Este estudo foi financiado pelo National Institutes of Health (Grant 1UM1HL112856).

Todos os procedimentos e protocolos foram aprovados pelo Comitê de Cuidados e Uso da Universidade de Illinois, em Chicago animal Institucional. As experiências aqui descritas são experiências agudas não-sobrevivência, e não houve uso de pomada. Manutenção de condições estéreis só ocorre quando os instrumentos cirúrgicos são lavados com etanol 70% em DIH 2 O. Sacrifício de ratos no final da experiência ocorreu através de sobredosagem de IV cetamina / xilazina. 1. Preparação de Instrumentos e Produtos Químicos <ol><li> Preparar a solução de estoque de 0,05 M de 5-HT de HCl em PBS. Depois dilui-se estoque com PBS a uma concentração final de 5-HT de 0,203 mM. </li><li> Diluir dronabinol em óleo de sésamo com uma concentração de 20 ug / uL. </li><li> Corte de 20 cm de comprimento de polietileno (PE) -50 tubagem (ID 0,58 milímetros, 0,965 milímetros OD). Em uma extremidade do tubo, corte uma ponta bisel com uma tesoura, e na outra ponta quadrada, inserir uma agulha de 23 g. Ligue este 23 g agulha para um 1 mlseringa, e encha-o com 0,3 ml de 50 u / ml de heparina. </li><li> Corte quatro pedaços de fio de seda 4-0 trançado e definido para o lado. </li><li> Esterilizar tesouras cirúrgicas, 2 fórceps Graefe curvas, e 3 grampos de micro com etanol a 70% em DIH 2 O. </li></ol> 2. O cateterismo de veia femoral Protocolo para o cateterismo é modificado a partir Jespersen et al 21. <ol><li> Anestesiar uma ratazana Sprague-Dawley com intraperitoneal (IP) de cetamina / xilazina (100 mg / kg: 10 mg / kg). Aperte o dedo do pé do rato e observar qualquer movimento para confirmar um nível adequado de anestesia. Raspar o aspecto ventral da coxa esquerda, e do aspecto ventral do pescoço. Se necessário pelo Comitê de Cuidados e Uso de Animais Institucional do local, aplicar pomada para evitar o ressecamento. </li><li> Rat seguro em decúbito dorsal em uma placa cirúrgica. Utilize uma tesoura cirúrgica para cortar a pele na coxa posterior esquerda. </li><li> Utilizando uma pinça, conduta dissectio contuson do músculo superficial para expor a veia femoral. Utilizar as pinças para separar a veia a partir da artéria femoral, e colocar dois segmentos em torno da veia femoral. </li><li> Use a pinça Graefe curvas para puxar-up da veia para parar o fluxo de sangue. Usando uma seringa de agulha G 22, por punção da veia femoral e, em seguida, insira a ponta chanfrada do tubo PE-50 na veia. </li><li> Verificar se o tubo PE-50 foi inserida correctamente por retracção do êmbolo da seringa. O sangue deve ser visto de entrar no tubo de PE-50. Amarre 2 nós ao redor da veia e PE-50 tubos usando as duas threads. </li></ol> 3. 5-HT induzido por Apnea via IV Infusion <ol><li> Coloque um medidor de tensão piezoelétrico em torno do rato para medir a respiração. </li><li> Altere as configurações para o nível de amplificação do amplificador eletrônico via software amplificador no computador: para amplificar (100x) e filtro passa-banda (10/01 Hz), os sinais respiratórios obtidos a partir do medidor de tensão, ajuste &quot;filtro highpas&quot; at &amp; #8220; AC @ 1 HZ &quot;e&quot; Filter Lowpass &quot;at&quot; 10 Hz &quot;, e definir a amplificação através da introdução de&quot; 10 &quot;para o&quot; ganho inicial &quot;e&quot; 100 &quot;para&quot; Total Gain &quot;.. </li><li> Altere as configurações de taxa de amostragem, abrindo &quot;Entrada Analógica&quot; via software de gravação no computador. Para digitalizar (500 taxa de amostragem Hz) usando um conversor analógico-digital, &quot;sample&quot; at &quot;1.000 Hz&quot; e &quot;pular&quot; todos os outros pontos de gravação por &quot;1&quot; (taxa de amostragem eficaz 500 Hz), e depois gravar o sinal usando o software de gravação. </li><li> Remover a seringa de 1 ml a partir do cateter e inserir uma seringa de vidro de 500 mL cheio com precisão de 0,203 mM de 5-HT no interior do cateter. </li><li> Coloque a 500 ul seringa de vidro de precisão em uma bomba de infusão. Infundir 12,5 ug / kg por 350 ul / kg de uma solução de 5-HT a uma taxa de 63 ml / hr. Executar várias infusões e observar apnéia em rato, que é visto umsa pausa na respiração (≥ 2,5 seg) nos sinais respiratórios gravados no monitor do computador. </li><li> Antes de prosseguir com a cirurgia de pescoço, monitorar o padrão respiratório e verificar se há reflexo dor de toe pitada no rato. Se a respiração for irregular, ou se existe um reflexo dor de aperto do dedo do pé, administrar IP cetamina / xilazina (100 mg / kg: 5 mg / kg) e, em seguida, voltar a confirmar a um nível adequado de anestesia. </li></ol> 4. Neck Surgery para expor Nodoso Ganglia <ol><li> Rat seguro em decúbito dorsal em uma placa cirúrgica. Faça um corte longitudinal mediana usando uma tesoura cirúrgica no pescoço. </li><li> Usando dissecção romba no músculo platisma (usando dois micro grampos para manter esse músculo clara do sítio cirúrgico), expor os músculos sternohyoideus e omohyoideus. Separe esses músculos para expor a artéria carótida interna e um de seus ramos, a artéria pterigopalatina. </li><li> Observe o nervo vago, uma vez que corre ao longo da artéria carótida interna e, em seguida, ao longo th<html> e pterygopalatine artéria. Observe como o nervo vago e artéria pterigopalatina, juntamente com o nervo glossofaríngeo (IX nervo craniano) e do nervo acessório (XI nervo craniano), digite o forame lacerados posteriores na base do crânio. </li><li> Observe que o gânglio nodoso é exibido como um inchaço do nervo vago direito antes de entrar no forame posterior dilacerado. Note também que os ramos da faringe e da laringe nervosas saem os aspectos anterior e posterior, respectivamente, do gânglio nodoso. 5 </li><li> Usando a pinça Graefe, separar o nervo vago a partir das artérias, e coloque um pedaço de fio ao redor do nervo vago. Coloque um grampo na linha para aplicar uma leve tensão no nervo vago, e limpar o gânglio nodoso de qualquer tecido conjuntivo para proporcionar menor resistência ao injetar. </li><li> Repita apnéia 5-HT induzido conforme previsto no ponto 3 para confirmar que nenhum dano foi feito para o gânglio nodoso. </li></ol>itle &quot;&gt; 5. Intranodose Ganglia Injeção de Dronabinol <ol><li> Encha a 10 ul precisão gastight seringa de vidro afixado com um 28 g de meia polegada agulha de seringa feito por encomenda com um 35 ° chanfradas ponta com 5 mL de dronabinol em óleo de gergelim. </li><li> Coloque um grampo micro no segmento de aplicar uma leve tensão no nervo vago, e perfurar o gânglio nodoso tomando cuidado para não perfurar através dele. Pressione a seringa lentamente (≥ 60 seg) para injetar todo o conteúdo da seringa. Note-se que alguns dos conteúdos de seringas vai vazar para fora do gânglio. </li><li> Apneia Repita 5-HT-induzida como indicado na seção 3. </li></ol>

