Todos os ratos foram utilizados de acordo com protocolos aprovados pelo Comitê Experimental Animal do Beijing Friendship Hospital, Capital Medical University.
1. Preparação cirúrgica, anestesia e técnicas cirúrgicas
NOTA: Um total de 40 ratas Wistar, pesando 200–220 g, foram obtidas comercialmente para o presente estudo. Dos 40 ratos, 10 foram selecionados aleatoriamente como o grupo controle, e o restante foi tratado como o grupo teste. Todos os animais foram alojados em um ambiente estéril nas instalações de animais do Hospital da Amizade de Pequim, da Universidade Médica da Capital.
<ol><li>Realizar anestesia geral administrando pentobarbital sódico por via intraperitoneal (40 mg/kg). Alternativamente, induza a anestesia usando isoflurano a 3%-4% e mantenha-o em 1%-3% (inalado). Aplique pomada oftálmica nos olhos para evitar o ressecamento. Em seguida, coloque o rato na plataforma cirúrgica e forneça suporte térmico. NOTA: Administrar analgésicos como buprenorfina, 0,05 mg/kg, SC, 0,1-0,2 mL no início do procedimento.</li>
	<li>Verifique a profundidade da anestesia pela falta de resposta à pinça do dedo do pé. Raspe a pele de toda a área das costas com uma navalha.</li>
	<li>Esterilizar o local cirúrgico com pelo menos 3 ciclos de um esfoliante de dois estágios, como clorexidina ou iodopovidona, seguido de álcool isopropílico. Prenda os membros com fita cirúrgica e faça uma incisão mediana de cerca de 3 cm nas costas com tesoura cirúrgica.</li>
	<li>Aprofunde a incisão através dos tecidos subcutâneos usando tesoura cirúrgica e corte os músculos ligados à coluna vertebral.</li>
	<li>Identifique e exponha visualmente a13ª costela (o espaço intervertebral conectado a essa costela é o intervalo T13\u2012L1). Marque a13ª costela usando uma sutura.</li>
	<li>Após a identificação, ressecte cuidadosamente os músculos ligados à coluna vertebral e exponha a coluna vertebral. Ressecção o ligamento supraespinhoso e o ligamento interespinhoso para uma identificação precisa da coluna vertebral. Exponha o nível de L4\u2012L5 com tesoura cirúrgica e pinça. NOTA: O ligamento supraespinhoso pode ser facilmente identificado devido à presença de tecido subcutâneo fino. Após a ressecção do ligamento supraespinhoso, o ligamento entre o processo espinhoso é o ligamento interespinhoso.</li>
	<li>Dissecar cuidadosamente o processo espinhoso vertebral L4\u2012L5 e partes do processo transversal usando pinça de Kelly para expor a medula espinhal (Figura 1).</li>
	<li>Expor completamente o cone medular ao nível de L4\u2012L5 e transecto o cone medular totalmente com tesoura de iridectomia. Insira um pouco de embalagem de tecido para bloquear a recuperação da medula espinhal.</li>
	<li>Feche o músculo e a pele sobrejacentes na camada externa da pele usando sutura não absorvível 4-0.</li>
	<li>Para o grupo controle, execute as etapas 1.1 a u20121.7 e deixe o cone medular intacto. Feche a incisão de acordo com o passo 1.9.</li>
</ol>2. Recuperação de animais
<ol><li>Manter os ratos numa incubadora com temperatura controlada (37 °C) durante a primeira hora após a operação e monitorizá-los até que estejam esternais ou em movimento ativo. NOTA: Demora cerca de meia hora para a recuperação total.</li>
	<li>Transfira o animal para uma gaiola limpa com comida e água suficientes. Mantenha os ratos em gaiolas separadas. NOTA: O sucesso da transecção é indicado quando os ratos do grupo de teste se movem apenas com a ajuda das patas dianteiras, enquanto os ratos do grupo controle podem andar normalmente.</li>
</ol>3. Gestão pós-operação
<ol><li>Injete Penicilina G, um antibiótico (50.000 U/mL por animal) por via intraperitoneal. Administrar analgésicos como buprenorfina, 0,05 mg/kg, SC, 0,1-0,2 mL a cada 6-12 h por 48 horas após a operação.</li>
	<li>Comprima a bexiga urinária no hipogástrio para ajudar com a micção. Realize isso duas vezes ao dia no mesmo horário (8h e 20h) por seis semanas. NOTA: A perda de constrição normal do detrusor é o símbolo de DU.</li>
	<li>Coloque todos os ratos em gaiolas metabólicas, cada uma contendo um funil de coleta de urina colocado sobre um papel absorvente previamente pesado para monitorar a micção e a incontinência.</li>
	<li>Coletar e observar a mudança de peso do papel absorvente, que indica o volume anulado (VV) e o volume residual de urina separadamente.</li>
</ol>4. Teste urodinâmico
<ol><li>Às seis semanas de pós-operatório, realizar um cistometrograma, utilizando o equipamento de medição urodinâmica da seguinte forma.<ol><li>Anestesiar ratos injetando 10% de hidrato de cloral na cavidade peritoneal (3 mL/kg).</li>
		<li>Comprima a bexiga para micção e, em seguida, fixe o rato à plataforma cirúrgica usando uma fita.</li>
		<li>Insira o cateter peridural (3F) na bexiga e conecte o equipamento de medição urodinâmica, o cateter peridural e a bomba de infusão pelo tubo de três membros.</li>
		<li>Bombear solução salina fisiológica a uma velocidade de 0,2 mL/min para medição urodinâmica (ver Tabela de Materiais). Registre o CCM e o DOP, e a complacência da bexiga (calculada dividindo δ volume da bexiga com δ pressão do detrusor).</li>
	</ol></li>
</ol>5. Análise estatística
<ol><li>Realizar análises estatísticas utilizando software comercialmente disponível.</li>
	<li>Use o teste de Kolmogorov-Smirnov para testar a normalidade dos dados.</li>
	<li>Expresse as variáveis normalmente distribuídas como valores médios com desvios-padrão. Use os testes t de Student pareados bicaudais para comparar os parâmetros do cistometrograma em ambos os grupos. NOTA: p < 0,05 indica que a diferença teve significância estatística.</li>
</ol>

