Magnetic Microbeads Research Articles

Assisting dementia diagnosis through the electrochemical immunosensing of glial fibrillary acidic protein

Glial fibrillary acidic protein (GFAP) is a member of the intermediate filament family of proteins with increased levels in serum and cerebrospinal fluid of patients with Alzheimer disease (AD) and other neurodegenerative diseases (NDs), such as vascular dementia (VD). This work describes the first magnetic microbeads (MBs)-based electrochemical immunoplatform for GFAP determination. The platform design comprises a sandwich immunoassay implemented on the MBs surface and amperometric transduction at single-use screen-printed carbon electrodes (SPCEs). Micro-sized carboxylic acid magnetic particles (COOH-MBs) were modified with a specific capture antibody (CAb) to selectively link the target protein, which was sandwiched with a biotinylated detector antibody (btn-DAb) further conjugated with a streptavidin-peroxidase (Strep-HRP) conjugate. Amperometric transduction was performed at SPCEs upon capturing the magnetic bioconjugates on their surface and through the hydrogen peroxide/hydroquinone (H2O2/HQ) system. The immunoplatform achieved a limit of detection of 67 pg mL−1 for the amperometric detection of standards and selectivity compatible with clinical applicability to assist in minimally invasive NDs diagnosis and prognosis. The MBs-based immunoplatform was applied with good results to determine the endogenous content of GFAP in protein brain extracts without matrix effect and using just 6.25 ng of sample per determination. Furthermore, the developed methodology was capable of differentiating between healthy subjects and patients diagnosed with VD and AD in only 2 h, providing accurate results in line with those obtained by an ELISA kit that used the same immunoreagents.

Exploring the Neuroprotective Role of Myo/Nog Cells in a Glaucomatous Mouse

IntroductionMyo/Nog cells express MyoD, Noggin and brain‐specific angiogenesis inhibitor 1 (BAI1). They are critical for eye development and respond to injury and stress in the neonatal and adult retina. Their depletion in the embryo results in eye and retinal malformations. Myo/Nog cells increase in number and migrate to areas of injury in response to stress. Cell death in the retina increases with Myo/Nog cell depletion and decreases with their addition to the vitreous. Here we examine the effects and locations of endogenous and exogenous Myo/Nog cells in mice eyes following the induction of glaucoma.MethodsThe anterior chamber of the right eye of C57BL/6J mice was injected with magnetic microbeads and guided to the iridocorneal angle using a handheld magnet, preventing the outflow of aqueous. Intraocular pressure was measured in both eyes following microbead injection on Days 0, 4, 7, 14, D21, and 28. On D7, three of the four groups were given bilateral injections into the anterior chamber of either PBS, unsorted brain cells, or Myo/Nog cells isolated from the brain. The fourth group did not receive any follow‐up injections. Eyes were collected on D32 and labeled with an antibody to BAI1 to locate Myo/Nog cells. H&E staining was used to count living cells in the retinal ganglion layer (RGC) and measure the thickness of the nerve fiber layer and inner nuclear layer (NFL:INL).ResultsEndogenous and exogenous Myo/Nog cells increased in glaucomatous eyes around areas of stress, including the cornea, trabecular meshwork, ciliary body and retina. The highest concentration of Myo/Nog cells was present around the RGC layer and cornea. Some Myo/Nog cells had phagocytized microbeads. Glaucomatous eyes treated with Myo/Nog cells displayed a lower concentration of endogenous Myo/Nog cells when compared to PBS and mixed population treatment groups. Addition of Myo/Nog cells also decreased loss of RGC cells and increased NFL:INL thickness in glaucomatous eyes compared to the control groups.ConclusionConsistent with other types of ocular injury, Myo/Nog cells accumulate in areas of stress following damage from increased intraocular pressure. Addition of Myo/Nog cells was neuroprotective in the glaucomatous retina.

Ultrasensitive detection of soy traces by immunosensing of glycinin and β-conglycinin at disposable electrochemical platforms.

