Didn't your competent? doctor start using remote ischemic conditioning years ago? NO? So, INCOMPETENCE REIGNED, and you suffer from that incompetence! And your board of directors is so incompetent they don't recognize it when it occurs!
Remote ischemic conditioning
(20 posts to July 2018)
Oops, I'm not playing by the polite rules of Dale Carnegie, 'How to Win Friends and Influence People'.
Telling stroke medical persons they know nothing about stroke is a no-no even if it is true.
Politeness will never solve anything in stroke. Yes, I'm a bomb thrower and proud of it. Someday a stroke 'leader' will try to ream me out for making them look bad by being truthful , I look forward to that day.
Multicenter Translational Trial of Remote Ischemic Conditioning in Acute Ischemic Stroke (TRICS BASIC)
Simone Beretta, MD, PhD https://orcid.org/0000-0002-9417-2748 simone.beretta@unimib.it, Mauro Tettamanti, PhD https://orcid.org/0000-0001-7345-0887, Jacopo Mariani, PhD https://orcid.org/0000-0001-9715-1291, Susanna Diamanti, MD, PhD https://orcid.org/0000-0002-6201-1536, Alessia Valente, PhD https://orcid.org/0000-0002-6917-9278, Ornella Cuomo, PhD https://orcid.org/0000-0002-6459-2530, Chiara Di Santo, PhD, … Show All … , and Carlo Ferrarese, MD, PhD https://orcid.org/0000-0002-7499-6600Author Info & Affiliations
Abstract
BACKGROUND:Basic science studies have reported remote ischemic conditioning (RIC) as neuroprotective in acute ischemic stroke, although clinical evidence remains conflicting. The TRICS BASIC study (Multicenter Translational Trial of Remote Ischemic Conditioning in Acute Ischemic Stroke) investigated the efficacy and safety of RIC in experimental ischemic stroke using a rigorous clinical trial methodology.METHODS:Multicenter, multispecies, parallel group, randomized, controlled, preclinical trial of transient femoral artery clipping to induce RIC in female and male rats and mice subjected to transient endovascular occlusion of the middle cerebral artery. Animals were randomized to receive RIC, or sham surgery, after reperfusion. The primary end point was a good functional outcome at 48 hours, assessed using a composite functional neuroscore. Secondary end points were infarct volume at 48 hours and safety, assessed using a standardized health report at 24 and 48 hours. Preenrollment harmonization, centralized monitoring, allocation concealment, blinded outcome assessment, and intention-to-treat analysis were applied.The trial enrolled 164 rodents (82 mice and 82 rats) of both sexes (53% females), across 7 laboratories. A greater proportion of RIC-treated rodents achieved a favorable functional outcome compared with controls, at 48 hours postischemia (55% versus 36%; odds ratio, 2.2 [95% CI, 1.23–4.4]; P=0.009). RIC was associated with a small reduction in infarct volume (standardized mean difference, −0.38 [95% CI, −0.70 to −0.05];>P=0.024). Health monitoring indicated no major safety concerns, and postoperative analgesia requirements were lower in RIC-treated mice.CONCLUSIONS:Surgically induced RIC provided a modest but evident neuroprotective effect in experimental ischemic stroke, underscoring the potential of this strategy as an adjunctive treatment in stroke care. The findings of the TRICS BASIC study highlighted the importance of multicenter preclinical trials in addressing variability and enhancing translational validity.URL:https://www.preclinicaltrials.eu; Unique identifier: PCTE0000177.Graphical AbstractStroke is one of the leading causes of death and disability at a global level,1 with the only effective treatment being time-dependent recanalization of cerebral arteries through intravenous thrombolysis or endovascular mechanical thrombectomy.2
The economic impact of cardiovascular risk factors is anticipated to rise substantially in the coming decades, concomitant with a projected increase in the incidence of stroke, and stroke-related costs.3 This underscores an urgent need to develop and integrate novel therapeutic strategies. Such approaches could complement current recanalization therapies or serve as alternatives in cases where these interventions are contraindicated, with the ultimate goal of improving clinical outcomes in stroke management.
