Flow Reperfusion Research Articles

Inhibition of microRNA-181c-5p rescues diabetes-impaired angiogenesis through activation of key mediators of angiogenesis, cell survival and novel genes, elmo3 and trib1

Abstract Introduction Patients with diabetes have impaired angiogenesis and poor coronary collateral vessel formation post-myocardial infarction (MI), which associates with higher mortality. There is a significant unmet clinical need for new agents that stimulate angiogenesis in response to ischaemia in diabetes. We have identified that miR-181c-5p has anti-angiogenic properties, but it’s role in diabetes remains unknown. Aim To elucidate the role of miR-181c-5p in diabetes-impaired angiogenesis. Methods Human coronary artery endothelial cells were transfected with a miR-181c-5p inhibitor (antimiR-181c-5p) or negative control (antimiR-Neg), exposed to glucose (5–25mM, 48h) then underwent Matrigel tubulogenesis assay or Boyden Chamber migration assay. Levels of proteins important for angiogenesis (e.g., VEGFA, p-ERK2, p-eNOS) were determined by Western Blot. Whole transcriptome sequencing was performed in vitro to identify novel gene targets of miR-181c-5p. In vivo, diabetic mice underwent hindlimb ischaemia or wound healing surgery and were injected with antimiR-181c-5p or antimiR-Neg. Tissues were extracted early (day 3) and late (days 10-14) post-surgery. Hindlimb blood-flow reperfusion was measured by Laser Doppler imaging. Hindlimb apoptosis was assessed by TUNEL and necrotic area was assessed by H&E. Wound area was calculated daily. Neovascularisation was assessed by CD31 (capillaries) and α-actin (arterioles) immunostaining. Results Inhibition of miR-181c-5p increased endothelial tubule number (+28%, P<0.01), tubule length (+12%, P<0.01) and cell migration (+67%, P<0.05). This associated with increased VEGFA (+21%, P<0.05) and p-ERK2 (+32%, P<0.05). Whole transcriptome and pathway analysis revealed changes to angiogenesis pathways and identified a first-time involvement of genes Elmo3 and Trib1 in the pro-angiogenic action of antimiR-181c-5p in diabetes. In vivo, inhibition of miR-181c-5p increased blood flow reperfusion to ischaemic hindlimbs (+30%, P<0.001) and arteriolar density (+45%, P<0.05) in diabetic mice. Mechanistically, this was associated with early changes to mediators of angiogenesis, Erk2 mRNA (+35%, P<0.05), p-ERK2 (+35%, P<0.05) and Trib1 mRNA (+80%, P<0.05); cell survival, Bcl-2 mRNA (+44%, P<0.05); and late apoptotic clearance, Elmo3 mRNA (+57%, P<0.001). Furthermore, this was also associated with an increase in late stage hindlimb apoptosis (+94%, P<0.05) and reduced necrotic area (-90%, P<0.05) in diabetic mice. Inhibition of miR-181c-5p increased diabetic wound closure (+22%, P<0.01), wound capillaries (+61%, P<0.05), Bcl-2 mRNA (+52%, P<0.05) and Elmo3 mRNA (+50%, P<0.05) in diabetic wounds. Conclusions Inhibition of miR-181c-5p rescues diabetes-impaired angiogenesis by activation of angiogenesis and cell survival mediators, and through novel genes, Trib1 and Elmo3. Our findings have implications for a novel miRNA-based strategy that improves myocardial neovascularisation and the prognosis of diabetic patients post-MI.

