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Journal of Visualized Experiments : JoVE Nov 2019Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such...
Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.
Topics: Animals; Cochlea; Epithelium; Hair Cells, Auditory; Hair Cells, Auditory, Inner; Mice; Microdissection; Organ of Corti
PubMed: 31762458
DOI: 10.3791/60299 -
Methods in Molecular Biology (Clifton,... 2018Laser Capture Microdissection has earned a permanent place among modern techniques connecting histology and molecular biology. Laser Capture Microdissection has become...
Laser Capture Microdissection has earned a permanent place among modern techniques connecting histology and molecular biology. Laser Capture Microdissection has become an invaluable tool in medical research as a means for collection of specific cell populations isolated from their environment. Such genomic sample enrichment dramatically increases the sensitivity and precision of downstream molecular assays used for biomarker discovery, monitoring disease onset and progression, and in the development of personalized medicine. The diversity of research targets (cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, frozen tissues, archival repository slides, etc.) and scientific objectives present a challenge in establishing standard protocols for Laser Capture Microdissection. In the present chapter, we share our experiences in design and successful execution of numerous diverse microdissection projects, and provide considerations to be taken into account in planning a microdissection study. Our workflow and protocols are standardized for a wide range of animal and human tissues and adapted to downstream analysis platforms.
Topics: DNA; Frozen Sections; Humans; Laser Capture Microdissection; Tissue Fixation; Workflow
PubMed: 29344854
DOI: 10.1007/978-1-4939-7558-7_3 -
Pathobiology : Journal of... 2023Histopathology has historically been the critical technique for the diagnosis and treatment of human disease. Today, genomics, transcriptomics, and proteomics from... (Review)
Review
Histopathology has historically been the critical technique for the diagnosis and treatment of human disease. Today, genomics, transcriptomics, and proteomics from specific cells, rather than bulk tissue, have become key to understanding underlying disease mechanisms and rendering useful diagnostic information. Extraction of desired analytes, i.e., nucleic acids or proteins, from easily accessible formalin-fixed paraffin-embedded tissues allows for clinically relevant activities, such as sequencing biomarker mutations or typing amyloidogenic proteins. Genetic profiling has become routine for cancers as varied as non-small cell lung cancer and prostatic carcinoma. The five main tissue dissection techniques that have been developed thus far include: bulk scraping, manual macrodissection, manual microdissection, laser-capture microdissection, and expression microdissection. In this review, we discuss the importance of tissue dissection in clinical practice and research, the basic methods, applications, as well as some advantages and disadvantages for each modality.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Prognosis; Lung Neoplasms; Dissection; Microdissection; Tissue Fixation; Paraffin Embedding
PubMed: 35952628
DOI: 10.1159/000525979 -
Journal of Visualized Experiments : JoVE Apr 2020Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular...
Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma's growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.
Topics: Animals; Brain Neoplasms; Glioma; Humans; Laser Capture Microdissection; Mice; Neoplasm Invasiveness; Sequence Analysis, RNA; Staining and Labeling
PubMed: 32338655
DOI: 10.3791/60939 -
Medecine Sciences : M/S Nov 2019One of the most fascinating aspects of the use of a laser beam in the field of biology has emerged with the development of devices able to perform fine dissections of... (Review)
Review
One of the most fascinating aspects of the use of a laser beam in the field of biology has emerged with the development of devices able to perform fine dissections of biological tissues. Laser microdissection can collect phenotypically identical cells from tissue regions laid on a microscope slide in order to make differential molecular analyses on these microdissected cells. Laser microdissection can be used many areas including oncology to specify molecular mechanisms that enable to adapt a treatment related to diagnosis and research in biology, but also forensic science for tissue selection, neurology for post-mortem studies on patients with Alzheimer's disease, for clonality studies from cell cultures and cytogenetics to decipher chromosomal rearrangements. This technology represents the missing link between clinical observations and the intrinsic physiological mechanisms of biological tissues and its major applications will be addressed here.
Topics: Histological Techniques; Humans; Laser Capture Microdissection; Molecular Diagnostic Techniques
PubMed: 31845879
DOI: 10.1051/medsci/2019166 -
International Journal of Impotence... Dec 2022Microdissection testicular sperm extraction (mTESE) has been proposed as a salvage treatment option for men with a previously failed classic TESE (cTESE), but data are...
Outcomes and predictive factors of successful salvage microdissection testicular sperm extraction (mTESE) after failed classic TESE: results from a multicenter cross-sectional study.