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Os autores não têm nada a revelar.

O DU é uma causa comum de sintomas do trato urinário inferior em homens e mulheres. É uma constelação complexa de sintomas com poucas opções de tratamento que podem diminuir significativamente a qualidade de vida (QV) dos acometidos18. Embora se acredite que o DU seja multifatorial, a compreensão de sua patogênese permanece rudimentar. Estudos têm demonstrado que a patogênese do DU pode estar relacionada a fatores miogênicos e neurogênicos.
Nas hipóteses m...

A subatividade do detrusor (UD) é uma disfunção típica do trato urinário inferior que permaneceu sob estudo. Embora a DU tenha sido definida pela International Continence Society (ICS)1, inúmeras terminologias diferentes são usadas para se referir a essa doença, por exemplo, "falha do detrusor", "bexiga acontrátil", "arreflexia do detrusor"2. A DU, conforme definida pela International Continence Society (ICS) em 2002, é uma contração de força e duração reduzidas, o que resulta em aumento prolongado no tempo de esvaziamento da bexiga, resultando assim na incapacidade de alcançar o esvaziamento completo da bexiga dentro de um período normal.
A DU pode acometer 48% dos homens e 12% das mulheres (com idade entre >70 anos)3 com sintomas do trato urinário inferior. Parece ser multifatorial, e não existe tratamento eficaz. É relatado que o DU é onipresente em pacientes com disfunção vesical neurogênica, como esclerose múltipla4, diabetes mellitus5, doença de Parkinson6 ou acidente vascular cerebral7. A DU também pode ser causada por danos nos nervos iatrogênicos, como histerectomia laparoscópica, prostatectomia ou outras intervenções cirúrgicas na pequena pelve8. As alterações fisiopatológicas e os tratamentos disponíveis de DU ainda são confusos devido à falta de um modelo animal apropriado para estudo.
O reflexo miccional é controlado por vias espino-bulboespinhais que combinam o centro de micção pontina, o núcleo parassimpático sacral e os centros do córtex mais sênior9. A ativação e a manutenção do reflexo da micção dependem principalmente do transporte regular de sinais sensoriais da bexiga para os centros do córtex mais sênior. Pode-se postular que a disfunção sensorial contribui para a UD.
A maioria dos estudos experimentais em animais relacionados a disfunções do trato urinário inferior tem se concentrado em modelos de bexiga hiperativa (OAB)10. Esses modelos fornecem uma compreensão razoável da fisiopatologia e do prognóstico da OAB. No entanto, apenas alguns modelos de DU foram relatados, por exemplo, lesão supraespinhal (lesões locais, descerebração e oclusão da artéria cerebral média), transecção ou lesão por contusão da medula espinhal, administração sistêmica (por exemplo, ciclofosfamida) ou intravesical de agentes irritantes ou inflamatórios (por exemplo, ácido, acroleína e lipopolissacarídeo)11,12,13,14 . Dentre esses métodos, apenas o método de transecção da medula espinhal ou lesão por contusão pode ser utilizado no estabelecimento de um modelo animal de DU13. Tentativas envolvendo a lesão do centro de micção pontino e dos centros do córtex superior foram abandonadas por causa do trauma grave. Assim, uma maior atenção está sendo dada para encontrar uma localização precisa no centro reflexo da micção para induzir o DU com efeitos colaterais mínimos.
Como mencionado anteriormente, um dos mecanismos de indução de DU é ferir a medula espinhal para danificar a via de sinalização do reflexo miccional. O método de queda de peso de Allen foi desenvolvido para estabelecer animais de laboratório com medula espinhal lesada15. No entanto, não existem mais dados experimentais disponíveis sobre este método. Além disso, uma vez que partes dos animais recuperaram a função espinhal após acidente vascular cerebral sem UD, não pode ser considerado como um método perfeito para gerar um modelo animal de DU16.
