Name: induction of diffuse axonal brain injury in rats based on rotational acceleration
Uploaded: 2020-05-09T15:00:00
Description: Watch this Scientific Journal Video about Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration at JoVE.com

Forfatterne taknemmeligt anerkende Dr. Nathan Kleeorin (Institut for Maskinteknik, Ben-Gurion University of the Negev) for hans hjælp med biomekaniske målinger. Vi takker også professor Olena Severynovska, Maryna Kuscheriava, Maksym Kryvonosov, Daryna Yakumenko og Evgenia Goncharyk fra Institut for Fysiologi, Fakultetet for Biologi, Økologi og Medicin, Oles Honchar Dnipro University, Dnipro, Ukraine for hendes støtte og nyttige bidrag til vores drøftelser.

Forsøgene blev udført på grundlag af anbefalingerne i Helsingfors- og Tokyo-erklæringerne og retningslinjerne for anvendelse af forsøgsdyr i Det Europæiske Fællesskab. Forsøgene blev godkendt af Animal Care Committee af Ben-Gurion University of the Negev.
1. Forberedelse af rotter til forsøgsproceduren
BEMÆRK: Vælg voksne hanner Sprague-Dawley-rotter, der vejer 300-350 g.
<ol><li>Få godkendelse til at udføre disse forsøg fra Institutional Animal Care...

<ol>
	<li>Faul, M., Wald, M. M., Xu, L., Coronado, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury+in+the+United+States;+emergency+department+visits,+hospitalizations,+and+deaths,+2002-2006.">Traumatic brain injury in the United States; emergency department visits, hospitalizations, and deaths, 2002-2006.</a> US Government. , (2010).</li><li>Taylor, C. A., Bell, J. M., Breiding, M. J., Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+Brain+Injury-Related+Emergency+Department+Visits,+Hospitalizations,+and+Deaths+-+United+States,+2007+and+2013.">Traumatic Brain Injury-Related Emergency Department Visits, Hospitalizations, and Deaths - United States, 2007 and 2013.</a> MMWR Surveillance Summaries. 66, 1-16 (2017).</li><li>Peterson, A. 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M., Esiri, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+damage:+a+key+predictor+of+outcome+in+human+CNS+diseases.">Axonal damage: a key predictor of outcome in human CNS diseases.</a> Brain. 126, 515-530 (2003).</li><li>Tang-Schomer, M. D., Johnson, V. E., Baas, P. W., Stewart, W., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+interruption+of+axonal+transport+due+to+microtubule+breakage+accounts+for+the+formation+of+periodic+varicosities+after+traumatic+axonal+injury.">Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury.</a> Experimental Neurology. 233, 364-372 (2012).</li><li>Johnson, V. E., Stewart, W., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury+and+amyloid-beta+pathology:+a+link+to+Alzheimer's+disease.">Traumatic brain injury and amyloid-beta pathology: a link to Alzheimer's disease.</a> Nature Reviews Neuroscience. 11, 361-370 (2010).</li><li>Sherriff, F. E., Bridges, L. R., Sivaloganathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+of+axonal+injury+after+human+head+trauma+using+immunocytochemistry+for+beta-amyloid+precursor+protein.">Early detection of axonal injury after human head trauma using immunocytochemistry for beta-amyloid precursor protein.</a> Acta Neuropathologica. 87, 55-62 (1994).</li><li>Reichard, R. R., White, C. L., Hladik, C. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapy+development+for+diffuse+axonal+injury.">Therapy development for diffuse axonal injury.</a> Journal of Neurotrauma. 30, 307-323 (2013).</li><li>McKenzie, K. 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Denne protokol beskriver en gnavermodel af DAI. I DAI forårsager rotationsacceleration på hjernen en forskydningseffekt, der udløser axonale og biokemiske ændringer, der fører til tab af aksonal funktion i en progressiv proces. Sekundære aksonale ændringer skyldes en hurtig aksonale strækskade og varierer i omfang og alvor4,5,10. Inden for få timer til dage efter den primære skade vil biokemiske ændringer føre til ta...