<ol>
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Physiol. 588 (21), 4303-4315 (2010).</li><li>Zhang, Z., Zhang, C., Zhou, M., Xu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+opioid+mu-receptors,+but+not+delta-+or+kappa-receptors,+switches+pulmonary+C-fiber-mediated+rapid+shallow+breathing+into+an+apnea+in+anesthetized+rats).">Activation of opioid mu-receptors, but not delta- or kappa-receptors, switches pulmonary C-fiber-mediated rapid shallow breathing into an apnea in anesthetized rats).</a> Respir. Physiol. Neurobiol. 183 (3), 211-217 (2012).</li><li>Calik, M. W., Radulovacki, M., Carley, D. 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Ther. 260 (2), 917-924 (1992).</li><li>Kopczynska, B., Szereda-Przestaszewska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5HT2+and+5HT3+receptors'+contribution+to+modeling+of+post-serotonin+respiratory+pattern+in+cats.">5HT2 and 5HT3 receptors' contribution to modeling of post-serotonin respiratory pattern in cats.</a> Life Sci. 75 (19), 2281-2290 (2004).</li><li>Fan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoid+agonists+inhibit+the+activation+of+5-HT3+receptors+in+rat+nodose+ganglion+neurons.">Cannabinoid agonists inhibit the activation of 5-HT3 receptors in rat nodose ganglion neurons.</a> J. Neurophysiol. 73 (2), 907-910 (1995).</li><li>Jespersen, B., Knupp, L., Northcott, C. 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The authors declare that they have no competing financial interests.

