deformability, RBC NOS3, plasma nitrite and other biomarkers over time 
(baseline, +2 hours and +7 days post RIC) between RIC groups (yes/no) will be examined using 
mixed models.  Fixed effects in the model are RIC group, time, and the two-factor interaction 
between RIC and time.  Subjects nested within RIC groups will be considered a random effect. 
6. Ethical considerations 
The RIC intervention is associated with mild to moderate discomfort and minimal risk for adverse 
events (10-12). The additional blood samples will however result in slight discomfort associated 
with venipuncture. The number of additional venipunctures will range from one to three, 
depending the number of routine planned blood samples. Every effort will be made to have study 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
15 
blood samples taken when routine samples are taken.  Use of sham is necessary for 
methodological reasons and is not associated with any risk/discomfort. 
 
Advantages and disadvantages of participating in the study 
 
a. Potential advantages: Participants diagnosed with acute ischemic stroke will receive extra 
follow-up including an extra neurological examination. 
 
b. Potential disadvantages: mild/moderate discomfort and minor bleeding in the skin can 
occur on the RIC treated arm. The RIC treatment has otherwise proven safe and without 
serious adverse effects. Sham-conditioning will not cause discomfort or side effects. Extra 
blood withdrawals will cause discomfort for the patient. 
 
Results from this pilot study will help establish whether a rheo-erythrocrine dysfunction in the 
RBC’s is a biomarker for stroke and RIC.  The establishment of a biomarker will help predict 
responders to RIC and serve as a preliminary investigation of a novel stroke biomarker and to 
better understand causes of microcirculatory dysfunction in stroke. 
 
The pilot study will need approval from The Central Denmark Region Committees on 
Health Research Ethics and the Danish Medicines Agency before the research project begins. 
7. Assessment of safety  
Emergency unblinding procedure 
All on call neurologists will have 24/7 telephone access to study investigators who can perform 
emergency unblinding if necessary. All changes will leave an audit-trail.  
 
Adverse events 
Patients are monitored in the stroke unit with NIHSS scoring and Scandinavian Stroke Scale (SSS) 
at close intervals, and adverse events are treated according to clinical guidelines.  
 
During the acute in-hospital phase the patients will be asked if they have experienced any 
deterioration of health or new symptoms during or after treatment with the investigational 
device. Furthermore, all patients will have a 24/7 contact number to the stroke center and a 
contact to a study research physician and will be instructed to report any deterioration of health 
or new symptoms. All discharged patients (and relatives) will be given contact details to study 
personnel and be instructed to report any deterioration of health or new symptoms. The patients 
with acute ischemic stroke will once again be asked for adverse events at the interview on day 7. 
End of study is the interview on day 7. 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
16 
Adverse events and adverse device events are defined according to ISO 14155:2011 and 
European Commission guideline on medical devices (MEDDEV 2.7/3 revision 3). 
 
Adverse event (AE) 
Any untoward medical occurrence, unintended disease or injury or any untoward clinical signs 
(including an abnormal laboratory finding) in subjects, users or other persons whether or not 
related to the investigational medical device. 
 
Serious Adverse Event (SAE)  
Adverse event that: 
a) led to a death, injury or permanent impairment to a body structure or a body function.  
b) led to a serious deterioration in health of the subject, that either resulted in: - a life-threatening 
illness or injury, or - a permanent impairment of a body structure or a body function, or - in-
patient hospitalization or prolongation of existing hospitalization, or - in medical or surgical 
intervention to prevent life threatening illness. 
 
Device Deficiency 
Inadequacy of an investigational medical device related to its identity, quality, durability, 
reliability, safety or performance. This could be malfunctions, use error, or inadequacy in the 
information supplied by the manufacturer. 
 
Adverse Device Effect 
Adverse event related to the use of an investigational medical device. This includes any adverse 
event resulting from insufficiencies or inadequacies in the instructions for use, the deployment, 
the implantation, the installation, the operation, or any malfunction of the investigational medical 
device. This includes any event that is a result of a use error or intentional abnormal use of the 
investigational medical device. 
 
Serious Adverse Device Effect (SADE) 
Adverse device effect that has resulted in any of the consequences characteristic of a serious 
adverse event. 
 