Traumatisk hjerneskade (TBI) er en væsentlig årsag til død og invaliditet i USA. Tuberkulose bidrager til ca. 30 % af alle personskader1,2. De førende årsager til TBI varierer mellem aldersgrupper og omfatter fald, højhastighedskollisioner under sport, forsætlig selvskade, bilulykker og overfald1,2,3.
Hjernediffus axonal skade (DAI) er en specifik type TBI induceret af roterende acceleration, omrystning eller blast skade af hjernen som følge af ubegrænset hoved bevægelse i det øjeblik efter skade4,5,6,7,8. DAI indebærer omfattende aksonale skader, der fører til langvarig neurologisk svækkelse, der er forbundet med dårligt resultat, byrdefulde udgifter til sundhedspleje og en dødelighed på 33-64 %1,2,4,5,9,10,11. På trods af betydelig nyere forskning i DAI's patogenese har der ikke været enighed om de bedste behandlingsmuligheder11,12,13,14.
I løbet af de seneste årtier har talrige forsøgsmodeller forsøgt præcist at kopiere forskellige aspekter af DAI11,12,15,16. Men disse modeller har begrænsninger i betragtning af den unikke præsentation af DAI i forhold til andre fokale skader. Disse tidligere modeller ikke kun forårsage axonal skade i hvide stof regioner, men også resultere i fokale hjerneskader. Klinisk, DAI er ledsaget af mikro blødninger, som kan udgøre en væsentlig årsag til skader på hvidt stof.
Kun to dyremodeller har vist sig at kopiere de vigtigste kliniske træk ved DAI. Gennarelli og kolleger produceret den første laterale hoved rotation enhed i 1982, ved hjælp af nonimpact hoved rotationsacceleration at fremkalde koma med DAI i en ikke-menneskelig primat model15. Denne primatmodel anvendte kontrolleret enkelt rotation for acceleration og deceleration for at fortrænge hovedet gennem 60° inden for 10-20 ms. Denne teknik var i stand til at efterligne nedsat bevidsthed og udbredt aksonal skade, der lignede virkningerne af svær TBI observeret i menneskelige hjerner. Men primat modeller er meget dyre4,11,16. Baseret på en delvis på den tidligere model, en gris model af roterende acceleration hjerneskade blev designet i 1994 (Ross et al.) med lignende resultater14.
Disse to dyremodeller, selv om de producerede forskellige præsentationer af typisk patologi, har tilføjet meget til begreberne DAI patogenese. Hurtig hovedrotation er generelt accepteret som den bedste metode til at fremkalde DAI, og gnavere giver en billigere model for den hurtige hovedrotationsundersøgelser11,16. Her validerer vi en enkel, reproducerbar og pålidelig gnavermodel af DAI, der forårsager udbredt skade på hvidt stof uden kraniebrud eller kontusioner. Denne nuværende model vil give en bedre forståelse af patosiologi af DAI og udvikling af mere effektive behandlinger.