Os passos críticos para o sucesso da injecção de neurotransmissores na gânglios nodosos são: 1) a identificação e a limpeza do tecido conjuntivo fora do gânglios nodosos; 2) confirmando a integridade do gânglio nodoso antes da injecção; 3) e usando uma agulha de calibre pequeno para injectar delicadamente em, mas não completamente, através de punção, os gânglios nodosos. O nervo vago inerva muitos órgãos no pescoço e abdômen, e transmite informações importantes, tais co...

Afferent signaling via the vagus nerve (cranial nerve X) transmits important general visceral information to the central nervous system (CNS) from baro-, chemo-, hepatic osmo-, cardiac, pulmonary, and gastric receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract (NTS) of the medulla. The cell bodies of the pseudounipolar neurons of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority is found in the former. Nodose ganglion cells contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that when activated, can modify afferent vagus nerve activity.1 Numerous innervations of the afferent vagus nerves coupled with the diversity of receptors located on the nodose ganglia illustrate the biological importance of this cranial nerve, and systemic drugs that do not cross into the CNS targeted at receptors on nodose ganglia can be used to treat various diseases, such as sleep apnea, gastroesophageal reflux disease, or chronic cough.2-4
The ease of access to the nodose ganglia lends itself to experimental manipulation by midline longitudinal incision made at the neck. The vagus nerve emerges from the posterior lacerated foramen at the base of the skull, and immediately displays a swelling of the nerve that is the nodose ganglion. The nodose ganglion is easily recognizable due to two nerve branches that arise from it: anteriorly the pharyngeal branch; and posteriorly superior laryngeal branch.5 Previous experimental manipulations of the nodose ganglia involved electrophysiological recordings,6 injections of immunohistochemical or immunofluorescent compounds for nerve tracings,7-10 superfusion or injections of neuroexcitotoxins,11-13 injections of adeno-associated virus to knockdown receptors,14,15 and injections of receptor-specific neurochemicals to change the activity of the vagus nerve.16,17
Systemic injections of neurochemicals are problematic in that systemic treatment affects both peripheral and central nervous systems. Thus, systemic treatment does not isolate the effect of neurochemicals on afferent vagal nerve activity. This protocol describes a method using readily available equipment of intranodose injections in the Sprague-Dawley rat that modulates vagus nerve activity without affecting the central nervous system. Stimulation of serotonin type 3 (5-HT3) receptors on nodose ganglia by intravenous (IV) infusion of serotonin (5-HT) induces the Bezold-Jarisch reflex, a vagal response trifecta of bradycardia, hypotension, and apnea, which can be abolished by supranodose vagatomy.11,17-19 Apnea is easily measured by placing a respiratory transducer around the abdomen of the rat.17,18 Cannabinoids decrease 5-HT-induced current in nodose ganglia cells,20 and intranodose ganglia injections of dronabinol attenuate 5-HT-induced apnea.

