A multicentre, randomised, sham-controlled trial on REmote iSchemic conditioning In patients with acute STroke (RESIST) – Rationale and study design

Short abstract

Rationale

Remote ischaemic conditioning, applied in the prehospital setting and continued in-hospital, may improve functional outcome in patients with acute ischaemic stroke and intracerebral haemorrhage.

Aims

To evaluate whether combined remote ischaemic per- and postconditioning can improve long-term functional outcome in acute ischaemic stroke and intracerebral haemorrhage patients.

Methods and design

Danish multicentre, prospective, randomised, patient-assessor blinded, sham-controlled study. Adult patients with a putative stroke identified prehospital with symptom duration <4 h, who are independent in daily activities will be randomised 1:1 to remote ischaemic conditioning or Sham-remote ischaemic conditioning. The treatment protocol will be five cycles, each consisting of 5 min with a blood pressure cuff inflation and 5 min with a deflated cuff placed on the upper extremity. The cuff pressure for remote ischaemic conditioning will be 200 mmHg–285 mmHg according to the individual systolic blood pressure and 20 mmHg sham-remote ischaemic conditioning during inflation. The study is approved as an acute study and consent is waived in the acute phase.

Sample size estimation: For a 7% increased odds for a beneficial shift on the modified Rankin Scale at a significance level of 5% and power of 90%, 1000 patients with a target diagnosis of acute ischaemic stroke and intracerebral haemorrhage and a total of 1500 patients with a prehospital presumed stroke will be included.

Study outcomes: The primary outcome will be the modified Rankin Scale score measured at three-month follow-up (analysed using ordinal logistic regression). ClinicalTrials.gov Identifier: NCT03481777.

Rationale

Intravenous alteplase (IV tPA) and endovascular treatment (EVT) are approved acute reperfusion treatments for acute ischaemic stroke (AIS) to be started as soon as possible.^1–3 Infarct progression can continue for days following a stroke in some patients, and failure of the collateral flow is a critical factor determining infarct growth.⁴ The pre-eminent therapeutic aim is therefore to restore blood flow, improve collateral perfusion and prevent ischaemic brain injury.⁵

On the other hand, in intracerebral haemorrhage (ICH), the pathophysiological mechanism is an eruption of blood into the brain parenchyma, causing tissue destruction with mass effect on adjacent brain tissues. Haematoma expansion as well as inflammatory pathways that are activated lead to further tissue damage, oedema and penumbral hypoperfusion.⁶

Novel therapeutics and neuroprotective strategies that can be started ultra-early after symptom onset are urgently needed to reduce disability in both AIS and ICH.^7,8

Ischaemic conditioning is one of the most potent activators of endogenous protective mechanisms against ischaemic injury.^9–11 Remote ischaemic conditioning (RIC) can be applied as repeated short-lasting ischaemia in tissue remote to the brain that results in protection against subsequent long-lasting ischaemic brain injury.¹² It can be applied prior to or during a prolonged ischaemic event as remote ischaemic preconditioning and perconditioning, respectively, or immediately after reperfusion as remote ischaemic postconditioning.¹³ RIC is commonly achieved by inflation of a blood pressure cuff to induce 5-min cycles of limb ischaemia alternating with 5 min of reperfusion. RIC activates several protective mechanisms, through humoral and neuronal pathways.¹⁴

Results from a recent proof-of-concept study at our institution indicated that RIC applied during ambulance transportation (perconditioning) as an adjunctive therapy to in-hospital IV tPA increases brain tissue survival after one month, although no effect on acute infarct growth or clinical outcome was found.¹⁵ No detrimental effect on haematoma expansion was found in patients with ICH, and emerging preclinical data indicate a significant increased haematoma reabsorption rate and an improved outcome after RIC-treated ICH.¹⁶

To-date, no serious adverse events have been documented in RIC. RIC is a feasible, non-pharmacologic and safe non-invasive treatment without significant discomfort that can be initiated prehospital worldwide.^8,15,17–20

The aim of the current study is to evaluate whether combined remote ischaemic per- and postconditioning can improve long-term functional outcome in AIS and ICH patients.

Methods and design

Design

REmote iSchemic conditioning In patients with acute STroke (RESIST) is a multicentre, prospective, randomised, patient-assessor blinded, sham-controlled study.

