Biodegradable Redox-Responsive AIEgen-Based-Covalent Organic Framework Nanocarriers for Long-Term Treatment of Myocardial Ischemia/Reperfusion Injury.

Timely restoration of blood supply after myocardial ischemia is imperative for the treatment of acute myocardial infarction but causes additional myocardial ischemia/reperfusion (MI/R) injury, which has not been hitherto effectively targeted by interventions for MI/R injury. Hence, the development of advanced nanomedicine that can reduce apoptosis of cardiomyocytes while protecting against MI/R in vivo is of utmost importance. Herein, a redox-responsive and emissive TPE-ss covalent organic framework (COF) nanocarrier by integrating aggregation-induced emission luminogens and redox-responsive disulfide motifs into the COF skeleton is developed. TPE-ss COF allows for efficient loading and delivery of matrine, a renowned anti-cryptosporidial drug, which significantly reduces MI/R-induced functional deterioration and cardiomyocyte injury when injected through the tail vein into MI/R models at 5 min after 30 min of ischemia. Moreover, TPE-ss COF@Matrine shows a drastic reduction in cardiomyocyte apoptosis and improvements in cardiac function and survival rate. The effect of the TPE-ss COF carrier is further elucidated by enhanced cardiomyocyte viability and triphenyltetrazolium chloride staining in vitro. This work demonstrates the cardioprotective effect of TPE-ss COFs for MI/R injury, which unleashes the immense potential of using COFs as smart drug carriers for the peri-reperfusion treatment of ischemic heart disease with low cost, high stability, and single postoperative intervention.

Nuclear receptor coactivator 4-mediated ferritinophagy contributes to cerebral ischemia-induced ferroptosis in ischemic stroke.

Ischemic stroke poses a significant health risk due to its high rate of disability and mortality. To address this problem, several therapeutic approaches have been proposed, including interruption targeting programmed cell death (PCD). Ferroptosis is a newly defined PCD characterized by iron-dependent accumulation of lipid peroxidation, and is becoming a promising target for treating numerous diseases. To explore the underlying mechanisms of the initiation and execution of ferroptosis in ischemic stroke, we established stroke models in vivo and in vitro simulating ischemia/reperfusion (I/R) neuronal injury. Different from previous reports on stroke, we tested ferroptosis by measuring the levels of core proteins, such as ACSL4, 15-LOX2, Ferritin and GPX4. In addition, I/R injury induces excessive degradation of ferritin via the autophagy pathway and subsequent increase of free iron in neurons. This phenomenon has recently been termed ferritinophagy and reported to be regulated by nuclear receptor coactivator 4 (NCOA4) in some cell lines. Increased NCOA4 in cytoplasm was detected in our study and then silenced by shRNA to investigate its function. Both in vivo and in vitro, NCOA4 deletion notably abrogated ferritinophagy caused by I/R injury and thus inhibited ferroptosis. Furthermore, we found that NCOA4 was upregulated by ubiquitin specific peptidase 14 (USP14) via a deubiquitination process in damaged neurons, and we found evidence of pharmacological inhibition of USP14 effectively reducing NCOA4 levels to protect neurons from ferritinophagy-mediated ferroptosis. These findings suggest a novel and effective target for treating ischemic stroke.

Protection of the myocardium against ischemia/reperfusion injury by angiotensin-(1-9) through an AT2R and Akt-dependent mechanism.

