Evans Blue Research Articles

Early hyperbaric oxygen therapy through regulating the HIF-1α signaling pathway attenuates Neuroinflammation and behavioral deficits in a mouse model of Sepsis-associated encephalopathy

BackgroundSepsis-associated encephalopathy (SAE) presents a significant clinical challenge, associated with increased mortality and healthcare expenses. Hyperbaric oxygen therapy (HBOT), involving inhaling pure or highly concentrated oxygen under pressures exceeding one atmosphere, has demonstrated neuroprotective effects in various conditions. However, the precise mechanisms underlying its protective actions against sepsis-associated brain injury remain unclear. This study aimed to determine whether HBOT protects against SAE and to elucidate the impact of the hypoxia-inducible factor-1α (HIF-1α) signaling pathway on SAE. MethodsThe experiment consisted of two parts. In the first part, C57BL/6 J male mice were divided into five groups using a random number table method: control group, sham surgery group, sepsis group, HBOT + sepsis group, and HBOT + sham surgery group. In the subsequent part, C57BL/6 J male mice were divided into four groups: sepsis group, HBOT + sepsis group, HIF-1α + HBOT + sepsis group, and HIF-1α + sepsis group. Sepsis was induced via cecal ligation and puncture (CLP). Hyperbaric oxygen therapy was administered at 1 h and 4 h post-CLP. After 24 h, blood and hippocampal tissue were collected for cytokine measurements. HIF-1α, TNF-α, IL-1β, and IL-6 expression were assessed via ELISA and western blotting. Microglial expression was determined by immunofluorescence. Blood-brain barrier permeability was quantified using Evans Blue. Barnes maze and fear conditioning were conducted 14 days post-CLP to evaluate learning and memory. ResultsOur findings reveal that CLP-induced hippocampus-dependent cognitive deficits coincided with elevated HIF-1α and increased TNF-α, IL-1β, and IL-6 levels in both blood and hippocampus. Observable activation of microglial cells in the hippocampus and increased blood-brain barrier (BBB) permeability were also evident. HBOT mitigated HIF-1α, TNF-α, IL-1β, and IL-6 levels, attenuated microglial activation in the hippocampus, and significantly improved learning and memory deficits in CLP-exposed mice. Additionally, these outcomes were corroborated by injecting a lentivirus that overexpressed HIF-1α into the hippocampal region of the mice. ConclusionHIF-1α escalation induced peripheral and central inflammatory factors, promoting microglial activation, BBB impairment, and cognitive dysfunction. However, HBOT ameliorated these effects by reducing HIF-1α levels in Sepsis-Associated Encephalopathy.

Modulation of the capillary leakage by exogenous albumin in a rat model of endothelial glycocalyx damage.

Endothelial glycocalyx (EG) plays a crucial role in maintaining the plasma proteins within the intravascular space. We studied whether exogenous albumin protects the EG in an experimental model of EG enzymatic damage in rats. Rats were divided into three groups of 10 animals that received (1) Evans blue (2) Evans blue + hyaluronidase, or (3) Evans blue + hyaluronidase + 20% human albumin via the tail vein. Spectrophotometric analysis was performed 2 h later to quantify the leakage of Evans blue-labeled albumin into the heart, lungs, brain, kidneys, liver, small intestine, spleen, and skeletal muscle. Administration of hyaluronidase numerically increased the capillary leakage of Evans blue in all examined tissues. Co-administration of albumin decreased the leakage of albumin in all tissues except the heart. In the lungs, the ratio between the absorbance and dry organ weight decreased from 5.3 ± 2.4 to 1.7 ± 0.5 (mean ± SD) (P < 0.002), and in the liver, the absorbance decreased from 2.2 ± 0.7 to 1.5 ± 0.4 (P < 0.011). Exogenous albumin decreased the capillary leakage of albumin which was interpreted as a sign of maintained EG integrity.

Protein Kinase C Beta II Inhibitor Conjugated with Myristic Acid and Trans-Activator of Transcription improves intracellular delivery and cardiovascular protection in porcine myocardial ischemia/reperfusion injury

