Tripedal DNA Walker as a Signal Amplifier Combined with a Potential-Resolved Multicolor Electrochemiluminescence Strategy for Ultrasensitive Detection of Prostate Cancer Staging Indicators.

A multicolor electrochemiluminescence (ECL) biosensor based on a closed bipolar electrode (BPE) array was proposed for the rapid and intuitive analysis of three prostate cancer staging indicators. First, [Irpic-OMe], [Ir(ppy)2(acac)], and [Ru(bpy)3]2+ were applied as blue, green, and red ECL emitters, respectively, whose mixed ECL emission colors covered the whole visible region by varying the applied voltages. Afterward, we designed a simple Mg2+-dependent DNAzyme (MNAzyme)-driven tripedal DNA walker (TD walker) to release three output DNAs. Immediately after, three output DNAs were added to the cathodic reservoirs of the BPE for incubation. After that, we found that the emission colors from the anode of the BPE changed as a driving voltage of 8.0 V was applied, mainly due to changes in the interfacial potential and faradaic currents at the two poles of the BPE. Via optimization of the experimental parameters, cutoff values of such three indicators at different clinical stages could be identified instantly with the naked eye, and standard precision swatches with multiple indicators could be prepared. Finally, in order to precisely determine the prostate cancer stage, the multicolor ECL device was used for clinical analysis, and the resulting images were then compared with standard swatches, laying the way for accurate prostate cancer therapy.

Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

Evaluation of size-exclusion chromatography, multi-angle light scattering detection and mass photometry for the characterization of mRNA.

The recent approval of messenger ribonucleic acid (mRNA) as vaccine to combat the COVID-19 pandemic has been a scientific turning point. Today, the applicability of mRNA is being demonstrated beyond infectious diseases, for example in cancer immunotherapy, protein replacement therapy and gene editing. mRNA is produced by in vitro transcription (IVT) from a linear DNA template and modified at the 3' and 5' ends to improve translational efficiency and stability. Co-existing impurities such as RNA fragments and double-stranded RNA (dsRNA), amongst others, can drastically impact mRNA quality and efficacy. In this study, size-exclusion chromatography (SEC) is evaluated for the characterization of IVT-mRNA. The effect of mobile phase composition (ionic strength and organic modifier), pH, column temperature and pore size (300 Å, 1000 Å, and 2000 Å) on the separation performance and structural integrity of IVT-mRNA varying in size is described. Non-replicating, self-amplifying (saRNA), temperature degraded, and ribonuclease (RNase) digested mRNA, the latter to characterize the 3' poly(A) tail, were included in the study. Beyond ultraviolet (UV) detection, refractive index (RI) and multi-angle light scattering (MALS) detection were implemented to accurately determine molecular weight (MW) of mRNA. Finally, mass photometry is introduced as a complementary methodology to study mRNA under native conditions.

Dual-microRNA-Controlled Electrochemiluminescence Biosensor for Breast Cancer Diagnosis and Supplemental Identification of Breast Cancer Metastasis.

Breast cancer remains the most frequently diagnosed cancer globally, and the metastasis of this malignancy is the primary cause of mortality in breast cancer patients. Hence, prompt diagnosis and timely detection of metastatic breast cancer are critical for effective therapeutic intervention. Both progression and metastasis of this malignancy are closely associated with aberrant expression of specific microRNAs (miRNAs) and enzymes. To facilitate breast cancer diagnosis and concomitant identification of metastatic breast cancer, we have engineered an innovative electrochemiluminescence (ECL)-based sensing platform integrated with enzyme-free DNA amplification circuits for dual functionality. Specifically, microRNA-21 (miR-21) is employed as a biomarker for breast cancer, and miR-21 induces the quenching of the ECL signal from luminophores via a strategically designed catalytic three-hairpin assembly (CTHA) circuit. Subsequently, miR-105 levels are measured via toehold-mediated strand displacement reactions (TSDR). Here, miR-105 restores the initially quenched ECL signal, enabling the assessment of the metastatic propensity. Our experimental data demonstrate that the devised ECL biosensor offers broad linear detection ranges and low detection limits for both miR-21 and miR-105. Importantly, our novel platform was also successfully validated by using cellular and serum samples. This biosensor not only discriminates breast cancer cell lines MCF-7 and MDA-MB-231 from nonbreast cancer cells like HepG2, TPC-1, and HeLa, but it also distinguishes between malignant MCF-7 and metastatic MDA-MB-231 cells. Consequently, our novel approach holds significant promise for clinical applications and precise cancer screening.

