Methods and Protocols最新文献_第9页

I-TEP: A Simple and Affordable Method to Measure Permeability in Reconstructed Tissues Combined with DAMO-TSC-Based Urea Assay. I-TEP：一种简单和负担得起的方法来测量重建组织的通透性结合damo - tsc为基础的尿素测定。

IF 2.5

Methods and Protocols Pub Date : 2026-05-03 DOI: 10.3390/mps9030073

Yudaï Sahuc, Elissa Elia, Christophe Caneparo, Nathan Félix, Marilou Hardy, Stéphane Chabaud, Stéphane Bolduc

{"title":"I-TEP: A Simple and Affordable Method to Measure Permeability in Reconstructed Tissues Combined with DAMO-TSC-Based Urea Assay.","authors":"Yudaï Sahuc, Elissa Elia, Christophe Caneparo, Nathan Félix, Marilou Hardy, Stéphane Chabaud, Stéphane Bolduc","doi":"10.3390/mps9030073","DOIUrl":"10.3390/mps9030073","url":null,"abstract":"Trans-epithelial permeability is a critical functional parameter for reconstructed tissues, particularly in genitourinary tissue engineering, where urine leakage must be avoided. Although Franz diffusion cells are considered the gold standard for permeability measurements, their cost and limited accessibility restrict their widespread use. In parallel, the reliable quantification of urea in culture media remains challenging due to protein interference and assay cost. The Inexpensive Trans-Epithelial Permeability (I-TEP) test is a simple and a low-cost Franz-like permeability system which can be combined with an optimized diacetyl monoxime-thiosemicarbazide (DAMO-TSC) colorimetric urea assay. I-TEP system relies on readily available laboratory components to create physically separate donor and receiver compartments, with the tissue acting as the sole diffusion interface. The DAMO-TSC assay was optimized through systematic evaluation of deproteinization, incubation time, storage conditions, and serum interference. The I-TEP test showed a strong correlation with conventional Franz diffusion cells when testing similar tissue samples. Deproteinization was identified as a mandatory step for accurate urea quantification in serum-containing media. The combined approach was successfully applied on engineered genitourinary tissues, demonstrating sensitivity to tissue maturation and cellular composition. This protocol provides a proof of concept for an affordable, robust, and autonomous method for routine permeability assessment, bridging the gap between costly commercial systems and high-throughput experimental needs.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13214736/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148032979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Collection and Lipidomic Analysis of Murine Knee Synovium and Infrapatellar Fat Pad. 小鼠膝关节滑膜及髌下脂肪垫的收集及脂质组学分析。

IF 2.5

Methods and Protocols Pub Date : 2026-05-02 DOI: 10.3390/mps9030070

Tong Yang, Luke Stasikelis, Alexander J Knights

{"title":"Collection and Lipidomic Analysis of Murine Knee Synovium and Infrapatellar Fat Pad.","authors":"Tong Yang, Luke Stasikelis, Alexander J Knights","doi":"10.3390/mps9030070","DOIUrl":"10.3390/mps9030070","url":null,"abstract":"Intra-articular soft connective tissues such as synovium and adipose tissue play a crucial role in governing joint homeostasis and disease progression in various forms of arthritis. In the knee, like many synovial joints, adipose tissue forms an integrated anatomic and functional unit with the joint-lining synovium, and the most prominent adipose depot is the infrapatellar fat pad (IFP). With growing evidence that lipid profiles in the synovium-IFP unit shift during progression of joint diseases like osteoarthritis (OA), there is strong impetus for consistent tissue collection approaches and reproducible subsequent lipid characterization. Here, we present a standardized dissection and low-input untargeted lipidomics workflow optimized for mouse knee synovium and IFP, to enable comprehensive lipid profiling. Synovium/IFP from multiple joints are pooled to increase input mass and guarantee robust lipid yield, followed by lipid extraction and high-resolution liquid chromatography-mass spectrometry (LC-MS) acquisition for global, untargeted lipidomic profiling. The analysis workflow encompasses robust feature detection, accurate lipid annotation, data transformation and normalization. These steps enhance comparability across samples, particularly those with low input amounts, while minimizing technical variance and batch effects. Using this approach, we detect a broad spectrum of lipid species spanning the major lipid categories. As expected for untargeted discovery, a subset of non-lipid species is also observed. This protocol provides a practical framework for robust, reproducible lipidomics in murine intra-articular soft tissues to support future disease-specific biomarker and drug target discovery in OA and other joint diseases.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13214787/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148032868","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Morphological Assessment of Stage HH38 of the Japanese Quail (Coturnix japonica) Heart by Micro-Sonogram. 日本鹌鹑（Coturnix japonica）心脏HH38期的显微超声形态学评价。