Angiotensin-(19), a peptide of the non-classical renin angiotensin system, has been shown to prevent and revert hypertension and cardiac hypertrophy. We hypothetized that systemic delivery of angiotensin-(1-9) following myocardial infarction will also be protective and extend to provide protection during reperfusion of the ischemic heart. Adult Sprague Dawley rats were subjected to left anterior descending artery ligation and treated with angiotensin-(1-9) via osmotic mini-pump for 2 weeks in the presence or absence of Mas receptor or AT2R antagonists (A779 and PD123319, respectively). Myocardial death and left ventricular function were evaluated after infarction. Infarct size and functional parameters were determined in isolated rat hearts after global ischemia/reperfusion in the presence of angiotensin-(1-9) plus receptor antagonists or Akt inhibitor at reperfusion. in vitro, neonatal rat ventricular cardiomyocytes underwent simulated ischemia/reperfusion and angiotensin-(1-9) was co-incubated with A779, PD123319 or Akt inhibitor. Systemic delivery of angiotensin-(1-9) significantly decreased cell death and improved left ventricular recovery after in vivo myocardial infarction. Perfusion with the peptide reduced the infarct size and improved functional recovery after ex vivo ischemia/reperfusion. In vitro, angiotensin-(1-9) decreased cell death in isolated neonatal rat ventricular cardiomyocytes subjected to simulated ischemia/reperfusion. The cardioprotective effects of angiotensin-(1-9) were blocked by PD123319 and Akti VIII but not by A779. Angiotensin-(1-9) limits reperfusion-induced cell death by an AT2R- and Aktdependent mechanism. Angiotensin-(1-9) is a novel strategy to protect against cardiac ischemia/reperfusion injury.

A minimally invasive catheter-based ultrasound technology for therapeutic interventions in brain: initial preclinical studies.

OBJECTIVE Minimally invasive procedures may allow surgeons to avoid conventional open surgical procedures for certain neurological disorders. This paper describes the iterative process for development of a catheter-based ultrasound thermal therapy applicator. METHODS Using an ultrasound applicator with an array of longitudinally stacked and angularly sectored tubular transducers within a catheter, the authors conducted experimental studies in porcine liver, in vivo and ex vivo, in order to characterize the device performance and lesion patterns. In addition, they applied the technique in a rodent model of Parkinson's disease to investigate the feasibility of its application in brain. RESULTS Thermal lesions with multiple shapes and sizes were readily achieved in porcine liver. The feasibility of catheter-based focused ultrasound in the treatment of brain conditions was demonstrated in a rodent model of Parkinson's disease. CONCLUSIONS The authors show proof of principle of a catheter-based ultrasound system that can create lesions with concurrent thermode-based measurements.

Pneumatic hydrodynamics influence transplastomic protein yields and biological responses during in vitro shoot regeneration of Nicotiana tabacum callus: Implications for bioprocess routes to plant-made biopharmaceuticals.

Transplastomic plants are capable of high-yield production of recombinant biopharmaceutical proteins. Plant tissue culture combines advantages of agricultural cultivation with the bioprocess consistency associated with suspension culture. Overexpression of recombinant proteins through regeneration of transplastomic Nicotiana tabacum shoots from callus tissue in RITA® temporary immersion bioreactors has been previously demonstrated. In this study we investigated the hydrodynamics of periodic pneumatic suspension of liquid medium during temporary immersion culture (4 min aeration every 8 h), and the impact on biological responses and transplastomic expression of fragment C of tetanus toxin (TetC). Biomass was grown under a range of aeration rates for 3, 20 and 40-day durations. Growth, mitochondrial activity (a viability indicator) and TetC protein yields were correlated against the hydrodynamic parameters, shear rate and energy dissipation rate (per kg of medium). A critical aeration rate of 440 ml min-1 was identified, corresponding to a shear rate of 96.7 s-1, pneumatic power input of 8.8 mW kg-1 and initial 20-day pneumatic energy dissipation of 127 J kg-1, at which significant reductions in biomass accumulation and mitochondrial activity were observed. There was an exponential decline in TetC yields with increasing aeration rates at 40 days, across the entire range of conditions tested. These observations have important implications for the optimisation and scale-up of transplastomic plant tissue culture bioprocesses for biopharmaceutical production.

Novel, selective EPO receptor ligands lacking erythropoietic activity reduce infarct size in acute myocardial infarction in rats.