This work reports the first electrochemical bioplatform for the determination of soy traces in food. The bioplatform involves sandwich-type immunoassays using specific antibodies for β-conglycinin and glycinin, which are the main allergenic soy proteins, and carboxylic acid-modified magnetic microbeads. Amperometric detection at -0.20V (vs. an Ag pseudo-reference electrode) was performed using single or dual screen-printed carbon electrodes and the H2O2/hydroquinone (HQ) system. The measured variation in the cathodic current was directly proportional to the concentration of target allergenic proteins. The developed bioplatforms exhibit a good selectivity and sensitivity providing limits of detection (LOD) values of 0.03 and 0.02ngmL-1 for β-conglycinin and glycinin, respectively. The determination of both proteins can be carried out in only 1.5h. The electrochemical bioplatforms allow their accurate determinations (with results statistically comparable to those provided by ELISA methodologies) in raw cookie dough and baked cookies enriched with soy flour. The results obtained confirm, in a pioneering way with electrochemical biosensors, the possibility of discriminating samples incurred with as little as 0.0005ppm of a food allergen in model cookie extracts.

Detection Efficiency of Ag Nanoparticle Labels for a Heart Failure Marker Using Linear and Square-Wave Anodic Stripping Voltammetry.

In this article, we compare linear sweep anodic stripping voltammetry (LASV) and square-wave anodic stripping voltammetry (SWASV) for detection of a nano metalloimmunoassay. Two separate immunoassays were examined: a model assay, based on interactions between antibodies, and a sandwich assay for the heart failure marker NT-proBNP. In both cases, one antibody is linked to a magnetic microbead, and one is linked to a spherical Ag nanoparticle label. Electrochemical detection is carried out on a paper device. The three analytical figures of merit studied were the precision of the measurements, the calibration sensitivity, and the limit of detection (LOD). For the NT-proBNP assay, the results show that after optimization of the pulse amplitude and frequency of the potential input for SWASV, the detection efficiency is substantially higher compared to LASV. Specifically, the calibration sensitivity increased by up to ~40 fold, the average coefficient of variation decreased by ~40%, and the (LOD) decreased to 300.0 pM. Finally, for a model immunoassay, a ~10-fold decrease in the LOD was observed for SWASV compared to LASV.

Paving the Way for Reliable Alzheimer's Disease Blood Diagnosis by Quadruple Electrochemical Immunosensing

AbstractAlzheimer's disease (AD), the most common neurodegenerative disorder, demands new cost‐effective and easy‐to‐use strategies for its reliable detection, mainly in the preclinical stages. Here, we report the first immunoplatform for the electrochemical multidetermination of four candidate protein biomarkers in blood, neurofilament light chain (NfL), Tau, phosphorylated Tau (p‐Tau) and TAR DNA‐Binding Protein 43 (TDP‐43). It involves implementation of sandwich‐type immunoassays and enzymatic labelling with horseradish peroxidase (HRP) on the surface of magnetic microbeads (MBs). Amperometric detection is performed after depositing the magnetic immunoconjugates on disposable quadruple transduction platforms by monitoring the enzymatic reduction of H2O2 mediated by hydroquinone (HQ). The immunoplatform achieved LOD values smaller than the content of target biomarkers in plasma of healthy subjects, with RSD values<5 %, and lower cost and shorter assay time (60–90 min) than other available methodologies and was applied to the analysis of plasma from healthy controls and AD patients.

Platelets Purification Is a Crucial Step for Transcriptomic Analysis.