Salvaging myocardial infarction with nanoenzyme-loaded hydrogels: Targeted scavenging of mitochondrial reactive oxygen species

Myocardial infarction resulting from coronary artery atherosclerosis is the leading cause of heart failure, which represents a significant global health burden. The limitations of conventional pharmacologic thrombolysis and flow reperfusion procedures highlight the urgent need for new therapeutic strategies to effectively treat myocardial infarction. In this study, we present a novel biomimetic approach that integrates polyphenols and metal nanoenzymes, inspired by the structure of pomegranates. We developed tannic acid-coated Mn-Co3O4 (MCT) nanoparticles in combination with an injectable collagen hydrogel for the effective treatment of myocardial infarction. The hydrogel enhanced the infarct microenvironment, while the slow-released MCT targets mitochondria to inhibit the post-infarction surge of reactive oxygen species, providing anti-apoptotic and anti-inflammatory effects. RNA sequencing revealed the potential of hydrogels to serve as an interventional mechanism during the post-infarction inflammatory phase. Notably, we found that the hydrogel, when combined with the nanopomegranate-based therapy, significantly improves adverse ventricular remodeling and restores cardiac function in early infarction management. The MCT hydrogel leverages the unique benefits of both MCT nanopomegranates and collagen, demonstrating a synergistic effect. This approach provides a promising example of the potential cooperation between nanomimetic structures and natural biomaterials in therapeutic applications.

Assessment of Hyperacute Cerebral Ischemia Using Laser Speckle Contrast Imaging.

Background: Accurate diagnosis of cerebral ischemia severity is crucial for clinical decision making. Laser speckle contrast imaging-based cerebral blood flow imaging can help assess the severity of cerebral ischemia by monitoring changes in blood flow. Method: In this study, we simulated hyperacute ischemia in rats, isolating arterial and venous flow-related signals from cortical vasculature. Pearson correlation was used to examine the correlation between damaged vessels. Granger causality analysis was used to investigate causality correlation in ischemic vessels. Results: Resting state analysis revealed a negative Pearson correlation between regional arteries and veins. Following cerebral ischemia induction, a positive artery-vein correlation emerged, which vanished after blood flow reperfusion. Granger causality analysis demonstrating enhanced causality coefficients for middle artery-vein pairs during occlusion, with a stronger left-right arterial effect than that of right-left, which persisted after reperfusion. Conclusions: These processing approaches amplify the understanding of cerebral ischemic images, promising potential future diagnostic advancements.

Unraveling the differential mechanisms of revascularization promoted by MSCs & ECFCs from adipose tissue or umbilical cord in a murine model of critical limb-threatening ischemia

BackgroundCritical limb-threatening ischemia (CLTI) constitutes the most severe manifestation of peripheral artery disease, usually induced by atherosclerosis. CLTI patients suffer from high risk of amputation of the lower extremities and elevated mortality rates, while they have low options for surgical revascularization due to associated comorbidities. Alternatively, cell-based therapeutic strategies represent an effective and safe approach to promote revascularization. However, the variability seen in several factors such as cell combinations or doses applied, have limited their success in clinical trials, being necessary to reach a consensus regarding the optimal “cellular-cocktail” prior further application into the clinic. To achieve so, it is essential to understand the mechanisms by which these cells exert their regenerative properties.Herein, we have evaluated, for the first time, the regenerative and vasculogenic potential of a combination of endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) isolated from adipose-tissue (AT), compared with ECFCs from umbilical cord blood (CB-ECFCs) and AT-MSCs, in a murine model of CLTI.MethodsBalb-c nude mice (n:32) were distributed in four different groups (n:8/group): control shams, and ischemic mice (after femoral ligation) that received 50 µl of physiological serum alone or a cellular combination of AT-MSCs with either CB-ECFCs or AT-ECFCs. Follow-up of blood flow reperfusion and ischemic symptoms was carried out for 21 days, when mice were sacrificed to evaluate vascular density formation. Moreover, the long-term molecular changes in response to CLTI and both cell combinations were analyzed in a proteomic quantitative approach.ResultsAT-MSCs with either AT- or CB-ECFCs, promoted a significant recovery of blood flow in CLTI mice 21 days post-ischemia. Besides, they modulated the inflammatory and necrotic related processes, although the CB group presented the slowest ischemic progression along the assay. Moreover, many proteins involved in the repairing mechanisms promoted by cell treatments were identified.ConclusionsThe combination of AT-MSCs with AT-ECFCs or with CB-ECFCs promoted similar revascularization in CLTI mice, by restoring blood flow levels, together with the modulation of the inflammatory and necrotic processes, and reduction of muscle damage. The protein changes identified are representative of the molecular mechanisms involved in ECFCs and MSCs-induced revascularization (immune response, vascular repair, muscle regeneration, etc.).