Automated Mass Photometry of Adeno-Associated Virus Vectors from Crude Cell Extracts.

Mass photometry (MP) is a fast and simple analysis method for the determination of the proportions of subpopulations in an AAV sample. It is label-free and requires minimal sample volumes between 5-10 µL, which makes it a promising candidate over orthogonal techniques such as analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (Cryo-TEM) or charge-detection mass spectrometry (CDMS). However, these methods are limited in their application to purified samples only. Here we developed a purification step based on single-domain monospecific antibody fragments immobilised on either a poly(styrene-divinylbenzene) resin or on magnetic beads prior to MP analysis that allows the quantification of empty, partially filled, full and overfull AAV vectors in crude cell extracts. This is aimed at identifying potentially promising harvest conditions that yield large numbers of filled AAV vectors during the early stages of the viral vector development platform, e.g., the type of transfection reagent used. Furthermore, we provide a direct comparison of the automated and manual handling of the mass photometer with respect to the quantities of AAV subspecies, molar mass of the capsid and payload, and highlight the differences between the "buffer-free" sample measurement and the "buffer-dilution" mode. In addition, we provide information on which candidates to use for calibration and demonstrate the limitations of the mass photometer with respect to the estimation of the capsid titer.

Quantification of Postoperative Inflammation in Laser and Surgical Procedures for Glaucoma with Laser Flare Photometry: A Review of the Literature.

PURPOSE: To review the application of laser flare photometry (LFP) in the objective quantification of aqueous flare (anterior chamber inflammation) post laser and surgical procedures for glaucoma. METHODS: A search was undertaken using the following: PubMed (all years), the Web of Science (all years), Ovid MEDLINE (R) (1980 to 30 March 2023), Ovid MEDLINE (R) Daily Update 30 March 2023, MEDLINE and MEDLINE non-indexed items, Embase (1980-2021, week 52), Ovid MEDLINE (R) and Epub Ahead of Print, in-Process & Other Non-Indexed Citations and Daily (1980 to 30 March 2023), CENTRAL (including Cochrane Eyes and Vision Trials Register), metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrial.gov) and the World Health Organization (WHO) International Clinical Trials Registry Platform (www.who.int/ictrp/search/en). Search terms included "aqueous flare," "anterior chamber inflammation," "tyndallometry," "laser flare photometry" combined with "laser," "iridotomy," "trabeculoplasty," "cataract surgery," "phacoemulsification," "glaucoma surgery," "minimally invasive glaucoma surgery," "trabeculectomy," "aqueous shunt," "glaucoma drainage" and "cyclophotocoagulation." RESULTS: The majority of studies utilizing laser flare photometry in grading flare have been post laser trabeculoplasty. The degree of flare produced varies according to the type of glaucoma laser or surgery performed, with filtration procedures and glaucoma drainage devices having marked and prolonged detectable levels. Aqueous flare in cyclodestructive procedures positively correlated with intraocular pressure (IOP) reduction. CONCLUSION: In comparison to clinician grading, laser flare photometry provides a more objective measure of post-surgical inflammation in eyes that have undergone laser and surgery for glaucoma. Further research is warranted into how this instrument can be utilized to identify eyes at high risk of failure and other adverse outcomes after glaucoma surgery.

Protocol for the integration of fiber photometry and social behavior in rodent models.