Unanticipated Serious Adverse Device Effect (USADE)  

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
17 
Serious adverse device effect which by its nature, incidence, severity or outcome has not been 
identified in the current version of the risk analysis report.  
 
Anticipated SADE (ASADE) 
An effect which by its nature, incidence, severity or outcome has been previously identified in the 
risk analysis report 
 
Event report and causality assessment 
The relationship between the use of the medical device and the occurrence of each adverse event 
will be assessed and categorized. All events will be registered in the electronic CRF and reported to 
the authorities at the interim analysis. A yearly safety report, containing a list of all SAEs/SADEs 
and near-miss incidents, thorough evaluation of individual events, risk/benefit analysis of the 
investigational medical device and safety conclusions, will be submitted to the Danish Health and 
Medicines Authority and the Independent Ethics Committee by the Sponsor as required by Danish 
law.  
 
Exceptions to and special considerations about reporting procedures are stated below: 
Acute stroke is an acute life-threatening disease with a high risk of neurological deterioration and 
mortality. The natural history of stroke is associated with a high risk of complications (stroke in 
progression, hemorrhagic transformation, dysphagia, cardiac arrhythmia, pneumonia, and other 
serious infections). These complications all relate to the index stroke (occurring before 
randomization) and is foreseeable. If an event occurs that based on the assessment by the 
investigator is a result of the natural history of the disease it will not be reported by the sponsor to 
the Danish medicines agency and regional ethics committee within 7 days/2 days (see list of 
foreseeable events/complications below). 
 
All expected serious adverse events will be reported to the Danish Medicines Agency (and regional 
ethics committee) every 3 months. 
All new serious events (Serious Adverse Events), complications that are not foreseeable or device 
near incidents or malpractice will be reported by the sponsor to the authorities (within 7 days, or 2 
days if there is a risk of event reoccurrence) according to ISO 14155:2011 and European 
Commission guideline on medical devices (MEDDEV 2.7/3 revision 3). Investigators will report the 
event to sponsor (within 24 hours). Sponsor will as fast as possible and no later than 7 days, or 2 
days if there is a risk of event reoccurrence, report the event to the Danish Medicines Agency and 
regional ethics committee.  
 
All Serious Adverse Device Effect (SADE) will be reported to the Danish Medicines Agency (and 
regional ethics committee) within 7 days, or 2 days if there is a risk of event reoccurrence 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
18 
 
List of foreseeable adverse events/complications 
Foreseeable events/complications that will not be reported by the sponsor to the Danish 
Medicines Agency and Regional Ethics Committee within 7 days/2days. 
Patient 
Population 
Event 
Frequency  
Mitigation/treatment 
TIA and AIS 
Recurrent stroke. 
10% at 90 
days a 
Standard medical treatment, 
intravenous 
thrombolysis/mechanical 
thrombectomy according to 
patient characteristics 
AIS 
Symptomatic intracerebral 
hemorrhage (related to IV 
tPA or EVT) 
7% b 
Antithrombotic/anticoagulation 
therapy cessation. 
Antifibrinolytic agent. 
Bloodpressure control.  
AIS, ICH 
Early (<24hours) and late 
(>24hours) neurological 
deterioration 
(∆NIHSS ≥2 or death) 
7-22% c 
 