Microdissection testicular sperm extraction (mTESE) has been proposed as a salvage treatment option for men with a previously failed classic TESE (cTESE), but data are scarce. We aimed to assess the outcome of and potential predictors of successful salvage mTESE in a cohort of men previously submitted to unfruitful cTESE. Data from 61 men who underwent mTESE after a failed cTESE between 01/2014 and 10/2020, at 6 tertiary-referral centres in Italy were analysed. All men were investigated with semen analyses, testicular ultrasound, hormonal and genetic blood testing. Pathological diagnosis from TESE was collected in every man. Descriptive statistics and logistic regression models were used to investigate potential predictors of positive sperm retrieval (SR+) after salvage mTESE. Baseline serum Follicle-Stimulating hormone (FSH) and total testosterone levels were 17.2 (8.6-30.1) mUI/mL and 4.7 (3.5-6.4) ng/mL, respectively. Sertoli-cell-only syndrome (SCOS), maturation arrest (MA) and hypospermatogenesis were found in 24 (39.3%), 21 (34.4%) and 16 (26.2%) men after cTESE, respectively. At mTESE, SR+ was found in 30 (49.2%) men. Patients with a diagnosis of hypospermatogenesis had a higher rate of SR+ (12/16 (75%)) compared to MA (12/21 (57.1%)) and SCOS (6/24 (25%)) patients at mTESE (p < 0.01). No clinical and laboratory differences were observed between SR+ and SR- patients at mTESE. There were no significant complications after mTESE. At multivariable logistic regression analysis, only hypospermatogenesis (OR 9.5; p < 0.01) was independently associated with SR+ at mTESE, after accounting for age and FSH.In conclusion, salvage mTESE in NOA men with previous negative cTESE was safe and promoted SR+ in almost 50%. A baseline pathology of hypospermatogenesis at cTESE emerged as the only independent predictor of positive outcomes at salvage mTESE.
Topics: Humans; Male; Azoospermia; Cross-Sectional Studies; Follicle Stimulating Hormone; Microdissection; Oligospermia; Retrospective Studies; Semen; Spermatozoa
PubMed: 34743195
DOI: 10.1038/s41443-021-00487-8 -
Journal of Visualized Experiments : JoVE Nov 2021Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However,...
Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 μm, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.
Topics: Animals; Cell Separation; Fallopian Tubes; Female; Humans; Immunohistochemistry; Mice; Microdissection; Oviducts
PubMed: 34806701
DOI: 10.3791/63168 -
Journal of Neurochemistry Oct 2021Over the last 10 years, considerable technical advances in mass spectrometry (MS)-based bioanalysis have enabled the investigation of lipid signatures in... (Review)
Review
Over the last 10 years, considerable technical advances in mass spectrometry (MS)-based bioanalysis have enabled the investigation of lipid signatures in neuropathological structures. In Alzheimer´s Disease (AD) research, it is now well accepted that lipid dysregulation plays a key role in AD pathogenesis and progression. This review summarizes current MS-based strategies, notably MALDI and ToF-SIMS imaging as well as laser capture microdissection combined with LC-ESI-MS. It also presents recent advances to assess lipid alterations associated with Amyloid-β plaques, one of the hallmarks of AD. Collectively, these methodologies offer new opportunities for the study of lipids, thus pushing forward our understanding of their role in such a complex and still untreatable disease as AD.
Topics: Animals; Humans; Laser Capture Microdissection; Lipids; Mass Spectrometry; Microglia; Neuroimaging; Plaque, Amyloid; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 33048341
DOI: 10.1111/jnc.15216 -
Urologiia (Moscow, Russia : 1999) Sep 2021Patients with non-obstructive azoospermia (NOA), which accounts for up to 10-15% of all cases of male infertility, until recently could only become parents using donor... (Review)
Review
Patients with non-obstructive azoospermia (NOA), which accounts for up to 10-15% of all cases of male infertility, until recently could only become parents using donor sperm or through adoption. Modern technical capabilities of sperm extraction in combination with the use of assisted reproductive technologies, make it possible to effectively overcome infertility in this group of patients. A number of highly effective techniques have been proposed for spermatozoa retrieval. However, surgical intervention is associated with certain risks, and therefore, the choice of the optimal treatment method is under discussion. A total of 52 articles were analyzed using the MEDLINE database (PubMed) to form an overview of the current principles of examination and preparation of a patient with NOA for the surgical sperm retrieval. This review is dedicated to the role of diagnostic testicular biopsy. In addition, a comparative information on the efficacy and safety of percutaneous, fine-needle aspiration, open multifocal and microdissection (micro-TESE) testicular biopsies is presented. Of the currently available sperm retrieval techniques in the urologic armamentarium, micro-TESE seems to be both the most effective and the safest. Micro-TESE can be a cumbersome procedure, however, it provides successful treatment in situations previously associated with zero chance of pregnancy.
Topics: Azoospermia; Female; Humans; Male; Microdissection; Pregnancy; Retrospective Studies; Sperm Retrieval; Testis
PubMed: 34486283
DOI: No ID Found -
Journal of Visualized Experiments : JoVE Aug 2022Increasing rates of addiction behavior have motivated mental health researchers and clinicians alike to understand antireward and recovery. This shift away from reward...
Increasing rates of addiction behavior have motivated mental health researchers and clinicians alike to understand antireward and recovery. This shift away from reward and commencement necessitates novel perspectives, paradigms, and hypotheses along with an expansion of the methods applied to investigate addiction. Here, we provide an example: A systems biology approach to investigate antireward that combines laser capture microdissection (LCM) and high-throughput microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). Gene expression network dynamics were measured and a key driver of neurovisceral dysregulation in alcohol and opioid withdrawal, neuroinflammation, was identified. This combination of technologies provides anatomic and phenotypic specificity at single-cell resolution with high-throughput sensitivity and specific gene expression measures yielding both hypothesis-generating datasets and mechanistic possibilities that generate opportunities for novel insights and treatments.
Topics: Gene Expression; Gene Regulatory Networks; Laser Capture Microdissection; Real-Time Polymerase Chain Reaction; Reward
PubMed: 35993753
DOI: 10.3791/64014