Name: in vitro functional characterization of mouse colorectal afferent endings
Uploaded: 2015-01-21T13:00:00.000+00:00
Duration: 14 min 9 s
Description: Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

NOTA: Este protocolo foi revisto e aprovado pela Universidade de Pittsburgh Institutional Animal Care e do Comitê Use. 1. Preparação da solução de Krebs modificado e teste de droga Alíquotas <ol><li> Adicione 6 L de solução de Krebs modificada, que contém (em mM): NaCl 117,9, KCl 4,7, NaHCO3 25, 1.3 NaH 2 PO 4, 1,2 MgSO4, 2,5 CaCl2, 11,1 de D-glucose, 2 butirato de sódio, acetato de sódio 20 , 0,004 nifedipina (para bloquear as contracções espontâneas do músculo), 0,003 e indometacina (para bloquear a síntese de prostaglandinas endógenas). Utilizar soluções ice-frias e quentes Krebs para dissecção dos tecidos e gravação de fibra única, respectivamente. </li><li> Preparar todas as soluções químicas (por exemplo, IS, capsaicina, ATP) em alíquotas a concentrações desejadas. </li></ol> 2. dissecção do tecido colorectum nervo- <ol><li> Anestesiar e eutanásia ratos machos (6-8 semanas de idade, 20 - 30 g) emuma câmara de CO 2 a um caudal que desloca 10 - 30% do volume da câmara por minuto até que os ratinhos parar de respirar completamente, como indicado pela ausência do movimento do tórax. </li><li> Imediatamente após a eutanásia, desangrar, cortando abrir a caixa torácica, perfurando o átrio direito, e imergindo a carcaça do mouse em um amplo volume de (~ 500 ml) de gelado Krebs solução (4 ° C) aeradas com carbogênio (95% O 2 , 5% de CO 2). </li><li> Remova cuidadosamente todas as vísceras, mas os dois pontos e pélvicas órgãos. Transecto o mouse pela metade em todo o segmento da coluna vertebral T12 ligeiramente acima do diafragma torácico e transferir a metade caudal para uma câmara de dissecção contendo gelo frio, borbulhar solução Krebs. </li><li> Sob um microscópio estereoscópico, remover a bexiga e os órgãos reprodutivos, ao seccionar os seus cruzamentos para a uretra, e remova o descendente / aorta abdominal até que se bifurca em artérias ilíacas comuns. Livre o PN ou o LSN de seu surrounding tecidos por dissecção romba e siga o nervo do lado de fora da crista ilíaca até o seu ponto de entrada ventral na L6 e S1 coluna vertebral (por PN) ou T13 e L1 coluna vertebral (por LSN). </li><li> Corte a sínfise púbica e à direita e articulações do acetábulo esquerda, e remova o osso ilíaco. Cuidadosamente livre tanto o PN ou LSN do músculo ligado e do tecido conjuntivo de perto da colorectum até onde o nervo entra na coluna vertebral. </li><li> Ressecar cuidadosamente o osso ilíaco para expor o colorectum distal. Dissecar o colorectum distal com o PN anexado ou LSN no continuum. </li><li> Transferir a colorectum com o nervo ligado ao compartimento da câmara de banho de tecido. Remover excesso de tecido conjuntivo por mais dissecação, e abrir o colorectum longitudinalmente ao longo da fronteira anti-mesentérica. </li><li> Com o lado da mucosa voltada para cima, o pino da borda mesentérica do colorectum adjacente ao compartimento de gravação para a base de silicone da chamber e anexar o comprimento antimesentérica do colorectum a um ancinho de ganchos ligados a um actuador de força (ilustrada na Figura 1 e fotografado na Figura 2A). </li><li> Estender o LSN PN ou no compartimento de gravação, o qual está ligado ao compartimento de banho por um buraco de rato e portão. Cuidadosamente, coloque o tronco do nervo sobre um espelho de vidro pequeno no compartimento de gravação, o que proporciona uma superfície hidrófila para o nervo para aderir. Superfuse compartimento do banho com água morna (30-32 ° C), solução de Krebs oxigenada e encher o compartimento de gravação com óleo mineral. </li></ol> Gravação 3. Single-fibra e localização do campo receptivo <ol><li> Retire cuidadosamente de volta o epineuro (bainha do nervo) do PN ou LSN, ao microscópio estereoscópico em alta ampliação (50 - 60X). Usando uma pinça fina, provocar o tronco do nervo em 5-8 feixes de nervos de ~ 100 m de espessura. </li><li> Coloque a referência de platina-irídioeléctrodo em contacto com a solução de Krebs na câmara de tecido. Sequencialmente colocar os feixes de nervos individuais sobre o eléctrodo de gravação feitos do mesmo material. </li><li> Use um pincel macio para evocar APs dos aferentes colorretais por acariciando acima e abaixo da superfície colorectal. Localize o pacote (s) de nervos que inervam o cólon através AP detectável (potencial de ação) gravações. NOTA: A PN e LSN também inervam a bexiga e outros órgãos