Name: an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices
Uploaded: 2018-04-11T15:00:00
Description: Watch this Scientific Journal Video about An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices at JoVE.com

Dette arbeidet ble finansiert av Nevro-Bio Ltd. Vi ønsker å takke Dr. Giovanni Ferrati og Dr. Sergio Rotondo (Neuro-Bio) for deres kommentarer og råd på manuskriptet.

Alle dyrestudier har utført under godkjent protokoller.
Merk: I denne delen, en rekke hovedfasene utført under den eksperimentelle prosedyren og foreslåtte tidsintervallet angis (figur 1). Videre er en detaljert beskrivelse av protokollen supplert med en illustrasjon panel, viser kritiske handlinger fra hjernen fjerning til vev homogenisering etter inkubasjonstiden (figur 2). Detaljer om materialer og instruksjoner for å bygge app...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+neurone+loss+in+Alzheimer's+disease:+cortical+or+subcortical?.">Early neurone loss in Alzheimer's disease: cortical or subcortical?.</a> Acta Neuropathol Commun. 3, 10 (2015).</li><li>Mesulam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cholinergic+Lesion+of+Alzheimer's+Disease:+Pivotal+Factor+or+The+Cholinergic+Lesion+of+Alzheimer's+Disease:+Pivotal+Factor+or+Side+Show?.">The Cholinergic Lesion of Alzheimer's Disease: Pivotal Factor or The Cholinergic Lesion of Alzheimer's Disease: Pivotal Factor or Side Show?.</a> Learn Mem. , 43-49 (2004).</li><li>Schliebs, R., Arendt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cholinergic+system+in+aging+and+neuronal+degeneration.">The cholinergic system in aging and neuronal degeneration.</a> Behavioural Brain Research. 221 (2), 555-563 (2011).</li><li>Mesulam, M. M., Mufson, E. J., Wainer, B. H., Levey, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+cholinergic+pathways+in+the+rat:+An+overview+based+on+an+alternative+nomenclature+(Ch1-Ch6).">Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1-Ch6).</a> Neuroscience. 10 (4), 1185-1201 (1983).</li><li>Mesulam, M., Mufson, E. J., Levey, A. I., Wainer, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+innervation+of+cortex+by+the+basal+forebrain:+cytochemistry+and+cortical+connections+of+the+septal+area,+diagonal+band+nuclei,+nucleus+basalis+(substantia+innominata),+and+hypothalamus+in+the+rhesus+monkey.">Cholinergic innervation of cortex by the basal forebrain: cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata), and hypothalamus in the rhesus monkey.</a> J Comp Neurol. 214 (2), 170-197 (1983).</li><li>Greenfield, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+and+targeting+the+basic+mechanism+of+neurodegeneration:+The+role+of+peptides+from+the+C-terminus+of+acetylcholinesterase:+Non-hydrolytic+effects+of+ache:+The+actions+of+peptides+derived+from+the+C-terminal+and+their+relevance+to+neurodegenerat.">Discovering and targeting the basic mechanism of neurodegeneration: The role of peptides from the C-terminus of acetylcholinesterase: Non-hydrolytic effects of ache: The actions of peptides derived from the C-terminal and their relevance to neurodegenerat.</a> Chemico-Biological Interactions. 203 (3), 543-546 (2013).</li><li>Garcia-Rat&#233;s, S., 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=(I)+Pharmacological+profiling+of+a+novel+modulator+of+the+&#945;7+nicotinic+receptor:+Blockade+of+a+toxic+acetylcholinesterase-derived+peptide+increased+in+Alzheimer+brains.">(I) Pharmacological profiling of a novel modulator of the &#945;7 nicotinic receptor: Blockade of a toxic acetylcholinesterase-derived peptide increased in Alzheimer brains.</a> Neuropharmacology. 105, 487-499 (2016).</li><li>Eimerl, S., Schramm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantity+of+calcium+that+appears+to+induce+neuronal+death.">The quantity of calcium that appears to induce neuronal death.</a> Journal of neurochemistry. 62 (3), 1223-1226 (1994).</li><li>Greenfield, S., Vaux, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commentary+Parkinson's+Disease,+Alzheimer's+Disease+and+Motor+Neurone+Disease:+Identifying+a+Common+Mechanism.">Commentary Parkinson's Disease, Alzheimer's Disease and Motor Neurone Disease: Identifying a Common Mechanism.