Name: a novel method to model chronic traumatic encephalopathy in drosophila
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1. Montagem do Dispositivo de Strike ( Figura 1 ) <ol><li> Remova o êmbolo de uma seringa de tuberculina de 1 ml. Corte o barril na marca de 1 mL. </li><li> Remova uma barreira de aerossol (3 mm de altura x 4 mm de diâmetro) de uma ponta de pipeta de 200 μL e use-a como pêndulo. Coloque o pêndulo dentro do tambor da seringa. Toque suavemente o cano para mover o pêndulo para a extremidade da ponta, com o lado plano cobrindo a abertura do bico. </li><li> Conecte a extremidade da ponta...

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+closed+head+traumatic+brain+injury.">A Drosophila model of closed head traumatic brain injury.</a> Proc Natl Acad Sci USA. 110 (44), E4152-E4159 (2013).</li><li>Barekat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Drosophila+as+an+integrated+model+to+study+mild+repetitive+traumatic+brain+injury.">Using Drosophila as an integrated model to study mild repetitive traumatic brain injury.</a> Sci Rep. 6, 25252 (2016).</li><li>Katzenberger, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+to+Inflict+Closed+Head+Traumatic+Brain+Injury+in+Drosophila.">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila.</a> J Vis Exp. (e52905), (2015).</li><li>Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ethomics+in+large+groups+of+Drosophila.">High-throughput ethomics in large groups of Drosophila.</a> Nat Methods. 6 (6), 451-457 (2009).</li><li>Straw, A. D., Dickinson, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motmot,+an+open-source+toolkit+for+realtime+video+acquisition+and+analysis.">Motmot, an open-source toolkit for realtime video acquisition and analysis.</a> Source Code Biol Med. 4 (5), 1-10 (2009).</li><li>Talavage, T. 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Modelos de animais que modelam fielmente as características de CTE, incluindo alterações neurofisiológicas, características neuropatológicas e déficits neurocomportamentais, são essenciais para descobrir mecanismos de doenças e para desenvolver alvos diagnósticos e terapêuticos. É compreensível que nenhum modelo animal de uma doença humana seja perfeito na imitação de todos os pontos finais clinicamente relevantes. No entanto, acreditamos que um modelo CTE robusto deve satisfazer os seguintes três requi...