pélvicos. </li><li> Use um par de 30 g pontas de agulha para dividir ainda mais o feixe de nervos em filamentos finos fascículo de ~ 10 mm de espessura e coloque um filamento indivíduo para o eletrodo de registro. </li><li> Coloque o eletrodo concêntrico rodada de ponta perpendicular à superfície da mucosa para excitar eletricamente terminações aferentes na intensidade suprathreshold estímulo (10 mA magnitude, 0,5 duração ms @ 0,3 Hz), que produz um raio de propagação mm atual ~ 2. Mova o eletrodo sistematicamente (~ 1,5 passos mm) Ao longo do comprimento e largura do colorectum achatadas para localizar terminações receptivas. </li><li> Quando um final aferentes está animado, ajustar a posição do eletrodo para identificar o local da ativação (campo receptivo, RF) que requer intensidade mínima de estímulo (limiar de estímulo). Descarte terminações com um limiar de estímulo&gt; 3 mA 19. </li><li> Calcula-se a velocidade de condução (CV) a partir de 1) a distância entre o eléctrodo estimulante no campo receptivo (RF) e o local de gravação e 2) o atraso na condução (por exemplo, a Figura 2B) entre o artefacto de estímulo e o aparecimento do potencial de acção . CV (m / s) = distância (mm) / atraso de condução (ms). </li></ol> 4. Classificação Funcional de Mechanosensitive colorretais Aferentes <ol><li> Depois de localizar um RF pela estimulação elétrica, aplicam-se as seguintes três estímulos mecânicos para a RF: <ol><li> Conduzir o estímulo sondagem pressionando a ponta de uma calibrated von Frey-like monofilamento de nylon (0,4 e 1 g vigor) perpendicularmente ao RF na colorectum achatada. </li><li> Conduzir o estímulo acariciando acariciando gentilmente a mucosa retal com uma multa fio de nylon de filamento (10 mg vigor) para gerar uma pequena tensão de corte superficial na RF. </li><li> Conduzir o trecho circular usando uma força atuador controlado por computador, o que proporciona uma força trecho em rampa (0-170 mN em 5 mn / seg) em direção circunferencial ao longo da borda anti-mesentérica do colorectum via o rake de ganchos descritos no passo 2.8 . </li></ol></li><li> Classifique aferentes como serosa (responder apenas a despontar sondagem), mucosa (responder a mucosa acariciando e neutralizar sondagem), muscular (responder ao estiramento circunferencial e neutralizar sondagem) muscular / mucosa (responder ao estiramento circunferencial, mucosa acariciando e neutralizar sondagem), ou MIA (não responsivos a qualquer um dos três estímulos mecânicos). </li><li> Para aferentes mesentéricos (apenas eminervação LSN) que são difíceis de ativar seletivamente pela estimulação elétrica, localize suas terminações receptivas acariciando mecânica / sondagem do mesentério. </li><li> Para aferentes sensíveis ao estiramento muscular e muscular (-mucosas), determinar o limiar de resposta, que é definida como a força que evoca o primeiro AP durante trecho em rampa. </li><li> Para aferentes serosa, gravar as suas respostas a ascendente níveis de pontuada sondagem do campo receptivo impulsionado pela força do atuador controlado por computador. </li></ol> 5. Chemical Aplicação / Modulação da Endings Receptivo <ol><li> Grave uma resposta de linha de base a um estímulo mecânico (ou seja, a resposta ao trecho em rampa, pontuada de sondagem, ou mucosa carícias). </li><li> Revestir a borda inferior de um pedaço de tubo (latão ou aço inoxidável, 10 mm de altura e 4 x 4 mm 2 quadrado ou 4 - 5 mm de diâmetro) com vaselina e colocá-lo sobre o campo receptivo no colorectum. </li><li> Removera solução de Krebs no interior do tubo, e exponha o fim receptivo para o 3 - 5 min a 170 ul da solução que contém o produto químico (s) a ser testado. </li><li> Monitorar a resposta das vias aferentes durante a aplicação de produtos químicos (aferentes são alguns quimiosensível). </li><li> Remover a solução química e tubos para terminar a acção do produto químico. Dentro de 4-6 min, testar a resposta de aferentes ao mesmo estímulo mecânico como na resposta de linha de base. </li><li> Reaplique o estímulo mecânico novamente após período suficiente de wash-out (&gt; 15 min). </li></ol> 6. spikes Gravação e Discriminando AP <ol><li> Digitalizar os sinais elétricos gravados a partir de axônios em 20 kHz e salvar os dados para um computador. Monitorar o sinal on-line por um monitor de áudio. </li><li> Analisar os pontos AP off-line e discriminar unidades individuais com base em análise de componentes principais das formas de onda de pico individuais 25. NOTA: Um registro não deve conter mais thum duas unidades activas facilmente discrimináveis. </li></ol>