Fiber photometry offers insight into cell-type-specific activity underlying social interactions. We provide a protocol for the integration of fiber photometry recordings into the analysis of social behavior in rodent models. This includes considerations during surgery, notes on synchronizing fiber photometry with behavioral recordings, advice on using multi-animal behavioral tracking software, and scripts for the analysis of fiber photometry recordings. For complete details on the use and execution of this protocol, please refer to Dawson et al. (2023).1.

Intravitreal Injection of Bevacizumab for the Prevention of Postoperative Proliferative Vitreoretinopathy in High-Risk Patients Selected by Laser Flare Photometry.

INTRODUCTION: To evaluate the effect of an intravitreal injection of bevacizumab at the time of rhegmatogenous retinal detachment (RRD) surgery, on postoperative proliferative vitreoretinopathy (PVR) in high-risk patients selected by laser flare photometry. METHODS: This single-center observational retrospective cohort study included 137 consecutive patients who underwent pars plana vitrectomy and gas tamponade for primary RRD with increased aqueous flare between July 2016 and June 2021. From June 2019, an intravitreal injection of bevacizumab was administered as an adjunct to RRD repair. Patients who underwent surgery before this time and who did not receive intravitreal bevacizumab served as controls. The main outcome was the rate of retinal redetachment due to PVR. RESULTS: The median flare value was 22.0 (16.5-36.5) pc/ms in the control group and 28.2 (19.7-41.0) pc/ms in the bevacizumab group (p = 0.063). Eyes treated with bevacizumab were more likely to have macula-off RRD (p = 0.003), grade B PVR (p = 0.038), and worse visual acuity (p = 0.004) than controls. The rate of PVR redetachment was significantly lower in the bevacizumab group (11.1%) than in the control (30.1%) (p = 0.012). This difference was more pronounced after adjusting for potential confounding factors (p = 0.005); the risk of developing PVR was 4.5-fold higher in controls (95% CI, 1.6-12.8). After adjustment, the final median visual acuity was also significantly higher in eyes treated with bevacizumab (p = 0.025). CONCLUSION: This pilot study provides preliminary evidence that bevacizumab may reduce the risk of PVR-related recurrent RRD and improve visual outcomes in high-risk patients selected by laser flare photometry.

AuPt Nanodonuts as a Novel Coreaction Accelerator and Luminophore for a Label-free Electrochemiluminescence Aptasensor.

Novel and effective coreaction accelerators are of great importance in electrochemiluminescence (ECL) systems. In this work, novel AuPt nanodonuts, i.e., SnS2 quantum dots (QDs)/Cys-AuPt heterogeneous nanorings (NRs), serve as both a highly effective coreaction accelerator and the luminophore in a label-free ECL aptasensor. The novel AuPt nanodonuts were formed by decorating SnS2 QDs onto AuPt NR surfaces, which would promote the production of more coreactant intermediate in the SnS2 QDs/K2S2O8 system. As a result, the ECL performance was greatly improved. Meanwhile, l-cysteine (l-Cys) played an important role in the combination between AuPt NRs and SnS2 QDs, and the nanodonuts served as the matrix to load numerous lincomycin (Lin) aptamers. Under optimal conditions, the ECL aptasensor exhibited ultrasensitive detection of Lin from 1 fg/mL to 0.1 pg/mL with a limit of detection (LOD) of 0.7 fg/mL (1.72 fM).

A fluorescence-electrochemiluminescence dual-mode sensor based on a "switch" system for highly selective and sensitive K-ras gene detection.

Herein, an fluorescence (FL)-electrochemiluminescence (ECL) dual-mode biosensor is constructed based on the dual-signal "turn-on" strategy of functionalized metal-organic frameworks nanosheets (RuMOFNSs)-tetraferrocene for K-ras gene detection, and the mechanism of bursting through front-line orbital theory is explained for the first time. Amino-functionalized tetraferrocene-labeled probe DNA molecules are linked to RuMOFNSs by covalent amide bonds, acting as FL and ECL intensity switches. The target DNA, complementary to the probe DNA, triggers cyclic amplification of the target by nucleic acid exonuclease III (Exo III), repelling tetraferrocene reporter groups away from RuMOFNSs and inhibiting the electron transfer process and photoinduced electron transfer (PET) effect. These phenomena induce a double turn-on of FL and ECL signals with a high signal-to-noise ratio. The developed FL-ECL dual-mode sensing platform provides sensitive detection of the K-ras gene with detection limits of 0.01 fM (the detection range is 1 fM to 1 nM) and 0.003 fM (the detection range is 0.01 fM to 10 pM), respectively. In addition, the proposed dual-mode sensor can be easily extended to detect other disease-related biomarkers by changing the specific target and probe base sequences, depicting potential applications in bioanalysis and early disease diagnosis.

