Ischemic stroke accounts for approximately 80% of all stroke cases worldwide and remains a leading cause of long‑term neurological disability and mortality. Intravenous thrombolysis and endovascular thrombectomy constitute the mainstay of acute reperfusion therapies, yet their clinical utility is severely constrained by narrow therapeutic time windows. Even when large‑vessel recanalization is successfully achieved, a considerable proportion of patients still suffer poor functional outcomes due to futile reperfusion and ischemia‑reperfusion injury. Historically, preclinical studies have largely focused on direct neuronal protection against calcium overload, oxidative stress and ferroptosis. Unfortunately, most promising neuroprotective agents that exhibited robust benefits in rodent models have failed to demonstrate satisfactory efficacy in phase‑Ⅱ and phase‑Ⅲ clinical trials. One critical contributor to such translational failure is the disregard for the pathological disturbance within the cerebral vascular microenvironment. Instead of merely regarding neuronal death as an autonomous cellular event, accumulating evidence indicates that sequential biochemical disruption of perivascular microenvironment acts as an initiating amplifier that propagates damage toward brain parenchyma after ischemic insult. The cerebral vascular microenvironment centers on brain microvessels and comprises endothelial cells, endothelial glycocalyx, basement membrane, pericytes, astrocytic end‑feet, perivascular microglia, as well as interstitial ions, inflammatory mediators, reactive oxygen species and extracellular matrix components. Distinct from the concept of neurovascular unit which emphasizes functional coupling between neurons, glia and vessels, the vascular microenvironment highlights dynamic biochemical homeostasis in perivascular interstitial space. Following cerebral ischemia, injured brain tissue can be spatially divided into the irreversibly damaged infarct core, metabolically‑stressed ischemic penumbra and mildly‑affected peripheral regions. These three zones lack rigid anatomical boundaries and evolve dynamically depending on reperfusion timing, presenting divergent features in blood perfusion, blood‑brain barrier integrity, cellular viability and immune‑inflammatory profiles. Four interrelated pathological cascades jointly disrupt microenvironmental homeostasis: calcium‑overload‑triggered cytotoxic cascades; blood‑brain barrier breakdown resulting from glycocalyx shedding, tight‑junction dissociation and matrix metalloproteinase‑driven basement‑membrane degradation; inflammatory‑immune dysregulation dominated by microglial phenotypic reprogramming; and mutual crosstalk between coagulation disturbance and neuroinflammation that exacerbates secondary brain injury. Multiple molecular layers orchestrate these pathological alterations. Excessive activation of HIF‑1α, NF‑κB and MAPK/ERK signaling pathways promotes barrier leakage, pro‑inflammatory factor secretion and extracellular‑matrix remodeling. Epigenetic modulation mediated by non‑coding RNAs including miRNAs and lncRNAs reshapes vascular microenvironment via three major patterns: modulating endothelial‑pericyte barrier integrity, switching microglia‑astrocyte inflammatory phenotypes, and driving metabolic reprogramming among multiple cell populations within microdomains. In addition, metabolic shift toward glycolysis among endothelial cells, microglia, astrocytes and compromised neurons establishes self‑amplifying vicious cycles of energy depletion and oxidative stress in perivascular niches. Targeted theranostic approaches oriented toward vascular microenvironment bring new opportunities for precision medicine of ischemic stroke. Magnetic nanoprobes and near‑infrared fluorescent probes respond to elevated reactive oxygen species, pH shift and inflammatory signals, enabling in situ visualization of pathological signatures of microenvironment and risk evaluation of hemorrhagic transformation. Function‑tailored nanodelivery systems exert multi‑modal protective effects: reactive oxygen species‑responsive polymeric micelles deliver calcium chelators to block calcium‑overload‑initiated injury cascades; nanozymes scavenge reactive oxygen/nitrogen species and revert microglial pro‑inflammatory polarization; biomimetic membrane‑coated nanoparticles accumulate at micro‑thrombosis sites to break thrombo‑inflammatory loops and mitigate no‑reflow phenomenon; ferroptosis‑inhibitor‑loaded nanocarriers eliminate excessive labile iron within perivascular toxic microdomains. During subacute and recovery phases, spatiotemporally controlled release of angiogenic factors by nanocarriers facilitates maturation of newly‑formed microvessels rather than blind vascular sprouting, which helps reconstruct intact blood‑brain barrier and restore microenvironmental homeostasis. Nevertheless, substantial translational bottlenecks still impede clinical transformation of these nanomedicines, including potential biosafety risks caused by in vivo material accumulation, protein‑corona‑induced impairment of targeting capacity, intrinsic blood brain barrier transport obstacles, lack of unified quality‑control criteria for large‑scale manufacture, reliance on high‑end clinical imaging instruments, and prominent interspecies discrepancies between experimental animals and human patients. On the basis of summarizing spatiotemporal pathological characteristics, multi‑level regulatory mechanisms and cutting‑edge microenvironment‑oriented diagnostic and interventional strategies, the present review advocates a conceptual shift from isolated neuronal protection to comprehensive reconstruction of perivascular vascular microenvironment homeostasis. Such perspective provides theoretical foundations for expanding therapeutic time windows and inspires future development of novel therapeutic modalities for ischemic stroke.