Introduction: While timely reperfusion of oxygenated blood is critical during resuscitation of ischemic myocardium, oxidative stress during reperfusion still leads to ischemia/reperfusion (I/R) injury and ultimately, cardiomyocyte death. The major sources of oxidative stress targeted in this study are NADPH oxidase (NOX-2) and mitochondria, which are both activated by protein kinase C beta II (PKCβII). In previous studies with an ex-vivo rat heart I/R model, Myristic Acid (Myr) and Trans-Activator Transcription (Tat; YGRKKRRQRRR) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-; N-Myr-Tat-CC-SLNPEWNET) showed cardioprotective effects and decreased infarct size. In this study, we hypothesized that Myr-Tat-PKCβII- will offer cardioprotective effects in comparison with a scrambled peptide control (Myr-Tat-PKCβII-scram; N-Myr-Tat-CC-WNPESLNTE) in an in-vivo porcine myocardial I/R model. Methods: Regional I(1 hour)/R(3 hours) was induced in Male Yorkshire pigs (38-50kg) by inflating a balloon to occlude the Left Anterior Descending Artery (LAD) at the level of the second diagonal branch (supplies 40% of anterior left ventricle) and then deflating it to allow for reperfusion. Then, a bolus of either Myr-Tat-PKCβII- or Myr-Tat-PKCβII-scram (20ng/kg; ~100pmol in blood) was immediately administered into the LAD. Cardiac function and injury were measured by monitoring changes in ejection fraction (EF) and through routine measurements of serum creatine phosphokinase (CPK), troponin I, and myoglobin, respectively. After I/R, the hearts were stained with 1% Evans Blue dye to identify the area at risk (AR) and 1% triphenyltetrazolium chloride to determine the area of necrosis (AN). Infarct size was then quantified (AN/AR) and all data was analyzed via Student’s t-test. Results: Analysis showed that Myr-Tat-PKCβII- significantly restored EF to within 1.40.7% of baseline compared to controls which only restored EF to within 6.42.1% (p&lt;0.05) of baseline EF. Myr-Tat-PKCβII- showed a significant increase in serum myoglobin levels at 1 hr of reperfusion (1362.1 83.4 μg/mL, n=4) compared to scrambled control (202.1197.3 μg/mL, n=3 p&lt;0.05). Myr-Tat-PKCβII- reduced infarct size to 10.0±2.8%; n=4; compared to scrambled control hearts (28.5±8.3%; n=6; p&lt;0.05). CPK and Troponin I levels were comparable in both groups. These results suggest that Myr-Tat-PKCβII- can help prevent cardiac injury when given immediately after an ischemic event. Conclusions: Data from ex-vivo rat heart I/R model, coupled with data from this in-vivo porcine myocardial I/R model indicate the effcacy of Myr-Tat-PKCβII- in preventing oxidative stress-induced myocardial I/R injury when given at the beginning of reperfusion. These findings suggest that Myr-Tat-PKCβII- could be useful in the clinical setting when administered immediately after cardiac resuscitation. Future studies include the treatment of rat cardiomyocyte cells with Myr-Tat-PKCβII- prior to 24 hr hypoxia/24 hr reoxygenation, followed by cell viability assays compared to untreated control. This additional data can help determine the optimal dose to use in a 12 week survival study using the same porcine myocardial I/R protocol. Funding: This study was funded by the Philadelphia College of Osteopathic Medicine, Department of Biomedical Sciences, Division of Research, and the Center for Chronic Diseases of Aging, the National Heart, Lung &amp; Blood Institute at NIH (grant # 1R43HL160338-01) and Young Therapeutics, LLC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PNA5, a Novel Glycosylated Angiotensin 1-7 Treatment Improves Infarct Size and Fibrotic Remodeling in Ischemic Reperfusion Injury

Background: Reperfusion during an acute ischemic event is of paramount importance to rescue cardiac tissue. Paradoxically, reperfusion of the ischemic tissue promotes further cardiac injury. Increased reactive oxidative species (ROS) and inflammation contribute to ischemic reperfusion injury (IR). PNA5, a glycosylated analog of angiotensin-(1-7), decreases ROS and inflammation. The purpose of this study is to indicate if PNA5 treatment has the ability to reduce infarct (IN) size and improve cardiac remodeling in IR. We hypothesize that PNA5 will improve fibrosis and diminish IN size in IR. Methods: 3-month-old male mice (C57BI/6J) underwent IR surgery. Mice were anesthetized and the left anterior descending artery was litigated for 45 minutes. Ischemic areas were then reperfused. Mice were immediately injected with PNA5 (subcutaneously s.c. at 100μg/kg, n=11) or with saline as control (n=11). Injections were continued daily with PNA5 or saline for 8-weeks. At the end of treatment, mice were sacrificed. Upon sacrifice, hearts were re-ligated and perfused with Evans Blue to identify the areas at risk (AAR). Hearts were stained with 2-3-4-triphenyltetrazolium chloride to show IN area. IN size and AAR were determined using Image J. Paraffn-embedded hearts were stained with Picrosirius Red for fibrosis. Fibrosis was measured as a ratio of collagen to total tissue area for 17 regions in the left ventricle (LV). Values are represented as mean ± standard error of mean. Results: PNA5-treated mice IN size was significantly decreased compared to saline-treated mice. (IN/AAR: IR-Saline 0.17± 0.02; PNA5 0.10± 0.01, p=0.005; IN/LV: IR-Saline 0.08± 0.01; PNA5 0.05±0.01; p=0.002, student’s T-test). PNA5 reduced fibrosis in the LV compared to IR-saline mice Conclusions: These results indicate PNA5 as a potential therapeutic treatment for IR as it improves IN size and fibrotic remodeling in IR. [Formula: see text] Supported by NIA U01AG066623 to MH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel preclinical model of fentanyl-induced cardiorespiratory collapse to investigate resuscitation outcomes