Platelets are small anucleate cells derived from the fragmentation of megakaryocytes and are involved in different biological processes especially hemostasis, thrombosis, and immune response. Despite their lack of nucleus, platelets contain a reservoir of megakaryocyte-derived RNAs and all the machinery useful for mRNA translation. Interestingly, platelet transcriptome was analyzed in health and diseases and led to the identification of disease-specific molecular signatures. Platelet contamination by leukocytes and erythrocytes during platelet purification is a major problem in transcriptomic analysis and the presence of few contaminants in platelet preparation could strongly alter transcriptome results. Since contaminant impacts on platelet transcriptome remains theoretical, we aimed to determine whether low leukocyte and erythrocyte contamination could cause great or only minor changes in platelet transcriptome. Using microarray technique, we compared the transcriptome of platelets from the same donor, purified by common centrifugation method or using magnetic microbeads to eliminate contaminating cells. We found that platelet transcriptome was greatly altered by contaminants, as the relative amount of 8274 transcripts was different between compared samples. We observed an increase of transcripts related to leukocytes and erythrocytes in platelet purified without microbeads, while platelet specific transcripts were falsely reduced. In conclusion, serious precautions should be taken during platelet purification process for transcriptomic analysis, in order to avoid platelets contamination and result alteration.

Ratiometric electrochemical detection of miRNA based on DNA nanomachines and strand displacement reaction.

MicroRNAs (miRNAs) play an important role in regulating gene expression in cells. Abnormal expression of miRNAs has been associated with a variety of diseases. A ratiometric electrochemical method for miRNA detection based on DNA nanomachines and strand displacement reaction was developed. Signal probe with ferrocene label and reference probe with methylene blue label were immobilized on gold nanoparticle (AuNP)-coated magnetic microbeads (AuNP-MMBs). The miRNA triggers the strand displacement reaction and forms a duplex with the protect probe, releasing one end of the DNA walker (DW); the released DW hybridizes with the ferrocene (Fc)-labeled signal probe. The signal probe detached from AuNP-MMBs upon cleavage of the Nb.BbvCI enzyme. The oxidation peak of MB moieties on the reference probe remains unchanged and the signals of Fc moieties on the signal probe are inversely proportional to the concentrations of miRNA. The ratio between Fc moieties at 0.35 V and MB moieties at -0.22 V (vs. Ag/AgCl) was used to quantify the expression level of miRNA with a detection limit down to 0.12 fM. The ratiometric assay possesses a strong ability to eliminate interference from environmental changes, thus offering the high selectivity of miRNA from the complexed biosystems, holding great significance for miRNA sensing. A ratiometric assay with high selectivity of miRNA has been developed based on DNA nanomachines and strand displacement reaction.

Engineered tracrRNA for enabling versatile CRISPR-dCas9-based biosensing concepts

In recent years, CRISPR-Cas (stands for: clustered regularly interspaced short palindromic repeats - CRISPR associated protein) based technologies have gained increasing attention in the biosensing field. Thanks to excellent sequence specificity, their use is of particular interest for detecting nucleic acid (NA) targets. In this context, signal generation and amplification can be realized by employing the cis-cleavage activity of the Cas9 protein, although other options involving the catalytically inactive dead Cas9 (dCas9) are increasingly explored. The latter are however mostly based on complex protein engineering processes and often lack efficient signal amplification. Here we showed for the first time that flexible signal generation and amplification properties can be integrated into the CRISPR-dCas9 complex based on a straightforward incorporation of a DNA sequence into the trans-activating CRISPR RNA (tracrRNA). The intrinsic nuclease activity of the engineered complex remained conserved, while the incorporated DNA stretch enabled two modes of amplified fluorescent signal generation: (1) as an RNA-cleaving DNA-based enzyme (DNAzyme) or (2) as hybridization site for biotinylated DNA probes, allowing subsequent enzyme labeling. Both signal generation strategies were demonstrated in solution as well as while coupled to a solid surface. Finally, in a proof of concept bioassay, we demonstrated the successful detection of single stranded DNA on magnetic microbeads using the engineered CRISPR-dCas9 complex. Thanks to the flexibility of incorporating different NA-based signal generation and amplification strategies, this novel NA engineering approach holds enormous promise for many new CRISPR-based biosensing applications.

Thermally Responsive Alkane Partitions for Assay Automation.