</a> Science. 113 (3), 485-492 (2002).</li><li>Badin, A. S., Morrill, P., Devonshire, I. M., Greenfield, 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=(II)+Physiological+profiling+of+an+endogenous+peptide+in+the+basal+forebrain:+Age-related+bioactivity+and+blockade+with+a+novel+modulator.">(II) Physiological profiling of an endogenous peptide in the basal forebrain: Age-related bioactivity and blockade with a novel modulator.</a> Neuropharmacology. 105, 47-60 (2016).</li><li>Brai, E., Stuart, S., Badin, A. -. S., Greenfield, 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=A+Novel+Ex+Vivo+Model+to+Investigate+the+Underlying+Mechanisms+in+Alzheimer's+Disease.">A Novel Ex Vivo Model to Investigate the Underlying Mechanisms in Alzheimer's Disease.</a> Frontiers in Cellular Neuroscience. 11, 291 (2017).</li><li>Cho, S., Wood, A., Bowlby, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+slices+as+models+for+neurodegenerative+disease+and+screening+platforms+to+identify+novel+therapeutics.">Brain slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics.</a> Current neuropharmacology. 5 (1), 19-33 (2007).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annual review of physiology. 46, 455-472 (1984).</li><li>Jensen, M. S., Lambert, J. D. C., Johansen, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+rat+hippocampus+slices+following+in+vivo+brain+ischemia.">Electrophysiological recordings from rat hippocampus slices following in vivo brain ischemia.</a> Brain Research. 554 (1-2), 166-175 (1991).</li><li>Ferrati, G., Martini, F. J., Maravall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+Adenosine+Receptor-Mediated+Regulation+of+Diverse+Thalamocortical+Short-Term+Plasticity+in+the+Mouse+Whisker+Pathway.">Presynaptic Adenosine Receptor-Mediated Regulation of Diverse Thalamocortical Short-Term Plasticity in the Mouse Whisker Pathway.</a> Frontiers in Neural Circuits. 10, 1-9 (2016).</li><li>Grinvald, A., Hildesheim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSDI:+a+new+era+in+functional+imaging+of+cortical+dynamics.">VSDI: a new era in functional imaging of cortical dynamics.</a> Nature Reviews Neuroscience. 5 (11), 874-885 (2004).</li><li>Badin, A. S., John, E., Susan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+spatio-temporal+bioactivity+of+a+novel+peptide+revealed+by+optical+imaging+in+rat+orbitofrontal+cortex+in+vitro:+Possible+implications+for+neurodegenerative+diseases.">High-resolution spatio-temporal bioactivity of a novel peptide revealed by optical imaging in rat orbitofrontal cortex in vitro: Possible implications for neurodegenerative diseases.</a> Neuropharmacology. 73, 10-18 (2013).</li><li>Greenfield, S. A., Badin, A. S., Ferrati, G., Devonshire, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+rat+brain+suggests+a+previously+missing+link+between+top-down+and+bottom-up+nervous+system+function.">Optical imaging of the rat brain suggests a previously missing link between top-down and bottom-up nervous system function.</a> Neurophotonics. 4, 31213 (2017).</li><li>Opitz-Araya, X., Barria, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures.">Organotypic hippocampal slice cultures.</a> Journal of visualized experiments: JoVE. (48), (2011).</li><li>Gong, C. -. X., Lidsky, T., Wegiel, J., Grundke-Iqbal, I., Iqbal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolically+active+rat+brain+slices+as+a+model+to+study+the+regulation+of+protein+phosphorylation+in+mammalian+brain.">Metabolically active rat brain slices as a model to study the regulation of protein phosphorylation in mammalian brain.</a> Brain Research Protocols. 6 (3), 134-140 (2001).</li></ol>

Det viktigste aspektet ved denne protokollen, basert på veletablerte ex vivo hjernen teknikken, kan synkront teste to speilende hemislices, fra samme anatomiske plan, overvåke deres svar etter et bestemt tilstand (kontrollere eller behandlet); Dette gir derfor en eksperimentell paradigme like strengt kontrollert som mulig. Muligheten for å vurdere i en tid - dose- og områdespesifikk måte forskjellige nevrokjemiske knyttet til nevronale svekkelse, sett i løpet av nevrodegenerative hendelser<sup class="xref"...