Em 1987, Bregman excogitou um processo de transectação da medula espinhal para geração do modelo animal de DU e adquiriu dados experimentais17. No entanto, esse método não foi aplicado para estabelecer o modelo animal de urânio empobrecido. Naquela época, os pesquisadores ainda estavam confusos sobre a patogênese da DU. Como os locais na medula espinhal associados à indução de OAB ou DU são adjacentes entre si, eles não conseguiram encontrar o local exato de dano à medula espinhal para induzir o DU17. A OAB e o DU foram introduzidos em conjunto ou separadamente por este método. Assim, embora esse método tenha introduzido a UD, ela era imprecisa e não podia ser usada para a compreensão da ocorrência e do processamento da UD.
Como dito acima, a falta de um modelo animal adequado de DU é um dos principais obstáculos para o estudo de DU. Os pesquisadores estão continuamente procurando um modelo preciso e gerenciável que possa simular a patologia da DU. Mesmo as opções de tratamento para DU não melhoraram significativamente durante os últimos 20 anos. Coletivamente, há uma grande necessidade de descrever um protocolo padrão para estabelecer um modelo animal de DU.
Assim, neste artigo, descrevemos um método para estabelecer com sucesso um modelo de DU em ratos por transecção do cone medular. A transecção foi realizada no nível L4\u2012L5 para separar o cone medular. A capacidade cistométrica máxima (CCM), a pressão de abertura do detrusor (POD) e a complacência da bexiga foram registradas e analisadas para validação do protocolo. O protocolo a seguir combinado viabilidade e confiabilidade de forma padronizada para estabelecer o modelo animal de DU, simulando a ocorrência e o processamento de DU. O protocolo pode ser utilizado como técnica para posterior estudo da UD.

<table>
	<tbody>
		<tr>
			<td>0.9% saline</td>
			<td>Wuhan Prosai Company</td>
			<td>EY-C1178</td>
			<td>pump for urodynamic measurement</td>
		</tr>
		<tr>
			<td>10% chloral hydrate</td>
			<td>Shandong Yulong Co., Ltd</td>
			<td>H37022673</td>
			<td>3mL/kg&#65292;administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Buprenorphine Hydrochloride Injection</td>
			<td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co. LTD</td>
			<td>H12020275</td>
			<td>0.05mg/kg subcutaneously 24h and 48h postoperation</td>
		</tr>
		<tr>
			<td>Epidural Catheter</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VABR3L</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Alta Technology Co., Ltd</td>
			<td>1ST5637</td>
			<td>50,000 unit/ml per animal</td>
		</tr>
		<tr>
			<td>pentobarbital</td>
			<td>Beijing solabo Technology Co., Ltd</td>
			<td>NK-WF0001</td>
			<td>40 mg/kg, administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Suture line(4-0)</td>
			<td>ETHICON</td>
			<td>VCP422H</td>
			<td>suture the injury</td>
		</tr>
		<tr>
			<td>Three-limb tube</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VAB3T</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Trace infusion pump</td>
			<td>Zhejiang Smith Medical Instrument Co., Ltd</td>
			<td>20162540335</td>
			<td>Pump the saline at a speed of 0.2ml/min for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Urodynamic measurement equipment</td>
			<td>Medical Measurement SystemsB.V.</td>
			<td>08-0467</td>
			<td>urodynamic measurement equipment can not only help the diagnosis of dysuria, but also provide objective materials for treatment and therapeutic effect. It is the most commonly used examination method in clinical diagnosis and treatment of lower urinary tract functional diseases</td>
		</tr>
		<tr>
			<td>Wistar Rats</td>
			<td>HFK Biotechnology Co.Ltd,Beijing ,China</td>
			<td>SCXK2012-0023</td>
			<td>200-220g</td>
		</tr>
	</tbody>
</table>

