Yazarlar Hartwig Wieboldt'a mükemmel teknik yardımı için ve Hamburg-Eppendorf Üniversitesi Tıp Merkezi UKE Mikroskopi Görüntüleme Tesisi'ne (Umif) mikroskop ve destek sağladığı için teşekkür ediyor. Bu araştırma DZHK (Alman Kardiyovasküler Araştırmalar Merkezi) [FKZ 81Z4710141] tarafından finanse edildi.

Yazarların açıklayacak bir şeyi yok.

Va başlangıcından önce sempatik sinir sisteminin nöronlarında ve glial hücrelerinde hücresel ve moleküler süreçlerin anlaşılması, ani kalp durması dünya çapında en yaygın ölüm nedeni olmaya devam ettiği için yüksek ilgi çekicidir5. Bu nedenle, mevcut yazıda, bu ağdaki bir murine elemanı olan murine SG'yi tanımlamak ve RNA, protein ve hücresel düzeyde sonraki analizleri yapmak için temel bir yöntem repertuarı sunuyoruz.
Murine SG'nin bir...

Kardiyak otonom sinir sistemi, kalbin uygun fizyolojik yanıtla çevresel değişikliklere uyum sağlamasını sağlayan sempatik, parasempatik ve duyusal bileşenlerin sıkı bir şekilde düzenlenmiş bir dengesidir1,2. Bu dengedeki bozukluklar, örneğin sempatik aktivitenin artması, başlangıç için önemli bir sürücü olarak ventrikül aritmilerinin (VA) bakımı olarak belirlenmiştir3,4. Bu nedenle, beta-blokerlerle sempatik aktivitenin farmakolojik olarak azaltılması yoluyla elde edilen otonom modülasyon, on yıllardırVA'lıhastaların tedavisinde bir temel taşı olmuştur 5,6. Ancak farmakolojik ve kateter bazlı müdahalelere rağmen, ilgili sayıda hasta hala tekrarlayan VA7'denmuzdariptir.
Kalbe sempatik giriş çoğunlukla, beyin sapından kalbe çok sayıda intratorasik sinir yoluyla bilgi aktaran sempatik zincirin yıldız gangliyonlarındaki (SG) çift taraflı yıldız şeklindeki yapılarındaki nöronal hücre cisimleri aracılığıylaaracılıkeder 8 ,9,10. Yaralanmadan sonra SG'den filizlenen sinir VA ve ani kardiyak ölüm ile ilişkilidir11,12, SG'yi otonom modülasyon için bir hedef olarak vurgulayarak13,14. Kalbe sempatik girişin azaltılması geçici olarak lokal anesteziklerin perkütan enjeksiyonu ile veya video destekli torakoskopi ile SG'nin kısmen çıkarılması ile kalıcı olarak elde edilebilir15,16. Kardiyak sempatik denervasyon, umut verici sonuçlarla tedavi-refrakter VA'lı hastalar için bir seçenek sunar14,16,17. Bu hastaların eksiz SG'lerinden nöronal ve nörokimyasal remodeling, nöro-inflamasyon ve glial aktivasyonun otonomik disfonksiyona katkıda bulunabilecek veya ağırlaştırabilecek sempatik yeniden şekillendirmenin ayırt edici özellikleri olduğunu öğrendik18,19. Yine de, bu nöronlardaki altta yatan hücresel ve moleküler süreçler, örneğin, nöronal transdifferentiasyonun kolinerjik fenotip20,21'erolü gibi bugüne kadar belirsizliğini korumamaktadır. Deneysel çalışmalar VA'yı tedavi etmek için yeni yaklaşımlar sunar, örneğin, optogenetik22yoluyla sempatik sinir aktivitesinin azaltılması , ancak SG'nin derinlemesine karakterizasyonu VA ile birlikte giden birçok kardiyak patolojide hala eksiktir. Bu patolojileri taklit eden fare modelleri, aritmilerin başlangıcından önce potansiyel olarak12,23olan nöronal yeniden şekillendirmeyi incelemeye izin verir. Bunlar, kalbin ve sinir sisteminin otonom karakterizasyonu için daha fazla morfolojik ve fonksiyonel analizlerle tamamlanabilir. Mevcut protokolde, VA'nın anlaşılmasını geliştirmek için murine SG'yi parçalamaya ve karakterize etmeye izin veren temel bir yöntem repertuarı sunuyoruz.