According to etiology  
AIS, ICH 
Aspiration pneumonia 
5-20% d 
Antibiotic treatment according 
to hospital SOP 
AIS, ICH 
Urinary tract infections 
12% e 
Antibiotic treatment according 
to local hospital SOP 
AIS, ICH 
Severe infections (sepsis) 
13% f 
Antibiotic treatment according 
to local hospital SOP 
AIS, ICH 
Cardiac arrhythmia 
29% g 
Depending on arrhythmia type. 
Treatment according to 
cardio.dk 
AIS, ICH 
Acute myocardial infarction 
6% h 
Treatment according to 
cardio.dk and conference call 
with cardiologist 
AIS, ICH 
Deep venous 
Thrombosis(DVT)/pulmonary 
embolism(PE) 
DVT: 8% i 
PE:1% j 
Treatment according to 
cardio.dk 
AIS, ICH 
Falls (in-hospital phase) 
14-65% k, l 
Treatment according to type of 
injury 
AIS, ICH 
Seizure 
AIS:3-10% m 
ICH:8% n 
Treatment according to local 
SOP 
AIS/ICH 
Mortality 30 days 
AIS:10-20% m 
ICH:30% n 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
19 
a)  Wang Y et al. Clopidogrel with Aspirin in Acute Minor Stroke or Transient Ischemic Attack. NEJM. 2013; 369:11-19 
b) Berkhemer OA. A Randomized Trial of Intraarterial Treatment for Acute Ischemic Stroke. NEJM. 2015; 372:11-20 
c) Kwan J et al. Early neurological deterioration in acute stroke: clinical characteristics and impact on outcome Journal of the Association of 
Physicians, 2006, Sept; 99:9: 625-633 
d) Hannawi Y et al. Stroke-Associated Pneumonia: Major Advances and Obstacles. Cerebrovasc Dis. 2013;35:430-443 
e) Bogason E. Urinary Tract Infections in Hospitalized Ischemic Stroke Patients: Source and Impact on Outcome. 2017;9(2):e1014. 
doi:10.7759/cureus.1014. 
f) BergerB. Epidemiologic features, risk factors, and outcome of sepsis in stroke patients treated on a neurologic intensive care unit. Journal of 
Critical care. 2013;29(2)  
g)Daniele O et al. Stroke and cardiac arrhythmias. J Stroke Cerebrovasc Dis. 2002 jan-feb;11(1):28-33 
h) Johnston KC et al. Medical and neurological complications of ischemic stroke: experience from the RANTTAS trial. RANTTAS Investigators. Stroke. 
1998;29(2):447 
i) Bembenek J. Early stroke-related deep venous thrombosis: risk factors and influence on outcome. J thromb Thrombolysis. 2011 Jul; 32(1):96-102 
j) Pongmoragot J. et al. Pulmonary Embolism in Ischemic Stroke: Clinical Presentation, Risk Factors, and Outcome. J Am Heart Ass. 2013 Dec;2(6) 
k) Davenport RJ, Dennis MS, Wellwood I, Warlow CP. Complications after acute stroke. Stroke. 1996; 27: 415–420. 
l Teasell R, McRae M, Foley N, Bhardwaj A. The incidence and consequences of falls in stroke patients during inpatient rehabilitation: Factors 
associated with high risk. Arch Phys Med Rehabil. 2002; 83: 329–333. 
m) Bladin C, Alexandrov AV, Bellavance A et al:  Seizures After Stroke: A Prospective Multicenter Study.Arch Neurol. 2000;57(11):1617-1622.  
n) Woo K-M, Yan S-Y, Cho K-T. Seizures after Spontaneous Intracerebral Hemorrhage. J. Korean Neurosurg Soc. 2012 Oct;52(4)312-319 
O)Schwartz J et al. Incorporating Stroke Severity Into Hospital Measures of 30-Day Mortality After Ischemic Stroke Hospitalization. Stroke. 2017. 
Ahead of print: https://doi.org/10.1161/STROKEAHA.117.017960 
p) Lichtman JH et al. 30-Day Mortality and Readmission after Hemorrhagic Stroke among Medicare 
 
 
List of anticipated adverse device effects 
RIC device 
Frequency 
Mitigation/treatment 
Local petechiae 
4-5% a 
none 
Discomfort/pain 
20-30%  
Treatment stopped if the non-competent patient 
verbally or non-verbally demonstrates intolerable 
discomfort or if the competent patient verbally 
wants to discontinue the treatment 
Near events (device related) 
None known in 
the literature 
 
Acute limb ischemia (upper 
extremity) – theoretical 
complication. 
None known in 
the literature 
Treatment stopped. Treatment according local 
hospital SOP’s for acute limb ischemia.  
Known atherosclerotic stenosis in upper 
extremities are an exclusion criterion. 
See Investigators Brochure for further details. 
Sham RIC device 
 
 
None known 
 
 
 
a) Hausenloy D et al. Remote Ischemic Preconditioning and Outcomes of Cardiac Surgery. New England J. Medicine. 2015 Oct;373(15):1408-17 
 