Introduction: Opioid-related morbidities have become a critical public health concern, with ultrapotent synthetic opioids (UPSO) such as fentanyl contributing to the rise in opioid-related fatalities. Respiratory depression is the cardinal trait of fentanyl overdose, leading to systemic hypoxia and subsequent cardiorespiratory collapse (CRC). There is growing recognition that fentanyl induced CRC (fiCRC) is unique from other forms of sudden CRC. Yet, the consequences of fiCRC and its reversal remain poorly understood. This is due in part to the limited preclinical studies investigating rescue from fiCRC. Objective: using a novel model of fiCRC and subsequent reversal, this ongoing study aims to define the impact of UPSO and naloxone rescue on in vivo survival outcomes. Methods and Results: Male and female C57BL/6J mice were used. UPSO fentanyl i.v. administration (30 to 500 mcg/kg) suppressed breathing and subsequently led to fiCRC. The return of spontaneous circulation (ROSC) was achieved by performing manual chest compressions in combination of epinephrine administration after 8 minutes of fiCRC. In subset of mice, naloxone was administered post ROSC. Outcomes after 72 hours were accessed. The latency of fiCRC (mean arterial pressure at 40 mmHg) exhibited a negative correlation with the doses of fentanyl administered. Resuscitation from fiCRC was unreliable across the dose spectrum, while naloxone consistently rescued the majority of fiCRC animals. Naloxone increased ROSC rate from 54% to 86% when the dose of fentanyl was 30 mcg/Kg (n=22). With naloxone rescue, we compared fiCRC with non-opioid-induced cardiac arrest followed by potassium chloride injection (0.8 mg/g). Both models showed severe lung edema as evidenced by Evans Blue staining. However, the lung wet/dry weight ratio was higher in fiCRC group than in the non-opioid treatment group (5.50±0.05 vs. 6.60±0.60, n=4, p&lt;0.05). Survival in fiCRC group was significantly worse than in non-opioid treatment group (survival rate: 25% vs. 83%, n=17, p&lt;0.05). Conclusions: Our study highlights two important findings. First, the reliable resuscitation of fiCRC necessitates the use of naloxone. Second, the likelihood of injury is significant following successful rescue from fiCRC by naloxone. Our continued work using this fiCRC model will lead to insights that can be leveraged for strategies to mitigate injury and improve survival following fentanyl overdose. R01 HL163965 &amp; R01 DA057767. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

WITHDRAWN: Recombinant osteopontin stabilizes the blood brain barrier following subarachnoid hemorrhage in rats

Background and PurposeRecombinant osteopontin (rOPN) decreases neuronal cell death and brain edema and improves the neurological scores following subarachnoid hemorrhage (SAH) in rats. The molecular mechanisms of the preservation of blood-brain barrier of rOPN following SAH in rats were examined in this study. MethodsAdult rats were divided into 3 groups: sham, vehicle, and treatment groups. Each animal in the SAH group underwent a surgical procedure to induce SAH, and the brains was harvested at 24 hours for analysis. For the mechanism part of the study two additional groups were operated and treated with Fib-14 and Wortmannin additional to rOPN. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 hours after SAH induction. The blood brain barrier (BBB) permeability was evaluated using Evans Blue dye extravasation. The expressions of tight junction proteins claudin-3, claudin-5, occludin and the phosphorylation of β-catenin were examined using Western blot analysis. ResultsrOPN improved neurological scores and reduced brain edema at 24 hours after SAH. The BBB preservation action of rOPN is mediated in part through stabilizing β-catenin and increasing expression of tight junction proteins, claudin-3, claudin-5, and occludin. rOPN is thought to exert its effects through FAK activated PI3K-Akt pathway. ConclusionSince BBB disruption is one of the major pathophysiology of EBI after SAH, rOPN may be a promising drug to maintain BBB integrity and functions in SAH treatment.

Cystatin C Attenuates Perihematomal Secondary Brain Injury by Inhibiting the Cathepsin B/NLRP3 Signaling Pathway in a Rat Model of Intracerebral Hemorrhage.

Secondary brain injury (SBI) is a noticeable contributor to the high mortality and morbidity rates associated with intracerebral hemorrhage (ICH), and effective treatment options remain limited. Cystatin C (CysC) emerges as a novel candidate for SBI intervention. The therapeutic effects and underlying mechanisms of CysC in mitigating SBI following ICH were explored in the current research. An in vivo ICH rat model was established by injecting autologous blood into the right caudate nucleus. Western blotting (WB) was utilized to assess the levels of CysC, cathepsin B (CTSB), and the NLRP3 inflammasome. Subsequently, the ICH rat model was treated with exogenous CysC supplementation or CysC knockdown plasmids. Various parameters, including Evans blue (EB) extravasation, brain water content, and neurological function in rats, were examined. RT-qPCR and WB were employed to determine the expression levels of CTSB and the NLRP3 inflammasome. The co-expression of CTSB, CysC, and NLRP3 inflammasome with GFAP, NeuN, and Iba1 was assessed through double-labeled immunofluorescence. The interaction between CysC and CTSB was investigated using double-labeled immunofluorescence and co-immunoprecipitation. The findings revealed an elevation of CysC expression level, particularly at 24h after ICH. Exogenous CysC supplementation alleviated severe brain edema, neurological deficit scores, and EB extravasation induced by ICH. Conversely, CysC knockdown produced opposite effects. The expression levels of CTSB and the NLRP3 inflammasome were significantly risen following ICH, and exogenous CysC supplement attenuated their expression levels. Double-labeled immunofluorescence illustrated that CysC, CTSB, and the NLRP3 inflammasome were predominantly expressed in microglial cells, and the interaction between CysC and CTSB was evidenced. CysC exhibited potential in ameliorating SBI following ICH via effectively suppressing the activation of the NLRP3 inflammasome mediated by CTSB specifically in microglial cells. These findings underscore the prospective therapeutic efficacy of CysC in the treatment of ICH-induced complications.