The study is approved as an acute study (consent is waived in the acute phase) by the Danish regional research ethics committees (ID:1–10-72–97-17), Danish Medicines Agency (ID:2017114177, EUDAMED: CIV-17–11-022324) and the Danish Data Protection Agency (ID: 1–16-02–16-18).

The trial will be conducted in accordance with good clinical practice guidelines (ICH-GCP), declaration of Helsinki (as revised in 2013) and is monitored by regional Good Clinical Practice units. The trial protocol is in accordance with SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) guidelines.²¹ The ClinicalTrials.gov Identifier is: NCT03481777.

Patient population

Patients with a prehospital putative stroke from participating stroke centres in Denmark will be recruited.

All Danish stroke centres and their prehospital regions will be invited to participate (currently three initiated stroke centres and one planned). Inclusion and exclusion criteria are listed in Figure 1 and baseline assessments and study procedures in Table 1.

Figure 1.

Inclusion and exclusion criteria and target population definition.

RIC: remote ischaemic conditioning.

Table 1.

Study procedures for patients with a target diagnosis of AIS and ICH.

	Pre-allocation	Post-allocation				Follow-up
Time points	Prehospital	Admission	+6 h	+24 h	+7 days	3 Months	12 Months
Eligibility screen	X
Acute waiver of consent	X
Informed consent		X------- X
In-hospital inclusion criteria		X a
Randomisation	X
Intervention
Remote ischaemic perconditioning (RIC/Sham)	X
Remote ischaemic postconditioning (RIC/Sham)			X	X-----X b
Assessment
Prehospital stroke score	X			X
NIHSS		X		X
Modified Rankin Scale						X
Physical activity (PASE)				X
Mood and quality of life				X		X
MACCE						X	X
Biomarkers
Brain MRI/CTc		X		X	X
Blood biomarkersd	X	X		X

Note: Patients are randomised in the acute prehospital phase to RIC/Sham-RIC.

RESIST: Remote ischemic conditioning in patients with acute stroke; RIC: remote ischaemic conditioning; NIHSS: National Institute of Health Stroke Scale; PASE: Physical Activity Scale for the Elderly; MACCE: Major Adverse Cardiac and Cerebrovascular Events; MRI: magnetic resonance imaging; CT: computer tomography.

^aPatients without evident vascular diagnosis (non-target diagnosis) will be followed until discharge and through registries.

^bPatients (target diagnosis) admitted at the Aarhus University Hospital will be treated with RIC: Acute, +6 h and twice daily for seven days; Patients from other centres only acute and after 6 h.

^cAIS patients treated with IV tPA and or/EVT will have 24-h MRI and ICH patients 24 h and 7 days CT (only at the Aarhus University Hospital).

^dBlood biomarkers (Table 2).

Randomisation

A prehospital stroke screening and severity assessment (PreSS) will be performed in the ambulance by paramedics or emergency medical technicians. The PreSS consists of the Cincinnati Prehospital Stroke Scale with an additional opportunity to report other neurological symptoms (e.g. ataxia, sensory disturbances and visual field loss) and PASS (Prehospital Acute Stroke Severity Scale).^22,23 The patient will be randomised to standard treatment with RIC or sham-RIC by the on-call neurologist at the receiving stroke centre using a randomised block design with random sized blocks. Lists of randomisation keys are computer generated and provided by a secure website. Patients are stratified by the receiving stroke centre, age (strata of age: ≥18 to 65, 66 to 80 and >80 years) and prehospital stroke severity (strata of PreSS: 1–2, 3–4 and 5–6 present symptoms/deficits).

Intervention

RIC and sham-RIC devices were developed in collaboration with Aarhus University, Faculty of Biomedical Engineering (Aarhus, Denmark) and produced by Seagull Healthcare (Slagelse, Denmark). Both RIC and sham devices are programmed for five cycles (50 min), each consisting of five minutes of cuff inflation followed by 5 min with a deflated cuff. For the RIC-device, default cuff pressure will be 200 mmHg; however, if the initial systolic blood pressure is above 175 mmHg, the cuff is automatically inflated to 35 mmHg above the systolic blood pressure to ensure complete arterial occlusion (maximum cuff pressure 285 mmHg). Cuff-pressure variability below 10 mmHg is accepted. The sham-RIC device will only inflate to a pressure of 20 mmHg. Every ambulance and helicopter in the participating Danish regions will be equipped with two investigational devices (RIC and Sham-RIC). The initial RIC/sham-RIC treatment protocol will be applied in the acute prehospital phase.