Annonsen er en kronisk patologi preget av gradvis nevrodegenerative verdifall påvirker andre hjernen områder, som entorhinal cortex (EC), BF, hippocampus (HC) og Luktelappen (SR)1,2,3, 4,5. På slutten stadier av AD utvikling føre til en progressiv kognitiv svikt, gjør denne sykdommen den vanligste formen for demens, ca sto for 70% av alle tilfeller6. Til tross for omfattende forsøk på å forstå den innledende stadiet forårsaker annonse, er det ikke en definert eksperimentelle indikasjon Klargjørende dem. Dessuten, er den mest populære teorien - "amyloid hypotesen" - stadig spurt siden det ikke gir en fullstendig profil forklarer AD pathobiology, heller en farmasøytisk mål som har vist seg effektiv7,8 ,9.
En alternativ teori som får økende oppmerksomhet antyder at første mekanismer forekommer under neurodegeneration er knyttet til en neuronal klynge primært utsatt i AD3,10,11 , 12 , 13 , 14. denne heterogene mobilnettet huben omfattet BF, mellomhjernen og hjernestammen, prosjektene til flere områder, slik som EU, HC og OB15,16. Til tross for sitt mangfold i neuronal morfologi og nevrotransmitter syntese aksjer denne kjernen av cellene en vanlig funksjon i å uttrykke verke, som kan også ha en ikke-enzymatisk funksjonen17,18. Denne ikke-klassiske rollen som en roman Signalering molekyl formidler kalsium (Ca2 +) strømme inn nerveceller som kan gjennomgå trophic eller giftig begivenheter i forbindelse Ca2 + dose, tilgjengelighet og neuronal alder17,18 , 19.
Under neurodegeneration, kan observerte mobilnettet tap derfor være tilknyttet denne ikke-enzymatisk funksjonen17,18,20, som skyldes et 30mer peptid (T30) kløyvde verke C-terminalen 20. i tråd med tidligere resultater, gjennomført på cellekultur og optisk tenkelig18,21 forberedelser, vi vist, gjennom en roman tilnærming basert på ex vivo rotte hjernen skiver med BF strukturer, som T30 indusert en AD-lignende profil22. Spesielt tilbyr denne nye metodikken et mer fysiologiske scenario enn cellekultur siden det opprettholder mange av egenskapene til en intakt vev, alt fra anatomisk til krets bevaring, om enn for et tidsvindu timer. Vi brukt denne protokollen for å utforske hendelser som skjer i de tidlige fasene av neurodegeneration, overvåking akutt svaret på T30 program.
Til tross for stor mengde litteratur om bruker hjernen skiver undersøke molekylær trasé underforstått i neuronal skade eller neurogenesis23,24, denne protokollen gir for første gang en mer umiddelbare og følsom lese ut sammenlignet til vanlig bruk av organotypic skiver. Men som er tilfellet for organotypic hjernen deler, kan denne akutt skive prosedyren også adoptert for flere formål, for eksempel evaluering av neuroprotective eller nevrotoksiske molekyler, oppdagelsen av primære molekylær omveltningene i en bestemt prosess, Immunohistochemical analyse og farmakologiske analyser for sentralnervesystemet relatert patologi.