Electrochemically assisted preparation of graphitic carbon nitride nanosheet membranes for efficient water purification

The development of a highly effective nanofiltration membrane for water purification is crucial for wastewater recovery. The graphitic carbon nitride (g-C3N4) membrane can be an ideal candidate for advanced water treatment due to its intrinsic porous structure and superior physicochemical stability. However, the developments of the g-C3N4 membranes are limited by their traditional preparation methods involving complicated processes, long reaction time and high-temperature conditions. A simple electrochemical-assisted strategy for the rapid preparation of the g-C3N4 nanosheet membranes at room temperature has been developed in this study. The ultra-thin g-C3N4 membrane with a uniform structure can be synthesized within 40 min, which is at least 7 times quicker compared to the traditional methods. The as-prepared g-C3N4 membrane shows a high water flux rate of ∼ 90 L m−2 h−1 bar−1. Simultaneously, an excellent rejection rate of 99.1% toward Evans blue is achieved based on the synergistic effect of size sieving and electrostatic repulsion. The breakthrough in the preparation method of g-C3N4 nanosheet membranes in this work is of great significance to the development of g-C3N4 membranes in the field of water purification.

Investigation of the effects of cilostazol on the myocardial ischemia-reperfusion injury of rats.

&lt;b&gt;&lt;i&gt;Background&lt;/i&gt;&lt;/b&gt;. Myocardial ischemia, occurring as a consequence of imbalance between oxygen supply and demand, causes a rapid metabolic and structural&lt;br /&gt; impairment within the tissue. After a period of ischemia, sudden onset of reperfusion causes a transition to aerobic metabolism within living cells. Afterwards, emerging substrates initiate a chain of reactions leading to tissue injury. This situation is called “ischemia reperfusion injury”. Despite all technical advancements in anesthesia, myocardial protection and cardiac surgical techniques, we still face the clinical reflections of ischemia reperfusion (IR) injury.&lt;br /&gt; &lt;b&gt;&lt;i&gt;Materials and methods&lt;/i&gt;&lt;/b&gt;. The protective effect of cilostasole on IR injury in an animal model of experimental myocardial ischemia and reperfusion was investigated. In this regional myocardial ischemia model, male Wistar-Albino rats were used as subjects and they were allocated into three groups; ischemia (n=8), sham (n=8), and cilostazole (n=8). LAD was occluded for 45 minutes, and then reperfused for three hours. Rats received Cilostazole 20 mg/kg/d by gastric gavage once daily. During IR hemodynamic parameters were recorded. Serum analysis for CK-MB and Troponin T were analysed at 180th minute of ischemia. Ischemic zone was measured by dying with Evans Blue and infarct area was measured by dying with triphenyltetrazolium chloride.&lt;br /&gt; &lt;b&gt;&lt;i&gt;Results.&lt;/i&gt;&lt;/b&gt; Before the onset of LAD occlusion, as well as at 25th, 60th and 120th minutes of occlusion, all groups were similar in terms of blood pressure and pulse rate.&lt;br /&gt; The total area, affected area and necrotic area were calculated by using formulas; affected area ratio= affected area/total area X 100, necrotic area ratio = necrotic area/total affected area X 100, necrotic area and affected area ratio = necrotic area /affected area X 100.&amp;nbsp; Affected area and total area ratio was significantly higher in IR group, compared with cilostazole group (t=8.965; p&amp;lt;0.001). Similarly, necrotic area and total area ratio was higher in IR group, compared with cilostazole group (t=8.965; p&amp;lt;0.001). The necrotic area and affected area ratios were similar in IR and cilostazole groups (t=0.245; p=0.810). CK-MB level differences were not statistically significant between two groups (Z=0.382; p=0.721).&lt;br /&gt; Troponin levels were similar between IR and cilostazole groups and the difference was not statistically significant (Z=0.630; p=0.574). Pathological specimens of the heart were scanned for myocytolysis, PMNL and hemorrhage.&amp;nbsp; The difference between mean value of MDA enzyme levels were statistically significant (p&amp;lt;0.001) between all groups.&amp;nbsp; MDA enzyme levels, from higher to lower was IR, cilostazole and Sham group.&amp;nbsp; SOD levels (F=5.910; p=0.009) were significantly lower in Sham group when compared with IR group (p=0.008). The differences between Sham and cilostazole groups and IR and cilostazole gropus were not statistically significant (p=0.008). According to planimetric values and enzyme levels, cilostazole was found to be effective in reducing the ischemic zone, without effecting the necrotic zone in cardiac ischemia reperfusion damage. Therefore cilostazole has protective effects agains ischemia reperfusion damage.&lt;br /&gt; &lt;br /&gt; &lt;b&gt;Conclusion&lt;/b&gt;.&amp;nbsp;This study explored how cilostazol affects myocardial ischemia-reperfusion injury in rats, finding that cilostazol administration during reperfusion may protect against such injury. Through various analyses, we observed positive outcomes associated with cilostazol treatment, suggesting its potential in reducing myocardial damage. Further research is needed to understand the underlying mechanisms and optimize therapeutic strategies, but our findings highlight cilostazol's promise in improving clinical outcomes in cardiac interventions.