The development of neuroprotective strategies has encountered significant translational barriers, with the majority of candidate therapies demonstrating promising preclinical efficacy but failing to achieve successful outcomes in clinical trials.4 To bridge this gap, the concept of multicenter preclinical randomized controlled trials has emerged as a critical intermediary step, mirroring the rigor and design of phase 3 clinical trials, and addressing variability across experimental settings.5,6
Remote ischemic conditioning (RIC) is a promising neuroprotective strategy that aims to induce systemic ischemic tolerance through brief episodes of ischemia and reperfusion applied to distant organs. Initially demonstrated in the heart, RIC has been shown to activate endogenous protective mechanisms against ischemic brain injury.7–10
The encouraging results observed in single-laboratory studies,11,12 require confirmation through multicenter preclinical randomized controlled trials to robustly evaluate the efficacy and safety of RIC in stroke models.
Currently completed phase 2 and 3 clinical randomized controlled trials on RIC in patients with acute ischemic stroke were conducted using different application protocols and provided conflicting results.13–20
The TRICS BASIC (Multicenter Translational Trial of Remote Ischemic Conditioning in Acute Ischemic Stroke) multicenter preclinical randomized controlled trial was designed to investigate efficacy and safety of surgical RIC, by transient femoral artery clipping, in 2 animal species (mice and rats) and both sexes, using the stroke model of transient middle cerebral artery (MCA) occlusion. This preclinical trial was realized within a network of 7 Italian laboratories with extensive expertise in the transient MCA occlusion model, aiming to generate high-quality, reproducible data to inform future clinical translation.
METHODS
Study Design
The study protocol was registered on preclinicaltrials.eu (identifier: PCTE0000177). Detailed methodology of the TRICS BASIC study has been published as a protocol article21 before starting randomization of experimental animals.
The data that support the findings of this study are available from the corresponding author on reasonable request.
Authorization for animal use was granted under project licenses issued by the Italian Ministry of Health (1056/2020-PR) and the Committee on Animal Care of the University of Milano Bicocca. All experimental procedures adhered to national regulations governing the use of laboratory animals (D.L. 26/2014) and conformed to the European Union Directive on the protection of animals used for scientific purposes (2010/63/EU). The experimental design and conduct were aligned with the National Institutes of Health guidelines and complied with the Animal Research: Reporting of In Vivo Experiments guidelines to ensure transparency and reproducibility in animal research.
The TRICS BASIC study was designed as a multicenter, multispecies, randomized, controlled, preclinical trial, to investigate efficacy and safety of RIC in experimental ischemic stroke and was performed in 7 Italian centers between September 2021 and January 2023.
There were 2 successive randomizations: (1) allocation to receive an occlusion of the MCA (MCA+) or its sham equivalent (MCA−), with a 5:1 ratio, and (2) allocation to receive a RIC (RIC+) via transient femoral artery occlusion, or its sham equivalent (RIC−), with a 1:1 ratio.
To ensure allocation concealment, each center received sealed, nontransparent, and nonresealable envelopes for the 2-step randomization, marked with codes for species (mouse or rat), sex, and progressive numbers. Upon opening an envelope, a sheet detailing center identification, sex, and MCA+/MCA− assignment was signed and dated. A second internal envelope containing the RIC+/RIC− treatment was opened only after MCA surgery to ensure allocation concealment for surgeons. Randomization lists and envelopes were prepared by personnel not involved in animal procedures and securely stored.
Animals of both sexes were equally represented. Animals that either scored below the prespecified threshold of 2 of the intraischemic clinical score (see below) or with no ischemic lesion detected at brain histology were excluded from the analyses (ie, animals without stroke). Animals that died before RIC randomization were excluded (ie, animals not reaching the target randomization) and replaced by other animals, up to 3 per center. Conversely, animals that died after RIC randomization were retained in the prespecified (intention-to-treat) primary analyses and given the worst score: this was true for naturally dying animals and for animals euthanized after showing signs of extreme distress.