IF 2.5

Methods and Protocols Pub Date : 2026-05-02 DOI: 10.3390/mps9030071

Jaden Roe, Ashlyn Benavides, Michael B Filla, Douglas C Bittel, Whitney Shae, Geetha Haligheri, James E O'Brien, Nataliya Kibiryeva

{"title":"Morphological Assessment of Stage HH38 of the Japanese Quail (Coturnix japonica) Heart by Micro-Sonogram.","authors":"Jaden Roe, Ashlyn Benavides, Michael B Filla, Douglas C Bittel, Whitney Shae, Geetha Haligheri, James E O'Brien, Nataliya Kibiryeva","doi":"10.3390/mps9030071","DOIUrl":"10.3390/mps9030071","url":null,"abstract":"A challenge of studying mammalian cardiac embryogenesis is the limited ability to perform experimental manipulations in animal models. The avian embryo is widely accepted as a model for mammalian heart developmental studies. In this study, we establish the methodology and protocols for studying the Japanese quail (Coturnix japonica) heart at embryonic day 10 (HH38) using the FUJIFILM VisualSonics Vevo 3100 ultrasound system equipped with a MX550D small animal cardiology transducer. These protocols were designed to measure right ventricular wall thickness, pulmonary artery diameter, and the outflow velocities of the right ventricular outflow tract (RVOT) and the pulmonary artery (PA), thereby establishing baseline parameters of the normally developing quail morphology. Quail embryos are an ideal model for cardiovascular research due to their short incubation period (16-17 days), experimental accessibility, and strong similarities to mammalian heart development. These developmental similarities include, but are not limited to, looping, chamber septation, and the development of a true four-chamber heart. High-resolution imaging modalities, including ultrasound and optical coherence tomography, enable noninvasive, real-time visualization of cardiac morphology and function throughout development. Echocardiography allows for quantitative and qualitative assessments of myocardial structure and cardiac hemodynamics. The similarity to the mammalian heart, combined with rapid embryogenesis, makes quail embryos a valuable model for investigating congenital heart defects, genetic modifications, and fundamental cardiac developmental processes. In this study, we describe reproducible incubation protocols and baseline echocardiographic parameters used to evaluate morphological and physiological changes in the developing embryonic quail heart on embryonic day 10.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13214935/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148032908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Comparative Analysis of Streptozotocin, Streptozotocin-Nicotinamide and Alloxan-Based Diabetes Models in Female Wistar Rats. 链脲佐菌素、链脲佐菌素-烟酰胺和四氧嘧啶型雌性Wistar大鼠糖尿病模型的比较分析。

IF 2.5

Methods and Protocols Pub Date : 2026-05-02 DOI: 10.3390/mps9030072

Sabrina-Gabriela Mîndruț, Cristina Pop, Sorin-Marian Mârza, Alexia-Teodora Hoța, Flaviu-Alexandru Tăbăran, Ibrahima Mamadou Sall, Ana Uifălean, Emilia-Laura Mogoșan, Oliviu Voștinaru, Cristina-Ionela Mogoșan