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion.

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

Combination Administration of Heparin and Nitroglycerin for the Treatment of Polycaprolactone-Induced Intravascular Embolism: A Preclinical Investigation.

As a new-generation collagen stimulator, polycaprolactone (PCL) containing filler has been extensively applied in facial dermal fillers and other medical aesthetic fields. However, inadvertent intravascular injection of PCL may result in complications such as tissue edema, flap necrosis, and even blindness. To date, there is no effective treatment for PCL-induced intravascular embolism. The aim of this study was to identify a viable resolution for the embolism resulting from intravascular administration of PCL-containing fillers. Two different animal experiments were performed: (1) PCL-induced rat inferior epigastric arteries embolism, followed by gross observation, histological evaluation, and cytokines analysis from serum; and (2) PCL-induced rabbit auricular artery embolism, immediately treated with heparin and nitroglycerin. The ears were then evaluated by gross observation, Laser speckle imaging, in vivo imaging system (IVIS) imaging, and histological evaluation. Saline and hyaluronic acids (HA) were used as controls, hyaluronidase was used as a positive drug. In a rat model of inferior epigastric arteries embolism, both intravascular injection of HA and PCL resulted in flap necrosis, indicating that the filler-induced intravascular embolism can lead to serious complications. In a rabbit model of auricular artery embolism, the combination treatment of heparin and nitroglycerin resulted in a relative blood reperfusion recovery of 80% in the ischemic area of the PCL group on day 7 post-operation, which was comparable to that of the HA group treated with hyaluronidase. Histological analysis revealed that the administration of heparin and nitroglycerin significantly attenuated intravascular thrombosis formation and inflammatory cell aggregation. The combination of heparin and nitroglycerin effectively restores blood flow reperfusion in the intravascular embolization caused by PCL filler injection, alleviates local tissue edema and flap necrosis. These findings offer a novel approach for future clinical management of intravascular embolization with PCL-containing filler injection. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Long-Term Region-Specific Mitochondrial Functionality Changes in Both Cerebral Hemispheres after fMCAo Model of Ischemic Stroke.

Cerebral ischemia/reperfusion (I/R) refers to a secondary brain injury that results in mitochondrial dysfunction of variable extent, leading to neuronal cell damage. The impact of this process has mainly been studied in the short term, from the early hours up to one week after blood flow reperfusion, and in the ischemic hemisphere only. The focus of this study was to assess the long-term impacts of I/R on mitochondrial functionality using high-resolution fluorespirometry to evaluate state-dependent activities in both ischemic (ipsilateral) and non-ischemic (contralateral) hemispheres of male mice 60, 90, 120, and 180 days after I/R caused by 60-min-long filament-induced middle cerebral artery occlusion (fMCAo). Our results indicate that in cortical tissues, succinate-supported oxygen flux (Complex I&II OXPHOS state) and H2O2 production (Complex II LEAK state) were significantly decreased in the fMCAo (stroke) group ipsilateral hemisphere compared to measurements in the contralateral hemisphere 60 and 90 days after stroke. In hippocampal tissues, during the Complex I&II ET state, mitochondrial respiration was generally lower in the ipsilateral compared to the contralateral hemisphere 90 days following stroke. An aging-dependent impact on mitochondria oxygen consumption following I/R injury was observed 180 days after surgery, wherein Complex I&II activities were lowest in both hemispheres. The obtained results highlight the importance of long-term studies in the field of ischemic stroke, particularly when evaluating mitochondrial bioenergetics in specific brain regions within and between separately affected cerebral hemispheres.