Erythropoietin (EPO) has been shown to protect the heart against acute myocardial infarction in pre-clinical studies, however, EPO failed to reduce infarct size in clinical trials and showed significant safety problems. Here, we investigated cardioprotective effects of two selective non-erythropoietic EPO receptor ligand dimeric peptides (AF41676 and AF43136) lacking erythropoietic activity, EPO, and the prolonged half-life EPO analogue, darbepoetin in acute myocardial infarction (AMI) in rats. In a pilot study, EPO at 100U/mL significantly decreased cell death compared to vehicle (33.8±2.3% vs. 40.3±1.5%, p<0.05) in rat neonatal cardiomyocytes subjected to simulated ischemia/reperfusion. In further studies (studies 1-4), in vivo AMI was induced by 30min coronary occlusion and 120min reperfusion in male Wistar rats. Test compounds and positive controls for model validation (B-type natriuretic peptide, BNP or cyclosporine A, CsA) were administered iv. before the onset of reperfusion. Infarct size (IS) was measured by standard TTC staining. In study 1, 5000U/kg EPO reduced infarct size significantly compared to vehicle (45.3±4.8% vs. 59.8±4.5%, p<0.05). In study 2, darbepoetin showed a U-shaped dose-response curve with maximal infarct size-reducing effect at 5µg/kg compared to the vehicle (44.4±5.7% vs. 65.9±2.7%, p<0.01). In study 3, AF41676 showed a U-shaped dose-response curve, where 3mg/kg was the most effective dose compared to the vehicle (24.1±3.9% vs. 44.3±2.5%, p<0.001). The positive control BNP significantly decreased infarct size in studies 1-3 by approximately 35%. In study 4, AF43136 at 10mg/kg decreased infarct size, similarly to the positive control CsA compared to the appropriate vehicle (39.4±5.9% vs. 58.1±5.4% and 45.9±2.4% vs. 63.8±4.1%, p<0.05, respectively). This is the first demonstration that selective, non-erythropoietic EPO receptor ligand dimeric peptides AF41676 and AF43136 administered before reperfusion are able to reduce infarct size in a rat model of AMI. Therefore, non-erythropoietic EPO receptor peptide ligands may be promising cardioprotective agents.

Retinoic acid ameliorates blood-brain barrier disruption following ischemic stroke in rats.

The intact blood-brain barrier (BBB) is essential in maintaining a stabilized milieu for synaptic and neuronal functions. Disruptions of the BBB have been observed following ischemia and reperfusion, both in patients and in animal models. Retinoic acid (RA), which plays crucial roles during vertebrate organogenesis, has been reported to participate in BBB development. However, it remains unclear whether RA could prevent BBB disruption in ischemic stroke. In this study, we determined that the injection of RA for 4 consecutive days resulted in increases in zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) expression, which are crucial components of the BBB structure. We demonstrated that RA pretreatment could alleviate the ischemic stroke-induced enlargement of vascular permeability, which is related to the up-regulated expression of ZO-1 and VE-cadherin proteins in rat models of middle cerebral artery occlusion (MCAO). Our findings further corroborated that the RA protective effect on BBB is dependent on RA receptor α in vitro oxygen-glucose deprivation (OGD) treatment. Significantly, RA administration immediately after MCAO reduced tissue plasminogen activator (tPA)-induced intracerebral hemorrhage (ICH) and ameliorated neurological deficits 24h after ischemic stroke. Taken together, our results suggest that RA may become a new therapeutic approach to prevent BBB dysfunction and tPA-induced ICH in ischemic stroke.

Ex situ germination as a method for seed viability assessment in a peatland orchid, Platanthera blephariglottis.