Treatment and mitigation of any adverse events will be according to regional guidelines. Adverse 
events as a result of standard treatment will be handled according to local guidelines. Adverse 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
20 
events may also occur as a suspected reaction to RIC/Sham RIC – in these cases, the conditioning 
stimulus will be stopped immediately, and appropriate treatment will be initiated. In cases of SAE 
and device related near events all patients will be followed until resolution of symptoms/signs 
and/or all clinically relevant treatment have been performed and/or until the symptoms/signs are 
in a stable phase.  
8. Data handling and record keeping 
All study data are recorded in an electronic CRF with blinded data and identification via a study 
identification number. The study will apply to the specifications of act on processing of personal 
data. The database(e-CRF) is handled by REDCAP at Aarhus University, Aarhus, Denmark. Patient 
participation will be recorded in the medical record. Data will be stored at the Department of 
Neurology, Aarhus University Hospital, for 15 years, after which the documents will be shredded. 
Blood-samples in the research biobank will be stored until completion of the trial (no later than 
31. january 2023).  
 
Methodological experts from collaborators at Medical College Georgia, Augusta, USA (Dr. Hess’ 
and Dr. Baban’s lab) will remotely supervise the technical aspects of flowcytometry and 
ektacytometry and assist in the interpretation of results (only anonymized flowcytometry and 
ektacytometry data). Flowcytometry and ektacytometry results reflects only snapshots of the 
function and deformability of the red blood cells and change rapidly.  
 
During the entire study period the local GCP unit will perform quality assurance control including 
source data verification. A complete list of all source data will be made and approved by the local 
GCP unit before study initiation. The investigator permits direct access to all source 
data/documents (including electronic patient record) at monitoring visits, audits and/or 
inspections by the regional ethics committee and Danish Medicines Agency. 
9. Further documentation 
 
• The study will respect and protect the patients’ physical and mental integrity as well as 
their privacy. The data protection regulations as well as the law on data protection will 
abide. 
 
• When the patient signs the consent form, the patient grants the study personnel access to 
information within the medical record necessary for the completion of the study. It also 
allows supervisory authorities in the Danish Medicines Agency access to the medical record 
in order to inspect and monitor the study. Information regarding previous admissions as 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
21 
well as diagnoses are retrieved from national registers including the National Patient 
Registry, the Danish Stroke Registry and the Central Person Register. 
• The study will be notified to the Region’s Internal List of research projects 
 
• The study is monitored by the GCP-unit Aalborg/Aarhus. 
 
• The study is covered by the Danish Patient Compensation Association. 
 
10. Economics 
 
• Initiator: Grethe Andersen, Professor, Senior Consultant, MD, DMSc, Department of 
Neurology & Danish Stroke Center, Aarhus University Hospital. 
 
• The ENOS trial has received funding from the Lundbeck Foundation on 140.000 dkr. (study 
investigator salary and current expenses) 
 
• Equipment and supplies are supplied by the “mother trial”, RESIST, which received funding 
from the following: 
 
o The Danish foundation Vilhelm Petersens mindelegat has supported the RESIST study 
with 1.240.000 dkr (research nurse salary and “stroke RIC” devices).  
o The Danish foundation TrygFonden has supported the RESIST study with 4.000.000 dkr 
(research personnel salaries, current expenses and investigational devices).  
o Aarhus University has supported the RESIST study with 550.000 dkr (RESIST study 
investigator salary) 
o Novo Nordisk has supported the RESIST study with 2.600.000 dkr (Rheo-erythrocrine 
dysfunction in stroke) 
 
• Financial supporters have no influence on study design, data collection, 
analysis/interpretation of results or publication of results. 
 
• The devices (Stroke-RIC and Sham-RIC) are produced by Seagull Aps. The company has no 
influence on study design, data collection, analysis/interpretation of results or publication 
of results. 
 
• Financial contributions will be deposited into a fund account administered by Aarhus 
University Hospital’s financial department. None of the physicians nor nurses involved in 
the study will benefit economically from their participation. Participation in the study is not 
linked to any remuneration.  