{"title":"Comparative Analysis of Streptozotocin, Streptozotocin-Nicotinamide and Alloxan-Based Diabetes Models in Female Wistar Rats.","authors":"Sabrina-Gabriela Mîndruț, Cristina Pop, Sorin-Marian Mârza, Alexia-Teodora Hoța, Flaviu-Alexandru Tăbăran, Ibrahima Mamadou Sall, Ana Uifălean, Emilia-Laura Mogoșan, Oliviu Voștinaru, Cristina-Ionela Mogoșan","doi":"10.3390/mps9030072","DOIUrl":"10.3390/mps9030072","url":null,"abstract":"Experimental diabetes models induced by streptozotocin (STZ) and alloxan are widely used in preclinical research; however, direct standardized comparisons in female rodents remain limited. The present study evaluated multiple chemical induction protocols in female Wistar rats, including STZ (40 and 65 mg/kg), STZ at the same doses combined with nicotinamide (110 mg/kg), and alloxan (130 mg/kg). Glycemic progression, oral glucose tolerance test, body weight evolution, oxidative stress markers, and multi-organ histopathology were assessed over a 14-day period. High-dose STZ (65 mg/kg) and alloxan produced rapid, sustained hyperglycemia (p < 0.0001), significant body weight reduction, increased lipid peroxidation (elevated MDA), nitric oxide overproduction, thiol depletion, and pronounced pancreatic and renal structural damage. In contrast, STZ-nicotinamide protocols generated moderate but stable hyperglycemia with partial preservation of islet architecture, attenuated oxidative imbalance, and improved systemic tolerability. Oral glucose tolerance test confirmed impaired glucose handling in the STZ-nicotinamide group, consistent with a type 2 diabetes-like phenotype rather than complete insulin deficiency. These results demonstrate that induction strategy critically determines metabolic stability, oxidative stress burden, and tissue remodeling patterns, supporting model selection according to specific experimental objectives.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13214971/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148032830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Large Language Models for Clinical Narrative Processing: Methods, Applications, and Challenges. 临床叙事处理的大型语言模型：方法、应用和挑战。

IF 2.5

Methods and Protocols Pub Date : 2026-05-01 DOI: 10.3390/mps9030069

Achilleas Livieratos, Junjing Lin, Paraskevi Chasani, Mina Gaga, Fotios S Fousekis, Charalambos Gogos, Karolina Akinosoglou, Konstantinos H Katsanos, Margaret Gamalo

{"title":"Large Language Models for Clinical Narrative Processing: Methods, Applications, and Challenges.","authors":"Achilleas Livieratos, Junjing Lin, Paraskevi Chasani, Mina Gaga, Fotios S Fousekis, Charalambos Gogos, Karolina Akinosoglou, Konstantinos H Katsanos, Margaret Gamalo","doi":"10.3390/mps9030069","DOIUrl":"10.3390/mps9030069","url":null,"abstract":"Large language models (LLMs) have rapidly advanced natural language processing and are increasingly used to analyze clinical narratives. Their ability to extract information, summarize records, and support clinical workflows makes them potential tools for enhancing documentation efficiency and the secondary application in the analysis of electronic health record (EHR) data. The aim of this work is to synthesize recent evidence on methodological approaches and applications of LLMs for clinical narrative processing, and to assess their performance, benefits, limitations, and implications for clinical practice. Across 2022-2026 studies, LLMs demonstrated strong performance in information extraction, summarization, triage prediction, section classification, and synthetic text generation, often surpassing traditional machine-learning models. Overall, LLMs improved the conversion of unstructured notes into actionable clinical insights, reduced documentation burden, and supported decision-making tasks. Key challenges included hallucinations, variable reproducibility, sensitivity to prompting, domain adaptation gaps, and limited transparency. Our findings indicate that LLMs show substantial promise for transforming clinical narrative processing, but safe adoption requires rigorous evaluation and continuous model auditing. This work provides a structured, non-systematic synthesis of representative studies and is intended as a high-level overview of emerging applications rather than a comprehensive systematic review.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13214763/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148032974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Simultaneous In Vivo Electrophysiology, Two-Photon Imaging, and Optogenetics for Probing Neurovascular Coupling. 同时在体内电生理，双光子成像，和光遗传学探测神经血管耦合。