A Encefalopatia Traumática Crônica (CTE) recentemente foi reconhecida como um distúrbio neurodegenerativo distinto, separado de outras tauopatias, como a doença de Alzheimer 1 . Ao contrário da doença de Alzheimer e outras tauopatias comuns - cujos fatores de risco mais importantes estão avançando em idade e história familiar de demência, o CTE, como indicado pelo seu nome, implica uma estreita associação com história de trauma cerebral, muito provavelmente visto em atletas de esportes de contato, Como boxers e jogadores de futebol, bem como em veteranos militares 2 , 3 , 4 , 5 . É pensado para ser iniciado por golpes repetidos de concussão e subconcussão na cabeça. Os pacientes podem apresentar sintomas e sinais como déficits cognitivos, mudanças de humor e comportamento e disfunção do movimento, que se sobrepõem significativamente com a doença de Alzheimer, frontotemporalDemência, demência do corpo de Lewy e doença de Parkinson 6 . Em contrapartida, os exames pós-mortem do tecido cerebral revelam um padrão distinto de acumulação de tau hiperfosforilada em torno de pequenos vasos sanguíneos nas profundezas dos sulcos corticais, uma característica patognomônica não observada nas outras condições degenerativas 7 . No entanto, até agora, muito pouco se sabe sobre a patogênese que leva à manifestação da doença. Isto é em grande parte devido à falta de um modelo animal fiel - apenas recentemente foram gerados modelos de roedores 5 , 8 . Esses organismos modelo apresentam as desvantagens dos cuidados intensivos em custos e uma vida útil relativamente longa, que não são adequadas para estudos de doenças neurodegenerativas. Em comparação com homólogos de mamíferos, animais invertebrados, como Drosophila, são uma excelente alternativa, com sua manutenção econômica,Ferramentas extensivas para dissecar determinantes genéticos e vida útil relativamente curta 9 . Notavelmente, a mosca e os cérebros humanos compartilham caminhos molecular e celular evolutivamente conservados, bem como semelhanças anatômicas 10 , 11 , 12 . Dois modelos engenhosos de Drosophila para estudar lesão cerebral traumática foram relatados anteriormente 13 , 14 . O primeiro dispositivo &quot;High Impact Trauma&quot; (HIT) projetado por Katzenberger e colegas continha moscas de movimento livre em um frasco de plástico que estava ligado à extremidade livre de uma mola metálica 13 , 15 . Quando o frasco de plástico foi inclinado verticalmente e liberado, ele atingiu uma almofada de poliuretano e transmitiu trauma para as moscas enquanto eles saltavam para a parede do frasco e se recuperaram. Em contraste, Barekat e colegas desenharam um método de entrega diferente, nósNa plataforma de homogeneizador Omni Bead Ruptor-24 14 . As moscas foram incapacitadas com CO 2 e colocadas em um tubo de rosca de rosca de 2 mL que foi fixado ao homogeneizador e submetido a condições de agitação pré-programadas. Um dos benefícios do uso do sistema de homogeneizador de tecido é que o experimentador poderia modular a intensidade do ferimento, a duração da lesão e o número de episódios de lesões. No entanto, ambos os regimes sofrem a mesma desvantagem: lesões primárias na cabeça são inflacionadas aleatoriamente em termos de localização e força de impacto. Além disso, ambos os métodos resultaram em mortalidade considerável, causada por danos colaterais inevitáveis ​​a outras partes do corpo e órgãos internos. Aqui, descrevemos um novo método para induzir o mTBI nas moscas da fruta. Nosso aparelho consiste em um pêndulo balístico a gás. Comparado com os modelos Drosophila existentes 14 , 15 , nosso método tem a vantagem única de fornecer medidasImpacto de urable, dirigido apenas para a cabeça de mosca de movimento livre, permitindo o controle preciso de vários fatores, como a gravidade do impacto, o intervalo de tempo entre os impactos e o número total de impactos sofridos.

<table border="0" cellpadding="0" cellspacing="0" width="692">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Aerosol Barrier</td>
			<td style="width:151px;">USA Scientific</td>
			<td style="width:119px;">1120-8810</td>
			<td style="width:207px;">Used as an impactor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">200 ul Pipette Tip</td>
			<td style="width:151px;">USA Scientific</td>
			<td style="width:119px;">1111-0706</td>
			<td>Used as a fly head holder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">1000 ul Pipette Tip</td>
			<td style="width:151px;">USA Scientific</td>
			<td style="width:119px;">1122-1830</td>
			<td>Used as a connector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">1 ml Tuberculin Syringe</td>
			<td style="width:151px;">Becton Dickinson</td>
			<td style="width:119px;">309625</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">60 mm Petri Dishes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td>FB0875713A</td>
			<td>Used as a tracking arenas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Flow Regulator</td>
			<td>Genesee Scientific</td>
			<td style="width:119px;">59-122WC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Standard Clamp Holder/stand</td>
			<td style="width:151px;">EISCO Scientific</td>
			<td style="width:119px;">CH0688</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Fine Brush</td>
			<td style="width:151px;">Genesee Scientific</td>
			<td style="width:119px;">59-204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Flypad</td>
			<td style="width:151px;">Genesee Scientific</td>
			<td style="width:119px;">59-114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sylgard Silicone Elastomer</td>
			<td style="width:151px;">Dow Corning</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCD Camera</td>
			<td style="width:151px;">Microsoft&#160;</td>
			<td style="width:119px;">HD-5000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ctrax Walking Fly Tracker</td>
			<td style="width:151px;">Caltech</td>
			<td style="width:119px;">Ctrax 0.2.11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MATLAB Image Processing Toolbox</td>
			<td style="width:151px;">MATLAB</td>
			<td style="width:119px;">R2015b</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel method to model chronic traumatic encephalopathy in drosophila