Focused ultrasound on the substantia nigra enables safe neurotensin-polyplex nanoparticle-mediated gene delivery to dopaminergic neurons intranasally and by blood circulation

Neurotensin-polyplex nanoparticles provide efficient gene transfection of nigral dopaminergic neurons when intracerebrally injected in preclinical trials of Parkinson’s disease because they do not cross the blood–brain barrier (BBB). Therefore, this study aimed to open BBB with focused ultrasound (FUS) on the substantia nigra to attain systemic and intranasal transfections and evaluate its detrimental effect in rats. Systemically injected Evans Blue showed that a two-pulse FUS opened the nigral BBB. Accordingly, 35 μL of neurotensin-polyplex nanoparticles encompassing the green fluorescent protein plasmid (79.6 nm mean size and + 1.3 mV Zeta-potential) caused its expression in tyrosine hydroxylase(+) cells (dopaminergic neurons) of both substantiae nigrae upon delivery via internal carotid artery, retro-orbital venous sinus, or nasal mucosa 30 min after FUS. The intracarotid delivery yielded the highest transgene expression, followed by intranasal and venous administration. However, FUS caused neuroinflammation displayed by infiltrated lymphocytes (positive to cluster of differentiation 45), activated microglia (positive to ionized calcium-binding adaptor molecule 1), neurotoxic A1 astrocytes (positive to glial fibrillary acidic protein and complement component 3), and neurotrophic A2 astrocytes (positive to glial fibrillary acidic protein and S100 calcium-binding protein A10), that ended 15 days after FUS. Dopaminergic neurons and axonal projections decreased but recuperated basal values on day 15 after transfection, correlating with a decrease and recovery of locomotor behavior. In conclusion, FUS caused transient neuroinflammation and reversible neuronal affection but allowed systemic and intranasal transfection of dopaminergic neurons in both substantiae nigrae. Therefore, FUS could advance neurotensin-polyplex nanotechnology to clinical trials for Parkinson’s disease.

415 Intraoperative Molecular Imaging of Gliomas using Indocyanine-Conjugated Choline Kinase Alpha Inhibitor

OBJECTIVES/GOALS: Distinguishing tumor tissue from normal brain parenchyma remains a major challenge during the resection of gliomas, leading to the persistence of tumor cells. This study aims to assess the choline kinase alpha-targeting fluorophore JAS239 as a novel fluorescent agent to intraoperatively visualize gliomas in an orthotopic murine model. METHODS/STUDY POPULATION: The human glioblastoma-derived U87 MG-Luc2 cell line will be intracranially implanted in nude mice and tumor growth will be assessed using bioluminescence imaging. After 14 days, the mice will be treated with either antiangiogenic therapy (10 mg/kg bevacizumab, twice/week) or saline (control). Tumor growth will be monitored until 21-28 days after initial implantation, at which point JAS239 (4.0 mg/kg, 90 min before sacrifice) and Evans Blue (4 ml/kg, 60 min before sacrifice) will be administered. The mice will be sacrificed, and their brains will be harvested and sectioned for near-infrared imaging. The brain sections will be processed for histopathologic analysis, allowing for the correlation of observed fluorescence with the distribution of tumor and comparison of signal-to-background ratios. RESULTS/ANTICIPATED RESULTS: JAS239 is an indocyanine-based choline mimetic (excitation 745 nm, emission 775 nm) that has been shown to cross the blood-tumor barrier (BTB) in rodent glioblastoma studies. PET imaging with choline-based radiotracers like 18F-choline has also been shown to delineate both contrast-enhancing tumor (CET) and non-contrast-enhancing tumor (NCET) regions, supporting the hypothesis that JAS239 will be able to visualize heterogeneous glioma tissue in our mouse model. Evans Blue is a passive dye in the visible light spectrum (excitation 620 nm, emission 680 nm) expected to only fluoresce in CET regions due to the disruption of the BTB. JAS239 is expected to fluoresce in both CET and NCET regions, which will be assessed by the fluorescence in mice treated with bevacizumab (expected to renormalize the BTB and model NCETs). DISCUSSION/SIGNIFICANCE: JAS239 may allow for real-time visualization of heterogeneous glioma tissue, which is important because there are no current intraoperative imaging agents for NCETs. Future research and clinical translation of this class of agents may allow surgeons to maximize the safe resection of gliomas, improving progression-free and overall survival rates.