Microfluidic Gradient Culture Arrays for Cell Pro-oxidation Analysis Using Bipolar Electrochemiluminescence.

A microfluidic gradient array is a widely used screening and analysis device, which has characteristics of high efficiency, high automation, and low consumption. Bipolar electrode electrochemiluminescence (BPE-ECL) has special value in microfluidic array chips. The combination of the microfluidic gradient and BPE arrays has potential for high-throughput screening. In this article, a microfluidic BPE array chip for gradient culture and conditional screening of cancer cells was designed. The generation of concentration gradients, continuous culture of cancer cells with high throughput, and drug screening through BPE-ECL of the Ru(bpy)32+/TPrA system can be performed in one chip. We tested gradient pro-oxidation of MCF-7 by ascorbic acid and the synergistic effect of pro-oxidation on doxorubicin. The method achieves high analysis efficiency through a BPE array while simplifying the tedious procedures required by cell culture methods.

PEG-functionalized black phosphorus quantum dots as stable and biocompatible electrochemiluminescence luminophores for sensitive detection of tumor biomarker.

Despite black phosphorous (BP) QDs possess the merits of size-tunable band-gap, high electron mobility, and intrinsic defects, the spontaneous agglomeration and rapid oxidation of BP QDs in aqueous solution caused low electrochemiluminescence (ECL) efficiency and unstable ECL signal, which confined its further application of biological analysis. Herein, polyethylene glycol-functionalized BP QDs (PEG@BP QDs) were prepared showing an efficient and stable ECL response, which is attributed to the fact that PEG as protectant not only effectively prevented the spontaneous agglomeration, but also restrained the rapid oxidation of BP QDs in aqueous solution. As proof-of-concept, PEG@BP QDs were used as an efficient ECL emitter to combine with palindrome amplification-induced DNA walker to construct a sensitive ECL aptasensing platform for detecting cancer marker mucin 1 (MUC1). Interestingly, with the aid of positively charged thiolated PEG, the reaction rate of DNA walker on the electrode interface was clearly increased for the recovery of the ECL signal. The ECL aptasensor provides sensitive determination with the detection limit of 16.5 fg/mL. The proposed strategy paves a path for the development of efficient and stable ECL nanomaterials to construct biosensors for biosensing and clinical diagnosis.

Ru(II)-modified metal organic framework as excellent electrochemiluminescence emitter for ultrasensitive nicotine detection.

The sensitive and selective nicotine detection in cigarette is necessary due to the cigarette addiction problem and the neurotoxicity of nicotine on human body. In this study, a novel electrochemiluminescence (ECL) emitter with excellent performance was prepared for nicotine analysis, by combining Zr-based metal organic framework (Zr-MOF) and branched polyethylenimine (BPEI)-coated Ru(dcbpy)32+ through electrostatic interaction. Ru(dcbpy)32+ integrated by Zr-MOF could be catalyzed by the reaction intermediates SO4•-, produced from the co-reactant S2O82-, resulting in a significant increase in ECL response. Interestingly, SO4•- with strong oxidizing ability could preferentially oxidize nicotine, leading to ECL quenching. The constructed ECL sensor based on the Ru-BPEI@Zr-MOF/S2O82- system displayed ultrasensitive determination of nicotine with a lower detection limit of 1.9 × 10-12 M (S/N = 3), which is three orders lower than previously reported ECL results and 4-5 orders lower than that of other types of method. This method puts forward a new approach for building efficient ECL system with greatly improved ECL sensitivity for nicotine detection.