UNLABELLED: • PREMISE OF THE STUDY: Assessing seed quality in orchids has been hindered by stringent germination requirements. Seed quality has traditionally been assessed in orchids using in vitro or in situ germination protocols or viability staining. However, these methods are not always well suited for rapid assessment of viability in the context of ecological studies.• METHODS: The potential of an ex situ protocol for seed viability assessment of orchids in ecological studies was investigated by sowing seeds of Platanthera blephariglottis on Sphagnum moss collected in the orchid's natural habitat. Ex situ germination results were compared with those obtained by viability staining using triphenyltetrazolium chloride (TTC), and the effect of seed testa color on staining and germination results was investigated.• KEY RESULTS: The ex situ protocol yielded high germination rates, with 66% of the seeds germinating after 9 wk. Depending on the seed testa color class, ex situ germination rates were about 1.4 to 2.5 times higher than viability rates determined using TTC, indicating that the TTC technique underestimated viability compared with the method using ex situ germination. The TTC estimates of viability rates were higher for seeds with dark-colored testae than for pale ones, whereas seed testa color had no effect on germination.• CONCLUSIONS: Our study showed promising results for the use of ex situ germination as an alternative to previously developed protocols for seed viability assessment of orchids in ecological studies. Staining using TTC might not be well suited for this purpose, since it introduced a bias with respect to seed testa color.

MiR-139-5p inhibits HGTD-P and regulates neuronal apoptosis induced by hypoxia-ischemia in neonatal rats.

Human growth transformation dependent protein (HGTD-P) is a newly identified protein that promotes neuronal apoptosis in hypoxia-ischemia brain damage (HIBD) in neonatal rats. However, the mechanisms regulating HGTD-P expression are not clear. Here we describe microRNAs targeted to HGTD-P and examine their effects on regulating neuronal apoptosis in HIBD. We use samples from cultured neurons after oxygen-glucose deprivation (OGD) and postnatal day 10 rat brains after hypoxia-ischemia (HI). RT-PCR, Western blotting, and immunostaining are used to detect the expression of HGTD-P and cleaved caspase 3, as well as real-time PCR detects microRNA expression. MicroRNA agomir is used to inhibit the expression of HGTD-P, and DAPI, TUNEL, and TTC staining are employed to detect cell apoptosis and brain damage. Moreover, in vitro processing assay is used to examine the mechanism by which HI down-regulates miR-139-5p expression. We found that miR-139-5p is down-regulated in neurons and rat brains after HI treatment. The expression pattern of miR-139-5p correlates inversely with that of HGTD-P. Furthermore, miR-139-5p agomir inhibits neuronal apoptosis and attenuates HIBD, which is concurrent with down-regulation of HGTD-P. Moreover, pre-miR-139 processing activity decreases in extracts from OGD neurons, and OGD neuronal extracts attenuates the processing of pre-miR-139 by Dicer. In conclusion, HI induces inhibitors which block the processing step of pre-miR-139, resulting in the down-regulation of mature miR-139-5p. The down-regulation of miR-139-5p plays a critical role in the up-regulation of HGTD-P expression. MiR-139-5p agomir attenuates brain damage when used 12h after HI, providing a longer therapeutic window than anti-apoptosis compounds currently available.

Diminution of 2,3,5-triphenyltetrazolium chloride toxicity on Listeria monocytogenes growth by iron source addition to the culture medium.

Tetrazolium salts (TTZ) such as 2,3,5-triphenyltetrazolium chloride (TTC) are readily reduced by bacterial populations of various genus. The reduced form of these redox indicators is conspicuously colored allowing a quick and easy detection of growth. The studies are mainly confined to Gram negative bacteria because of an important toxic effect of tetrazolium salts on Gram positive bacteria. Indeed, we observed an important impact of different tetrazolium salts on Listeria monocytogenes growth, curiously limited to an increase in the duration of the lag phase. In this study, we demonstrate that increasing the iron concentration in a medium containing TTC leads to a significant decrease of the lag phase. L. monocytogenes growth was kinetically measured and growth parameters were estimated using the Baranyi model. While lag phase diminution was found to be iron concentration dependent, growth rate was not affected. Addition of iron enables growth of some strains totally inhibited by a 0.4 g/l of TTC and for the other a significant reduction of the latency is observed. The nature of the mechanism resulting in a decrease of the observed lag phase remains unclear. Then, the use of iron supplementation may be proposed to overcome the inhibitory effect of TTC on L. monocytogenes.