465 Focused Ultrasound-Mediated Blood-Spinal Cord Barrier Permeabilization: A Promising Targeted Approach for Treating Chronic Spinal Cord Injury in Rat Models

INTRODUCTION: Focused ultrasound (FUS) with microbubble contrast agents can temporarily disrupt the blood-brain barrier (BBB), enabling drug delivery. FUS may also offer a minimally invasive method for delivering therapeutic agents to the affected area in chronic spinal cord injury (SCI) by transiently opening the blood-spinal cord barrier (BSCB). METHODS: Nine female Sprague-Dawley rats underwent a T9-T11 laminectomy with contusion SCI and were randomly allocated into FUS or control group. Three weeks later, FUS was administered intravenously with microbubbles. A pressure sweep was performed until ultra-harmonics were captured using a wideband hydrophone. We administered 2% Evans blue (EB) immediately after FUS confirmed the BSCB permeability. Transcardial perfusion was performed 2 hours later, and spinal cords were collected. Spectrophotometry was used to determine the EB concentration. Statistical analysis was performed using a student t-test. RESULTS: Five rats were treated with FUS while four were used as controls. Successful feedback control was achieved with FUS, and ultra-harmonics were observed in the FUS group at a mean peak pressure of 0.5 MPa. Macroscopic identification of EB in the spinal cord sections of animals exposed to FUS confirmed the localized opening of the BSCB. Spectrophotometry revealed an increase in EB concentration in the FUS group compared to controls: Mean EB concentration was 0.03 μg in the FUS group versus 0.016 μg in controls at 1-cm above the injury (p &lt; 0.001); 0.27 μg versus 0.093 μg at the injury site (p &lt; 0.001); and 0.25 μg versus 0.18 μg at 1-cm below the injury(p &lt; 0.001). CONCLUSIONS: FUS increases the permeability of the BSCB in a chronic contusion SCI rat model. This suggests that FUS-mediated BSCB permeabilization may hold promise as a targeted approach for improving therapeutic outcomes in the treatment of chronic SCI.

Vitamin D3 reduces the symptoms of ovarian hyperstimulation syndrome in mice and inhibits the release of granulosa cell angiogenic factor through pentraxin 3

It has been reported that the effective inhibition of vascular endothelial growth factor (VEGF) can prevent the progression of ovarian hyperstimulation syndrome (OHSS). The present study aimed to investigate the mechanism underlying the effect of vitamin D3 (VD3) on OHSS in mouse models and granulosa cells. The effects of VD3 administration (16 and 24 IU) on ovarian permeability were determined using Evans blue. In addition, ovarian pathology, corpus luteum count, inflammatory responses, and hormone and VEGFA levels were assessed using pathological sections and ELISA. Molecular docking predicted that pentraxin 3 (PTX3) could be a potential target of VD3, and therefore, the effects of human chorionic gonadotropin (hCG) and VD3 as well as PTX3 overexpression on the production and secretion of VEGFA in granulosa cells were also investigated using western blotting and immunofluorescence. Twenty-four IU VD3 significantly reversed the increase in ovarian weight and permeability in mice with OHSS. Additionally, VD3 diminished congestion and the number of corpus luteum in the ovaries and reduced the secretion levels of inflammatory factors and those of estrogen and progesterone. Notably, VD3 downregulated VEGFA and CD31 in ovarian tissues, while the expression levels of PTX3 varied among different groups. Furthermore, VD3 restored the hCG-induced enhanced VEGFA and PTX3 expression levels in granulosa cells, whereas PTX3 overexpression abrogated the VD3-mediated inhibition of VEGFA production and secretion. The present study demonstrated that VD3 could inhibit the release of VEGFA through PTX3, thus supporting the beneficial effects of VD3 administration on ameliorating OHSS symptoms.

Adjudin protects blood–brain barrier integrity and attenuates neuroinflammation following intracerebral hemorrhage in mice

Secondary brain injury exacerbates neurological dysfunction and neural cell death following intracerebral hemorrhage (ICH), targeting the pathophysiological mechanism of the secondary brain injury holds promise for improving ICH outcomes. Adjudin, a potential male contraceptive, exhibits neuroprotective effects in brain injury disease models, yet its impact in the ICH model remains unknown. In this study, we investigated the effects of adjudin on brain injury in a mouse ICH model and explored its underlying mechanisms. ICH was induced in male C57BL/6 mice by injecting collagenase into the right striatum. Mice received adjudin treatment (50 mg/kg/day) for 3 days before euthanization and the perihematomal tissues were collected for further analysis. Adjudin significantly reduced hematoma volume and improved neurological function compared with the vehicle group. Western blot showed that Adjudin markedly decreased the expression of MMP-9 and increased the expression of tight junctions (TJs) proteins, Occludin and ZO-1, and adherens junctions (AJs) protein VE-cadherin. Adjudin also decreased the blood–brain barrier (BBB) permeability, as indicated by the reduced albumin and Evans Blue leakage, along with a decrease in brain water content. Immunofluorescence staining revealed that adjudin noticeably reduced the infiltration of neutrophil, activation of microglia/macrophages, and reactive astrogliosis, accompanied by an increase in CD206 positive microglia/macrophages which exhibit phagocytic characteristics. Adjudin concurrently decreased the generation of proinflammatory cytokines, such as TNF-α and IL-1β. Additionally, adjudin increased the expression of aquaporin 4 (AQP4). Furthermore, adjudin reduced brain cell apoptosis, as evidenced by increased expression of anti-apoptotic protein Bcl-2, and decreased expression of apoptosis related proteins Bax, cleaved caspase-3 and fewer TUNEL positive cells. Our data suggest that adjudin protects against ICH-induced secondary brain injury and may serve as a potential neuroprotective agent for ICH treatment.

Lifibrate attenuates blood-brain barrier damage following ischemic stroke via the MLCK/p-MLC/ZO-1 axis.