TRIM2 Selectively Regulates Inflammation-Driven Pathological Angiogenesis without Affecting Physiological Hypoxia-Mediated Angiogenesis.

Angiogenesis is a critical physiological response to ischemia but becomes pathological when dysregulated and driven excessively by inflammation. We recently identified a novel angiogenic role for tripartite-motif-containing protein 2 (TRIM2) whereby lentiviral shRNA-mediated TRIM2 knockdown impaired endothelial angiogenic functions in vitro. This study sought to determine whether these effects could be translated in vivo and to determine the molecular mechanisms involved. CRISPR/Cas9-generated Trim2-/- mice that underwent a periarterial collar model of inflammation-induced angiogenesis exhibited significantly less adventitial macrophage infiltration relative to wildtype (WT) littermates, concomitant with decreased mRNA expression of macrophage marker Cd68 and reduced adventitial proliferating neovessels. Mechanistically, TRIM2 knockdown in endothelial cells in vitro attenuated inflammation-driven induction of critical angiogenic mediators, including nuclear HIF-1α, and curbed the phosphorylation of downstream effector eNOS. Conversely, in a hindlimb ischemia model of hypoxia-mediated angiogenesis, there were no differences in blood flow reperfusion to the ischemic hindlimbs of Trim2-/- and WT mice despite a decrease in proliferating neovessels and arterioles. TRIM2 knockdown in vitro attenuated hypoxia-driven induction of nuclear HIF-1α but had no further downstream effects on other angiogenic proteins. Our study has implications for understanding the role of TRIM2 in the regulation of angiogenesis in both pathophysiological contexts.

Intraoperative Anesthetic Strategies to Mitigate Early Allograft Dysfunction After Orthotopic Liver Transplantation: A Narrative Review.

Orthotopic liver transplantation (OLT) is the most effective treatment for patients with end-stage liver disease (ESLD). Hepatic insufficiency within a week of OLT, termed early allograft dysfunction (EAD), occurs in 20% to 25% of deceased donor OLT recipients and is associated with morbidity and mortality. Primary nonfunction (PNF), the most severe form of EAD, leads to death or retransplantation within 7 days. The etiology of EAD is multifactorial, including donor, recipient, and surgery-related factors, and largely driven by ischemia-reperfusion injury (IRI). IRI is an immunologic phenomenon characterized by dysregulation of cellular oxygen homeostasis and innate immune defenses in the allograft after temporary cessation (ischemia) and later restoration (reperfusion) of oxygen-rich blood flow. The rising global demand for OLT may lead to the use of marginal allografts, which are more susceptible to IRI, and thus lead to an increased incidence of EAD. It is thus imperative the anesthesiologist is knowledgeable about EAD, namely its pathophysiology and intraoperative strategies to mitigate its impact. Intraoperative strategies can be classified by 3 phases, specifically donor allograft procurement, storage, and recipient reperfusion. During procurement, the anesthesiologist can use pharmacologic preconditioning with volatile anesthetics, consider preharvest hyperoxemia, and attenuate the use of norepinephrine as able. The anesthesiologist can advocate for normothermic regional perfusion (NRP) and machine perfusion during allograft storage at their institution. During recipient reperfusion, the anesthesiologist can optimize oxygen exposure, consider adjunct anesthetics with antioxidant-like properties, and administer supplemental magnesium. Unfortunately, there is either mixed, little, or no data to support the routine use of many free radical scavengers. Given the sparse, limited, or at times conflicting evidence supporting some of these strategies, there are ample opportunities for more research to find intraoperative anesthetic strategies to mitigate the impact of EAD and improve postoperative outcomes in OLT recipients.

Laser-induced microbubble as an in vivo valve for optofluidic manipulation in living Mice's microvessels.