MCA Occlusion in Mice and Rats
Adult C57BL/6J mice (weight, 26 g ±5%; age, 10–12 weeks) and adult Sprague-Dawley rats (weight, 250 g ±5%; age, 8–10 weeks), male and female 1:1 (regardless of estrous cycle), housed in single cages, exposed to 12/12 hours light/dark cycle, at controlled room temperature, with free access to food and water, in specific pathogenfree facilities were used. Animals were anesthetized by 3% isoflurane in O2/N2O (1:3) and maintained by 1% to 1.5% isoflurane in O2/N2O (1:3). Transient occlusion of the origin of the right MCA was induced (60 minutes in mice and 100 minutes in rats), followed by a reperfusion period of 48 hours. In mice, a silicone-coated monofilament nylon suture (sized, 7–0; diameter, 0.06–0.09 mm; length, 10±1 mm; diameter with coating, 0.23 mm; coating length, 2 mm; Doccol Corporation, Redlands, CA) was introduced into the right common carotid artery and advanced to block the right MCA. In rats, a silicone-coated filament (sized, 5–0; diameter with coating, 0.33 mm; coating length, 5–6 mm; Doccol Corporation, Redlands, CA) was introduced in the right external carotid artery and pushed through the internal carotid artery to occlude the origin of the right MCA.
For both species, body temperature was kept at 37 °C by a heating pad during surgery. During MCA occlusion, animals were awakened from anesthesia, kept in a warm box, and tested for the intraischemic clinical score (see below) in single cages. After 60 minutes, blood flow was restored by carefully removing the filament under anesthesia. Sham rodents received the same anesthetic regimen and surgery as MCA occluded animals, that is, their common, external, and internal carotid arteries were isolated, but the filament was not introduced. After surgery, animals were returned to single cages.
Intraischemic Clinical Score
After MCA occlusion, the following intraischemic clinical score was applied to test the correct induction of cerebral ischemia. Animals were judged ischemic and included in the trial if presenting ≥3 of the following deficits after filament insertion:
1.
The palpebral fissure had an ellipsoidal shape (not the normal circular one).
2.
One or both ears extended laterally.
3.
Asymmetrical body bending on the ischemic side.
4.
Limbs extended laterally and did not align with the body.
RIC in Mice and Rats
RIC was induced via transient femoral artery occlusion. At a set time after reperfusion (20 minutes in rats and 10 minutes in mice), the femoral artery was identified, isolated, and transiently occluded with a microsurgical clip to halt blood flow for the specified duration (20 minutes in rats and 10 minutes in mice). Successful occlusion was confirmed by visually inspecting the distal femoral artery. Sham-treated animals (RIC−) underwent the same procedure without femoral artery clipping.
Outcomes
Outcomes were measured at the end of the study (48 hours after MCA occlusion). The primary outcome was the proportion of animals (separately by species) with good functional outcome assessed by the De Simoni neuroscore,22,23 which comprehensively assessed global and focal sensorimotor deficits. A good outcome was defined as a De Simoni neuroscore of ≤20, on a scale from 0 (no deficits) to 56 (all deficits present at maximum value).
The coordinating unit conducted centralized training on the correct administration of the De Simoni composite neuroscore through multiple prerandomization meetings and a detailed instructional video.24
Secondary outcomes were as follows:
Infarct volume measured by volumetric histology; brains were fixed in ice-cold 10% neutral buffer formalin and shipped to the coordinating center (University of Milano-Bicocca) for centralized processing and blinded evaluation; coronal sections (50 µm) were obtained and stained using Cresyl Violet 0.1% (Bioptica, Milano, Italy). Infarct areas were measured in consecutive sections with 250 µm interval (on average, 19 sections for rat brain and 9 sections for mouse brains). Infarct volume was calculated using ImageJ image processing software (National Institutes of Health, Bethesda, MD), corrected for interhemispheric asymmetries due to cerebral edema, and expressed in cubic millimeters.
De Simoni neuroscore, considered as a continuous value.