IF 2.5

Methods and Protocols Pub Date : 2026-04-25 DOI: 10.3390/mps9030068

Dalchand Ahirwar, Kun Xie, Philip O'Herron

{"title":"Simultaneous In Vivo Electrophysiology, Two-Photon Imaging, and Optogenetics for Probing Neurovascular Coupling.","authors":"Dalchand Ahirwar, Kun Xie, Philip O'Herron","doi":"10.3390/mps9030068","DOIUrl":"10.3390/mps9030068","url":null,"abstract":"Neuronal activity and cerebral blood flow are tightly coupled to support the high metabolic demands of the brain. Disruption of neurovascular coupling is a defining feature of many neurodegenerative disorders such as Alzheimer's disease, stroke, small vessel disease, Parkinson's disease, and aging. Progress in understanding the mechanisms underlying neurovascular coupling requires experimental approaches that can simultaneously measure neuronal activity and vascular dynamics with high spatial and temporal resolution, while also enabling targeted perturbations of the system. Here, we present a methodological framework that combines chronic electrophysiological recordings with two-photon imaging of cerebral blood flow and optogenetic manipulation of the vasculature in vivo. Using a chronically implanted flexible electrode array, we obtain measurements of the single- and multi-unit spiking activity, as well as local field potentials. Concurrently, two-photon microscopy enables high-resolution measurements of vessel diameter and blood flow within individual vascular segments. In addition, optogenetic control of vascular smooth muscle cells allows for rapid and reversible manipulation of the vessel diameter through the same cranial window while simultaneously recording the neural and vascular activity. We provide detailed protocols for surgical implantation, data acquisition, and analysis, and discuss experimental considerations and limitations. This combined platform offers a powerful tool for mechanistic studies of neurovascular coupling and its dysfunction in disease models.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13214910/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148032888","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Evaluation of Cryopreserved Primary Swine Macrophages as a Substrate for African Swine Fever Virus Replication. 冷冻原代猪巨噬细胞作为非洲猪瘟病毒复制底物的评价。

IF 2.5

Methods and Protocols Pub Date : 2026-04-24 DOI: 10.3390/mps9030067

Vivian K O'Donnell, Andrew Schoepke, Heather Petrowski, Leslie Blakemore, Douglas P Gladue, Bonto Faburay, Manuel V Borca

引用次数: 0

Proposal for a Protocol and a Handmade Arduino-Based and Open Source Device for Measuring the Residual Charge of Alkaline Batteries in View of an Attempt to Recharge Them. 针对尝试给碱性电池充电的问题，提出了一种测量碱性电池剩余电量的协议和基于arduino手工制作的开源设备。

IF 2.5

Methods and Protocols Pub Date : 2026-04-19 DOI: 10.3390/mps9020066

Giovanni Visco, Maria Pia Sammartino, Angela Marchetti, Mauro Castrucci, Mauro Tomassetti

{"title":"Proposal for a Protocol and a Handmade Arduino-Based and Open Source Device for Measuring the Residual Charge of Alkaline Batteries in View of an Attempt to Recharge Them.","authors":"Giovanni Visco, Maria Pia Sammartino, Angela Marchetti, Mauro Castrucci, Mauro Tomassetti","doi":"10.3390/mps9020066","DOIUrl":"10.3390/mps9020066","url":null,"abstract":"Portable devices are powered in direct current (DC) or by batteries (primary battery), accumulators (secondary battery), and now supercapacitors, which can also be used for energy storage. The European Portable Battery Association states that approximately 239,000 tons of batteries were placed on the market in the European Economic Area (EEA) plus Switzerland in 2022. Even if they were all disposed of correctly respecting the 3R paradigm (Reduce, Reuse and Recycle), non-rechargeable batteries create an environmental problem because they do not discharge completely with an obvious waste of energy. Secondary batteries and supercapacitors can be recharged because they use reversible chemical/physical processes while primary batteries cannot be recharged because they are based on irreversible redox reactions; nevertheless, it is possible to try to recover their residual charge if this is higher than a threshold beyond which the reactions can be reversible. The most used batteries are alkaline zinc/manganese dioxide and they are non-rechargeable; an inappropriate recharge attempt can lead to serious harm to the operator and the environment. This paper describes a simple Arduino-based circuit and the protocol to measure and graph the residual charge of an alkaline battery in order to establish if it can be recharged. The circuit, design, the Arduino Uno R3 sketch (i.e., microprocessor software) and the full protocol are here presented under the open source license (Copyright Creative Commons Public license, CC BY-NC-ND 4.0 EN) so that they could become a pilot system and then a commercial product. The residual charge of 158 batteries, obtained after discharging those that, by eye, appeared damaged, was measured. Results evidenced that 49% of batteries had a residual voltage, under low load, between 1.2 and 1.6 V, making them good candidates for a recharge attempt.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13118361/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776451","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Optimized Protocols to Extract Total Transcripts and Proteins from Lipid-Rich Tissues. 从富含脂质的组织中提取总转录本和蛋白质的优化方案。

