Effect of Rho-Kinase and Autophagy on Remote Ischemic Conditioning-Induced Cardioprotection in Rat Myocardial Ischemia/Reperfusion Injury Model

Objective. Remote ischemic conditioning (RIC) is a cardioprotective method in ischemia/reperfusion (I/R) injury. This study investigated the mechanism of Rho-kinase-mediated autophagy in RIC. Methods. Sixty male Sprague–Dawley rats were randomly divided into six groups: sham, I/R, RIC, I/R+fasudil, RIC+wortmannin, and RIC+fasudil+wortmannin. Throughout the experiment, mean arterial pressure and heart rate were continuously monitored. Histopathology and ultrastructure and myocardial enzymes’ expression were evaluated to determine the degree of cardiac injury. The protein expression of the Rho-kinase substrates myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1), autophagy-related protein light chain 3-II (LC3-II) and Beclin 1, and protein kinase B (AKT) was measured in the myocardial tissue. Results. Compared with the sham group, the mean arterial pressure and heart rate were decreased, myocardial enzyme levels were increased, and myocardial damage was aggravated in the I/R group; however, RIC improved these alterations. The expression of phosphorylated MLC and MYPT1 was lower, while LC3-II, Beclin 1, and phospho-AKT expression levels were higher in the RIC group compared with the I/R group. Obviously, treatment of the I/R group rats with fasudil, a Rho-kinase inhibitor, significantly ameliorated the I/R effects, whereas treatment of the RIC group rats with wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor, inhibited the RIC protective effects. Moreover, the rats in the RIC+fasudil+wortmannin group showed similar changes to those in the RIC+wortmannin group. Conclusion. These results showed that RIC protected the myocardium from I/R injury by suppressing Rho-kinase and the underlying mechanism may be related to enhancing autophagy via the PI3K/AKT pathway.

Sympathetic Nerve Innervation and Metabolism in Ischemic Myocardium in Response To Remote Ischemic Conditioning

Background Multiple potential interventions have been tested to protect the heart against myocardial ischemia/reperfusion (MIR) injury. Remote ischemic conditioning (RIC), an endogenous cardioprotective approach, could markedly improve cardiac function post-myocardial ischemia injury. In this study, we aimed to assess the effects of RIC on cardiac sympathetic nerve innervation and metabolism in the association with Chondroitin sulfate proteoglycans (GSPG). Methods Transient myocardial ischemia (30 min) is induced by ligature of the left anterior descending coronary artery ligation (LAD) in male Sprague Dawley rats (250-350 g), in vivo cardiac [11C]mHED and 2-[18F]FDG PET scans were performed at 14 days after ischemia. Remote ischemic preconditioning (RIPerc) was induced by three cycles of five-minute-long unilateral hind limb ischemia and intermittent five minutes of reperfusion during LAD occlusion period. The quantitative parameters were quantified in parametric polar maps. This standardized format facilitates the regional radioactive quantification of parameters in deficit regions to remote areas. The ex vivo radionuclide distribution was additionally identified using autoradiography. Myocardial neuron density and GSPG expression were assessed by immunohistochemistry. Results There was no significant difference in the metabolism-defected to the remote activity ratio (44.6±4.8% vs. 45.4±4.4%) between control rats (MIR) and treated (MIR+RIPerc) rats (P>0.05). Additionally, the mean nervous activity of denervated myocardium activity was significantly elevated in rats with RIPerc coupled with reduced denervated myocardium size compared to the rats MIR group (35.9±7.1% vs. 28.9±2.3% of the left ventricular (LV) remote area (P<0.05). These findings were associated with preserved LV systolic function and a significant reduction in GSPG expression in the myocardium. Conclusion RIPerc presented the effect on cardiac sympathetic nerve innervation following ischemia, but there is no significant effect on myocardial metabolism. A long-term outcome study is warranted.

Preventing Ischemic Cerebrovascular Events in High-Risk Patients With Non-disabling Ischemic Cerebrovascular Events Using Remote Ischemic Conditioning: A Single-Arm Study

Background: Secondary stroke prevention after a high-risk, non-disabling ischemic cerebrovascular event needs to be enhanced. The study was conducted to investigate whether remote ischemic conditioning (RIC) is effective in preventing recurrent ischemic events within 3 months.Methods: This was a four-center, single-arm, open-label Phase IIa futility trial (PICNIC-One Study). Adult patients (≥18 years of age) who had an acute minor ischemic stroke (AMIS) with a National Institutes of Health Stroke Scale score ≤ 3 or a transient ischemic attack (TIA) with moderate-to-high risk of stroke recurrence (ABCD score ≥ 4) within 14 days of symptom onset were recruited. Patients received RIC as adjunctive therapy to routine secondary stroke prevention regimen. RIC consisted of five cycles of 5-min inflation (200 mmHg) and 5-min deflation of cuffs (45 min) on bilateral upper limbs twice a day for 90 days.Results: A total of 285 patients met the study criteria, of which 167 provided signed informed consent and were enrolled. Data from 162 were analyzed with five subjects excluded. Recurrent AIS/TIA occurred in 6/162 (3.7%) patients within 3 months, with no occurrence of hemorrhagic stroke. The top three adverse events were upper limb pain (44/162, 27.2%), petechia (26/162, 16.0%), and heart palpitation (5/162, 3.1%). About 68 (42.0%) subjects completed ≥ 50% of 45-min RIC sessions.Conclusions: RIC is a safe add-on procedure and it has a potential benefit in reducing recurrent cerebrovascular events in patients with high-risk, non-disabling ischemic cerebrovascular events as the risk of stroke/TIA events is lower than expected; however, its compliance needs to be improved. Our study provides critical preliminary data to plan a large sample size, randomized controlled clinical study to systematically investigate the safety and efficacy of RIC in this population.