<table><tbody><tr><td>96-well plate</td><td>TPP</td><td>92097</td><td>RNAscope</td></tr><tr><td>Adhesion Slides SuperFrost plus&amp;nbsp; 25 x 75 x 1 mm</td><td>R. Langenbrinck</td><td>03-0060</td><td>Microscopy</td></tr><tr><td>Albumin bovine Fraction V receptor grade lyophil.</td><td>Serva</td><td>11924.03</td><td>Whole mount staining</td></tr><tr><td>bisBenzimide H33342 trihydrochloride (Hoechst)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>B2261</td><td>Whole mount staining</td></tr><tr><td>Chicken anti neurofilament</td><td>EMD Millipore</td><td>AB5539</td><td>Whole mount staining</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Merck, KGA, Darmstadt, Germany</td><td>D8418</td><td>Whole mount staining</td></tr><tr><td>Donkey anti chicken IgY Alexa 647&amp;nbsp;</td><td>Merck, KGA, Darmstadt, Germany</td><td>AP194SA6</td><td>Whole mount staining</td></tr><tr><td>Donkey anti goat IgG Alexa 568&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11057</td><td>Whole mount staining</td></tr><tr><td>Donkey anti rabbit IgG Alexa 488&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A21206</td><td>Whole mount staining</td></tr><tr><td>Drying block 37-100 mm</td><td>Whatman (Sigma Aldrich)</td><td>WHA10310992&amp;nbsp;</td><td>Whole mount staining</td></tr><tr><td>Eosin Y</td><td>Sigma Aldrich</td><td>E4009</td><td>Whole mount staining</td></tr><tr><td>Ethanol 99 % denatured with MEK, IPA and Bitrex (min. 99,8 %)</td><td>Th.Geyer</td><td>2212.5000</td><td>Whole mount staining</td></tr><tr><td>Eukitt mounting medium</td><td>AppliChem</td><td>253681.0008</td><td>Whole mount staining</td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td>Whole mount staining</td></tr><tr><td>Fluoromount-G + DAPI</td><td>Southern Biotech</td><td>0100-20</td><td>Whole mount staining</td></tr><tr><td>Goat anti choline acetyltransferase</td><td>EMD Millipore</td><td>AP144P</td><td>Whole mount staining</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; 30% (w/w)</td><td>Merck, KGA, Darmstadt, Germany</td><td>H1009</td><td>Whole mount staining</td></tr><tr><td>Heparin Sodium 25.000 UI / 5ml</td><td>Rotexmedica</td><td>PZN: 3862340</td><td>Preparation SG</td></tr><tr><td>High-capacity cDNA reverse transctiption kit</td><td>Life technologies</td><td>&amp;nbsp;4368813</td><td>RNA isolation</td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott Laboratories</td><td>2594.00.00</td><td>Preparation SG</td></tr><tr><td>Mayer&#039;s hemalum solution</td><td>Merck</td><td>1.09249.0500</td><td>Whole mount staining</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td>Whole mount staining</td></tr><tr><td>Microscope cover glasses 20x20 mm or smaller</td><td>Marienfeld</td><td>0101040</td><td>Whole mount staining</td></tr><tr><td>miRNeasy Mini Kit</td><td>Qiagen</td><td>217004</td><td>RNA isolation</td></tr><tr><td>NanoDrop 2000c</td><td>Thermo Fisher Scientific</td><td>ND-2000C</td><td>RNA isolation</td></tr><tr><td>Opal 570 Reagent Pack</td><td>Akoya Bioscience</td><td>FP1488001KT</td><td>RNAscope</td></tr><tr><td>Paraformaldehyde, 16% w/v aq. soln., methanol free&amp;nbsp;</td><td>Alfa Aesar</td><td>43368</td><td>Whole mount staining</td></tr><tr><td>Pasteur pipettes, LDPE, unsterile, 3 ml, 154 mm</td><td>Th.Geyer</td><td>7691202</td><td>Whole mount staining</td></tr><tr><td>Phosphate-buffered saline tablets</td><td>Gibco</td><td>18912-014</td><td>Whole mount staining</td></tr><tr><td>Pinzette Dumont SS Forceps</td><td>FineScienceTools</td><td>11203-25</td><td>Preparation SG</td></tr><tr><td>QIAzol Lysis Reagent</td><td>Qiagen&amp;nbsp;</td><td>79306</td><td>RNA isolation</td></tr><tr><td>Rabbit anti tyrosine hydroxylase</td><td>EMD Millipore</td><td>AB152</td><td>Whole mount staining</td></tr><tr><td>RNAlater</td><td>Merck</td><td>R0901-100ML</td><td>RNA isolation (optional)</td></tr><tr><td>RNAscope Multiplex Fluorescent Reagent Kit v2</td><td>biotechne (ACD)</td><td>323100</td><td>RNAscope</td></tr><tr><td>RNAscope Probe-Mm-S100b-C2</td><td>biotechne (ACD)</td><td>431738-C2</td><td>RNAscope</td></tr><tr><td>RNAscope Probe-Mm-Tubb3</td><td>biotechne (ACD)</td><td>423391</td><td>RNAscope</td></tr><tr><td>Stainless steel beads 7 mm&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>69990</td><td>RNA isolation</td></tr><tr><td>Sudan black B</td><td>Roth</td><td>0292.2</td><td>Whole mount staining</td></tr><tr><td>TaqMan Gene Expression Assay Cdkn1b (Mm00438168_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay Choline acetyltransferase (Mm01221880_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay MKi67 (Mm01278617_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay PTPCR (Mm01293577_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay S100b (Mm00485897_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay Tyrosin Hydroxylase (Mm00447557_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan mastermix</td><td>Applied biosystems</td><td>4370074</td><td>Gene Expression analysis&amp;nbsp;</td></tr><tr><td>Tissue Lyser II</td><td>Qiagen</td><td>85300</td><td>RNA isolation</td></tr><tr><td>Triton X-100 10% solution</td><td>Sigma-Aldrich</td><td>93443-100ml</td><td>Whole mount staining</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P9416-100ML</td><td>RNAscope</td></tr><tr><td>Wacom bamboo pen</td><td>Wacom</td><td>CTL-460/K</td><td>Cell size measurements</td></tr><tr><td>Whatman prepleated qualitative filter paper, Grade 595 1/2</td><td>Sigma-Aldrich</td><td>WHA10311647</td><td>Whole mount staining</td></tr><tr><td>Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate</td><td>Thermo Fisher Scientific</td><td>W21404</td><td>RNAscope</td></tr></tbody></table>