IF 2.5

Methods and Protocols Pub Date : 2026-04-10 DOI: 10.3390/mps9020065

Nicolas De Azevedo, Anthony Lozano, Ramon E Parsons, Tiphaine C Martin

{"title":"Optimized Protocols to Extract Total Transcripts and Proteins from Lipid-Rich Tissues.","authors":"Nicolas De Azevedo, Anthony Lozano, Ramon E Parsons, Tiphaine C Martin","doi":"10.3390/mps9020065","DOIUrl":"10.3390/mps9020065","url":null,"abstract":"Background: Highly lipidic tissues (e.g., adipose tissue, brain, and liver) are challenging for transcript and protein extraction and for next-generation sequencing. Lipids can clog filters, columns, and pipettes; cause autofluorescence and quenching in imaging; and interfere with centrifugation-based separation.Aim: To identify the most suitable method for extracting total RNA for RT-qPCR and an alternative method for extracting total protein for quantification in mice fed a regular or high-fat diet.Methods: We compared three total RNA extraction methods and two total protein extraction methods.Results: The highest total RNA yield and purity were obtained with TRIzol and chloroform, with optimized steps added to the original protocol to address the challenges posed by highly lipid-rich tissues. For total protein extraction, an adipose tissue-specific kit from Invent Biotechnologies yielded higher protein levels than the classical RIPA-based method. Among lipid-rich tissues, we observed that adipose tissue was more challenging to process than the brain and the liver.Conclusions: Adipose tissue, particularly under a high-fat diet, is the most challenging lipid-rich tissue, followed by the brain and then the liver. We highlight protocols that improve total RNA and protein yields and purity, which may benefit other researchers working with these tissues.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13118782/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776255","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Stereological Assessment of Locus Coeruleus in the Mouse: A Methodological Study in Pups and Adult Animals. 小鼠蓝斑体视学评价：幼鼠和成年动物的方法学研究。

IF 2.5

Methods and Protocols Pub Date : 2026-04-09 DOI: 10.3390/mps9020064

Marco Scotto, Alessandro Galgani, Marina Boido, Nooria Mohammady, Alessandro Vercelli, Filippo S Giorgi

{"title":"Stereological Assessment of Locus Coeruleus in the Mouse: A Methodological Study in Pups and Adult Animals.","authors":"Marco Scotto, Alessandro Galgani, Marina Boido, Nooria Mohammady, Alessandro Vercelli, Filippo S Giorgi","doi":"10.3390/mps9020064","DOIUrl":"10.3390/mps9020064","url":null,"abstract":"Unbiased stereology represents the most accurate approach for estimating the total number of neurons of specific brain regions; however, its reliability critically depends on the use of rigorously defined and anatomically appropriate sampling parameters. The brain nucleus Locus Coeruleus (LC) plays a key role in several brain functions. LC impairment has been associated with a range of disorders affecting individuals across the lifespan, from infancy to adulthood. In animal models of these conditions, precise estimation of LC neuronal number is essential. The LC analysis poses specific methodological challenges due to its small size, indistinct anatomical boundaries, and age-dependent changes in neuronal density. In this study, we present a detailed and reproducible stereological workflow for the quantification of LC neurons in the mouse brain across the lifespan. Using C57BL/6J mice at postnatal, adult, and aged stages, we optimized all key components of the Optical Fractionator method, LC neurons were identified by immunoperoxidase staining for tyrosine hydroxylase (TH) and quantified using systematic-random sampling implemented in Stereo Investigator® software. We show that age-specific adjustment of stereological parameters is necessary to obtain reliable estimates, particularly at early postnatal stages characterized by high neuronal packing density. With the optimized protocols described here, TH+ LC neuron counts consistently met accepted precision criteria, as assessed by the Gundersen coefficient of error.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13118734/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776551","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0