MoS2-Carbon Nanodots as a New Electrochemiluminescence Platform for Breast Cancer Biomarker Detection.

In this work, we present the combination of two different types of nanomaterials, 2D molybdenum disulfide nanosheets (MoS2-NS) and zero-dimensional carbon nanodots (CDs), for the development of a new electrochemiluminescence (ECL) platform for the early detection and quantification of the biomarker human epidermal growth factor receptor 2 (HER2), whose overexpression is associated with breast cancer. MoS2-NS are used as an immobilization platform for the thiolated aptamer, which can recognize the HER2 epitope peptide with high affinity, and CDs act as coreactants of the anodic oxidation of the luminophore [Ru(bpy)3]2+. The HER2 biomarker is detected by changes in the ECL signal of the [Ru(bpy)3]2+/CD system, with a low detection limit of 1.84 fg/mL and a wide linear range. The proposed method has been successfully applied to detect the HER2 biomarker in human serum samples.

Surface-Defect-Involved and Eye-Visible Electrochemiluminescence of Unary Copper Nanoclusters for Immunoassay.

A single-stabilizer-capped strategy is proposed for achieving highly efficient and surface-defect-involved electrochemiluminescence (ECL) from unary copper nanoclusters (NCs) via employing l-cysteine (Cys) as a capping agent of luminophore. The Cys-capped CuNCs (Cys-CuNCs) can be electrochemically injected with valence band (VB) holes and exhibit eye-touchable ECL processes around +0.95 and +1.15 V upon employing TPrA as a coreactant. Both accumulated ECL spectra and spooling ECL spectra demonstrated that the two ECL processes are of the same single waveband and spectrally identical to each other with the same maximum emission wavelength of 640 nm. Promisingly, ECL of the Cys-CuNCs/TPrA system is obviously red-shifted for ∼150 nm to PL of Cys-CuNCs, indicating that the bandgap-engineered routes for ECLs of Cys-CuNCs are completely blocked. The oxidative-reduction ECL process of the Cys-CuNCs/TPrA system is a kind of highly efficient, eye-visible, and single-color emission in surface-defect-involved route. The capping agent of Cys can enable the CuNCs/TPrA system with a stronger ECL than other thiol capping agents, so that Cys-CuNCs are utilized as ECL tags for sensitive and selective immunoassays, which exhibit a wide linear response range from 0.05 pg/mL to 0.5 ng/mL and a limit of detection of 0.01 pg/mL (S/N = 3) with carcinoembryonic antigen as the analyte. Moreover, both the luminophore Cys-CuNCs and conjugates Ab2-CuNCs can be safely stored in aqueous media without any protector, which is promising for the evolution and clinic application of metal NC ECL in the surface-defect-involved route.

Mesoporous Nanozyme-Enhanced DNA Tetrahedron Electrochemiluminescent Biosensor with Three-Dimensional Walking Nanomotor-Mediated CRISPR/Cas12a for Ultrasensitive Detection of Exosomal microRNA.

Exosomal microRNAs (exomiRNAs) have emerged as ideal biomarkers for early clinical diagnostics. The accurate detection of exomiRNAs plays a crucial role in facilitating clinical applications. Herein, an ultrasensitive electrochemiluminescent (ECL) biosensor was constructed using three-dimensional (3D) walking nanomotor-mediated CRISPR/Cas12a and tetrahedral DNA nanostructures (TDNs)-modified nanoemitters (TCPP-Fe@HMUiO@Au-ABEI) for exomiR-155 detection. Initially, the 3D walking nanomotor-mediated CRISPR/Cas12a strategy could effectively convert the target exomiR-155 into amplified biological signals for improving the sensitivity and specificity. Then, TCPP-Fe@HMUiO@Au nanozymes with excellent catalytic performance were used to amplify ECL signals because of the enhanced mass transfer and increased catalytic active sites, originating from its high surface areas (601.83 m2/g), average pore size (3.46 nm), and large pore volumes (0.52 cm3/g). Meanwhile, the TDNs as the scaffold to fabricate "bottom-up" anchor bioprobes could improve the trans-cleavage efficiency of Cas12a. Consequently, this biosensor achieved the limit of detection down to 273.20 aM ranging from 1.0 fM to 1.0 nM. Furthermore, the biosensor could discriminate breast cancer patients evidently by analyzing exomiR-155, and these results conformed to that of qRT-PCR. Thus, this work provides a promising tool for early clinical diagnostics.