location, dissection, and analysis of the murine stellate ganglion

Otonom sinir sistemi, kardiyak elektrofizyolojinin önemli bir sürücüsüdür. Özellikle sempatik dalının rolü, ventriküler aritmilerin (VA) patofizyolojisinde devam eden bir araştırma konusudur. Sempatik zincirin çift taraflı yıldız şeklindeki yapıları olan yıldız gangliyonlarındaki (SG)     nöronlar sempatik altyapının önemli bir bileşenidir. SG, tedavi-refrakter VA'lı hastalarda kardiyak sempatik denervasyon yoluyla tedavi için tanınan bir hedeftir. VA'lı hastalarda SG'de nöronal remodeling ve glial aktivasyon tanımlanmış olmakla birlikte, aritmi başlangıcından önce potansiyel olarak altta kalan hücresel ve moleküler süreçler sadece yeterince anlaşılmamıştır ve otonom modülasyonu iyileştirmek için aydınlatılmalıdır. Fare modelleri sempatik nöronal yeniden şekillendirmeyi incelememize izin verir, ancak murine SG'nin tanımlanması deneyimsiz araştırmacı için zordur. Bu nedenle, murine SG'nin derinlemesine hücresel ve moleküler biyolojik çalışmaları birçok yaygın kardiyak hastalık için eksiktir. Burada, yetişkin farelerde SG'nin RNA düzeyinde analizler (gen ekspresyon analizleri için RNA izolasyonu, yerinde hibridizasyon), protein seviyesi (immünofluoresan bütün montaj lekelenmesi) ve hücresel düzeyde (temel morfoloji, hücre büyüklüğü ölçümü) incelenmesi ve incelenmesi için temel bir repertuar açıklıyoruz. Hazırlık tekniğindeki zorlukların üstesinden gelmek için potansiyel çözümler sunuyoruz ve otofluoresansların söndürülerek boyamanın nasıl iyileştirileceği. Bu, hücre kompozisyonunu ve yeniden şekillendirme süreçlerini belirlemek için nöronların yanı sıra glial hücrelerin yerleşik belirteçler aracılığıyla görselleştirilmesine izin verir. Burada sunulan yöntemler, SG'nin VA'ya eğilimli farelerde otonomik disfonksiyon hakkında daha fazla bilgi edinmesine izin verir ve kalpteki otonom sinir sisteminin nöronal ve glial bileşenlerini araştıran ek tekniklerle tamamlanabilir.

Şekil 1, SG'nin nasıl tanımılacağını ve inceldiğini görselleştirir. Şekil 1A, konumun şematik bir çizimini gösterirken, Şekil 1B, kalp-akciğer paketinin çıkarılmasından sonra görünümü toraksa sunar. SG ve göğüs kafesinden gelen sol ve sağ longus colli kasları oryantasyon için önemli yerlerdir. Diseksiyon kaslar ve ilk kaburga arasındaki noktalı çizgiler boyunca gerçekleştirilir. SG ve sempatik zin...