Optofluidic regulation of blood microflow in vivo represents a significant method for investigating illnesses linked to abnormal changes in blood circulation. Currently, non-invasive strategies are limited to regulation within capillaries of approximately 10 μm in diameter because the adaption to blood pressure levels in the order of several hundred pascals poses a significant challenge in larger microvessels. In this study, using laser-induced microbubble formation within microvessels of the mouse auricle, we regulate blood microflow in small vessels with diameters in the tens of micrometers. By controlling the laser power, we can control the growth and stability of microbubbles in vivo. This controlled approach enables the achievement of prolonged ischemia and subsequent reperfusion of blood flow, and it can also regulate the microbubbles to function as micro-pumps for reverse blood pumping. Furthermore, by controlling the microbubble, narrow microflow channels can be formed between the microbubbles and microvessels for assessing the apparent viscosity of leukocytes, which is 76.9 ± 11.8 Pa·s in the in vivo blood environment. The proposed design of in vivo microbubble valves opens new avenues for constructing real-time blood regulation and exploring cellular mechanics within living organisms.

Apelin prevents diabetes-induced poor collateral vessel formation and blood flow reperfusion in ischemic limb.

Peripheral arterial disease (PAD) is a major risk factor for lower-extremity amputation in diabetic patients. Unfortunately, previous clinical studies investigating therapeutic angiogenesis using the vascular endothelial growth factor (VEGF) have shown disappointing results in diabetic patients, which evokes the necessity for novel therapeutic agents. The apelinergic system (APJ receptor/apelin) is highly upregulated under hypoxic condition and acts as an activator of angiogenesis. Apelin treatment improves revascularization in nondiabetic models of ischemia, however, its role on angiogenesis in diabetic conditions remains poorly investigated. This study explored the impact of Pyr-apelin-13 in endothelial cell function and diabetic mouse model of hindlimb ischemia. Nondiabetic and diabetic mice underwent femoral artery ligation to induce limb ischemia. Diabetic mice were implanted subcutaneously with osmotic pumps delivering Pyr-apelin-13 for 28 days. Blood flow reperfusion was measured for 4 weeks post-surgery and exercise willingness was assessed with voluntary wheels. In vitro, bovine aortic endothelial cells (BAECs) were exposed to normal (NG) or high glucose (HG) levels and hypoxia. Cell migration, proliferation and tube formation assays were performed following either VEGF or Pyr-apelin-13 stimulation. Following limb ischemia, blood flow reperfusion, functional recovery of the limb and vascular density were improved in diabetic mice receiving Pyr-apelin-13 compared to untreated diabetic mice. In cultured BAECs, exposure to HG concentrations and hypoxia reduced VEGF proangiogenic actions, whereas apelin proangiogenic effects remained unaltered. Pyr-apelin-13 induced its proangiogenic actions through Akt/AMPK/eNOS and RhoA/ROCK signaling pathways under both NG or HG concentrations and hypoxia exposure. Our results identified the apelinergic system as a potential therapeutic target for angiogenic therapy in diabetic patients with PAD.

Myocardial‐Targeting Tannic Cerium Nanocatalyst Attenuates Ischemia/Reperfusion Injury

AbstractIschemic heart disease (IHD) is one of the leading causes of death worldwide. Medications or surgery have been considered as effective protocols to treat IHD for decades. Yet the reperfusion of the blood flow frequently leads to the generation of excessive reactive oxygen species (ROS), causing prominent and irreversible damage to the cardiomyocytes. In the present work, tannic acid‐assembled tetravalent cerium (TA−Ce) nanocatalysts with appealing cardiomyocyte‐targeting and antioxidation capability have been synthesized and applied for the effective and biocompatible ischemia/reperfusion injury therapeutics. TA−Ce nanocatalysts could effectively rescue the cardiomyocytes from oxidative stress induced by H2O2 challenge as well as oxygen‐glucose deprivation in vitro. In the murine ischemia/reperfusion model, cardiac accumulation and intracellular ROS scavenging could be achieved against the pathology, substantially reducing the myocardial infarct area and recovering heart functionality. This work illuminates the design of nanocatalytic metal complexes and their therapeutic prospects in ischemic heart diseases with high effectiveness and biocompatibility, paving the way for the clinical translation from bench to bedside.