Dysfunction of tight junction proteins-associated damage to the blood-brain barrier (BBB) plays an important role in the pathogenesis of ischemic stroke. Lifibrate, an inhibitor of cholinephosphotransferase (CPT), has been used as an agent for serum lipid lowering. However, the protective effects of Lifibrate in ischemic stroke and the underlying mechanism have not been clearly elucidated. Here, we employed an in vivo mice model of MCAO and an OGD/R model in vitro. In the mice models, neurological deficit scores and infarct volume were assessed. Evans Blue solution was used to detect the BBB permeability. The TEER was examined to determine brain endothelial monolayer permeability. Here, we found that Lifibrate improved neurological dysfunction in stroke. Additionally, increased BBB permeability during stroke was significantly ameliorated by Lifibrate. Correspondingly, the reduced expression of the tight junction protein ZO-1 was restored by Lifibrate at both the mRNA and protein levels. Using an in vitro model, we found that Lifibrate ameliorated OGD/R-induced injury in human bEnd.3 brain microvascular endothelial cells by increasing cell viability but reducing the release of LDH. Importantly, Lifibrate suppressed the increase in endothelial monolayer permeability and the reduction in TEER induced by OGD/R via the rescue of ZO-1 expression. Mechanistically, Lifibrate blocked activation of the MLCK/ p-MLC signaling pathway in OGD/R-stimulated bEnd.3 cells. In contrast, overexpression of MLCK abolished the protective effects of Lifibrate in endothelial monolayer permeability, TEER, as well as the expression of ZO-1. Our results provide a basis for further investigation into the neuroprotective mechanism of Lifibrate during stroke.

Electroacupuncture stimulation enhances the permeability of the blood-brain barrier: A systematic review and meta-analysis of preclinical evidence and possible mechanisms.

An important cellular barrier to maintain the stability of the brain's internal and external environment is the blood-brain barrier (BBB). It also prevents harmful substances from entering brain tissue through blood circulation while providing protection for the central nervous system. It should be noted, however, that the intact BBB can be a barrier to the transport of most drugs into the brain via the conventional route of administration, which can prevent them from reaching effective concentrations for the treatment of disorders affecting the central nervous system. Electroacupuncture stimulation has been shown to be effective at opening the BBB in a series of experimental studies. This study systematically analyzes the possibility and mechanism by which electroacupuncture opens the BBB. In PubMed, Web of Science, VIP Database, Wanfang Database, and the Chinese National Knowledge Infrastructure, papers have been published for nearly 22 years aimed at opening the BBB and its associated structures. A comparison of EB content between electroacupuncture and control was selected as the primary outcome. There were also results on vascular endothelial growth factor (VEGF), nerve growth factor (NGF), P-Glycoprotein (P-gp), Matrix Metalloproteinase 9 (MMP-9), and glial fibrillary acidic protein (GFAP). We utilized Review Manager software analysis to analyze correlations between studies with a view to exploring the mechanisms of similarity. Evans Blue infiltration forest plot: pooled effect size of 2.04, 95% CI: 1.21 to 2.87, P < 0.01. These results indicate that electroacupuncture significantly increases EB penetration across the BBB. Most studies have reported that GFAP, MMP-9, and VEGF were upregulated after treatment. P-gp expression decreased as well. Electroacupuncture can open the BBB, and the sparse-dense wave is currently the most effective electroacupuncture frequency for opening the BBB. VEGF plays an important role in opening the BBB. It is also important to regulate the expression of MMP-9 and GFAP and inhibit the expression of P-gp.

Ginsenoside Rd enhances blood-brain barrier integrity after cerebral ischemia/reperfusion by alleviating endothelial cells ferroptosis via activation of NRG1/ErbB4-mediated PI3K/Akt/mTOR signaling pathway

The incidence of ischemic stroke is increasing year by year and showing a younger trend. Impaired blood-brain barrier (BBB) is one of the pathological manifestations caused by cerebral ischemia, leading to poor prognosis of patients. Accumulating evidence indicates that ferroptosis is involved in cerebral ischemia/reperfusion injury (CIRI). We have previously demonstrated that Ginsenoside Rd (G-Rd) protects against CIRI-induced neuronal injury. However, whether G-Rd can attenuate CIRI-induced disruption of the BBB remains unclear. In this study, we found that G-Rd could upregulate the levels of ZO-1, occludin, and claudin-5 in ipsilateral cerebral microvessels and bEnd.3 cells, reduce endothelial cells (ECs) loss and Evans blue (EB) leakage, and ultimately improve BBB integrity after CIRI. Interestingly, the expressions of ACSL4 and COX2 were upregulated, the expressions of GPX4 and xCT were downregulated, the levels of GSH was decreased, and the levels of MDA and Fe2+ were increased in ischemic tissues and bEnd.3 cells after CIRI, suggesting that ECs ferroptosis occurred after CIRI. However, G-Rd can alleviate CIRI-induced BBB disruption by inhibiting ECs ferroptosis. Mechanistically, G-Rd prevented tight junction loss and BBB leakage by upregulating NRG1, activating its tyrosine kinase ErbB4 receptor, and then activating downstream PI3K/Akt/mTOR signaling, thereby inhibiting CIRI-induced ferroptosis in ECs. Taken together, these data provides data support for G-Rd as a promising therapeutic drug for cerebral ischemia.