Activation of invariant natural killer T cells by α-galactosylceramide ameliorates myocardial ischemia/reperfusion injury in mice.

Invariant natural killer T (iNKT) cells orchestrate tissue inflammation via regulating various cytokine productions. However the role of iNKT cells has not been determined in myocardial ischemia/reperfusion (I/R) injury. The purpose of this study was to examine whether the activation of iNKT cells by α-galactosylceramide (α-GC), which specifically activates iNKT cells, could affect myocardial I/R injury. I/R or sham operation was performed in male C57BL/6J mice. I/R mice received the injection of either αGC (I/R+αGC, n=48) or vehicle (I/R+vehicle, n=49) 30 min before reperfusion. After 24h, infarct size/area at risk was smaller in I/R+αGC than in I/R+vehicle (37.8 ± 2.7% vs. 47.1 ± 2.5%, P<0.05), with no significant changes in area at risk. The numbers of infiltrating myeloperoxidase- and CD3-positive cells were lower in I/R+αGC. Apoptosis evaluated by TUNEL staining and caspase-3 protein was also attenuated in I/R+αGC. Myocardial gene expression of tumor necrosis factor-α and interleukin (IL)-1ß in I/R+αGC was lower to 46% and 80% of that in I/R+vehicle, respectively, whereas IL-10, IL-4, and interferon (IFN)-γ were higher in I/R+αGC than I/R+vehicle by 2.0, 4.1, and 9.6 folds, respectively. The administration of anti-IL-10 receptor antibody into I/R+αGC abolished the protective effects of αGC on I/R injury (infarct size/area at risk: 53.1 ± 5.2% vs. 37.4 ± 3.5%, P<0.05). In contrast, anti-IL-4 and anti-IFN-γ antibodies did not exert such effects. In conclusion, activated iNKT cells by αGC play a protective role against myocardial I/R injury through the enhanced expression of IL-10. Therapies designed to activate iNKT cells might be beneficial to protect the heart from I/R injury.

Role of hypoxia inducible factor-1α in remote limb ischemic preconditioning.

Remote ischemic preconditioning (RIPC) has emerged as a feasible and attractive therapeutic procedure for heart protection against ischemia/reperfusion (I/R) injury. However, its molecular mechanisms remain poorly understood. Hypoxia inducible factor-1α (HIF-1α) is a transcription factor that plays a key role in the cellular adaptation to hypoxia and ischemia. This study's aim was to test whether RIPC-induced cardioprotection requires HIF-1α upregulation to be effective. In the first study, wild-type mice and mice heterozygous for HIF1a (gene encoding the HIF-1α protein) were subjected to RIPC immediately before myocardial infarction (MI). RIPC resulted in a robust HIF-1α activation in the limb and acute cardioprotection in wild-type mice. RIPC-induced cardioprotection was preserved in heterozygous mice, despite the low HIF-1α expression in their limbs. In the second study, the role of HIF-1α in RIPC was evaluated using cadmium (Cd), a pharmacological HIF-1α inhibitor. Rats were subjected to MI (MI group) or to RIPC immediately prior to MI (R-MI group). Cd was injected 18 0min before RIPC (Cd-R-MI group). RIPC induced robust HIF-1α activation in rat limbs and significantly reduced infarct size (IS). Despite Cd's inhibition of HIF-1α activation, RIPC-induced cardioprotection was preserved in the Cd-R-MI group. RIPC applied immediately prior to MI increased HIF-1α expression and attenuated IS in rats and wild-type mice. However, RIPC-induced cardioprotection was preserved in partially HIF1a-deficient mice and in rats pretreated with Cd. When considered together, these results suggest that HIF-1α upregulation is unnecessary in acute RIPC.

Tetrazolium reduction allows assessment of biofilm formation by Campylobacter jejuni in a food matrix model.