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The authors have nothing to disclose.

A preparação in vitro colorectum-nervo descrito aqui tem provado ser uma abordagem poderosa para estudar funções de codificação neural de aferentes colorretais individuais, que bem complementa outras abordagens não-funcionais (por exemplo, estudos celulares, moleculares e histológicos) sobre os neurônios sensoriais viscerais ( veja o comentário 27 para detalhes). Mecanismos neuronais que contribuem para a nocicepção e hipersensibilidade colorectal longo prazo foram revelados e ma...

Dor e hipersensibilidade são predominantes as queixas dos pacientes que sofrem de desordens gastrointestinais funcionais, incluindo o síndrome do intestino irritável (IBS), que existem na ausência de pathobiological causa aparente ou danos nos tecidos. Por exemplo, pacientes com SII exibem hipersensibilidade, incluindo respostas intensificadas para distensão rectal balão e aumento da sensibilidade na função normal do intestino, bem como a hipersensibilidade de referência somáticas (isto é, sensibilidade à palpação da zona abdominal) 1. Porque direccionamento aferentes colorectal tem provado ser eficaz no alívio da dor e hipersensibilidade em pacientes com IBS (por exemplo, a instilação intra-rectal de anestésicos locais 2,3; ingestão oral da guanilato ciclase C-agonista linaclotide 4-6), uma melhor compreensão do inervação aferente do colorectum é importante. Aferentes viscerais, incluindo aferentes colorretais, São capazes de responder às modalidades químicos / em nutrientes e térmicos (por exemplo, 7-9). No entanto, aferentes viscerais que respondem a estímulos mecânicos (ou seja, aferentes mechanosensitive) ter sido o mais estudado porque estímulos mecânicos (por exemplo, distensão luminal, estiramento) são aqueles que normalmente dão origem a sensações conscientes, incluindo desconforto e dor 10-16. Além disso, as vísceras também são inervados por aferentes insensíveis mecanicamente (MIAs), vulgarmente designados por silenciosas ou dormir nociceptors 17. Em condições fisiológicas normais, MIAs não respondem ao estímulo mecânico ou ter resposta muito alta limiares 18, mas pode se tornar ativo e adquirir mecanosensibilidade em condições fisiopatológicas e contribuir para a hipersensibilidade. Usando a preparação in vitro e protocolo descrito aqui, nós desenvolvemos e empregou uma estratégia de estímulo elétrico para o mar<html> RCH para terminações receptivas, permitindo a identificação imparcial de ambas as terminações mechanosensitive e MIA no colorectum 19. A inervação colorectal é derivado splanchnic lombar (LSN) e do nervo pélvico (PN) vias, e inclui aferentes colorretal que podem ser classificados em cinco classes mechanosensitive (serosa, mucosa, muscular, músculo-mucosa, mesentérica) e uma classe MIA 20. Usando esta preparação in vitro, verificou-se que MIAs colorretais adquirido mecanosensibilidade (sensibilizar) após uma breve exposição de seus campos receptivos a uma sopa inflamatória (IS), que sensibilizados 71% dos MIAs na via PN e 23% das MIAs na via LSN 19. Nós também documentada sensibilização a longo prazo (até 28 dias) de MI As no contexto da hipersensibilidade visceral comportamentais de longa duração (isto é, nos ratos que receberam os tratamentos com zimosan intracolónica 21 ou ácido 2,4,6-trinitrobenzenossulfónico (TNBS) 22) . jove_content &quot;&gt; Entre aferentes mechanosensitive, aferentes musculares e muscular-mucosas são as únicas classes que tonicamente codificam trecho circunferencial da colorectum (ou seja, são stretch-sensitive) e servir à codificação de nocivo distensão colorectal 23,24. Usando um computador controlado- força do actuador, foi aplicado um padrão, homogénea e reprodutível em rampa estiramento na direcção circunferencial do tecido colorectal achatada e mais categorizados aferentes sensíveis ao estiramento tão baixo limiar e de alto limiar 23. Além disso, o curso de tempo da sensibilização de estiramento aferentes sensíveis ao após zymosan intracolônica 21 ou 22 TNBS tratamento corresponde ao início, persistência e / ou recuperação de hipersensibilidade visceral comportamental, sugerindo um papel de aferentes colorretais sensíveis ao estiramento na dor visceral e hipersensibilidade.

