Background and Purpose: Isoleucine is a branched-chain amino acid serving as an essential nutrient resource and metabolic. However， its role in cerebral ischemic stroke remains unknown. Experimental Approach: Middle cerebral artery occlusion (MCAO) was used to mimic in vivo model of stroke. Oxygen-glucose deprivation insult (OGD) was used to injure cultured cortical neurons. High-Performance Liquid Chromatography (HPLC) was used to measure the level of isoleucine. A western blot assay and immunofluorescent staining were used to measure the level of CBFB and PTEN. TTC staining was used to measure the infarct size. Cell death and viability were assessed by LDH and CCK8 assays. DCS was used to stimulate cortical neurons. tDCS was used to stimulate the cortex. Key Results: Extraneuronal isoleucine is decreased and intraneuronal isoleucine is increased after rat cerebral I/R injury. Reducing intraneuronal isoleucine via inhibition of its transporter, LAT1 promotes neuronal survival whereas supplementing isoleucine aggravates neuronal damage. Isoleucine downregulates the expression of CBFB, and that acts upstream of PTEN to mediate isoleucine-induced neuronal damage after OGD insult. To identify the therapeutic approach that suppresses the ischemia-induced increase of intraneuronal isoleucine, we tested the effect of tDCS on isoleucine. Our data suggest that Cathodal tDCS can reduce cerebral infarct size. And such neuroprotection is mediated through reducing LAT1-dependent increase of intraneuronal isoleucine. Conclusions and Implications: This study identifies LAT1- dependent increase of intraneuronal isoleucine promotes neuronal death after rat cerebral I/R injury. Our results indicate that tDCS protects against rat cerebral I/R injury through regulating LAT1-isoleucine-CBFB-PTEN signaling.
Abstract Isoleucine is a branched chain amino acid. The role of isoleucine in cerebral ischemia–reperfusion injury remains unclear. Here, we show that the concentration of isoleucine is decreased in cerebrospinal fluid in a rat model of cerebral ischemia–reperfusion injury, the rat middle cerebral artery occlusion (MCAO). To our surprise, the level of intraneuronal isoleucine is increased in an in vitro model of cerebral ischemia injury, the oxygen–glucose deprivation (OGD). We found that the increased activity of LAT1, an L‐type amino acid transporter 1, leads to the elevation of intraneuronal isoleucine after OGD insult. Reducing the level of intraneuronal isoleucine promotes cell survival after cerebral ischemia–reperfusion injury, but supplementing isoleucine aggravates the neuronal damage. To understand how isoleucine promotes ischemia‐induced neuronal death, we reveal that isoleucine acts upstream to reduce the expression of CBFB (core binding factor β, a transcript factor involved in cell development and growth) and that the phosphatase PTEN acts downstream of CBFB to mediate isoleucine‐induced neuronal damage after OGD insult. Interestingly, we demonstrate that direct‐current stimulation reduces the level of intraneuronal isoleucine in cortical cultures subjected to OGD and that transcranial direct‐current stimulation (tDCS) decreases the cerebral infarct volume of MCAO rat through reducing LAT1‐depencent increase of intraneuronal isoleucine. Together, these results lead us to conclude that LAT1 over activation‐dependent isoleucine‐CBFB‐PTEN signal transduction pathway may mediate ischemic neuronal injury and that tDCS exerts its neuroprotective effect by suppressing LAT1 over activation‐dependent signalling after cerebral ischemia–reperfusion injury.
Thrombosis and neointimal hyperplasia are the main causes for the failure of small diameter vascular grafts, and a complete and functional endothelium is essential in preventing these problems. Therefore, grafts that could be endothelialized rapidly are highly desirable. This study constructed a vascular graft with catalytic nitric oxide (NO) generation and promoted endothelial cell (EC) adhesion for rapid in situ endothelialization, and examined the in vivo performance of an NO-generating vascular graft for the first time. A macroporous electrospun polycaprolactone (PCL) graft was prepared and modified via layer-by-layer self-assembly. Organoselenium immobilized polyethyleneimine was loaded onto the graft for in situ catalytic NO generation, while hyaluronic acid was grafted with an EC specific peptide Arg-Glu-Asp-Val and deposited to promote EC adhesion. This dual-modified material generated a strong and sustained flow of NO from S-nitrosoglutathione and significantly enhanced EC adhesion in vitro. In a co-culture experiment of ECs and smooth muscle cells (SMCs), this material promoted the adhesion of ECs and increased the EC/SMC ratio. After implantation in rats, the modified grafts showed a remarkably promoted endothelialization compared to PCL ones with an endothelium coverage of 89% versus 55% after 4 weeks, and the ECs on modified grafts were better organized in a pattern similar to that of the native vessel. The results indicated that the combination of catalytic NO generation and promoted EC adhesion proposed in this work may be a promising method for rapid endothelialization of small diameter vascular grafts.
Hypoxia, induced by inadequate oxygen supply, is a key indication of various major illnesses, which necessitates the need to develop new nanoprobes capable of sensing hypoxia environments for the targeted system monitoring and drug delivery. Herein, we report a hypoxia-responsive, periodic mesoporous organosilica (PMO) nanocarrier for repairing hypoxia damage. β-cyclodextrin (β-CD) capped azobenzene functionalization on the PMO surface could be effectively cleaved by azoreductase under a hypoxia environment. Moreover, the nanosystem is equipped with fluorescence resonance energy transfer (FRET) pair (tetrastyrene derivative (TPE) covalently attached to the PMO framework as the donor and Rhodamine B (RhB) in the mesopores as the receptor) for intracellular visualization and tracking of drug release in real-time. The design of intelligent nanocarriers capable of simultaneous reporting and treating of hypoxia conditions highlights a great potential in the biomedical domain.
Experiments were performed in 42 urethane-anaesthetized(0.5g/kg,iv)rab-bits of both sexes.Discharge of 60 units correlated with respiration was recordedwith glass microelectrodes in the septum.The units could be divided into 3 types:30 related to inspiratory phase,16 with expiratory phase,and 14 being phase-span-ning.The respiratory unit discharges in septum were shown to exist after bilateralvagotomy or injection of nekethamide.Some of them could be induced by asphy-xia.The others emerged spontaneously respiratory discharges which disappearedimmediately after minute injection of 2% sod.pentothal into the fourth ventricle.It is suggested that the respiratory information on to the septum of the limbicsystem might be considered to play a significant role in the coordination of autono-mic functions and emotional activities.
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