Blinding
Functional and clinical assessments were conducted locally by researchers blinded to MCA occlusion and RIC treatment allocation, that is, by a person not participating in/assisting the surgical practices. Centrally conducted histological procedures and data analysis (RIC+ versus RIC−) were conducted blinded to the group allocation.
Data Collection
Study data were collected and managed using REDCap electronic data capture tools hosted at the Istituto di Ricerche Farmacologiche Mario Negri IRCCS on behalf of the coordinating unit. REDCap served as a secure web-based platform for research data management, offering validated data entry, audit trails, automated data exports, and integration with external sources for statistical analysis.
Health and Safety Assessment
An MCA occlusion health and safety report documented data on animal identification, procedural details, and postsurgical outcomes. Evaluations occurred presurgery, intraoperatively, and at 24 and 48 hours postsurgery, following the IMPROVE guidelines.25 Key metrics included weight changes, isoflurane exposure, respiratory rates, surgical times, and postoperative distress categorized as mild, moderate, or severe. Buprenorphine (0.05–0.1 mg/kg every 8–12 hours) was the only analgesic permitted during the study. This systematic approach ensured comprehensive tracking of health parameters and adverse outcomes to promote animal welfare during the conduct of the study.
Sample Size Calculation
A 20% rate of good functional outcomes was used as the baseline for animals subjected to MCA occlusion without effective treatment. An improvement of at least 30% (20%–50%) was established as the minimum effect size of translational significance. Statistical significance (α) was set at 0.050 (2-tailed). Using a χ2 test for data analysis, a total of 80 animals equally randomized between RIC+ and RIC− groups yielded a statistical power of 82%. The same calculation was applied to both species, requiring 160 animals to undergo MCA occlusion. Accounting for a 30% exclusion rate, a target of 120 animals randomized per species was set. No power calculation was conducted for nonoccluded animals, as they served solely as internal controls.
Statistical Methods
Descriptive analyses were conducted on baseline and procedural characteristics. The primary outcome, the dichotomized De Simoni neuroscore, was analyzed using a logistic regression model, which included randomization strata (RIC, center [as a random effect], and sex). To explore potential sex-specific effects of RIC, models incorporating a sex-by-RIC interaction were also tested, but were not selected as the final models due to nonsignificant results (P>0.20 from likelihood ratio tests). An intention-to-treat approach was applied, with animals that died after RIC being assigned a negative outcome. The 2 parallel trials were analyzed separately by species, and results were combined in an overall analysis if 1 trial showed a borderline significant P value.
Secondary outcomes (infarct volume and continuous De Simoni neuroscore) were analyzed using mixed linear regressions, with sex and RIC as covariates, and center as a random effect. To address species differences in infarct size, the infarct volume was expressed as a percentage of the ipsilateral hemispheric volume and standardized by subtracting the group mean and dividing by the group SD.
Health and safety monitoring data were presented as numbers and percentages for the 2 groups.
Comparisons were made in the MCA+ groups, whereas the MCA− (sham) group was analyzed descriptively. All analyses were conducted blind using Stata/IC versus 15 (Statacorp).
RESULTS
Study Flow Diagram
Between October 2021 and January 2023, a total of 216 animals (110 mice and 106 rats) were used. Of these, 52 animals were excluded based on eligibility criteria, which included death before randomization, major surgical complications, randomization to sham surgery, or absence of visible ischemic injury on histological analysis. This resulted in a final sample of 164 randomized animals (82 mice and 82 rats). The study flow diagram is shown in Figure 1.
Baseline Characteristics of the Study Population
The baseline characteristics of the 164 animals included were well-balanced across treatment groups in terms of sex, weight, body temperature during MCA occlusion or RIC surgery, isoflurane exposure duration, and respiratory rate throughout the surgical procedure (Table 1). Intraischemic clinical assessments demonstrated a 92% accuracy in identifying ongoing cerebral ischemia, correctly diagnosing ischemia in 164 animals, although yielding a false-positive rate of 8% (14 animals with no ischemic lesions confirmed by histology; Table 2). As prespecified in the study protocol, animals with false-positive diagnosis of ischemia were excluded from the final analysis.
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