Name: location, dissection, and analysis of the murine stellate ganglion
Uploaded: 2020-12-22T14:45:00.000+00:00
Duration: 7 min 50 s
Description: Watch this Scientific Journal Video about Location, Dissection, and Analysis of the Murine Stellate Ganglion at JoVE.com

저자는 그의 우수한 기술 지원에 대한 하트 위비히 Wieboldt 감사하고 싶습니다, 그리고 현미경및 지원을 제공하는 대학 의료 센터 함부르크 - Eppendorf의 UKE 현미경 이미징 시설 (Umif). 이 연구는 DZHK에 의해 투자되었다 (심장 혈관 연구를위한 독일 센터) [FKZ 81Z4710141].

저자는 공개 할 것이 없습니다.

VA의 개시에 선행된 교감 신경계의 신경세포 및 분자 프로세스에 있는 세포 및 분자 프로세스의 이해는, 급격한 심장 정지가 전 세계적으로 죽음의 일반적인 원인 남아 있기 때문에, 높은 관심사입니다5. 따라서, 현재 원고에서, 우리는 뮤린 SG를 식별하는 방법의 기본 레퍼토리를 제공합니다 - 이 네트워크 내의 뮤린 원소 - RNA에 후속 분석을 수행, 단백질, 및 세포 수준.
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심장 자율 신경계는 심장이 적절한 생리적 반응1,2로환경 변화에 적응할 수 있도록 교감, 부교감 및 감각 성분의 엄격하게 조절된 평형이다. 이러한 평형의 교란은 예를 들어, 교감 활동의 증가, 발병의 핵심 동인으로서 확립된 뿐만 아니라 심실 부정맥(VA)3,4의유지보수로 확립되었다. 따라서, 베타 차단제와의 교감 활동의 약리학적 감소를 통해 달성된 자율 변조는 수십 년 동안 VA를 가진 환자의 치료에 초석이 되어왔다5,6. 그러나 약리학 및 카테터 기반 내정간섭에도 불구하고, 환자의 관련 수는 아직도 재발성 VA7때문에 손해를 입습니다.
심장에 대한 공감 입력은 주로 스텔레이트 신경리아(SG)의 신경 세포 체를 통해 중재되며, 교감 사슬의 양자 별 모양 구조는 뇌간에서 심장8,9,10까지수많은 내트라토라시크 신경을 통해 정보를 전달한다. 부상 후 SG로부터 발아하는 신경은 VA및 갑작스런심장사멸(11,12)과관련이 있으며, SG를 자율변조의 대상으로강조한다(13,14). 심장에 대한 공감 입력의 감소는 비디오 보조 흉부검사(15,16)를통해 SG를 부분적으로 제거함으로써 국소 마취제의 경피 주입또는 영구적으로 영구적으로 얻을 수 있다. 심장 동정 성 서성 검사는 유망한 결과 와 치료 불응성 VA 환자에 대한 옵션을 제공합니다14,16,17. 우리는 신경 및 신경 화학적 리모델링, 신경 염증 및 신경교 활성화가 자율 기능 장애18,19에기여하거나 악화 시킬 수있는 공감 리모델링의 특징이라는 이러한 환자의 이식 SG에서 배웠습니다. 여전히, 이러한 뉴런의 기본 세포 및 분자 과정은 현재까지 모호하게 남아, 예를 들어, 해저 현상형으로 뉴런 전이의 역할20,21. 실험 연구는 VA를 치료하는 새로운 접근 방식을 제시, 예를 들어, 광유전학을 통해 교감 신경 활동의 감소(22)하지만 SG의 심층 특성은 여전히 VA와 함께 가는 많은 심장 병리학에서 부족. 이러한 병리를 모방 마우스 모델은 잠재적으로 부정맥의 발병 앞에 신경 리모델링을 연구 할 수 있습니다12,23. 이들은 심혼과 신경계의 자율 특성화를 위한 추가 형태학 및 기능적인 분석에 의해 완료될 수 있습니다. 본 프로토콜에서, 우리는 VA의 이해를 향상시키기 위해 뮤린 SG를 해부하고 특성화 할 수있는 방법의 기본 레퍼토리를 제공합니다.