A encefalopatia traumática crônica (CTE) é uma doença neurodegenerativa estabelecida que está intimamente associada à exposição a lesões cerebrais traumáticas leves repetitivas (mTBI). Os mecanismos responsáveis ​​por suas complexas mudanças patológicas permanecem bastante evasivos, apesar de um consenso recente para definir os critérios neuropatológicos. Aqui, descrevemos um novo método para desenvolver um modelo de CTE em Drosophila melanogaster ( Drosophila ), na tentativa de identificar os genes e caminhos chave que levam ao acúmulo de tau hiperfosforilizado característico e a morte neuronal no cérebro. Os impactos de resistência ajustável para infligir feridas fechadas leves são entregues diretamente na cabeça da mosca, submetendo a cabeça a aceleração e desaceleração rápidas. Nosso método elimina os problemas potenciais inerentes a outros modelos de mTBI de Drosophila ( por exemplo, a morte de animais pode ser induzida por danos aOutras partes do corpo ou órgãos internos). O menor cuidado de animais com uso intensivo de mão-de-obra e custo, vida curta e extensas ferramentas genéticas tornam a mosca da fruta ideal para estudar a patogênese do CTE e possibilitar a realização de telas genéticas e farmacológicas avançadas em grande escala, genômicas. Prevemos que a caracterização contínua do modelo gerará importantes insights mecanísticos sobre prevenção de doenças e abordagens terapêuticas.

Para estabelecer um modelo CTE usando Drosophila adulta, determinamos a eficácia do nosso dispositivo ao infligir um único ferimento na cabeça fechada. Para eliminar as variações relativas ao genótipo, ao sexo ou à idade, utilizamos moscas fêmeas Canton-S WT de 2 dias na experiência. Poderíamos facilmente controlar a força do pêndulo, regulando a taxa de fluxo de CO 2 a uma pressão de gás constante de 100 kPa. As moscas expostas a uma única greve com a ...

Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Aqui, descrevemos uma nova abordagem para infligir lesões cerebrais traumáticas de cabeça fechada em Drosophila melanogaster . Nosso método tem a vantagem de entregar diretamente impactos repetitivos com força ajustável para a cabeça sozinho. Uma maior exploração do sistema de invertebrados ajudará a iluminar a patogênese da encefalopatia traumática crônica.

Um novo método para modelar a encefalopatia traumática crônica em<em&gt; Drosophila</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