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
22 
11. Publication 
Results, both positive, negative and inconclusive will be published in scientific papers and during 
scientific meetings/congresses.  
12. Source data access and monitoring 
Trial-related audits and/or monitoring will be provided by direct access to source data/documents. Local 
monitors from the Unit for Good Clinical Practice, Aarhus University, will perform the audit and monitoring. 
Before enrollment can start a trial monitoring plan must be available.  The audit and monitoring process 
will involve a 100% monitoring of signed consent forms (“samtykkeerklæringer og fuldmagtserklæringer”) 
and serious adverse events.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
23 
13. Appendix  
 
Appendix 1: Baseline data and treatment characteristics 
 
Age 
Platelet inhibitor treatment 
Male/female 
Anticoagulation therapy 
Medical History 
New oral anticoagulation treatment (NOAC) 
Hypertension 
Opioid treatment 
Smoking 
SSRI treatment 
Alcohol 
Clinical and physiological data 
Hyperlipidemia 
Modified Rankin Scale prestroke 
Previous myocardial infarction 
Yes: within 3 months, yes: more than 3 months, no, unknown 
NIHSS (Baseline) 
Angina pectoris 
Symptom onset (time) 
             Recent angina pectoris (< 4 weeks)            
 If yes, date for latest episode 
Time of admission 
Atrial fibrillation 
RIC compliance data 
Previous ischemic stroke 
• 
Time (start/stop) for each RIC/sham cycle 
             Stroke < 3 month 
If yes, date 
• 
Bloodpressure and pulse at every RIC/sham RIC 
treatment 
Previous TIA 
NIHSS (7 days)  
             Recent TIA < 4 weeks 
If yes, date for latest episode 
Stroke etiology (TOAST) 
Previous ICH 
Montreal Cognitive assessment at baseline and +7 days 
Peripheral artery disease 
RIC questionnaire at +7 days 
Physical activity (PASE interview) 
IV tPA/EVT treated AIS  
Medication on admission 
Treatment initiation (time) and treatment characteristics 
Statins 
Quality indicators (Danish Stroke Registry) 
Calcium channel blockers 
 
ACE inhibitors/angiotensin receptor blockers 
 
Beta-blockers 
 
 
 
Baseline data obtained from treating physician, database of Danish stroke center (ddsc.dk), Danish stroke registry and 
electronic health records.  
 
 
 
 
 
 
 
 
 
 
 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
24 
Appendix 2: Study flowchart 
 
 
 
 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
25 
Appendix 3: Montreal Cognitive Assessment 
 
 
 
 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
26 
Appendix 4: RIC questionnaire (Danish) 
 
Example, minor changes may occur. Only patients with ischemic stroke. 
 
 
REMOTE ISKÆMISK KONDITIONINGS SPØRGESKEMA 
Patientens opfattelse af behandlingen 
 
1. Hvor meget smerte/ubehag oplevede du under behandlingen (afklemning af 
blodforsyningen til armen)? 
       (Sæt kryds på nedenstående skala) 
 
 
 
 
 
2. Har du under eller efter behandlingen (afklemning af blodforsyningen til armen) oplevet 
nogle bivirkninger til behandlingen? 
 
JA, beskriv hvilke?:__________________________________________________________                        
            NEJ 
 
 
3. Såfremt afklemning af blodforsyningen armen kan beskytte mod udvikling af visse hjerte-kar 
sygdomme, ville du så være villig til at fortsætte med behandlingen efter at forsøget er 
færdigt? 
 
JA 
 
NEJ 
 
 
4. Hvis du skulle fortsætte behandlingen i 1 år, hvor tit ville du være villig til at tage 
behandlingen? 
 
2 gange dagligt 
 
1 gang dagligt 
 
Hver anden dag 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
27 
2 gange om ugen 
 
1 gang om ugen 
 
Mindre end 1 gang pr uge. 
 
 
 
5. Hvor lang tid synes du hver behandling maksimalt måtte vare? (hver behandling varer nu 50 
minutter) 
 
1 time 
 
50 minutter 
 
40 minutter  
 
30 minutter  
 
20 minutter eller mindre  
 
 
 
6. Man kan i forsøget du har været med i enten blive udtrukket til at blive behandlet med  
én virksom eller én ikke-virksom form af behandlingen. Hvilken for form af behandlingen 
blev du behandlet med?  
 