<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

자율 신경계는 심장 전기 생리학의 실질적인 동인입니다. 특히 그 공감 분기의 역할은 심실 부정맥 (VA)의 병리 생리학에 있는 조사의 지속적인 문제입니다. 동정 사슬의 양측 별 모양 구조인 스텔레이트 신경리아(SG)의   뉴런은   공감 인프라의 중요한 구성 요소입니다. SG는 치료 내화 VA를 가진 환자에 있는 심장 교감 기질을 통해 처리를 위한 인정된 표적입니다. SG의 신경 리모델링 및 신경교 활성화는 VA 환자에서 설명되었지만, 잠재적으로 부정맥의 발병에 앞서 있는 근본적인 세포 및 분자 과정은 충분히 이해되지 않으며 자율 변조를 개선하기 위해 해명되어야 합니다. 마우스 모델은 우리가 공감 신경 리모델링을 연구 할 수 있습니다, 하지만 murine SG의 식별은 경험이 없는 조사자에게 도전이다. 따라서, 뮤린 SG의 심층 세포 및 분자 생물학적 연구는 많은 일반적인 심장 질환에 대한 부족하다. 여기서, RNA 수준(유전자 발현 분석을 위한 RNA 절연, 현장에서 혼성화), 단백질 수준(면역형성 전체 마운트 염색), 및 세포 수준(기본 형태학, 세포 크기 측정)에서 분석을 위해 성인 마우스에서 SG를 해부하고 연구하기 위한 기본 레퍼토리를 설명합니다. 우리는 준비 기술의 문제를 극복하기 위한 잠재적인 해결책과 자동 불발성 담금질을 통해 염색을 개선하는 방법을 제시합니다. 이를 통해 세포 조성 및 리모델링 과정을 결정하기 위해 확립된 마커를 통해 뉴런뿐만 아니라 신경교 세포의 시각화를 가능하게 한다. 여기에 제시된 방법은 SG가 VA에 걸리기 쉬운 마우스에 있는 자율 장애에 대한 추가 정보를 얻기 위하여 허용하고 심혼에 있는 자율 신경계의 신경 및 신경교 성분을 조사하는 추가 기술에 의해 보완될 수 있습니다.

도 1은 SG를 식별하고 해부하는 방법을 시각화합니다. 도 1A는 위치의 회로도 도면을 나타내고, 도 1B는 심장 폐 패키지를 제거한 후 흉부로 뷰를 제시한다. SG와 늑골 케이지의 왼쪽 및 오른쪽 긴 콜리 근육은 방향에 중요한 랜드마크입니다. 해부는 근육과 첫 번째 갈비뼈 사이의 점선을 따라 수행됩니다. SG와 공감 체인은 흰색<strong...

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

심장 자율 신경계의 병리학적 변화, 특히 교감 지점에서 심실 부정맥의 발병 및 유지 보수에 기여합니다. 본 프로토콜에서, 우리는 근본적인 분자 및 세포 프로세스의 이해를 향상시키기 위하여 murine stellate ganglia를 특성화하는 방법을 보여줍니다.

뮤린 스텔레이트 갠리온의 위치, 해부 및 분석

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

10.0K 조회수.  EVK Düsseldorf, cNEP, cardiac Neuro- and Electrophysiology Research Consortium. 심장 자율 신경계의 병리학적 변화, 특히 교감 지점에서 심실 부정맥의 발병 및 유지 보수에 기여합니다. 본 프로토콜에서, 우리는 근본적인 분자 및 세포 프로세스의 이해를 향상시키기 위하여 murine stellate ganglia를 특성화하는 방법을 보여줍니다.

비디오: 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.

전압에 민감한 염료와 크랩 Stomatogastric 신경절에서 뉴런의 광학 이미징

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

연구

신경과학

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 신경계의 해부

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

생물학

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.

암 보레알리스 Stomatogastric 신경계의 해부

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.

성장 Axons 함께 손쥐 Schwann 세포 개발 분석

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.

쥐 모델에서 우수한 자궁경부 신경절 절제술을 위한 수술 기법

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

암 연구학

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.

신선한 냉동 인간 조직에서 장의 중추의 격리에 대 한 레이저 캡처 서의 최적화

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.

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.

단일 핵 RNA 시퀀싱을 위한 마우스 교감신경 신경절의 바코드된 항체를 사용한 저입력 핵 분리 및 멀티플렉싱

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

의학

마우스 교감신경 신경 세포 표적화: 요추 교감신경 절제술 접근법

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.

저자 스포트라이트: 교감신경 뉴런의 가소성 탐구

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.

생쥐의 외과적 요추 교감신경 절제술

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