For point-of-care diagnostic tools to be impactful, they must be inexpensive, equipment-free, and sample-to-answer (i.e., require no user intervention). Here, we report a new approach to enable sample-to-answer diagnostics that utilizes thermally responsive alkane partitions (TRAPs) as automated pseudo-valves. When combined with the magnetic manipulation of microbeads, TRAPs enable the pumpless automation of all steps in complex assays. We demonstrate that in relatively narrow channel geometries, liquified alkane partitions continue to separate reagents on each side of the partition while enabling the transition of magnetic beads from one reagent to the next, replacing manual pipetting steps in conventional assays. In addition, we show that in relatively broader geometries, liquified partitions breach, enabling the addition/mixing of preloaded reagents. Through calculation and experimentation, we determine the geometric design rules for implementing the stationary and removable partitions in fluidic channels. In addition, we demonstrate that magnetic microbeads can be pulled through liquified stationary TRAPs without disrupting partition integrity and without disrupting bound protein complexes attached at the microbead surface. The TRAP technology introduced here can enable a new low-cost and equipment-free approach for fully automated sample-to-answer diagnostics.

Magnetic bead mixing in a microfluidic chamber induced by an in-plane rotating magnetic field

Magnetic microbeads have been widely used for the capturing of biomarkers, as well as for microfluidic mixing for point-of-care diagnostics. In magnetic micromixing, microbead motion is generated by external electromagnets, inducing fluid kinetics, and consequently mixing. Here, we utilize an in-plane rotating magnetic field to induce magnetic bead mixing in a circular microfluidic chamber that allows better access with (optical) readout than for existing micromixing approaches. We analyze the magnetic bead dynamics, the induced fluid profiles and we quantify the mixing performance of the system. The rotating field causes the combination of (1) a global rotating flow counter to the external field rotation induced by magnetic particles moving along the chamber side wall, with (2) local flow perturbations induced by rotating magnetic bead clusters in the central area of the chamber, rotating in the same direction as the external field. This combination leads to efficient mixing performance within 2 min of actuated magnetic field. We integrate magnetic mushroom-shaped features around the circumference of the chamber to generate significantly higher global fluid velocities compared with the no-mushroom configuration, but this results in less efficient mixing due to the absence of the central rotating bead clusters. To validate and understand the experimental results and to predict further enhancement of mixing, we carry out numerical simulations of induced fluid profiles and their corresponding mixing indices, and we explore the additional effect of integrating geometrical structures. The micromixing method we introduce here is particularly suitable for microfluidic devices in which the biochemical assay happens in a microfluidic chamber under no-flow conditions, i.e., with initially stagnant fluids, and for which the time-to-result is critical, such as in point-of-care diagnostics.

Effect of heating conditions on the magnetic properties of micron-sized carboxyl modified-magnetite particles synthesized by a spray pyrolysis and heating process

Superparamagnetic carboxyl (COOH) modified-magnetite (Fe3O4) (COOH-Fe3O4) micrometer-sized porous particles were synthesized by the spray pyrolysis of a 0.1 M Fe(NO3)3·9H2O and 0.2 M citric acid solution and a subsequent heating process in either an Ar + 1% H2 (PO2 = 10−20 Pa) or N2 (PO2 = 1 Pa) gas atmosphere. Fe3O4 formed due to carbon and hydrogen gas generation of thermal decomposition of citric acid, even under reduction conditions with a PO2 below 10−23 Pa. The COOH-Fe3O4 particles heated in an Ar + 1% H2 or N2 atmosphere were porous (40% porosity) and about 1.2 to 1.3 μm in diameter. The particles consist of nano-sized COOH-Fe3O4 crystallite about 7 nm in diameter. The specific surface area increased from 116 to 127 m2/g by increasing the heating time in an Ar + 1% H2 atmosphere from 10 to 20 h. The saturation magnetization of the COOH-Fe3O4 particles (38.7 A·m2/kg) heated in Ar + 1% H2 for 10 h was much higher than that of commercial magnetic microbeads (17 A·m2/kg) and the coercivity was 0 kA/m. These superparamagnetic COOH-Fe3O4 particles dispersed in distilled water were attracted to a NdFeB magnet for 7–10 s under a 400 Mt external magnetic field, compared with 10 s for commercial magnetic microbeads.