AIMS: To develop a staining method for specific detection of metabolically active (viable) cells in biofilms of the foodborne pathogen Campylobacter jejuni. METHODS AND RESULTS: Conversion of 2,3,5 triphenyltetrazolium chloride (TTC) to insoluble, red 1,3,5-triphenylformazan (TPF) was dependent on metabolic activity of Camp. jejuni. When used with chicken juice, TTC staining allowed quantification of Camp. jejuni biofilm levels, whereas the commonly used dye, crystal violet, gave high levels of nonspecific staining of food matrix components (chicken juice). The assay was optimized to allow for monitoring of biofilm levels and adapted to monitor levels of Camp. jejuni in broth media. CONCLUSIONS: Staining with TTC allows for the quantification of metabolically active Camp. jejuni and thus allows for quantification of viable cells in biofilms and food matrices. The TTC staining method can be adapted to quantify bacterial cell concentration in a food matrix model, where the accepted method of A600 measurement is not suitable due to interference by components of the food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: 2,3,5 Triphenyltetrazolium chloride (TTC) staining is a low-cost technique suitable for use in biofilm analysis, allowing rapid and simple imaging of metabolically active cells and increasing the methods available for biofilm assessment and quantification.

Legacy Effects in Buds and Leaves of European Beech Saplings (Fagus sylvatica) after Severe Drought.

Against the background of climate change, we studied the effects of a severe summer drought on buds of European beech (Fagus sylvatica L.) saplings and on leaves formed during the subsequent spring in trees attributed to different drought-damage classes. For the first time, we combined assessments of the vitality (assessed through histochemical staining), mass and stable carbon isotope ratios (δ13C) of buds from drought-stressed woody plants with morphological and physiological variables of leaves that have emerged from the same plants and crown parts. The number, individual mass and vitality of the buds decreased and δ13C increased with increasing drought-induced damage. Bud mass, vitality and δ13C were significantly intercorrelated. The δ13C of the buds was imprinted on the leaves formed in the subsequent spring, but individual leaf mass, leaf size and specific leaf area were not significantly different among damage classes. Vitality and δ13C of the buds are suitable indicators of the extent of preceding drought impact. Bud vitality may be used as a simple means of screening saplings for the flushing capability in the subsequent spring. European beech saplings are susceptible, but-due to interindividual differences-are resilient, to a certain extent, to a singular severe drought stress.

Re-evaluation and modification of dehydrogenase activity tests in assessing microbial activity for wastewater treatment plant operation.

Reliable and cost-effective methods for monitoring microbial activity are critical for process control in wastewater treatment plants. The dehydrogenase activity (DHA) test has been recognized as an efficient measure of biological activity due to its simplicity and broad applicability. Nevertheless, the existing DHA test methods suffer from imperfections and are difficult to implement as routine monitoring techniques. In this work, an accurate and cost-effective modified DHA approach was developed and the procedure for the DHA test was critically evaluated with respect to the standard construction, sample pretreatment, incubation and extraction conditions. The feasibility of the modified DHA test was demonstrated by comparison with the oxygen uptake rate and adenosine triphosphate in a sequencing batch reactor. The sensitivities of the two typical tetrazolium salts to toxicant inhibition by heavy metals and antibiotics were compared, revealing that 2,3,5-triphenyltetrazolium chloride (TTC) exhibited a higher sensitivity. Furthermore, the sensitivity mechanism of the two DHA tests was elucidated through electrochemical experiments, theoretical analysis and molecular simulations. Both tetrazolium salts were found to be effective artificial electron acceptors due to their low redox potentials. Molecular docking simulations revealed that TTC could outperform other tetrazolium salts in accepting electrons and hydrogens from dehydrogenase. Overall, the modified DHA approach presents an accurate and cost-effective way to measure microbial activity, making it a practical tool for wastewater treatment plants.

Bioactive properties and organosulfur compounds profiling of newly developed garlic varieties of Bangladesh.