A new method was developed for simultaneous detection of total 19 kinds of organophosphate esters(OPEs)and their diester metabolites(di-OPEs)in human serum(1.0 mL)and urine(1.5 mL)with low volume of samples.The target compounds were determined using ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)after acetonitrile liquid-liquid extraction combined with purification using an ENVI-18 solid-phase extraction(SPE)column.OPEs and di-OPEs were separated using a Shim-pack GIST C18 column(100 mm×2.1 mm,2 μm)with a Shim-pack GIST-HP(G)C18 guard column.An electrospray ionization source(ESI)was employed in mass spectrometry analysis,with positive/negative ion mode using the multiple reaction monitoring(MRM).All target compounds were separated within 15 min,and exhibited good linear relationships in the concentration range of 2-100 ng/mL,with correlation coefficients(R2)above 0.994.The method detection limits(MDL)in serum ranged from 0.001 to 0.178 ng/mL and the MDL in urine ranged from 0.001 to 0.119 ng/mL.The recoveries of the analytes spiked in serum and urine matrices at two concentration levels were 30.5%-126.8%,with the relative standard deviations(RSDs)ranged from 1%to 23%.In addition,paired serum and urine samples from 11 patients were analyzed.For all samples tested,the internal standards of OPEs exhibited recoveries between 61%and 114%,whereas the internal standards for di-OPEs had recoveries ranging from 43%to 103%.OPEs and di-OPEs exhibited high detection frequencies in 22 serum and urine samples.Triethyl phosphate(TEP),tributyl phosphate(TBP),tris(2-ethylhexyl)phosphate(TEHP),tris(2-butoxyethyl)phosphate(TBEP),tris(1-chloro-2-propyl)phosphate(TCIPP),triphenyl phosphate(TPHP),tri-m-tolyl-phosphate(TMTP)and 2-ethylhexyl diphenyl phosphate(EHDPP)were universally detected in all serum samples.TCIPP was identified at the highest concentrations(median 0.548 ng/mL)in serum samples.In urine samples,the detection frequency for 12 kinds of target compounds reached 100%.Notably,TBP emerged as the predominant OPE in urine,demonstrating a median concentration of 0.506 ng/mL.Regarding di-OPEs,bis(2-chloroethyl)phosphate(BCEP)and bis(2-butoxyethyl)hydrogen phosphate(BBOEP)were the most abundant in urine,with median concentrations of 6.404 and 2.136 ng/mL,respectively.The total concentrations of OPEs and di-OPEs in serum and urine were 1.580-3.843 ng/mL and 5.149-17.537 ng/mL,respectively.These results not only confirmed the effectiveness of the method in detection of OPEs and di-OPEs in biological matrices,but also revealed the widespread presence of OPE compounds in human body and pointed to potential exposure risks.

Objective To prepare an attenuated Listeria vaccine Lmdd-LMP2A expressing the Ep-stein-Barr virus latent membrane protein 2A ( EBV-LMP2A) and evaluate its specific anti-tumor effects on nasopharyngeal carcinoma.Methods The gene fragment encoding EBV-LMP2A was amplified by PCR analysis and then subcloned into the shuttle vector p1565.PCR restriction enzyme digestion and DNA se-quencing were performed to identify the recombinant shuttle vector p1565-LMP2A.The p1565-LMP2A vector was then transformed into competent strains of the attenuated Listeria monocytogenes ( Lmdd) .The recombi-nant attenuated Listeria vaccine strain Lmdd-LMP2A was verified by Western blot assay.The histological sections of spleen and liver tissues were stained by Haematoxylin and eosin ( H&E) for analysis of inflamma-tion.A tumor-bearing HLA-A2 transgenic mouse model was established by subcutaneous injection of CNE-1/HLA-A2/LMP2A nasopharyngeal carcinoma cell line.The prepared Lmdd-LMP2A vaccine was inoculated into the mice via tail intravenous injection for the evaluation of specific CTL induction and the in vivo anti-tumor effects.Results The shuttle vector p1565-LMP2A and the recombinant attenuated Listeria vaccine strain Lmdd-LMP2A with stable expression of LMP2A protein were successfully constructed.The immunized mice showed mild inflammations with no structural damage and necrosis as indicated by H&E staining.The growth of tumors in tumor-bearing HLA-A2 transgenic mice was significantly inhibited by the immunization of Lmdd-LMP2A vaccine as compared with mice without inoculation.The survival time of mice was prolonged with the immunization of Lmdd-LMP2A vaccine.Conclusion The prepared attenuated Listeria vaccine Lm-dd-LMP2A showed specific anti-tumor effects with the safety advantage, suggesting the possibility of using it for anti-tumor therapy in clinic.