Multimodal Locomotion and Cargo Transportation of Magnetically Actuated Quadruped Soft Microrobots.

Untethered microrobots have attracted extensive attention due to their potential for biomedical applications and micromanipulation at the small scale. Soft microrobots are of great research importance because of their highly deformable ability to achieve not only multiple locomotion mechanisms but also minimal invasion to the environment. However, the existing microrobots are still limited in their ability to locomote and cross obstacles in unstructured environments compared to conventional legged robots. Nature provides much inspiration for developing miniature robots. Here, we propose a bionic quadruped soft thin-film microrobot with a nonmagnetic soft body and 4 magnetic flexible legs. The quadruped soft microrobot can achieve multiple controllable locomotion modes in the external magnetic field. The experiment demonstrated the robot's excellent obstacle-crossing ability by walking on the surface with steps and moving in the bottom of a stomach model with gullies. In particular, by controlling the conical angle of the external conical magnetic field, microbeads gripping, transportation, and release of the microrobot were demonstrated. In the future, the quadruped microrobot with excellent obstacle-crossing and gripping capabilities will be relevant for biomedical applications and micromanipulation.

Both T and B cells are indispensable for the development of a PBMC transfer-induced humanized mouse model for SSc

BackgroundRecently, a novel humanized mouse model for systemic sclerosis (SSc) was established by transferring peripheral blood mononuclear cells (PBMC) from patients with SSc to Rag2−/−Il2rg−/− immunodeficient mice. Here, we aimed to investigate the role of T and B cells in this humanized mouse model.MethodsT and B cells were depleted in vitro from freshly isolated PBMC using anti-CD3 and anti-CD19 magnetic microbeads, respectively. Subsequently, PBMC and T or B cell-depleted PBMC were transferred into Rag2−/−/Il2rg−/− mice via intraperitoneal injection. Twelve weeks after the transfer, mice were sacrificed and evaluated.ResultsMice transferred with whole PBMC from SSc patients developed systemic inflammation in the lungs, kidneys, and liver, and 6 out of 11 mice died or had to be sacrificed during the experiment. By contrast, such inflammation and death were not observed in mice transferred with corresponding T or B cell-depleted PBMC. In line with this finding, transfer with whole PBMC restored the splenic white pulp composing of human T, B, and plasma cells and led to the production of a considerable amount of human autoantibodies in recipient mice, while those immunological features were rarely observed in mice that received T or B cell-depleted PBMC. In contrast to our previous findings demonstrating a transfer of the protective effect of a B cell therapy into the mouse, treatment of SSc patients with chemical immunosuppressive drugs did not affect the pathogenicity of PBMC.ConclusionsThis study demonstrates that both T and B cells are indispensable for the pathogenesis of the PBMC transfer-induced mouse model for SSc.

Fast Genotyping of K-Ras Codons 12 and 13 Based on Streptavidin Magnetic Microbeads Equipped with Biotin-Coupled Single Base-Pair Mismatch Pcr (Sm-Pcr)

Fast Genotyping of K-Ras Codons 12 and 13 Based on Streptavidin Magnetic Microbeads Equipped with Biotin-Coupled Single Base-Pair Mismatch Pcr (Sm-Pcr)

Protocol for Isolation and Culture of Mouse Hepatocytes (HCs), Kupffer Cells (KCs), and Liver Sinusoidal Endothelial Cells (LSECs) in Analyses of Hepatic Drug Distribution.