Studies are being carried out on achieving the maximum quality of garlic through various approaches. In Bangladesh, new garlic varieties (BARI 1-4, BAU-1, BAU-2, BAU-5) have been recently developed by artificial selection to enhance their quality. The present study aimed to evaluate their potency in terms of bioactive properties and organosulfur compounds content using different bioassay and GC-MS techniques while comparing them with other accessible varieties (Chinese, Indian, Local). The new variety, BARI-3 showed the highest antioxidant activity and total phenolic content. It was also found with the highest level of a potent blood pressure-lowering agent, 2-vinyl-4H-1,3-dithiine (78.15 %), which is never reported in any garlic at this percentage. However, the local variety exhibited greater inhibitory properties against the tested organisms including multidrug-resistant pathogens compared to other varieties. This study primarily shows the potential of these two kinds of garlic for their further utilization and development.

Potential protective effects of Azelnidipine against cerebral ischemia-reperfusion injury in male rats.

This study was performed to evaluate the neuroprotective effect of Azelnidipine in cerebral ischemia/reperfusion and to envisage its mechanisms. Twenty-eight adult male Sprague-Dawley rats weighing 200-300 g were randomized into 4 groups (7 rats in each group). Sham (neck dissection without bilateral common carotid artery occlusion), control (30 minutes of bilateral common carotid artery occlusion and reperfusion for 1 hour), vehicle (identical volume of 0.3% carboxymethylcellulose (CMC) orally every day then bilateral common artery occlusion and reperfusion), and Azelnipine-treated rats (7 days of Azelnidipine pretreatment 3 mg/kg/day followed by bilateral common carotid artery occlusion and reperfusion). In addition to brain infarct volume and histopathological assessment, the brain tissues were harvested to evaluate cerebral IL-6, IL-10, TNF-α, ICAM-1, NF-κB p65, and total antioxidant capacity levels. Cerebral levels of IL-6, IL-10, TNF-α, NF-κB p65, and ICAM-1, besides cerebral infarct volume, were significantly elevated in control and vehicle related to sham groups, while total antioxidant capacity was markedly reduced. Azelnidipine treatment resulted in remarkable upregulation of total antioxidant capacity; meanwhile, IL-6, TNF-α, NF-κB p65, and ICAM-1 showed a considerable reduction. Cerebral IL-10 levels were not affected by Azelnidipine pretreatment. Histologically, control and vehicle rats showed severe ischemic injury, which was greatly reversed by Azelnidipine treatment. The current study disclosed that Azelnidipine could markedly reduce cerebral infarct volume and ameliorate histopathological damage in male rats exposed to cerebral ischemia/reperfusion. The neuroprotective effects of Azelnidipine probably stemmed from its anti-inflammatory and antioxidative properties. Azelnidipine had no effect on cerebral IL-10 levels.

Brain-targeting, acid-responsive antioxidant nanoparticles for stroke treatment and drug delivery.

Stroke is the leading cause of death and disability. Currently, there is no effective pharmacological treatment for this disease, which can be partially attributed to the inability to efficiently deliver therapeutics to the brain. Here we report the development of natural compound-derived nanoparticles (NPs), which function both as a potent therapeutic agent for stroke treatment and as an efficient carrier for drug delivery to the ischemic brain. First, we screened a collection of natural nanomaterials and identified betulinic acid (BA) as one of the most potent antioxidants for stroke treatment. Next, we engineered BA NPs for preferential drug release in acidic ischemic tissue through chemically converting BA to betulinic amine (BAM) and for targeted drug delivery through surface conjugation of AMD3100, a CXCR4 antagonist. The resulting AMD3100-conjugated BAM NPs, or A-BAM NPs, were then assessed as a therapeutic agent for stroke treatment and as a carrier for delivery of NA1, a neuroprotective peptide. We show that intravenous administration of A-BAM NPs effectively improved recovery from stroke and its efficacy was further enhanced when NA1 was encapsulated. Due to their multifunctionality and significant efficacy, we anticipate that A-BAM NPs have the potential to be translated both as a therapeutic agent and as a drug carrier to improve the treatment of stroke.