<table border="0" cellpadding="0" cellspacing="0" width="660">
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Name of Material/ Equipment</td>
			<td style="width:155px;">Company</td>
			<td style="width:141px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">5-HT HCl</td>
			<td style="width:155px;">MP Biomedicals</td>
			<td style="width:141px;">215376591</td>
			<td style="width:175px;">12.5 &#181;g/kg per 350 &#181;l/kg</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Dronabinol (Marinol) 10 mg Capsules (80 &#181;g/&#181;l)</td>
			<td style="width:155px;">AbbVie</td>
			<td style="width:141px;">NDC 0051-0023-21</td>
			<td style="width:175px;">Dilute with sesame oil to 20 &#181;g/&#181;l</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Sesame Oil</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:141px;">S3547</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Intramedic Polyethylene-50</td>
			<td style="width:155px;">BD</td>
			<td style="width:141px;">427411</td>
			<td style="width:175px;">Ordered from VWR (Cat. # 63019-047)</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Graefe Forceps</td>
			<td style="width:155px;">Roboz</td>
			<td style="width:141px;">RS-5138</td>
			<td style="width:175px;">Two are needed</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Johns Hopkins Bulldog Clamp</td>
			<td style="width:155px;">Roboz</td>
			<td style="width:141px;">RS-7441</td>
			<td style="width:175px;">Three are needed</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Piezoelectric Strain Gauge</td>
			<td style="width:155px;">Ambu</td>
			<td style="width:141px;">813255-100</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Data Acquisition USB Subsystems</td>
			<td rowspan="2" style="width:155px;">DataWave Technologies</td>
			<td style="width:141px;">NA</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Sciworks Experimenter Software</td>
			<td style="width:141px;">NA</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">CyberAmp</td>
			<td style="width:155px;">Axon Instruments</td>
			<td style="width:141px;">NA</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Syringe, 500 &#181;L, Model 1750 TLL</td>
			<td rowspan="3" style="width:155px;">Hamilton Company</td>
			<td style="width:141px;">81220</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Syringe, 10 &#181;L, Model 1801 RN</td>
			<td style="width:141px;">7659-01</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Needle, 28 gauge, Small Hub RN</td>
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a method of nodose ganglia injection in sprague-dawley rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

A Figura 1 representa gravação respiração amostra em ratos que tinham infusão de 5-HT para provocar apneia antes e depois da injecção dos gânglios intranodose de dronabinol. 5-HT activa 5-HT3 sobre os gânglios nodosos que contribuem para o reflexo de Bezold-Jarisch de bradicardia, hipotensão, e apneia. 11,17-19 injecções gânglios Intranodose dronabinol de activar os receptores CB inibitórios, ou modula alostericamente 5-HT 3 receptores que inibem a excitaç...

Watch this Scientific Journal Video about A Method of Nodose Ganglia Injection in Sprague-Dawley Rat at JoVE.com

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

Um método de Nodoso Gânglios Injecção em ratos Sprague-Dawley

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

a-method-of-nodose-ganglia-injection-in-sprague-dawley-rat

Research

JoVE Journal

Neuroscience

18.5K visualizações.  University of Illinois at Chicago. Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

Vídeo: A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Entrega de genes ao cérebro pós-natal do rato por Non-ventricular Injection sHRNA e Eletroporação

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Neurociência

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Um método geral para a avaliação de Incubação de Craving Sacarose em Ratos

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Técnica minimamente invasiva para a injeção em Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

Um rato do modelo de fadiga Central usando um modificado método de plataforma múltipla

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

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.

Otimização de captura Laser Microdissection para o isolamento dos gânglios entéricos de tecido humano fresco congelado

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

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Injeção direta de um vetor de Lentivirus destaca várias vias motoras na medula espinhal de ratos

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Geração de neuroesferas do hipocampal preliminar misturado e dos neurônios corticais isolados de E14-E16 embrião do rato de Sprague Dawley

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine &#946;-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

This paper describes the isolation and culturing of embryonic rat sympathetic neurons from the superior cervical ganglia. It also provides detailed protocols for immunocytochemical staining and for preparing neuronal extracts for mass spectrometric analysis.

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Dupla injeção direta de sangue na Cisterna Magna como modelo de hemorragia subaracnóide

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Um método de limpeza de tecido hidrofóbico para tecido cerebral de rato

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...