Development of the new generation of drugs (e.g., oligo- and polynucleotides administered intravascularly either as free compounds or as nano-formulations) frequently encounters major challenges such as lack of control of targeting and/or delivery. Uncontrolled or unwanted clearance by the liver is a well-known and particularly important hurdle in this respect. Hence, reliable techniques are needed to identify the type(s) of liver cells, receptors, and metabolic mechanisms that are responsible for unwanted clearance of these compounds.We describe here a method for the isolation and culture of the major cell types from mouse liver: hepatocytes (HCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs). The presently described protocol employs perfusion of the liver with a collagenase-based enzyme preparation to effectively transform the intact liver to a single cell suspension. From this initial cell suspension HCs are isolated by specified centrifugation schemes, yielding highly pure HC preparations, and KCs and LSECs are isolated by employing magnetic-activated cell sorting (MACS). The MACS protocol makes use of magnetic microbeads conjugated with specific antibodies that bind unique surface antigens on either KCs or LSECs. In this way the two cell types are specifically and separately pulled out of the initial liver cell suspension by applying a magnetic field, resulting in high purity, yield, and viability of the two cell types, allowing functional studies of the cells.If the drug compound in question is to be studied with respect to liver cell distribution of intravascularly administered drug compounds the isolated cells can be analyzed directly after isolation. Detailed studies of receptor-ligand interactions and/or dynamics of intracellular metabolism of the compound can be conducted in primary surface cultures of HCs, LSECs, and KCs established by seeding the isolated cells on specified growth substrates.

Highly accurate multiprotein detection on a digital ELISA platform.

The emerging single-molecule detection platform digital enzyme-linked immunosorbent assay (ELISA) can detect numerous proteins simultaneously at serum concentrations as low as picograms per milliliter. We sought to improve cytokine detection with this platform to aid diagnosis of conditions such as allergy and asthma. We developed a multiple single-molecule detection digital ELISA approach, through the application of encoded magnetic microbeads to simultaneously detect three cytokines in one serum sample. We tested the approach's utility to distinguish asthma-related cytokines in children. Concentrations of interleukin-4 (IL-4) and IL-6 were significantly higher in children with asthma than in healthy controls, while the concentration of interferon-γ (IFN-γ) was significantly lower. Our method has higher accuracy than conventional methods, and our results indicate that the proposed improved high-sensitivity digital ELISA-based diagnosis approach can facilitate early detection and treatment of childhood asthma or related diseases.

Advances in Magnetic Microbead Affinity Selection Screening: Discovery of Natural Ligands to the SARS-CoV-2 Spike Protein.

Affinity selection-mass spectrometry, which includes magnetic microbead affinity selection-screening (MagMASS), is ideal for the discovery of ligands in complex mixtures that bind to pharmacological targets. Therapeutic agents are needed to prevent or treat COVID-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection of human cells by SARS-CoV-2 involves binding of the virus spike protein subunit 1 (S1) to the human cell receptor angiotensin converting enzyme-2 (ACE2). Like antibodies, small molecules have the potential to block the interaction of the viral S1 protein with human ACE2 and prevent SARS-CoV-2 infection. Therefore, a MagMASS assay was developed for the discovery of ligands to the S1 protein. Unlike previous MagMASS approaches, this new assay used robotics for 5-fold enhancement of throughput and sensitivity. The assay was validated using the SBP-1 peptide, which is identical to the ACE2 amino acid sequence recognized by the S1 protein, and then applied to the discovery of natural ligands from botanical extracts. Small molecule ligands to the S1 protein were discovered in extracts of the licorice species, Glycyrrhiza inflata. In particular, the licorice ligand licochalcone A was identified through dereplication and comparison with standards using HPLC with high-resolution tandem mass spectrometry.

Development of gold nanoparticle-based visual assay for rapid detection of Escherichia coli specific DNA in milk of cows affected with mastitis

For early diagnosis of infections, a rapid, sensitive and precise method to identify microbial DNA coming from veterinary clinical samples, such as milk of cows affected with mastitis, is fascinating. We developed an amplification-free visual assay for rapid and sensitive detection of specific DNA from Escherichia coli, particularly uid A gene encoding for beta-glucuronidase, isolated in milk of cows affected with mastitis, based on multiple gold nanoparticles (AuNPs) captured on a magnetic microbead surface, leading to plasmonic signal enhancement, thereby improving overall sensitivity of the assay. This test can be performed in 1–1.5 h after post template DNA preparation and can be visualized with naked eyes without the use of any expensive equipment. The visual assay detects a minimum microbial load of 102 CFU/μl and can identify bacterial DNA as low as 1 pg. This test provides a precise detection tool for E coli in clinical samples, such as milk of cows affected with mastitis, as a quick and user-friendly molecular detection approach.