Watch this Scientific Journal Video about Location, Dissection, and Analysis of the Murine Stellate Ganglion at JoVE.com

Location, Dissection, and Analysis of the Murine Stellate Ganglion

Kardiyak otonom sinir sistemindeki patofizyolojik değişiklikler, özellikle sempatik branşında, ventriküler aritmilerin başlamasına ve korunmasına katkıda bulunur. Mevcut protokolde, alttaki moleküler ve hücresel süreçlerin anlaşılmasını geliştirmek için murine stellat gangliyonunun nasıl karakterize edilir olduğunu gösteriyoruz.

Murine Stellate Ganglion'un Yeri, Diseksiyonu ve Analizi

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

location-dissection-and-analysis-of-the-murine-stellate-ganglion

Research

JoVE Journal

Medicine

9.9K Görüntüleme.  EVK Düsseldorf, cNEP, cardiac Neuro- and Electrophysiology Research Consortium. Kardiyak otonom sinir sistemindeki patofizyolojik değişiklikler, özellikle sempatik branşında, ventriküler aritmilerin başlamasına ve korunmasına katkıda bulunur. Mevcut protokolde, alttaki moleküler ve hücresel süreçlerin anlaşılmasını geliştirmek için murine stellat gangliyonunun nasıl karakterize edilir olduğunu gösteriyoruz.

Video: Location, Dissection, and Analysis of the Murine Stellate Ganglion

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Gerilim duyarlı Boyalar ile Yengeç Stomatogastric Ganglion Nöronlar Optik Görüntüleme

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Nörobilim

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

Homarus americanus Stomatogastric Sinir Sistemi Diseksiyon

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

Biyoloji

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

Kanser Borealis Stomatogastric Sinir Sistemi Diseksiyon

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

Büyüyen Aksonlar boyunca murin Schwann Hücresi Kalkınma Analizi

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary gland secretion, circadian rhythm, macular degeneration, immune function, and cardiac physiology. Murine surgical sympathectomy is a method for studying the effects of adrenergic innervation by allowing for complete, unilateral adrenergic ablation while avoiding the need for repeated pharmacologic intervention and the associated side effects. However, surgical sympathectomy in mice is technically challenging because of the small size of the superior cervical ganglion. This study describes a surgical technique for reliably identifying and resecting the superior cervical ganglion to ablate the sympathetic nervous system. The successful identification and removal of the ganglion are validated by imaging the fluorescent sympathetic ganglia using a transgenic mouse, identifying post-resection Horner's syndrome, staining for adrenergic markers in the resected ganglia, and observing diminished adrenergic immunofluorescence in the target organs following sympathectomy. This model enables future studies of cancer progression as well as other physiological processes regulated by the sympathetic nervous system.

The present protocol describes a mouse model of the ablation of adrenergic innervation by identifying and resecting the superior cervical ganglion.

Bir Murin Modelinde Superior Servikal Ganglionektomi için Cerrahi Teknik

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary ...

Kanser Araştırmaları

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

Lazer-yakalama mikrodiseksiyon optimizasyonu gelen insan dokusu taze donmuş enterik gangliyon yalıtım için

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

Erkek ve Dişi Sıçanlarda Pelvik Otonom Ganglia ve İlişkili Sinirlerin Diseksiyonu

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

Whole Central and Peripheral Nervous System Mice Dissection

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent nervous system, nor is there a complete schematic representing it that is freely available. Only methods to harvest the brain, the spinal cord, a specific dorsal root ganglion, and the sciatic nerve (separately) are available. Here, we provide detailed pictures and a schematic of the central and peripheral murine nervous system. More importantly, we outline a robust procedure to perform its dissection. The 30 min pre-dissection step allows isolating the intact nervous system within the vertebra with muscles free of viscera and skin. A 2-4 h dissection follows it under a micro-dissection microscope to expose the spinal cord and the thoracic nerves, and finally peel the whole central and peripheral nervous system off the carcass. This protocol represents a significant step forward in studying the anatomy and pathophysiology of the nervous system globally. For example, the dissected dorsal root ganglions from a neurofibromatosis type I mice model can be further processed for histology to unravel changes in tumor progression.

Here, we present a protocol to complete the dissection of central and peripheral nervous systems of mice. The dissected tissues are further analyzed in downstream applications, such as taking gross pictures or histology.

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent ...

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

Düşük Girişli Çekirdek İzolasyonu ve Tek Çekirdekli RNA Dizilimi için Fare Sempatik Ganglia'nın Barkodlu Antikorları ile Çoklama

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into ...

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Fare Sempatik Nöronlarının Hedeflenmesi: Lomber Sempatektomi Yaklaşımı

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

Yazar Spotlight: Sempatik Nöronların Plastisitesini Keşfetmek

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

Farelerde Cerrahi Lomber Sempatektomi

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...