Simultaneous dynamic glucose-enhanced (DGE) MRI and fiber photometry measurements of glucose in the healthy mouse brain.

Glucose is the main energy source in the brain and its regulated uptake and utilization are important biomarkers of pathological brain function. Glucose Chemical Exchange Saturation Transfer (GlucoCEST) and its time-resolved version Dynamic Glucose-Enhanced MRI (DGE) are promising approaches to monitor glucose and detect tumors, since they are radioactivity-free, do not require 13C labeling and are is easily translatable to the clinics. The main principle of DGE is clear. However, what remains to be established is to which extent the signal reflects vascular, extracellular or intracellular glucose. To elucidate the compartmental contributions to the DGE signal, we coupled it with FRET-based fiber photometry of genetically encoded sensors, a technique that combines quantitative glucose readout with cellular specificity. The glucose sensor FLIIP was used with fiber photometry to measure astrocytic and neuronal glucose changes upon injection of D-glucose, 3OMG and L-glucose, in the anaesthetized murine brain. By correlating the kinetic profiles of the techniques, we demonstrate the presence of a vascular contribution to the signal, especially at early time points after injection. Furthermore, we show that, in the case of the commonly used contrast agent 3OMG, the DGE signal actually anticorrelates with the glucose concentration in neurons and astrocytes.

Critical Calibration of Mass Photometry for Higher-Mass Samples Such as Adeno-Associated Virus Vectors.

Mass photometry (MP) is a label-free, single-molecule technique that can determine molecular mass distribution with very low sample consumption in a short time. Because of the established experimental instrument and analytical software, MP measurements may be readily obtained; thus, the application of MP is expanding, especially in the fields of bioscience and biotechnology. However, because the MP data quality is strongly focus-dependent, optical settings must be intrinsically strict. In this study, we report the importance of the critical calibration of the mass photometer, which is required for the accurate estimation of high-molecular mass samples, such as adeno-associated virus vectors. Additionally, a method for optimizing the instrument settings, including the calibration of the stage, is presented.

Association between laser flare photometry and symptom duration in primary rhegmatogenous retinal detachment.

PURPOSE: The purpose of this study was to investigate preoperative blood-ocular barrier disruption via laser flare photometry (LFP) in patients diagnosed with rhegmatogenous retinal detachment (RRD), and to analyse possible associations with symptom duration and anatomical parameters. METHODS: We retrospectively analysed consecutive patients presenting with RRD at a single centre between January 2016 and March 2020. LFP was performed in both eyes after pupillary dilatation prior to RRD surgery. Symptom duration, extent of retinal detachment, and lens status were assessed. For statistical analysis, we carried out the unequal variances t test and Welch's analysis of variance (ANOVA). RESULTS: We included 373 eyes of 373 patients (mean age 63.96 years ± 10.29; female:male ratio 1:1.8). LFP values quantified in photon count per millisecond (pc/ms) increased with longer symptom duration when comparing patients with a symptom duration of 0-3 days (n = 158; 9.25 ± 6.21 pc/ms) and ≥ 4 days (n = 215; 11.97 ± 11.58 pc/ms; p = 0.004). LFP values also rose with the number of retinal quadrants affected by RRD (1 quadrant, 6.82 ± 4.08 pc/ms; 2 quadrants, 10.08 ± 7.28 pc/ms; 3 quadrants, 12.79 ± 7.9 pc/ms; 4 quadrants, 31.57 ± 21.27 pc/ms; p < 0.001), macula off status (macula on, 8.89 ± 6.75 pc/ms; macula off, 12.65 ± 11.66 pc/ms; p < 0.001), and pseudophakic lens status (pseudophakia, 12.86 ± 9.52 pc/ms; phakia: 9.31 ± 9.67 pc/ms; p < 0.001). CONCLUSION: In RRD patients, blood-ocular barrier disruption quantified by LFP is associated with the duration of symptoms and the disease's anatomical extent. These results warrant further investigation of the potential clinical use of LFP in RRD.