A highly efficient technique to simultaneously remove acidic and basic dyes using magnetic ion-exchange microbeads

The purpose of this study was to examine the combination of magnetic anion-exchange microbeads (MAM) and magnetic cation-exchange microbeads (MCM) to remove crystal violet (CV; a basic dye) and acid green 9 (AG9; an acidic dye) from their individual and combined solutions. Adsorption kinetics and isotherms experiments were performed in batch mode. CV and AG9 displayed superior affinity towards MCM and MAM, respectively, and their combined solution was efficiently adsorbed by combining MCM and MAM. The pseudo-first-order, pseudo-second-order, Elovich and intra-particle diffusion models well described the adsorption kinetic data, and the pseudo-second-order model appeared a better fit for the two-component CV/AG9 system. The better fit of the Langmuir isotherm for CV adsorption indicated that CV adsorption occurred on active sites with equal affinity in the monolayer. In contrast, AG9 adsorption onto the heterogeneous MAM surface appeared to be multilayered adsorption. The adsorption capacities of the two dyes decreased with the increase in the co-existing salt concentration and increased only slightly at the high salt level due to the salting-out effect. Moreover, these microbeads maintained most of their initial capacity during five reuse cycles, indicating the great potential of MCM and MAM to remove basic and acidic dyes in practical applications.

TRPV4-induced M\xfcller cell gliosis and TNF-\u03b1 elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-\u03baB pathway

BackgroundGlaucoma, the leading cause of irreversible blindness worldwide, is a type of retinal disease characterized by the selective death of retinal ganglion cells (RGCs). However, the pathogenesis of glaucoma has not been fully elucidated. Transient receptor potential vanilloid 4 (TRPV4) is a pressure-sensitive and calcium-permeable cation channel. TRPV4 is widely distributed in the retina and its sustained activation leads to RGC death; indicating that TRPV4 may be a possible target for glaucoma treatment. Here, we investigated the effects of TRPV4 on RGC apoptosis in a rat model of chronic ocular hypertension (COH), then examined the mechanism underlying these effects.MethodsThe COH model was established by injection of micro-magnetic beads into the anterior chamber of adult male rats. The expression levels of TRPV4, glial fibrillary acidic protein, and inflammatory factors were assessed by immunohistochemistry and immunoblotting. RGC apoptosis and visual dysfunction were evaluated by TUNEL assay and photopic negative response. Functional expression of TRPV4 was examined by electrophysiology and calcium imaging. Real-time polymerase chain reaction and immunoblotting were employed to investigate the molecular mechanism underlying the effects of TRPV4 on tumor necrosis factor-α (TNF-α) release.ResultsWe found that TRPV4 played an essential role in glaucoma, such that high levels of TRPV4 expression were associated with elevated intraocular pressure. Furthermore, TRPV4 activation was involved in glaucoma-induced RGC apoptosis and RGC-related reductions in visual function. Mechanistic investigation demonstrated that TRPV4 activation led to enhanced Müller cell gliosis and TNF-α release via the JAK2/STAT3/NF-kB pathway, while TRPV4 inhibition could reverse these effects. Finally, TRPV4 activation could lead to elevated expression of TNF receptor 1 in RGCs, while inhibition of TNF-α could reduce TRPV4-mediated RGC apoptosis.ConclusionsTRPV4 activation induces Müller cell gliosis and TNF-α elevation via the JAK2/STAT3/NF-κB pathway, which may exacerbate RGC apoptosis in glaucoma; these results suggest that TRPV4 can serve as a therapeutic target in glaucoma treatment.