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			<td height="42" style="height:42px;width:216px;">0.01 M sodium citrate</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2.5% normal horse serum</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">H0146</td>
			<td>Liquid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4 % buffered formaldehyde solution</td>
			<td style="width:121px;"></td>
			<td style="width:161px;"></td>
			<td></td>
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			<td height="63" style="height:63px;width:216px;">Anti-Amyloid Precursor Protein, C - terminal antibodyproduced in rabbit</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">Lot 056M4867V</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">biotinylated secondary antibody</td>
			<td style="width:121px;">Vector</td>
			<td style="width:161px;">BA-1000-1.5</td>
			<td>10 mM sodium phosphate, pH 7.8, 0.15 M NaCl, 0.08% sodium azide, 3 mg/ml bovine serum albumin</td>
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			<td height="21" style="height:21px;width:216px;">bone-cutting forceps</td>
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			<td height="63" style="height:63px;width:216px;">DAB Peroxidase (HRP) Substrate Kit (with Nickel), 3,3&#8217;-diaminobenzidine</td>
			<td style="width:121px;">vector laboratory</td>
			<td style="width:161px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">embedding cassettes</td>
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			<td height="21" style="height:21px;width:216px;">ethanol 99.9 %</td>
			<td style="width:121px;">ROMICAL</td>
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			<td>Flammable Liquid</td>
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			<td height="21" style="height:21px;width:216px;">guillotine</td>
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			<td height="42" style="height:42px;width:216px;">Hematoxylin</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">H3136-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide solution</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">88597-100ML-F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isofluran, USP 100%</td>
			<td style="width:121px;">Piramamal Critical Care, Inc</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Olympus BX 40 microscope</td>
			<td style="width:121px;">Olympus</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">paraffine</td>
			<td style="width:121px;">paraplast plus leica biosystem</td>
			<td style="width:161px;"></td>
			<td>Tissue embedding medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">phosphate-buffered saline (PBS)</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">P5368-10PAK</td>
			<td>Contents of one pouch, when dissolved in one liter of distilled or deionized water, will yield 0.01 M phosphate buffered saline (NaCl 0.138 M; KCl - 0.0027 M); pH 7.4, at 25 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptavidin HRP</td>
			<td style="width:121px;">ABCAM</td>
			<td style="width:161px;">ab64269</td>
			<td>Streptavidin-HRP for use with biotinylated secondary antibodies during IHC / immunohistochemistry.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">xylene</td>
			<td style="width:121px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of diffuse axonal brain injury in rats based on rotational acceleration

Traumatisk hjerneskade (TBI) er en væsentlig årsag til død og invaliditet. Diffus aksonal skade (DAI) er den fremherskende mekanisme til skade i en stor procentdel af TBI patienter, der kræver hospitalsindlæggelse. DAI indebærer udbredt axonal skade fra rysten, rotation eller blast skade, fører til hurtig axonal stretch skade og sekundære axonal ændringer, der er forbundet med en langvarig indvirkning på funktionel genopretning. Historisk set har eksperimentelle modeller af DAI uden fokale skader været vanskelige at designe. Her validerer vi en enkel, reproducerbar og pålidelig gnavermodel af DAI, der forårsager udbredt skade på hvidt stof uden kraniebrud eller kontusioner.

Tabel 1 illustrerer protokollens tidslinje. Dødeligheden i denne model af DAI var 0%. En Mann-Whitney test viste, at neurologiske underskud var betydeligt større for de 15 DAI rotter i forhold til de 15 fingerede rotter på 48 timer efter intervention (Mdn = 1 vs 0), U = 22,5, p < 0,001, r = 0,78 (se tabel 2). Dataene måles i tal og præsenteres som median og 25-75 percentilinterval.
Repræsentative fotomikrografer af thalamic dele af hjernevæv er vist i <...

Watch this Scientific Journal Video about Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration at JoVE.com

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Denne protokol validerer en pålidelig, nem at udføre og reproducerbare gnavere model af hjernen diffus axonal skade (DAI), der inducerer udbredt hvide stof skader uden kraniebrud eller kvæstelser.

Induktion af diffus axonal hjerneskade hos rotter baseret på rotationsacceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