detrusor underactivity model in rats by conus medullaris transection

O objetivo do protocolo apresentado foi estabelecer um modelo de subatividade do detrusor (UD) em ratos através da transecção do cone medular. A laminectomia foi realizada em um total de 40 ratas Wistar (grupo controle: 10 ratos; grupo teste: 30 ratos) pesando 200–220 g, e o cone medular foi transectado no nível L4\u2012L5 no grupo teste. Todos os ratos foram alojados e alimentados sob as mesmas condições ambientais por seis semanas. No grupo teste, a micção da urina foi realizada duas vezes ao dia por seis semanas, e o volume residual médio de urina foi registrado. Um cistometrograma foi realizado em ambos os grupos. A capacidade cistométrica máxima (CCM), a pressão de abertura do detrusor (DOP) e a complacência da bexiga foram registradas e calculadas. O grupo teste apresentou retenção urinária significativa após a cirurgia, tanto durante quanto após o choque espinhal. No entanto, nenhuma anormalidade foi observada no grupo controle. Quando comparado ao grupo controle, o CCM e a complacência da bexiga no grupo teste foram significativamente maiores do que os do grupo teste (3,24 ± 2,261 mL versus 1,04 ± 0,571 mL; 0,43 ± 0,578 mL/cmH 2 O versus 0,032 ± 0,016 mL/cmH 2 O), enquanto o DOP no grupo teste foi menor que o controle (20,28 ± 14,022 cmH 2 O versus 35 ± 13,258 cmH2 O). Este método de estabelecer um modelo animal de DU pela transecção do cone medular oferece uma excelente oportunidade para entender a fisiopatologia da UD de uma maneira melhor.

Todo o procedimento da transecção do cone medular pode ser concluído dentro de 45 minutos por cirurgiões experientes. Nosso laboratório já realizou mais de 100 casos de cirurgias de transecção do cone medular. A taxa de sucesso é superior a 95%, conforme definido pela sobrevivência dos ratos e pela indução bem-sucedida de DU. O teste urodinâmico confirmou a indução de DU.
Com base em nossa experiência, a indução de DU pode ser avaliada preliminarmente pelo volume residual de ...

Watch this Scientific Journal Video about Detrusor Underactivity Model in Rats by Conus Medullaris Transection at JoVE.com

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Apresentamos um método para estabelecer um modelo de subatividade do detrusor por transecção do cone medular em ratos. A subatividade do detrusor foi estimulada com sucesso nesses animais. O modelo pode ser usado para estudar a função do trato urinário.

Modelo de Subatividade do Detrusor em Ratos por Transecção de Conus Medullaris

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

detrusor-underactivity-model-in-rats-by-conus-medullaris-transection

Research

JoVE Journal

Neuroscience

2.0K visualizações.  Capital Medical University. Apresentamos um método para estabelecer um modelo de subatividade do detrusor por transecção do cone medular em ratos. A subatividade do detrusor foi estimulada com sucesso nesses animais. O modelo pode ser usado para estudar a função do trato urinário.

Vídeo: Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Transecção do nervo óptico: Um Modelo de Apoptose Neuron Adultos no Sistema Nervoso Central

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Neurociência

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Métodos para manipulações experimentais após a transecção do nervo óptico no sistema nervoso central de mamíferos

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

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 ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Geração de topicamente transgênicas Rats por<em&gt; In utero</em&gt; Eletroporação e<em&gt; In vivo</em&gt; Triagem Bioluminescência

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

Motor Nerve transecção e Time-lapso de Comportamentos de células gliais em Zebrafish ao vivo

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Transecção medular nos larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Um Modelo de Aprendizagem lateralizados Odor em ratos recém-nascidos para Dissecando Formação Neural dos circuitos Consolidação da Memória

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Transecção parcial nervo óptico em ratos: um modelo estabelecido com uma nova abordagem operativa para avaliar secundária degeneração das células ganglionares da retina

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

Subaguda Microhemorrhages Cerebral induzida pela injeção de lipopolissacarídeo em ratos

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

Um modelo pré-clínico para avaliar a recuperação do cérebro após acidente vascular cerebral agudo em ratos

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...