<table><tbody><tr><td>Leica MZ16 stereo microscope</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Leica IC D camera</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Amplifier</td><td>World Precision instruments, Inc.</td><td>SYS-DAM80</td><td>Low-noise differential amplifier</td></tr><tr><td>Two-compartment tissue chamber</td><td>Custom made</td><td></td><td></td></tr><tr><td>Power1401</td><td>Cambridge Electronic Design Limited</td><td>Power1401</td><td>Data acquisition, analog signal input/out</td></tr><tr><td>Spike2 v5.02</td><td>Cambridge Electronic Design Limited</td><td></td><td>Software package that works with the Power1401</td></tr><tr><td>Audio monitor</td><td>Natus</td><td>Am 8</td><td></td></tr><tr><td>Square pulse stimulator</td><td>Natus</td><td>S48</td><td>To deliver electrical stimuli</td></tr><tr><td>Photoelectric isolation unit</td><td>Natus</td><td>PSIU6</td><td>Stimulus isolation to reduce noise</td></tr><tr><td>Concentric bipolar microelectrode</td><td>FHC Inc.</td><td>CBFFG75</td><td>To deliver electrical stimuli</td></tr><tr><td>Dual-mode lever system</td><td>Aurora Scientific Inc.</td><td>Series 300C</td><td>To deliver mechanical stimuli</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>Forceps with fine tips</td></tr></tbody></table>

in vitro functional characterization of mouse colorectal afferent endings

Este vídeo mostra em pormenor um protocolo de fibra única gravação electrofisiológico in vitro utilizando uma preparação colorectum rato-do nervo. A abordagem permite a identificação imparcial e caracterização funcional de aferentes colorretais individuais. Gravações extracelulares de potenciais de acção propagadas (APs) que se originam a partir de um ou alguns aferente (ou seja, de fibra única) campos receptivos (RFS) na colorectum fascículos são feitos de fibras nervosas provocavam. O colorectum é removido quer com o pélvica (PN) ou splanchnic lombar (LSN) do nervo ligado e abertos longitudinalmente. O tecido é colocado numa câmara de registo, fixado plana e perfundidas com solução de Krebs oxigenada. Estimulação elétrica Focal é usado para localizar as terminações aferentes colorretais, que são ainda mais testadas por três estímulos mecânicos distintos (sem corte sondagem, acariciando mucosa e stretch circunferencial) para categorizar funcionalmente os aferentes em cinco mechanosensiaulas operatório. Endings respondendo a nenhum desses estímulos mecânicos são classificados como aferentes mecanicamente insensíveis (MIAs). Ambos mechanosensitive e MIAs pode ser avaliado para a sensibilização (ou seja, a resposta melhorada, limiar reduzida e / ou aquisição de mecanosensibilidade) por exposição localizada entre os FR para produtos químicos (por exemplo, sopa inflamatória (IS), a capsaicina, trifosfato de adenosina (ATP)). Nós descrevemos o equipamento e preparação de gravação colorectum-nervo, colheita de colorectum com anexado PN ou LSN, identificação de FR no colorectum, gravação de fibra única de fascículos, e aplicação localizada de produtos químicos para a RF. Além disso, os desafios de preparação e aplicação de estimulação mecânica padronizada também são discutidos.