Traumatisk hjerneskade er en af de vigtigste årsager død og handicap. Diffus axonal hjerneskade er en udbredt axonal skade udvikle efter TBI. I denne forskning vil jeg gerne præsentere vores model om induktion diffus axonal hjerneskade baseret på roterende acceleration mekanisme resultat TBI.Vælg voksne mandlige Sprague-Dawley rotter vejer 300 til 350 gram. Giv rotte chow og vand ad libitum. Udfør alle eksperiment mellem kl. Enheden blev placeret på et tungt, stabilt laboratoriebord.Anordningen består af følgende komponenter: en cylinder, en jernvægt, en rotationsmekanisme bestående af et cylindrisk rør og hovedfikseringsstifter. Vægten er fastgjort til en streng og forhøjet til højden af 120 centimeter. Den frit faldende vægt rammer bolten, der aktiverer rotationsmekanismen.Den rotte lateral hoved rotation enhed blev brugt til at dreje hovedet hurtigt fra nul til 90 grader. Efter induktion, diffus axonal hjerneskade, rotte blev flyttet til opsøgningsrum. F er den kraft, der påføres dyrets hoved i kg.Capital M er kraftens øjeblik, K for kinetisk energi. Lille m er massen af den faldende vægt, g for gravitationel acceleration, h for højde i centimeter, og D er afstanden mellem ørestifterne i centimeter. Til vurdering af neurologiske underskud og grade model underskud, brugte vi Neurologisk Sværhedsgrad Score i rotter.Vi evaluerer mobilitet, hemiplegi, stråle gå opgave, fiasko i stråle gå opgave. Fejl i strålebalanceringsopgaven bred 1,5 centimeter. Stabilitet og strålebalancering, indsats på strålebalance 1,5 centimeter bred og reflekser.48 timer efter skaden, alle rotter, skade og kontrolgruppe, var dybt bedøvet og transcardiacally perfunderes med 0,9% hepariniseret saltvand efterfulgt af 500 milliliter 4% formaldehyd i 0,1 M fosfat buffer saltvand. Efter perfusion, halshugning blev produceret og hjernen blev straks fjernet, derefter fastsat i en 4% formaldehyd opløsning i 48 timer. Hjernen blev derefter blokeret i fem millimeter koronale sektioner fra olfaktoriske pære ansigt til den visuelle cortex, mens lillehjernen og hjernestammer blev dissekeret.Efter paraffin indlejring, koronal og sagittal sektioner blev foretaget fra thalamus. Producere immunkemiske farvning af beta-amyloid forløber protein. De forskellige grupper af rotter på forskellige tidspunkter er vist på ordningen.Diffus axonal hjerneskade i begyndelsen af eksperimentet. På tidspunktet 48 timer blev neurologisk sværhedsgradsscore undersøgt. På tidspunktet for 48 timer blev der udført immunkemisk farvning af beta-APP i alle to grupper.Illustreret neurologisk underskud 48 timer efter diffus axonal hjerneskade for to studiegrupper. En Mann-Whitney test viste, at neurologiske underskud var betydeligt større for 15 diffus axonal hjerneskade rotter sammenlignet med 15 naive rotter på 48 timer efter intervention. Repræsentative fotomikrografer, der afslører aksonale og neuronale immunoreactivities efter isoleret diffus axonal hjerneskade hos rotter 48 timer efter skaden.Mindre cellulære strenge påvises med beta-amyloid forløber protein. I sham gruppe, blev det ikke opdaget. I vores arbejde udviklede vi en enkel, reproducerbar og pålidelig ny model til diffus axonal hjerneskade baseret på rotationsaccelerationsmekanisme.En sådan model vil muliggøre en bedre forståelse af patofysiologi af diffus axonal hjerneskade og udvikling af mere effektive behandlinger. Mange tak for deres opmærksomhed.

Research

JoVE Journal

Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Preparation of Oligomeric ÃƒÅ½Ã‚Â²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

En<em&gt; Ex vivo</em&gt; Laser-induceret Rygmarvsskader model til opgørelse Mekanismer axon Degeneration i realtid

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

In vivo Funktionel Brain Imaging tilgang baseret på Bioluminescent Calcium Indikator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Virkningerne af Blast-induceret Neurotrauma på trykisoleret gnaver midt cerebrale arterier

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Aktivitet-baseret træning på et løbebånd med rygmarven skadet Wistar rotter

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

Generation af 3-D kollagen-baserede Hydrogels til at analysere Axonal vækst og adfærd under nervesystemet udvikling

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Vurdering af langvarig depression induktion i voksne cerebellar skiver

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Avanceret diffusion Imaging i hippocampus af rotter med mild traumatisk hjerneskade

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

Murine model af kontrolleret kortikale virkning for induktion af traumatisk hjerneskade

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...