<table>
	<tbody>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S7653</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P9333</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S5761</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Magnesium sulphate heptahydrate (MgSO4 (7H2O))</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>63138</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5655</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes salt</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H7006</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes acid</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H3375</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>G7528</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Calcium chloride dehydrate</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>223506</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>T30 peptide</td>
			<td>Genosphere Biotechnologies, France</td>
			<td></td>
			<td>AChE-derived peptide tested</td>
		</tr>
		<tr>
			<td>Surgical dissecting kit</td>
			<td>World Precision Instruments, USA</td>
			<td>Item #: MOUSEKIT</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Surgical blades</td>
			<td>Swann-Morton, UK</td>
			<td>BS 2982</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher Scientific, USA</td>
			<td>11566873</td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td></td>
			<td></td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica, Germany</td>
			<td>VT1000 S</td>
			<td>Slicing</td>
		</tr>
		<tr>
			<td>Brushes</td>
			<td></td>
			<td></td>
			<td>Tissue handling</td>
		</tr>
		<tr>
			<td>Oxygen canister</td>
			<td></td>
			<td></td>
			<td>Sectioning and incubation phase</td>
		</tr>
		<tr>
			<td>1x Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific, USA</td>
			<td>BP2438-4</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Phosphatase inhibitors</td>
			<td>Fisher Scientific, USA</td>
			<td>1284-1650</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche complete PIC, USA</td>
			<td>4693116001</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Pestles</td>
			<td>Starlab, UK</td>
			<td>I1415-5390</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660 nm Protein Assay</td>
			<td>Thermo Scientific, USA</td>
			<td>22660</td>
			<td>Protein concentration</td>
		</tr>
	</tbody>
</table>

an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices

Til tross for mange studier som forsøker å utvikle pålitelige dyremodeller som reflekterer primært behandler underliggende neurodegeneration, har svært få blitt allment akseptert. Her foreslår vi en ny prosedyre tilpasset fra velkjente ex vivo hjernen stykke teknikken, som tilbyr en nærmere i vivo-som scenariet enn i vitro preparater, for å undersøke tidlige hendelser utløser celle degenerasjon, som observert i Alzheimers sykdom (AD). Denne varianten består av enkle og å reprodusere skritt, som aktiverer bevaring av den anatomiske cytoarchitecture valgte hjernen regionen og lokale funksjonaliteten i en fysiologisk miljøet. Ulike anatomiske områder kan fås fra samme hjernen, gir muligheten til å utføre flere eksperimenter med behandlinger i spørsmålet i en område - dose- og tidsavhengige måte. Potensielle begrensninger som kan påvirke resultatene knyttet til denne metoden gjelder bevaring av vevet, dvs. vedlikehold av integriteten anatomiske slicing og inkubasjon trinnene og snittykkelsen, som kan påvirke biokjemiske og immunohistochemical analysen. Denne tilnærmingen kan anvendes for ulike formål, som utforsker molekylære mekanismer involvert i fysiologiske eller pathological betingelser, narkotikarelaterte screening eller dose-respons analyser. Til slutt kan denne protokollen også redusere antall dyr i atferdsmessige studier. Programmet rapporterte her er nylig beskrevet og testet for første gang på ex vivo rotte hjernen skiver som inneholder basale forebrain (BF), som er en av regionene cerebral hovedsakelig påvirket i Annonsen. Spesielt har det vist at administrasjon av et giftig peptid avledet fra C-terminus av acetylcholinesterase (verke) kan be en AD-lignende profil, utløser, langs antero-posterior akse BF, en differensiell uttrykk for proteiner er endret på Annonsen, som alpha7 nikotinsyre reseptoren (α7-nAChR), fosforylert Tau (p-Tau) og amyloid beta (Aβ).

Protokollen presenteres her angir at administrasjon av et giftig peptid, T30, modulerer på en områdeavhengige måte uttrykk for α7-nAChR, p-Tau og Aβ i BF inneholder inndelinger (figur 3A). Nikotinsyre reseptoren viser en betydelig økning i rostral-behandlet hemislice forhold til motparten kontroll (del 1, p = 0.0310) (figur 3B), mens middels skive ikke avslører endringer mellom to betingelser ( SKIVE 2, p...

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An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Vi presenterer en metode som gir ytterligere innsikt i tidlig hendelsene underliggende neurodegeneration og basert på etablerte ex-vivo hjernen teknikken, kombinerer fordelene i vivo og i vitro eksperimenter. Videre representerer en unik mulighet for direkte sammenligning av behandlet og ubehandlet i samme anatomiske plan.

En alternativ tilnærming til studere primære hendelser i Neurodegeneration bruker Ex Vivo rotte hjernen skiver

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

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An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

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