In cases of transport time under 50 min, the RIC/sham-RIC protocol continues during the initial assessment at the stroke centre until the 50-min treatment time has been achieved. The cuff will whenever possible by default be placed on the non-paretic upper extremity and if monosymptomatic aphasia on the left upper extremity.

At 6 h after completion of the first RIC/sham-RIC protocol, an additional series of RIC/sham-RIC will be performed (postconditioning) for all patients with a confirmed stroke diagnosis.

For patients with a stroke diagnosis admitted to Aarhus University Hospital, ischaemic postconditioning will continue twice daily (8 am and 8 pm) for seven days. Ischaemic postconditioning will continue at home/rehabilitation if the patient is discharged before day 7.

Patients in both groups will receive standard care, which includes acute reperfusion therapy (IV tPA (alteplase, 0.9 mg/kg, 10% as bolus) and/or EVT (stent retrievers and/or direct aspiration first-pass technique), according to national and international guidelines.

Study outcomes

Primary outcome

The primary outcome will be the three months modified Rankin Scale (mRS) score. The mRS score will be determined by either face-to-face assessment or based on structured telephone interviews performed by two different assessors who are blinded to the allocated intervention. If disagreement occurs, the patient will be contacted by a blinded third and final assessor. The structured telephone interview is based upon a validated Danish translation of the ‘slightly revised simplified modified Rankin Scale questionnaire’.²⁴

Every possible effort will be made to assess the outcome in patients who are unable to participate in the interview. The outcome will then be assessed by contact to a named relative or the general practitioner.

Secondary outcomes

There are three categories of secondary outcomes: clinical (I), neuroimaging (II) and biomarkers (III). For the clinical outcomes (I): analysis on early neurological improvement (NIHSS and PreSS), three months functional outcome (mRS) and Major Adverse Cardiac and Cerebral Events and Mortality will be performed. Neuroimaging (II): Acute (24-h) infarct growth (AIS), 24-h haematoma expansion and seven days haematoma reabsorption rate.

As for the biomarker endpoints (III): exploratory analysis on biomarkers for identifying RIC responders and in general as stroke diagnostics and prognostication tools will be performed. Selected secondary endpoints with planned statistical analysis are listed in Table 2.

Table 2.

Secondary endpoints.

Clinical (I)	Neuroimaging (II)
• mRS at three months by subgroups of: AIS(General ordinal logistic regression, significance level 5%)	• 24-h haematoma growth in ICH patients(Binomial regression analysis, significance level of 5%)
• mRS at three months by subgroups of: AIS tPA and/or EVT treated AIS(General ordinal logistic regression, significance level 5%)	• 24-h infarct growth in AIS patients (tPA and/or EVT treated) (Binomial regression analysis, significance level of 5%)
• mRS at three months by subgroups of: ICH(General ordinal logistic regression, significance level 5%)	• AIS patients with large vessel occlusion (LVO) eligible to EVT treatment(Binomial regression analysis. Significance level of 5%)
• Major Adverse Cardiac and Cerebral Events (MACCE) and recurrent ischaemic events 3 and 12 monthsa(Cox regression analysis, significance level of 5%)	• Differences in haematoma reabsorption rate (seven days) in patients with ICHb(Binomial regression analysis, significance level of 5%)
• Three-month and 12-month mortalitya (Two sample test of proportion (Chi-square test), significance level of 5%)	Blood-biomarkers (III)
• Proportion with complete remission of symptoms within 24 h (TIA) (Binomial regression analysis. Significance level of 5%)	• Rheo-erythrocrine dysfunction (RBC deformability, eryNOS3, AMPKα1 and plasma nitrite)
• 24-h Prehospital stroke score (PreSS) difference(All randomised and in subgroups: AIS, AIS tPA and/or EVT treated and ICH) (General ordinal logistic regression, significance level 5%)	• Coagulation profile of prehospital putative stroke patients
• Early and very early neurological improvementc(All randomised, all with target diagnosis and in subgroups: AIS, AIS tPA and/or EVT treated and ICH) (General ordinal logistic regression analysis, significance level of 5%)	• microRNA and extracellular vesicles in prehospital stroke diagnostic and as markers for RIC responders
• Prestroke physical activity level (PASE) as a predictor for early and long-term recovery(Target diagnosis and subgroups AIS and ICH) (General ordinal logistic regression, significance level 5%)	• Glial Fibrillary Acidic Protein (GFAP) and Occludin in prehospital stroke diagnostic

mRS: modified Rankin Scale; AIS: acute ischaemic stroke; ICH: intracerebral haemorrhage; TIA: transient ischaemic attack; NIHSS: National Institute of Health Stroke Score; PASE: Physical Activity for the Elderly; RBC: red blood cell; eryNOS3: erythrocyte nitric oxide synthase 3; AMPK1α: AMP-activated protein kinase (AMPK) 1α; MicroRNA: micro ribonucleic acid.