Alleviation of brain injury by applying TGN-020 in the supraoptic nucleus via inhibiting vasopressin neurons in rats of focal ischemic stroke.

AIMS: To understand mechanisms underlying vasopressin hypersecretion in stroke and its association with brain injury, we investigated effects of blocking aquaporin 4 (AQP4) in the supraoptic nucleus (SON) on vasopressin neuronal activity and cerebral injuries in male rats of unilateral middle cerebral artery occlusion (MCAO). MAIN METHODS: Establishing MCAO model without or with microinjection of TGN-020 into the SON, performing Western blots and immunohistochemistry and analyzing the expression levels and spatial distribution of functional proteins in the SON and/or the cerebral cortex. KEY FINDINGS: MCAO increased plasma vasopressin levels, caused neurological damage and increased glycogen synthase kinase 3ß (GSK-3ß) in the SON and the cortex of MCAO side. In the SON, MCAO significantly increased c-Fos in vasopressin neurons and astrocytic somata in the ventral glial lamina. MCAO significantly reduced glial fibrillary acidic protein (GFAP) and AQP4 around vasopressin neurons, which accompanied separation of GFAP from AQP4. By contrast, blocking AQP4 by microinjection of TGN-020 into the SON blocked MCAO-evoked GSK-3ß increase as well as the reduction of AQP4 relative to GFAP around vasopressin neurons in the SON. In the cortex, TGN-020 in the SON also blocked MCAO-evoked increase in GSK-3ß while reduced neurological damages. SIGNIFICANCE: These findings indicate that MCAO disrupts interactions of GFAP with AQP4 in astrocytic processes in the SON, which increases vasopressin neuronal activity. Blocking AQP4 in the SON can block abnormal activation of vasopressin neurons and alleviate ischemic brain injury, which provides novel targets for alleviating ischemic brain injury.

Lepidium meyenii (maca) and soy isoflavones reduce cardiac stunning of ischemia-reperfusion in rats by mitochondrial mechanisms.

BACKGROUND AND AIM: Phytoestrogens are traditionally used for cardiovascular risks but direct effects on the ischemic heart remain unclear. Plants with phytoestrogens are used for reducing menopausic symptoms and they could also be cardioprotectives. Here we investigated whether maca (Lepidium meyenii) contains isoflavones and prevents cardiac stunning, in comparison to soy isoflavones. EXPERIMENTAL PROCEDURE: Both products were orally and daily administered to rats during 1 week before exposing isolated hearts to ischemia/reperfusion (I/R). Young male (YM), female (YF) and aged female (AgF) rats treated with maca (MACA, 1 g/kg/day) or soy isoflavones (ISOF, 100 mg/kg/day) were compared to acute daidzein (DAZ, 5 mg/kg i.p.) and non-treated rat groups. Isolated ventricles were perfused inside a calorimeter to simultaneously measure contractile and calorimetrical signals before and during I/R. RESULTS AND CONCLUSIONS: Maca has genistein and daidzein. MACA and ISOF improved the post-ischemic contractile recovery (PICR) and muscle economy (P/Ht) in YM and YF hearts, but not in AgF hearts. DAZ improved PICR and P/Ht more in YM than in YF. The mKATP channels blockade reduced both PICR and P/Ht in DAZ-treated YM hearts, without affecting them in ISOF or MACA-treated YM hearts. In MACA treated YF hearts, the simultaneous blockade of NOS and mKATP channels, or the mNCX blockade reduced cardioprotection. Results show that subacute oral treatment with maca or with soy isoflavones was strongly preventive of cardiac ischemic dysfunction, more than the acute administration of a pure isoflavone (daidzein, genistein). Maca induced synergistic and complex mechanisms which prevented mitochondrial calcium overload.