The overall goal of this novel procedure is to inflict closed traumatic brain injuries in the fruit fly, Drosophila, to establish a new chronic traumatic encephalopathy model. This method can help answer key questions in the chronic traumatic encephalopathy field, such as how the repetitive trauma induces neuron degeneration. The main advantage of this technique is that it delivers the repetitive impacts of adjustable strengths to the fly head directly without causing damage to other parts of the animal body.Real world demonstration of this method is critical as assembly of the device and fly loading steps are difficult to learn, because the Drosophila head is so small and it is important to apply the impact only to the head. To make the apparatus used to concuss or strike the fruit flies, first, make the impactor portion of the apparatus. Remove the plunger from a one milliliter polycarbonate tuberculin syringe and then cut the barrel at the one milliliter mark.For an impactor, use the aerosol barrier found on a 200 microliter pipette tip. This impactor should be three millimeters long and four millimeters in diameter. Then, pack the impactor into the syringe barrel with the flat side towards the nozzle.To provide force, attach the syringe barrel to a carbon dioxide flow regulator. The regulator must have an On Off toggle switch and good control of the gas flow rate. Then, clamp the barrel vertically to an iron stand, such that the impactor will fall to the bottom of the barrel.Next, make the fly holder. Start with a 200 microliter pipette tip. Cut the pipette's tip five millimeters from the small end to make a 8 millimeter diameter opening.A fly head will fit through this hole, but the thorax will not. Lastly, make a for the fly holder and impactor device. First, cut a one milliliter pipette tip 44 millimeters from the small opening.Second, cut a six millimeter length from the needle cover for a one milliliter syringe. Then, push the two together. Anesthetize a single, two-day old adult female fly using carbon dioxide.From the carbon dioxide pad, use a fine brush to gently transfer the fly into the holder. Then, gently tap the holder until the head protrudes from the tip. Of the proboscis is exposed, use a blunted, one milliliter syringe needle to gently tuck it back inside the tip.It is critical to keep the fly body, especially the mouth part, inside the holder. Otherwise, the fly may die from damage to the internal organs or from not being able to ingest food. Next, set the gas pressure to 100 kilopascals and adjust the flow as needed.Once the fly is loaded, tighten the holder to the syringe barrel using the connector, such that the fly head faces downward. Then, using the toggle switch, send a burst of gas that moves the impactor to strike the fly once and only once. Now, detach the fly holder and dump the fly back onto the carbon dioxide pad.Brush the fly into an empty vial until it recovers. Keep one fly per vial. The recovery takes only a few minutes.Repeat the process to test four flies per experimental group. Processing two groups, a test and a sham, should only take 20 to 30 minutes. Treat shams just like test animals, except do not include an aerosol barrier in the impactor tube.To track fly movements in a Petri dish, first, fill a six centimeter dish with transparent silicon elastomer to make the tracking arena. Leave a three millimeter gap between the silicon and the lid so that flies can walk freely in the gap, but not take flight. Beneath the tracking arena, have a white background.Next, anesthetize four flies from the same treatment group and place them in the arena. Let the flies acclimate for an hour to the arena, which should be fully lit and at about 22 degrees Celsius. Not hotter than room temperature.After an hour, use a charge-coupled device camera positioned above the arena to record the fly's activity for five minutes. After the five minute recording, anesthetize the flies in the arena and return them to a new home vial. Then, dispose of the arena.Later, analyze the recordings using the freely available Ctrax software. This software generates tracking data, which can be exported in a programmable language compatible format, such as the MATLAB format. From the data, calculate the distance traveled per frame, the mean walking distance for each fly, and the average distance traveled per fly.For statistical significance, expect to test about 100 flies per treatment group. To establish a chronic traumatic encephalopathy model, the effectiveness of a single, closed-head injury made by the impacting apparatus at different speeds was analyzed using two-day old Canton-S females. The gas pressure was held at 100 kilopascals.Flies exposed to a single strike at the highest tested flow rate, exhibited minimal external defects. Despite no clear evidence for external damage, injuries at a flow rate of 15 liters per minute were acutely lethal, resulting in less than a 10%survival within 24 hours. Survival increased with lower flow rates.One hundred percent survival was achieved at five liter per minute or less, so five liters per minute was selected as the standard for the model. Just after being struck, flies gradually recovered their mobility within four minutes. Recovery for the shams was about a minute quicker.Post-impact, video-assisted movement tracking was used to measure locomotive function. During the two days after the impact, locomotion slowly returned to normal. This evidence for gradual recovery is in line with CTE recovery in humans.To evaluate long term effects, a repetitive traumatic brain injury protocol of five strikes over five days was applied. Twenty days post-treatment, the locomotion of surviving members in each group was compared and the treated group clearly walked less. The lifespan of the treated group was also substantially reduced, with a median at 26 days compared to 37 days for controls.After its development, this technique paves the way for researchers in the field of mild repetitive traumatic brain injury to explore pathogenic mechanisms in the classic animal model Drosophila melanogaster. While attempting this procedure, it is important to remember to shield the proboscis, thorax and abdomen from direct impact by the aerosol barrier.

Research

JoVE Journal

Neuroscience

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

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Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

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Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

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Um método de baixo custo para análise de Apreensão-como atividade e movimento em<em&gt; Drosophila</em

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

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Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

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A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Um Método para infligir Cabeça Fechada Lesão cerebral traumático em<em&gt; Drosophila</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

Gravação de EEG em Ratos Neonatais de Movimentação Livre Usando um Novo Método

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Novel Assay for Cold Nociception in Drosophila Larvae

Novel Assay for Cold Nociception in Drosophila Larvae

Romance Ensaio para Fria nocicepção em<em&gt; Drosophila</em&gt; Larvas

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

Um modelo de romance In Vitro de explosão traumatismo crânio-encefálico

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...