A configuração está ilustrada na Figura 1. Ela inclui uma câmara de tecido feito à medida que abriga o colorectum num compartimento de banho revestido de silicone e o nervo ligado num compartimento cheio com óleo mineral contígua. A câmara de dois compartimentos foi usinado a partir de um bloco sólido de plástico acrílico por uma máquina CNC; parte inferior de ambos os compartimentos foi posteriormente revestido com silicone firme para permitir a fácil pin para baixo do tecido colorretal. A...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

In vitro caracterização funcional de Rato Colorretal Aferente Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K visualizações.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

Vídeo: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

A colonoscopia flexível em camundongos para avaliar a gravidade da colite e colorretal Tumores Usando um sistema validado endoscópica Scoring

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Medicina

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Um<em&gt; In-vitro</em&gt; Preparação de neurônios entéricos isolados e células gliais do plexo mioentérico do Rato Adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Neurociência

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

Gravação combinado de saída Mecanicamente Stimulated aferentes e Nerve Terminal Rotulagem no mouse do folículo piloso Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

In Vitro A gravação de mesentérica aferentes a atividade do nervo no mouse jejuno e segmentos do cólon

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

In Vitro Caracterização das propriedades eletrofisiológicas das fibras aferentes do cólon em ratos

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

Intestinal epithelium confers not only nutrient absorption but protection against noxious substances. The apical-most epithelial intercellular junction, i.e., the tight junction, regulates paracellular solute and ion permeability. Here, a protocol for the preparation of mucosal sheets and assessment of the ion selectivity of tight junctions using Ussing chamber technique is described.

Avaliação funcional da barreira de junção apertada intestinal e permeabilidade de íons em tecido nativo pela técnica da câmara de ussing

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog ...

Biologia

Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intraluminal Injection

Source: Boerner, K. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/62093">Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.</a> J. Vis. Exp. (2021).
In this video, we describe a surgical procedure to generate mouse proximal colon loop or pcLoop segment followed by intraluminal injection of fluorescent markers into the segment to study the intestinal epithelial permeability.

Geração do modelo de alça de cólon proximal de camundongo: um procedimento cirúrgico para gerar alça de cólon proximal e medir a permeabilidade intestinal pela detecção de marcadores fluorescentes no soro após injeção intraluminal

Source: Boerner, K. et al. Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized ...

JoVE Encyclopedia of Experiments

Modelo de Roedores

Adulto

Electrophysiological Recording from Colonic Afferent Nerves in an Ex Vivo Rat Colon

Source: Meng, Y. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56090">In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats.</a> J. Vis. Exp. (2017)
This video demonstrates a method to record the activity of colonic afferent nerves in an ex-vivo rat colon, cannulated at both ends and infused with saline to increase intraluminal pressure. As pressure increases, the nerve signal rises, indicating its stimulation.

Registro eletrofisiológico de nervos aferentes colônicos em um cólon de rato ex vivo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Neuroscience

Neurophysiology

Electrophysiology Techniques

Recording of Mesenteric Afferent Nerve Activity in an Ex Vivo Mouse Jejunum Segment

In this procedure, using a micromanipulator, lower the tip of the suction electrode into the organ bath. Then, gently aspirate some Krebs solution from the organ bath so that the tip of the electrode is submerged in the Krebs solution. Ensure that the Krebs solution covers the wire electrode inside the suction electrode.
Next, position the tip of the suction electrode immediately next to the transected afferent nerve strand, and draw the transected nerve strand into the capillary over its entire length. Maneuver the tip of the electrode towards the adipose tissue, and aspirate it into the glass capillary with the plunger, thereby mechanically sealing the nerve in the capillary from the contents of the organ bath.
To verify the recording of afferent nerve activity, perform a ramp distension-induced increase in afferent firing by closing the output port to lead to a gradual rise in pressure in the intestinal segment. Only perform the desired experimental protocol when three consecutive ramp distensions yield a reproducible multi-unit discharge.

In vitro caracterização funcional de Rato Colorretal Aferente Endings

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