^aDeath and new vascular events in the 12 months’ follow-up period will be obtained from Danish Civil Registration System and The Danish National Patient Register.

^bΔHaematoma volume (seven days): Haematoma Volume _basenline–Haematoma _{volume day 7}.

^cVery early neurological improvement: Reduction in prehospital stroke score (PreSS) ≥ 1 points or resolution of symptoms at admission: Items on PreSS related to NIHSS baseline ΔPreSS = PreSS_prehospital – same items on NIHSS _baseline. Early Neurological Improvement: Reduction in NIHSS ≥ 4 (baseline versus 24-h NIHSS): ΔNIHSS = NIHSS_baseline–NIHSS₂₄.

Data monitoring and safety committee

An independent data monitoring and safety committee will meet at regular intervals during the trial and ensure the safety of enrolled patients. They will assist and advise the Trial Steering Committee, assessing the validity and credibility of the trial and ensure that risk/benefit ratio for participants remains acceptable. The Data monitoring and safety committee (DMSC) have complete access to all trial-related data and will meet at least once every 12 months. The committee will make recommendations to the sponsor regarding study modification, continuation or termination. Predefined special attention will be given to the distribution of serious adverse events, three months functional outcome, mortality, acute haematoma growth in ICH (24 h) and acute neurological deterioration (24 h).

Sample size estimates

The treatment effect of RIC on long-term functional outcome is unknown. We have assumed a small but clinical significant neuroprotective effect corresponding to a 7% increased odds for a beneficial shift on the mRS. The sample size calculation was based on a simulation-based approach to the analysis of statistical power when ordinal logistic regression analysis is performed (significance level of 5%).

The statistical power was simulated at different hypothetical sample sizes (on the target population ranging from 200 to 1900) with 2000 simulation-runs performed at each step. Unpublished data on IV-tPA and/or EVT-treated AIS and ICH patients from our institution were used (not shown).

Based on our previous trial experience with prehospital RIC, we estimate that a sample of 1000 subjects with target diagnosis (AIS and ICH) will be feasible to include during the study period (1500 with a prehospital putative stroke). Including 1000 patients with a target diagnosis provides sufficient power (90%) at a significance-level of 5% to detect RIC treatment effects of the estimated 7% (Table 3). We estimate that 85% of confirmed stroke patients will be AIS and the remaining 15% will be ICH. Reperfusion therapy rates in AIS patients is estimated to be 50%

Table 3.

Estimated sample size and composition of the prehospital randomised study population.

Treatment effect (assumed neuroprotective)	5%	6%		7%
Sample (target diagnosis), n	1000	1000		1000
Power	66%	80%		90%
Significance level	5%	5%		5%
The estimated prehospital, randomised, sample:
Sample size, prehospital			Proportion of randomised (%)			n
Target diagnosis (AIS and ICH), n			67			1000
Non-vascular diagnosis, n			27			403
TIA without DWI lesion, n			4			60
Lost to follow-up, n			2			30
Total, n						1492
Plan to include						1500

DWI: Diffusion-weighted imaging; AIS: acute ischaemic stroke; ICH: intracerebral haemorrhage; TIA: transient ischaemic attack.

Statistical analysis (Primary endpoint)

The primary endpoint will be three months mRS score in the population fulfilling the in-hospital inclusion criteria (target population, Figure 1). The primary trial analysis will be performed on the target population which consists of prehospital randomised patients with an in-hospital diagnosis of AIS (including TIA with a DWI lesion) or ICH regardless of adherence to investigational treatment (modified Intention-To-Treat).²⁵ The main analysis of the primary study endpoint will be performed using the entire range (‘shift analysis’) of the mRS (General ordinal logistic regression, with a random effect on the stratification groups as outlined in the section on randomisation).

