Name: a novel method to model chronic traumatic encephalopathy in drosophila
Uploaded: 2017-07-04T13:00:00
Description: Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

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1. Montering av streikapparatet ( figur 1 ) <ol><li> Fjern stemplet fra en 1 ml tuberkulinsprøyte. Klipp fat på 1 ml merket. </li><li> Fjern en aerosolbarriere (3 mm høyde x 4 mm diameter) fra en 200 μL pipettspiss og bruk den som en støtfanger. Plasser støtfangeren inne i sprøytekarmen. Trykk forsiktig på fatet for å flytte støtfangeren til spissen, med den flate siden som dekker dyseåpningen. </li><li> Fest tappens ende til plastrør som er koblet til karbondioksid (CO 2...

<ol>
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T., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+and+synaptic+organization:+analysis+of+the+Drosophila+genome.">Ion channels and synaptic organization: analysis of the Drosophila genome.</a> Neuron. 26 (1), 35-43 (2000).</li><li>Appel, L. F., 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=The+Drosophila+Stubble-stubbloid+gene+encodes+an+apparent+transmembrane+serine+protease+required+for+epithelial+morphogenesis.">The Drosophila Stubble-stubbloid gene encodes an apparent transmembrane serine protease required for epithelial morphogenesis.</a> Proc Natl Acad Sci USA. 90 (11), 4937-4941 (1993).</li><li>Piyankarage, S. C., Featherstone, D. E., Shippy, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoliter+hemolymph+sampling+and+analysis+of+individual+adult+Drosophila+melanogaster.">Nanoliter hemolymph sampling and analysis of individual adult Drosophila melanogaster.</a> Anal Chem. 84 (10), 4460-4466 (2012).</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+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. M., 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=Functionally-detected+cognitive+impairment+in+high+school+football+players+without+clinically-diagnosed+concussion.">Functionally-detected cognitive impairment in high school football players without clinically-diagnosed concussion.</a> J Neurotrauma. 31 (4), 327-338 (2014).</li><li>Theadom, 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=Frequency+and+impact+of+recurrent+traumatic+brain+injury+in+a+population-based+sample.">Frequency and impact of recurrent traumatic brain injury in a population-based sample.</a> J Neurotrauma. 32 (10), 674-681 (2015).</li><li>Drobysheva, D., 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=An+optimized+method+for+histological+detection+of+dopaminergic+neurons+in+Drosophila+melanogaster.">An optimized method for histological detection of dopaminergic neurons in Drosophila melanogaster.</a> J Histochem Cytochem. 56 (12), 1049-1063 (2008).</li></ol>

Dyremodeller som trofastmodellerer CTE-funksjoner, inkludert nevrofysiologiske endringer, nevropatologiske karakteristikker og neurobehavioral underskudd, er avgjørende for å avdekke sykdomsmekanismer og for å utvikle diagnostiske og terapeutiske mål. Det er forståelig at ingen dyremodell av en menneskelig sykdom er perfekt for å etterligne alle klinisk relevante endepunkter. Vi mener imidlertid at en robust CTE-modell skal tilfredsstille følgende tre krav: (1) virkningen må brukes direkte på et hode som har en...