Den virksomme behandling 
 
Den ikke-virksomme behandling 
 
 
Tak for din hjælp. Spørgeskemaet afleveres til forsøgspersonalet 
 
 
 
 
 
 
 
 
 
 
 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
28 
14. References 
 
 
1. 
Committee TEsOEcatEw. Guidelines for Management of Ischaemic Stroke and 
Transcient Ischaemic Attack 20082008 [cited 2017 3/8]. Available from: 
https://www.karger.com/Article/Pdf/131083. 
2. 
Hjernesagen. Facts om hjerneskader  [updated 28/4 2017; cited 2017 2/9 2017]. 
Available from: https://www.hjernesagen.dk/om-hjerneskader/facts-om-hjerneskader. 
3. 
Paulson OB, Gjerris, F., Sørensen, P. Klinisk Neurologi og neurokirurgi. 6 ed: 
FADL’s forlag; 2015. 394-432 p. 
4. 
Pan JJ. Remote ischemic conditioning for acute ischemic stroke: dawn in the 
darkness. Reviews in the neurosciences. 2016;27(5):501-10. 
5. 
Apopleksi DSf. Nationale retningslinjer for intravenøs trombolysebehandling ved akut 
iskæmisk apopleksiVersion 2. 2016. Available from: http://www.dsfa.dk/wp-
content/uploads/Nationale_retningslinier_trombolyse_2016_110216.pdf. 
6. 
Albers GW. Thrombectomy for Stroke at 6 to 16 Hours with Selection by Perfusion 
Imaging. The New England journal of medicine. 2018;378(8):708-18. 
7. 
Nogueira RG. Thrombectomy 6 to 24 Hours after Stroke with a Mismatch between 
Deficit and Infarct. The New England journal of medicine. 2018;378(1):11-21. 
8. 
Hess DCDC. Remote ischaemic conditioning-a new paradigm of self-protection in the 
brain. Nature reviews Neurology. 2015;11(12):698-710. 
9. 
Hess DCDC. Remote limb perconditioning [corrected] and postconditioning: will it 
translate into a promising treatment for acute stroke? Stroke (1970). 2013;44(4):1191-7. 
10. 
England TJTJ. RECAST (Remote Ischemic Conditioning After Stroke Trial): A Pilot 
Randomized Placebo Controlled Phase II Trial in Acute Ischemic Stroke. Stroke (1970). 
2017;48(5):1412-5. 
11. 
Hougaard KDKD. Remote ischemic perconditioning as an adjunct therapy to 
thrombolysis in patients with acute ischemic stroke: a randomized trial. Stroke (1970). 
2014;45(1):159-67. 
12. 
Meng RR. Upper limb ischemic preconditioning prevents recurrent stroke in 
intracranial arterial stenosis. Neurology. 2012;79(18):1853-61. 
13. 
Stamler JS. Blood flow regulation by S-nitrosohemoglobin in the physiological 
oxygen gradient. Science (American Association for the Advancement of Science). 
1997;276(5321):2034-7. 
14. 
Corti P. Is nitrite the circulating endocrine effector of remote ischemic 
preconditioning? Circulation research. 2014;114(10):1554-7. 
15. 
Stamler JS. Endocrine nitric oxide bioactivity and hypoxic vasodilation by inhaled 
nitric oxide. Circulation research. 2012;110(5):652-4. 
16. 
Grau M. RBC-NOS-dependent S-nitrosylation of cytoskeletal proteins improves RBC 
deformability. PloS one. 2013;8(2):e56759. 
17. 
Suhr F. Moderate exercise promotes human RBC-NOS activity, NO production and 
deformability through Akt kinase pathway. PloS one. 2012;7(9):e45982. 
18. 
Meram E. Shear stress-induced improvement of red blood cell deformability. 
Biorheology. 2013;50(3-4):165-76. 
19. 
Cortese-Krott MM. Endothelial nitric oxide synthase in red blood cells: key to a new 
erythrocrine function? Redox biology. 2014;2:251-8. 
20. 
Ozüyaman B. RBC NOS: regulatory mechanisms and therapeutic aspects. Trends in 
molecular medicine. 2008;14(7):314-22. 

ENOS stroke trial 
 
version 1.2 (October 9th, 2019) 
29 
21. 
Kleinbongard P. Red blood cells express a functional endothelial nitric oxide 
synthase. Blood. 2006;107(7):2943-51. 
22. 
Nagarajan S. Mechanical perturbations trigger endothelial nitric oxide synthase 
activity in human red blood cells. Scientific reports. 2016;6(1):26935. 
23. 
Hess D. Rheo-Erythrocrine Dysfunction in Stroke and Remote Ischemic Condiitoning 
(REDS). 2018.