All patients randomised in RESIST will have a focal neurological deficit at enrolment (documented on the prehospital stroke score). The presence or absence of specific focal neurological symptoms will be assessed again after 24 h using the same score as pre-randomisation (PreSS) in all randomised patients (PreSS 24 h/at discharge). The primary safety analysis will be made on the entire randomised population using the difference in prehospital stroke score (PreSS) to document change in neurological deficits between pre-randomisation and 24 h/discharge (General ordinal logistic regression). Furthermore, vascular events and mortality in the 12-month follow-up period will be analysed for all randomised subjects.

If RIC is associated with ultra-early treatment effect and reduced symptom severity (in the ambulances), this could affect the in-hospital indication for IV-tPA and/or EVT (in the case of rapid improvement of symptoms or mild symptoms). Because of this, a supplementary analysis using general ordinal logistic regression, adjusted for possible imbalances in reperfusion treatments (IV-tPA and EVT) between treatment arms will be made.

Study organisation and funding

The sponsor of the trial is Aarhus University and Aarhus University Hospital. The study is funded by TrygFonden, Novo Nordisk Foundation, Manufacturer Vilhelm Pedersen and Wife's foundation, Aase Ejnar Danielsen’s Foundation and Aarhus University. Neither the funding sources, nor the device manufactures, have had any influence on study design, data collection, analysis or interpretation, nor the decision to publish or the preparation, review and approval of the manuscript. The investigators have no financial interest in the investigational devices.

Current status of the trial

The study started recruitment in March 2018 and the estimated completion date is December 2022. As of 23 September 2019: 409 patients have been recruited.

Discussion

In the current trial, we use a combination of remote ischaemic perconditioning (prehospital) and postconditioning (in-hospital) to achieve possible synergistic effects and improve long-term recovery.^26–28 The primary endpoint for this trial will be functional outcome at three months (mRS) reflecting both possible acute and long-term effects associated with remote ischaemic per- and postconditioning.

Treatment is started in the early prehospital phase in patients with a presumed stroke. To prevent an obvious dilution of sample size, the primary efficacy (mRS) endpoint will be analysed on the target population with documented acute vascular lesions (AIS and ICH). For the safety endpoints, the analyses will be based on the entire randomised sample. This approach has been done in a recent comparative study and is considered reasonable given the excellent safety profile of RIC.^{15,17–20,25}

Several important lessons have been learned from the first RIC pilot study performed at our institution: Automatic devices will be used (sham-controlled), enabling the treatment to continue in-hospital and during rehabilitation/at home. Treatment compliance will be documented electronically on each device.

In our previous study, we found a possible early prehospital treatment effect (before administration of reperfusion treatment) and irrespective of recanalisation status.¹⁵ Furthermore, we found no indication of safety concerns in patients with ICH treated with RIC in prehospital field. Based on this, we have simplified the inclusion criteria to reflect the population of acute stroke patients presenting in the prehospital phase, not limiting it to AIS patients who receive reperfusion therapy.

Summary and conclusion

RESIST is a multicentre, prospective, randomised, patient-assessor blinded, sham-controlled study investigating whether RIC applied in the acute prehospital phase and continued in-hospital improves functional outcome in patients with AIS and ICH. RIC is feasible, safe and promising neuroprotective treatment that has first-aid potential worldwide. Enrolment started in 2018 and is expected to run until the end of 2022.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The study is funded by TrygFonden (ID: 120636), Novo Nordisk Foundation (ID: NNF00052924), Manufacturer Vilhelm Pedersen and Wife's foundation (ID: NNF16OC0023474), Aase Ejnar Danielsens Foundation (ID: 10–002120) and Aarhus University.

Ethical approval

Central Denmark Region Committees on Biomedical Research Ethics (ID:1–10-72–97-17). Danish Medicines Agency (ID: 2017114177, EUDAMED CIV-ID: CIV-17–11-022324).

Informed consent

Informed consent is waived in the acute phase. Written informed consent is obtained as soon as possible either by the patient or by his/her legal representative and an independent physician.

Guarantor

GA.

Contributorship

RAB, NH and GA researched literature and conceived the study. RAB, GA, NH, SPJ, JBV, MF, DCH, HK, MFG and ABB were involved in protocol development and study conduct. RAB wrote the first draft of the article. All authors provided significant input into reviewing, modifying and editing the article and approved the final version of the article. All authors contributed to writing of the article.

Acknowledgement

NA