Immune Modulation as a Key Mechanism for the Protective Effects of Remote Ischemic Conditioning After Stroke

Remote ischemic conditioning (RIC), which involves a series of short cycles of ischemia in an organ remote to the brain (typically the limbs), has been shown to protect the ischemic penumbra after stroke and reduce ischemia/reperfusion (IR) injury. Although the exact mechanism by which this protective signal is transferred from the remote site to the brain remains unclear, preclinical studies suggest that the mechanisms of RIC involve a combination of circulating humoral factors and neuronal signals. An improved understanding of these mechanisms will facilitate translation to more effective treatment strategies in clinical settings. In this review, we will discuss potential protective mechanisms in the brain and cerebral vasculature associated with RIC. We will discuss a putative role of the immune system and circulating mediators of inflammation in these protective processes, including the expression of pro-and anti-inflammatory genes in peripheral immune cells that may influence the outcome. We will also review the potential role of extracellular vesicles (EVs), biological vectors capable of delivering cell-specific cargo such as proteins and miRNAs to cells, in modulating the protective effects of RIC in the brain and vasculature.

Ischemic Conditioning in the Right Colon and Terminal Ileum: An Experimental Rat Model

Introduction: Preoperative gastric ischemic conditioning (IC) improves the outcome of esophageal replacement gastroplasty and is associated with low morbidity. However, when the stomach cannot be used for esophageal replacement, a colonic replacement is required. The study aim was to assess the viability of right colon and terminal ileum IC in a rat model, the histological damage/recovery sequence, and determine if neovascularization is a potential adaptive mechanism. Methods: The study was conducted in Rattus norvegicus with ileocolic vascular ligation. Seven groups of animals were established (six rats per group) with groups defined by the date of their post-IC euthanasia (+1, +3, +6, +10, +15, and +21 days). Comparisons were made with a sham group. Viability of the model was defined as <10% of transmural necrosis. The evaluation of histological damage used the Chiu score in hematoxylin and eosin sections of paraffin-embedded specimens with CD31 immunohistochemical assessment of neovascularization by the median of submucosal vessel counts in five high-magnification fields. Results: Transmural colon necrosis occurred in 1/36 animals (2.78%) with no animal demonstrating transmural ileal necrosis. The maximum damage was observed in the colon on +1 day post-IC (average Chiu score 1.67, P = 0.015), whereas in the ileum, it was on days +1, +3, and +6 (average Chiu score 1.5, 1.3, and 1.17; P = 0.015, 0.002, and 0.015, respectively). In the +21-day group, histological recovery was complete in the colon in four (66.7%) of the six animals and in the ileum in five (83.3%) of six animals. There were no significant differences in quantitative neovascularization in any of the groups when compared with the sham group or when comparisons were made between groups. Conclusions: The tested animal model for IC of the colon and terminal ileum appeared to be feasible. Histological damage was maximal between the 1st and 3rd day following IC, but by day 21, recovery was complete in two-thirds of the rats. There was no evidence in this preliminary IC model that would suggest neovascularization as an adaptive mechanism.

Preclinical evidence of remote ischemic conditioning in ischemic stroke, a metanalysis update

Remote ischemic conditioning (RIC) is a promising therapeutic approach for ischemic stroke patients. It has been proven that RIC reduces infarct size and improves functional outcomes. RIC can be applied either before ischemia (pre-conditioning; RIPreC), during ischemia (per-conditioning; RIPerC) or after ischemia (post-conditioning; RIPostC). Our aim was to systematically determine the efficacy of RIC in reducing infarct volumes and define the cellular pathways involved in preclinical animal models of ischemic stroke. A systematic search in three databases yielded 50 peer-review articles. Data were analyzed using random effects models and results expressed as percentage of reduction in infarct size (95% CI). A meta-regression was also performed to evaluate the effects of covariates on the pooled effect-size. 95.3% of analyzed experiments were carried out in rodents. Thirty-nine out of the 64 experiments studied RIPostC (61%), sixteen examined RIPreC (25%) and nine tested RIPerC (14%). In all studies, RIC was shown to reduce infarct volume (− 38.36%; CI − 42.09 to − 34.62%) when compared to controls. There was a significant interaction caused by species. Short cycles in mice significantly reduces infarct volume while in rats the opposite occurs. RIPreC was shown to be the most effective strategy in mice. The present meta-analysis suggests that RIC is more efficient in transient ischemia, using a smaller number of RIC cycles, applying larger length of limb occlusion, and employing barbiturates anesthetics. There is a preclinical evidence for RIC, it is safe and effective. However, the exact cellular pathways and underlying mechanisms are still not fully determined, and its definition will be crucial for the understanding of RIC mechanism of action.

Potential Therapies to Protect the Aging Heart Against Ischemia/Reperfusion Injury

Despite important advances in the treatment of myocardial infarction that have significantly reduced mortality, there is still an unmet need to limit the infarct size after reperfusion injury in order to prevent the onset and severity of heart failure. Multiple cardioprotective maneuvers, therapeutic targets, peptides and drugs have been developed to effectively protect the myocardium from reperfusion-induced cell death in preclinical studies. Nonetheless, the translation of these therapies from laboratory to clinical contexts has been quite challenging. Comorbidities, comedications or inadequate ischemia/reperfusion experimental models are clearly identified variables that need to be accounted for in order to achieve effective cardioprotection studies. The aging heart is characterized by altered proteostasis, DNA instability, epigenetic changes, among others. A vast number of studies has shown that multiple therapeutic strategies, such as ischemic conditioning phenomena and protective drugs are unable to protect the aged heart from myocardial infarction. In this Mini-Review, we will provide an updated state of the art concerning potential new cardioprotective strategies targeting the aging heart.