Myocardial-Targeting Tannic Cerium Nanocatalyst Attenuates Ischemia/Reperfusion Injury.

Ischemic heart disease (IHD) is one of the leading causes of death worldwide. Medications or surgery have been considered as effective protocols to treat IHD for decades. Yet the reperfusion of the blood flow frequently leads to the generation of excessive reactive oxygen species (ROS), causing prominent and irreversible damage to the cardiomyocytes. In the present work, tannic acid-assembled tetravalent cerium (TA-Ce) nanocatalysts with appealing cardiomyocyte-targeting and antioxidation capability have been synthesized and applied for the effective and biocompatible ischemia/reperfusion injury therapeutics. TA-Ce nanocatalysts could effectively rescue the cardiomyocytes from oxidative stress induced by H2 O2 challenge as well as oxygen-glucose deprivation in vitro. In the murine ischemia/reperfusion model, cardiac accumulation and intracellular ROS scavenging could be achieved against the pathology, substantially reducing the myocardial infarct area and recovering heart functionality. This work illuminates the design of nanocatalytic metal complexes and their therapeutic prospects in ischemic heart diseases with high effectiveness and biocompatibility, paving the way for the clinical translation from bench to bedside.

Case Report: Suspected Hyperacute Rejection During Living Kidney Transplantation

We report a case of suspected hyperacute rejection during living kidney transplantation. A 61-year-old man underwent kidney transplantation in November 2019. Before the transplantation, immunologic tests revealed the presence of anti-HLA antibodies but not donor-specific HLA antibodies. The patient was intravenously administered 500 mg of methylprednisolone (MP) and basiliximab before perioperative blood flow reperfusion. After blood flow restoration, the transplanted kidney turned bright red and then blue. Hyperacute rejection was suspected. After the intravenous administration of 500 mg of MP and 30 g of intravenous immunoglobulin, the transplanted kidney gradually changed from blue to bright red. The initial postoperative urine output was good. On the 22nd day after the renal transplantation, the patient was discharged with a serum creatinine level of 2.38 mg/dL, and the function of the transplanted kidney gradually improved. In this study, non-HLA antibodies may have been a cause of the hyperacute rejection, which was managed with additional perioperative therapies.

The Effects of Vagus Nerve Stimulation on Animal Models of Stroke-Induced Injury: A Systematic Review.

Ischemic stroke is one of the leading causes of death worldwide, and poses a great burden to society and the healthcare system. There have been many recent advances in the treatment of ischemic stroke, which usually results from the interruption of blood flow to a particular part of the brain. Current treatments for ischemic stroke mainly focus on revascularization or reperfusion of cerebral blood flow to the infarcted tissue. Nevertheless, reperfusion injury may exacerbate ischemic injury in patients with stroke. In recent decades, vagus nerve stimulation (VNS) has emerged as an optimistic therapeutic intervention. Accumulating evidence has demonstrated that VNS is a promising treatment for ischemic stroke in various rat models through improved neural function, cognition, and neuronal deficit scores. We thoroughly examined previous evidence from stroke-induced animal studies using VNS as an intervention until June 2022. We concluded that VNS yields stroke treatment potential by improving neurological deficit score, infarct volume, forelimb strength, inflammation, apoptosis, and angiogenesis. This review also discusses potential molecular mechanisms underlying VNS-mediated neuroprotection. This review could help researchers conduct additional translational research on patients with stroke.

Ischemia Reperfusion Injury Induced Blood Brain Barrier Dysfunction and the Involved Molecular Mechanism.