Investigating the role of Prefrontal Neurons in cognitive tasks through Fiber Photometry and Adapted Barnes Maze Protocols

The medial prefrontal cortex (mPFC) is essential in the execution of cognitive tasks, however very little is known on how these neurons are modulated during specific tasks and which subtype of neurons are responsible for so. Therego, with the intention of addressing this issue, we recorded mPFC gabaergic and glutamatergic activation patterns through fiber photometry (FIP) in mice, while simultaneously performing the Barnes Maze (BM) cognitive task (4 day behavioral trial). In addition, an altered structural and procedural protocol for BM was validated in this study due to necessary modifications allowing FIP and BM to happen simultaneously. A successful protocol validation was followed by our preliminary results, which showed that both glutamatergic and gabaergic neurons presented significant change in activation intensity and number of events in specific contexts throughout the task days. In addition, when stratified and crossed with BM performance parameters, such as latency to complete tasks and adopted strategy, glutamatergic and gabaergic neurons presented a significant decline in both activation patterns and number of activation events throughout the days. This data suggest not only an important role of glutamatergic and gabaergic mPFC neurons in learning, memory and decision making, but also that activation patterns of each of these groups may serve as markers for cognitive progression and/or dysfunction. KEY-WORDS: Memory, Learning, Decision Making, Medial Prefrontal Cortex (mPFC), Fiber Photometry (FIP), Barnes Maze (BM), Glutamatergic, Gabaergic, Neuronal Activity, Neuronal Activation Patterns, Neuronal Dynamics.

O córtex pré-frontal medial (mPFC) é essencial na execução de tarefas cognitivas, no entanto, pouco se sabe sobre como esses neurônios são modulados durante tarefas específicas e qual subtipo de neurônios é responsável por isso. Portanto, com a intenção de abordar essa questão, registramos os padrões de ativação de neurônios gabaérgicos e glutamatérgicos do mPFC por meio de fotometria de fibra (FIP) em camundongos, enquanto realizávamos simultaneamente a tarefa cognitiva do Labirinto de Barnes (BM) (ensaio comportamental de 4 dias). Além disso, um protocolo estrutural e procedimental alterado para o BM foi validado neste estudo devido a modificações necessárias que permitiram a realização simultânea de FIP e BM. Uma validação bem-sucedida do protocolo foi seguida pelos nossos resultados preliminares, que mostraram que tanto os neurônios glutamatérgicos quanto os gabaérgicos apresentaram mudanças significativas na intensidade de ativação e no número de eventos em contextos específicos ao longo dos dias da tarefa. Além disso, quando estratificados e cruzados com parâmetros de desempenho do BM, como latência para completar as tarefas e estratégia adotada, os neurônios glutamatérgicos e gabaérgicos apresentaram uma diminuição significativa nos padrões de ativação e no número de eventos de ativação ao longo dos dias. Esses dados sugerem não apenas um papel importante dos neurônios glutamatérgicos e gabaérgicos do mPFC na aprendizagem, memória e tomada de decisões, mas também que os padrões de ativação de cada um desses grupos podem servir como marcadores de progressão e/ou disfunção cognitiva. PALAVRAS-CHAVE: Memória, Aprendizagem, Tomada de Decisões, Córtex Pré-Frontal Medial (mPFC), Fotometria de Fibra (FIP), Labirinto de Barnes (BM), Glutamatérgico, Gabaérgico, Atividade Neuronal, Padrões de Ativação Neuronal, Dinâmica Neuronal.