Name: a rat model of ecohiv brain infection
Uploaded: 2021-01-21T14:30:00
Description: Watch this Scientific Journal Video about A Rat Model of EcoHIV Brain Infection at JoVE.com

Dit werk werd gefinancierd door NIH-subsidies HD043680, MH106392, DA013137 en NS100624.

Alle dierprotocollen werden herzien en goedgekeurd door de Animal Care and Use Committee van de Universiteit van South Carolina (federaal betrouwbaarheidsnummer: D16-00028). Zes volwassen mannelijke F344/N rat werd gekoppeld in een gecontroleerde omgeving onder een 12/12 licht: donkere cyclus met ad libitum toegang tot voedsel en water. Alle dieren werden verzorgd aan de hand van richtlijnen die zijn vastgesteld door de National Institutes of Health in de Guide for the Care and Use of Laboratory Animals.
<p class="jo...

<ol>
	<li>Illenberger, J. 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=HIV+Infection+and+Neurocognitive+Disorders+in+the+Context+of+Chronic+Drug+Abuse:+Evidence+for+Divergent+Findings+Dependent+upon+Prior+Drug+History.">HIV Infection and Neurocognitive Disorders in the Context of Chronic Drug Abuse: Evidence for Divergent Findings Dependent upon Prior Drug History.</a> Journal of Neuroimmune Pharmacology. 15 (4), 715-728 (2020).</li><li>Joseph, S. B., Swanstrom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+HIV-1+entry+phenotypes+as+a+guide+to+changing+target+cells.">The evolution of HIV-1 entry phenotypes as a guide to changing target cells.</a> Journal of Leukocyte Biology. 103 (3), 421-431 (2018).</li><li>Potash, M. 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+mouse+model+for+study+of+systemic+HIV-1+infection,+antiviral+immune+responses,+and+neuroinvasiveness.">A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness.</a> Proceedings of the National Academy of Sciences U S A. 102 (10), 3760-3765 (2005).</li><li>Albritton, L. M., Tseng, L., Scadden, D., Cunningham, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+putative+murine+ecotropic+retrovirus+receptor+gene+encodes+a+multiple+membrane-spanning+protein+and+confers+susceptibility+to+virus+infection.">A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection.</a> Cell. 57, 659-666 (1989).</li><li>Geraghty, P., Hadas, E., Kim, B. H., Dabo, A. J., Volsky, D. J., Foronjy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+infection+model+of+chronic+obstructive+pulmonary+disease+in+mice.">HIV infection model of chronic obstructive pulmonary disease in mice.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 312 (4), 500-509 (2017).</li><li>Gu, C. 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=EcoHIV+infection+of+mice+establishes+latent+viral+reservoirs+in+T+cells+and+active+viral+reservoirs+in+macrophages+that+are+sufficient+for+induction+of+neurocognitive+impairment.">EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment.</a> PLoS Pathogens. 14 (6), 1007061 (2018).</li><li>Ellenbroek, B., Youn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+in+neuroscience+research:+is+it+a+rat+race.">Rodent models in neuroscience research: is it a rat race.</a> Disease Models, Mechanisms. 9 (10), 1079-1087 (2016).</li><li>Feduccia, A. A., Duvauchelle, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+apparatus+and+method+for+drug+reinforcement.">Novel apparatus and method for drug reinforcement.</a> Journal of Visualized Experiments. (42), e1998 (2010).</li><li>Bertrand, S. J., Mactutus, C. F., Harrod, S. B., Moran, L. M., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+proteins+dysregulate+motivational+processes+and+dopamine+circuitry.">HIV-1 proteins dysregulate motivational processes and dopamine circuitry.</a> Scientific Reports. 8 (1), 7869 (2018).</li><li>McGaughy, J., Sarter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+vigilance+in+rats:+task+validation+and+effects+of+age,+amphetamine,+and+benzodiazepine+receptor+ligands.">Behavioral vigilance in rats: task validation and effects of age, amphetamine, and benzodiazepine receptor ligands.</a> Psychopharmacology. 117 (3), 340-357 (1995).</li><li>Li, H., Aksenova, M., Bertrand, S., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+filamentous+actin+(F-actin)+puncta+in+rat+cortical+neurons.">Quantification of filamentous actin (F-actin) puncta in rat cortical neurons.</a> Journal of Visualized Experiments. (108), (2016).</li><li>McLaurin, K. A., Li, H., Booze, R. M., Mactutus, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Timing:+NeuroHIV+Progression+in+the+Post-cART+Era.">Disruption of Timing: NeuroHIV Progression in the Post-cART Era.</a> Scientific Reports. 9 (1), 827 (2019).</li><li>McLaurin, K. A., Moran, L. M., Li, H., Booze, R. M., Mactutus, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Power+of+Interstimulus+Interval+for+the+Assessment+of+Temporal+Processing+in+Rodents.">The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents.</a> Journal of Visualized Experiments. (146), e58659 (2019).</li><li>Li, H., McLaurin, K. A., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ballistic+Labeling+of+Pyramidal+Neurons+in+Brain+Slices+and+in+Primary+Cell+Culture.">Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture.</a> Journal of Visualized Experiments. (158), (2020).</li><li>Ko, 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=Macrophages+but+not+Astrocytes+Harbor+HIV+DNA+in+the+Brains+of+HIV-1-Infected+Aviremic+Individuals+on+Suppressive+Antiretroviral+Therapy.">Macrophages but not Astrocytes Harbor HIV DNA in the Brains of HIV-1-Infected Aviremic Individuals on Suppressive Antiretroviral Therapy.</a> Journal of Neuroimmune Pharmacology. 14 (1), 110-119 (2019).</li><li>Sopper, 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=The+effect+of+simian+immunodeficiency+virus+infection+in+vitro+and+in+vivo+on+the+cytokine+production+of+isolated+microglia+and+peripheral+macrophages+from+rhesus+monkey.">The effect of simian immunodeficiency virus infection in vitro and in vivo on the cytokine production of isolated microglia and peripheral macrophages from rhesus monkey.</a> Virology. 220 (2), 320-329 (1996).</li><li>Llewellyn, G. N., Alvarez-Carbonell, D., Chateau, M., Karn, J., Cannon, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+infection+of+microglial+cells+in+a+reconstituted+humanized+mouse+model+and+identification+of+compounds+that+selectively+reverse+HIV+latency.">HIV-1 infection of microglial cells in a reconstituted humanized mouse model and identification of compounds that selectively reverse HIV latency.</a> Journal of NeuroVirology. 24 (2), 192-203 (2018).</li></ol>