Stroke is characterized by the abrupt failure of blood flow to a specific brain region, resulting in insufficient supply of oxygen and glucose to the ischemic tissues. Timely reperfusion of blood flow can rescue dying tissue but can also lead to secondary damage to both the infarcted tissues and the blood-brain barrier, known as ischemia/reperfusion injury. Both primary and secondary damage result in biphasic opening of the blood-brain barrier, leading to blood-brain barrier dysfunction and vasogenic edema. Importantly, blood-brain barrier dysfunction, inflammation, and microglial activation are critical factors that worsen stroke outcomes. Activated microglia secrete numerous cytokines, chemokines, and inflammatory factors during neuroinflammation, contributing to the second opening of the blood-brain barrier and worsening the outcome of ischemic stroke. TNF-α, IL-1β, IL-6, and other microglia-derived molecules have been shown to be involved in the breakdown of blood-brain barrier. Additionally, other non-microglia-derived molecules such as RNA, HSPs, and transporter proteins also participate in the blood-brain barrier breakdown process after ischemic stroke, either in the primary damage stage directly influencing tight junction proteins and endothelial cells, or in the secondary damage stage participating in the following neuroinflammation. This review summarizes the cellular and molecular components of the blood-brain barrier and concludes the association of microglia-derived and non-microglia-derived molecules with blood-brain barrier dysfunction and its underlying mechanisms.

Ischemic Conditioning to Reduce Fatigue in Isometric Skeletal Muscle Contraction

Ischemic preconditioning (IPC) is a non-invasive protective maneuver that alternates short periods of occlusion and reperfusion of tissue blood flow. Given the heterogeneity in the magnitude and frequency of IPC-induced improvements in physical performance, here we aimed to investigate, in a well-controlled experimental set-up, the local effects of IPC in exposed muscles in terms of tissue oxygenation and muscle fatigue. Nineteen subjects were enrolled in one of the two groups, IPC (3 × 5/5 min right arm ischemia/reperfusion; cuff inflations 250 mmHg) and SHAM (3 × 5/5 min pseudo ischemia/reperfusion; 20 mmHg). The subjects performed a fatiguing contraction protocol before and 30 min after the IPC treatment, consisting of unilateral intermittent isometric elbow flexions (3 s ON/OFF, 80% of maximal voluntary contraction) until exhaustion. While muscle strength did not differ between groups, post- vs. pre-treatment endurance was significantly reduced in the SHAM group (4.1 ± 1.9 vs. 6.4 ± 3.1 repetitions until exhaustion, p < 0.05) but maintained in IPC (7.3 ± 2.0 vs. 7.1 ± 4.3, n.s.). The decrease in tissue oxygenation and the increase in deoxygenated hemoglobin were significantly reduced post- vs. pre-IPC (p < 0.05), but not post- vs. pre-SHAM. The results suggest that IPC delays the onset of fatigue likely through improved metabolic efficiency of muscles.

Liraglutide Accelerates Ischemia‐Induced Angiogenesis in a Murine Diabetic Model

Background Severe hindlimb ischemia is a chronic disease with poor prognosis that can lead to amputation or even death. This study aimed to assess the therapeutic effect of liraglutide on hind-limb ischemia in type 2 diabetic mice and to elucidate the underlying mechanism. Methods and Results Blood flow reperfusion and capillary densities after treatment with liraglutide or vehicle were evaluated in a mouse model of lower-limb ischemia in a normal background or a background of streptozotocin-induced diabetes. The proliferation, migration, and tube formation of human umbilical vein endothelial cells were analyzed in vitro upon treatment with liraglutide under normal-glucose and high-glucose conditions. Levels of phospho-Akt, phospho-endothelial nitric oxide synthase, and phospho-extracellular signal-related kinases 1 and 2 under different conditions in human umbilical vein endothelial cells and in ischemic muscle were determined by western blotting. Liraglutide significantly improved perfusion recovery and capillary density in both nondiabetic and diabetic mice. Liraglutide also promoted, in a concentration-dependent manner, the proliferation, migration, and tube formation of normal glucose- and high glucose-treated human umbilical vein endothelial cells, as well as the phosphorylation of Akt, endothelial nitric oxide synthase, and extracellular signal-related kinases 1 and 2 both in vitro and in vivo. The liraglutide antagonist exendin (9-39) reversed the promoting effects of liraglutide on human umbilical vein endothelial cell functions.Furthermore, exendin (9-39), LY294002, and PD98059 blocked the liraglutide-induced activation of Akt/endothelial nitric oxide synthase and extracellular signal-related kinases 1 and 2 signaling pathways. Conclusions These studies identified a novel role of liraglutide in modulating ischemia-induced angiogenesis, possibly through effects on endothelial cell function and activation of Akt/endothelial nitric oxide synthase and extracellular signal-related kinases 1 and 2 signaling, and suggested the glucagon-like peptide-1 receptor may be an important therapeutic target in diabetic hind-limb ischemia.