Kronisk traumatisk encefalopati (CTE) har nylig blitt anerkjent som en distinkt nevrodegenerativ lidelse, adskilt fra andre tauopatier som Alzheimers sykdom 1 . I motsetning til Alzheimers sykdom og andre vanlige tauopatier - hvis viktigste risikofaktorer fremmer alderen og en familiehistorie av demens, innebærer CTE, som angitt av navnet, en nær tilknytning til en historie med hjerne traumer, sannsynligvis sett hos kontakt idrettsutøvere, Slik som boksere og fotballspillere, samt i militære veteraner 2 , 3 , 4 , 5 . Det antas å bli initiert av gjentatte hjernerystelser og subkoncussive slag mot hodet. Pasienter kan presentere symptomer og tegn som kognitive underskudd, humør og atferdsendringer og bevegelsesdysfunksjon, som overlapper betydelig med Alzheimers sykdom, frontotemporalDemens, Lewy body demens og Parkinsons sykdom 6 . I motsetning til dette viser etterforsøk av hjernevev et tydelig mønster av hyperfosforylert tauakkumulering som omgir små blodkar ved dybden av kortikale sulci, en patognomonisk funksjon som ikke er observert i de andre degenerative tilstander 7 . Imidlertid er så lite kjent om patogenesen som fører til sykdoms manifestasjon. Dette skyldes i stor grad mangelen på en trofast dyremodell - bare nylig har gnagermodeller blitt generert 5 , 8 . Disse modellorganismer har ulempene med kostnadskrevende omsorg og en relativt lang levetid, som ikke er velegnet for neurodegenerative sykdomsstudier. Sammenlignet med pattedyr-kolleger, er virvelløse dyr som Drosophila et utmerket alternativ, med kostnadseffektivt vedlikehold,Omfattende verktøy for dissektering av genetiske determinanter, og relativt kort levetid 9 . Bemerkelsesverdig deler fly og menneskelige hjerner evolusjonært konserverte molekylære og cellulære veier, så vel som anatomiske likheter 10 , 11 , 12 . To tidligere ingeniøse Drosophila- modeller for å studere traumatisk hjerneskade har blitt rapportert tidligere 13 , 14 . Den første &quot;High Impact Trauma&quot; (HIT) -enheten designet av Katzenberger og kolleger, inneholdt frittflygende fluer i et plastflaske som var bundet til den frie enden av en metallfjær 13 , 15 . Når plastflasken ble hevet oppreist og løslatt, rammet den en polyuretanpute og ga traumer til fluene da de hoppet til hetteglassveggen og gjenvunnet. I kontrast, designet Barekat og kolleger en annen leveringsmetode ossInn i Omni Bead Ruptor-24 homogeniseringsplattformen 14 . Fluer ble uegnet med CO 2 og plassert i et 2 ml skruekapslør som var sikret til homogenisatoren og underkastet forprogrammerte rystningsbetingelser. En fordel ved å bruke vevshomogenisatorsystemet er at eksperimentet kunne modulere intensiteten av skade, varighet av skade og antall skadeproblemer. Imidlertid har begge regimene den samme ulempen: primære skader på hodet er tilfeldig påført når det gjelder slagplassering og styrke. I tillegg resulterte begge metodene i betydelig dødelighet, forårsaket av uunngåelig sikkerhetsskade på andre deler av kroppen og indre organer. Her beskriver vi en ny metode for å indusere mTBI i fruktfluer. Apparatet vårt består av en gassdrevet ballistisk støtfanger. Sammenlignet med de eksisterende Drosophila- modellene 14 , 15 , har metoden vår enestående fordel ved å levere målingUvirkelig påvirkning, kun rettet mot det frie flygende hodehodet, og dermed muliggjøre nøyaktig kontroll av ulike faktorer, som for eksempel virkningsgrad, tidsintervallet mellom påvirkninger og det totale antall påvirkninger som opprettholdes.

<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

Kronisk traumatisk encefalopati (CTE) er en etablert neurodegenerativ sykdom som er nært forbundet med eksponering for gjentatt mild traumatisk hjerneskade (mTBI). Mekanismene som er ansvarlige for sine komplekse patologiske forandringer forblir stort sett unnvikende, til tross for en ny konsensus for å definere nevropatologiske kriterier. Her beskriver vi en ny metode for å utvikle en modell av CTE i Drosophila melanogaster ( Drosophila ) i et forsøk på å identifisere nøkkelgenene og banene som fører til den karakteristiske hyperfosforylerte tauakkumuleringen og nevronaldød i hjernen. Justerbar kraftpåvirkning for å forårsake mild lukket skade blir levert direkte til flyhodet, og utsetter hodet for rask akselerasjon og retardasjon. Vår metode eliminerer de potensielle problemene som er forbundet med andre Drosophila mTBI-modeller ( f.eks. Dyredød kan forårsakes av skade påAndre deler av kroppen eller til indre organer). Den mindre arbeids- og kostnadskrevende dyrpleie, kort levetid og omfattende genetiske verktøy gjør fruktfluen ideell til å studere CTE-patogenesen og gjøre det mulig å utføre store, genomgående fremadrettede og farmakologiske skjermbilder. Vi forventer at den pågående karakteriseringen av modellen vil gi viktige mekanistiske innsikter om sykdomsforebygging og terapeutiske tilnærminger.

For å etablere en CTE-modell ved hjelp av voksen Drosophila , fastslått vi effektiviteten av enheten vår ved å forårsake en enkelt lukkeskader. For å eliminere variasjonene knyttet til genotype, kjønn eller alder brukte vi 2-dagers Canton-S WT kvinnelige fluer i forsøket. Vi kunne enkelt kontrollere styrken av støtfangeren ved å regulere strømningshastigheten til CO 2 ved et konstant gasstrykk på 100 kPa. Fluer utsatt for en enkelt streik med høyest strø...

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

Her beskriver vi en ny tilnærming til å forårsake traumatisk hjerneskade i lukkede hoder i Drosophila melanogaster . Vår metode har fordelen av direkte å levere repeterende påvirkninger med justerbar styrke til hodet alene. Videre utforskning av ryggradssystemet vil bidra til å belyse patogenesen av kronisk traumatisk encefalopati.

En ny metode for å modellere kronisk traumatisk encefalopati i<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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Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

En metode å påføre Stengt Leder traumatisk hjerneskade i<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

Registrering av EEG i fritt bevegelige neonatale rotter ved hjelp av en ny metode

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

Novel analyse for Cold nociception i<em&gt; Drosophila</em&gt; Larver

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

Romanen In Vitro modell for Blast traumatisk hjerneskade

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