FGF17 protects cerebral ischemia reperfusion-induced blood-brain barrier disruption via FGF receptor 3-mediated PI3K/AKT signaling pathway

Maintaining blood-brain barrier (BBB) integrity is critical components of therapeutic approach for ischemic stroke. Fibroblast growth factor 17 (FGF17), a member of FGF8 superfamily, exhibits the strongest expression throughout the wall of all major arteries during development. However, its molecular action and potential protective role on brain endothelial cells after stroke remains unclear. Here, we observed reduced levels of FGF17 in the serum of patients with ischemic stroke, as well as in the brains of mice subjected to middle cerebral artery occlusion (MCAO) injury and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain microvascular endothelial cells (bEnd.3) cells. Moreover, treatment with exogenous recombinant human FGF17 (rhFGF17) decreased infarct volume, improved neurological deficits, reduced Evans Blue leakage and upregulated the expression of tight junctions in MCAO-injured mice. Meanwhile, rhFGF17 increased cell viability, enhanced trans-endothelial electrical resistance, reduced sodium fluorescein leakage, and alleviated reactive oxygen species (ROS) generation in OGD/R-induced bEnd.3 cells. Mechanistically, the treatment with rhFGF17 resulted in nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear accumulation and upregulation of heme oxygenase-1 (HO-1) expression. Additionally, based on in-vivo and in-vitro research, rhFGF17 exerted protective effects against ischemia/reperfusion (I/R) -induced BBB disruption and endothelial cell apoptosis through the activation of the FGF receptor 3/PI3K/AKT signaling pathway. Overall, our findings indicated that FGF17 may hold promise as a novel therapeutic strategy for ischemic stroke patients.

The nongenomic neuroprotective effects of estrogen, E2-BSA, and G1 following traumatic brain injury: PI3K/Akt and histopathological study.

Studies suggest that both genomic and nongenomic pathways are involved in mediating the salutary effects of steroids following traumatic brain injury (TBI). This study investigated the nongenomic effects of 17β-estradiol (E2) mediated by the PI3K/p-Akt pathway after TBI. Ovariectomized rats were apportioned to E2, E2-BSA (E2 conjugated to bovine serum albumin), G1 [G-protein-coupled estrogen receptor agonist (GPER)] or their vehicle was injected following TBI, whereas ICI (classical estrogen receptor antagonist), G15 (GPER antagonist), ICI+G15, and their vehicles were injected before the induction of TBI and injection of drugs. Diffuse TBI was induced by the Marmarou model. Evans blue (EBC, 5 h), brain water contents (BWC), histopathological changes, and brain PI3K and p-Akt protein expressions were measured 24 h after TBI. The veterinary comma scale (VCS) was assessed before and at different times after TBI. The results showed a reduction in BWC and EBC and increased VCS in the E2, E2-BSA, and G1 groups. Also, E2, E2-BSA, and G1 reduced brain edema, inflammation, and apoptosis. The ICI and G15 inhibited the beneficial effects of E2, E2-BSA, and G1 on these parameters. All drugs, following TBI, prevented the reduction of brain PI3K/p-Akt expression. The individual or combined use of ICI and G15 eliminated the beneficial effects of E2, E2-BSA, and G1 on PI3K/p-Akt expressions. These findings indicated that PI3K/p-Akt pathway plays a critical role in mediating the salutary effects of estradiol on histopathological changes and neurological outcomes following TBI, suggesting that GPER and classic ERs are involved in regulating the expression of PI3K/p-Akt.

Restraint stress induces blood-brain barrier injury in rat amygdala by activating the Rho/ROCK signaling pathway

To investigate the role of Rho/ROCK signaling pathway in mediating restraint stress-induced blood-brain barrier (BBB) injury in the amygdala of rats. Sixty male SD rats were randomized equally into control group (with food and water deprivation for 6 h per day), restraint stress group (with restraint for 6 h per day), stress + fasudil treatment (administered by intraperitoneal injection at 1 mg/100 g 30 min before the 6-h restraint) group, and fasudil treatment alone group. The elevated plus-maze test was used to detect behavioral changes of the rats, serum corticosterone and S100B levels were determined with ELISA, and Evans Blue leakage in the brain tissue was examined to evaluate the changes in BBB permeability. The changes in expression levels of tight junction proteins in the amygdala were detected using immunofluorescence assay and Western blotting, and Rho/ROCK pathway activation was detected by Pull-down test and Western blotting. Ultrastructural changes of the cerebral microvascular endothelial cells were observed using transmission electron microscopy. Compared with those in the control group, the rats in restrain stress group and stress+fasudil group showed obvious anxiety-like behavior with significantly increased serum corticosterone level (P<0.001). Compared with those in the control group and stress+fasudil group, the rat models of restrain stress showed more obvious Evans Blue leakage and higher S100B expression (P<0.01) but lower expressions of tight junction proteins in the amygdala. Pull-down test and Western blotting confirmed that the expression levels of RhoA-GTP, ROCK2 and P-MLC 2 were significantly higher in stress group than in the control group and stress + fasudil group (P<0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the cerebral microvascular endothelial cells in the rat models of restrain stress. Restraint stress induces BBB injury in the amygdala of rats by activating the Rho/ROCK signaling pathway.