Exaggerated blood pressure response to static exercise in hindlimb ischemia-reperfusion.

Peripheral artery disease (PAD) reduces the blood flow supply in the affected limbs as one of the significant cardiovascular concerns. Revascularization surgery in the femoral artery plays a central role in treating PAD. Exercise is also a rehabilitation strategy suggested for PAD patients to improve vascular functions. However, the effects of limb ischemia-reperfusion (IR), one of the most predominant complications in revascularization surgery, on exercise-induced arterial blood pressure (BP) response are poorly understood. In the present study, we determined 1) the blood flow status in the hindlimb muscles of rats (plantar muscle, red and white portions of gastrocnemius) with different time points of the hindlimb IR; and 2) the BP response to static muscle contraction in rats at different time points after the blood flow reperfusion procedure. Results of this study indicated that, compared with the Sham group, the blood flow in the hindlimb muscles evaluated by Evans blue concentration was significantly reduced at 6h of femoral artery occlusion (FAO 6h) (vs. sham control, p < 0.05). The decreased blood flow was gradually recovered after the blood flow reperfusion for 18 (IR 18h), 66 (IR 66h), and 114 (IR 114h) hours (p < 0.05 vs. FAO 6h for all IR groups). The response of mean arterial pressure was 20 ± 4mmHg in Sham rats (n = 7); 32 ± 10mmHg in IR 18h rats (n = 10); 27 ± 7mmHg in IR 66h rats (n = 13); 26 ± 4mmHg in IR 114h rats (n = 9) (p < 0.05 vs. Sham for all groups). No significant difference was observed in the peak-developed tension during muscle contraction among all the groups (p > 0.05). In conclusion, static exercise-induced BP response is exaggerated following IR. Whereas the BP response is not statistically significant but tends to decrease with a prolonged IR time, the exaggerated BP response remains through time points from post-IR 18h-114h.

Amelioration of Brain Damage after Treatment with the Methanolic Extract of Glycyrrhizae Radix et Rhizoma in Mice.

Glycyrrhizae Radix et Rhizoma (GR) is a traditional herbal medicine widely used in Asian countries. GR was the most frequently used medicine among stroke patients in Donguibogam, the most representative book in Korean medicine. In the present study, we investigated the neuroprotective effects of the GR methanolic extract (GRex) on an ischemic stroke mice model. Ischemic stroke was induced by a 90 min transient middle cerebral artery occlusion (MCAO), and GRex was administered to mice with oral gavage after reperfusion of MCA blood flow. The MCAO-induced edema and infarction volume was measured, and behavioral changes were evaluated by a novel object recognition test (NORT). Immunofluorescence stains and Western blotting identified underlying mechanisms of the protective effects of GRex. GRex post-treatment in mice with MCAO showed potent effects in reducing cerebral edema and infarction at 125 mg/kg but no effects when the dosage was much lower or higher than 125 mg/kg. GRex inhibited the decrease of spontaneous motor activity and novel object recognition functions. The neuroprotective effects of GRex on ischemic stroke were due to its regulation of inflammation-related neuronal cells, such as microglia and astrocytes.