In dit protocol hebben we een EcoHIV-geïnduceerd HIV-infectiemodel bij ratten vastgesteld. In het bijzonder beschreven we een bilaterale stereotaxic injectie van EcoHIV in de cortex die met succes een actieve HIV-infectie in de rattenhersenen veroorzaakte 7 dagen na de injectie. Bovendien tonen we aan dat EcoHIV-infectie bij ratten een goed biologisch systeem kan zijn om belangrijke aspecten van HAND te bestuderen. Acht weken na de EcoHIV-infectie vertoonden ratten significante neurocognitieve stoornissen, waaronder de ...

Biologische systemen hebben ons begrip van hiv-1 geassocieerde neurocognitieve aandoeningen (HAND) en hun onderliggende neurale mechanismen verbeterd2. Het bepalen welk biologisch systeem het meest geschikt is voor een bepaalde studie is vaak afhankelijk van de kwestie van belang2. De beperking van het scala aan modellen van gastdieren daagt studies uit naar de ontwikkeling van hiv-1-ziekten. Om hiv-1 virale replicatie en pathogenese te onderzoeken, creëerden Potash et al.3 een muismodel van actieve HIV-1-infectie, waarbij het coderingsgebied van HIV-oppervlakte-envelopglycoproteïne, gp120, werd vervangen door ecotropische MLV gp80, wat leidde tot succesvolle virale replicatie bij muizen4. Na staartaderinjecties bij chimerische HIV (EcoHIV) muizen werden veel kenmerken waargenomen die lijken op die van HIV-1 seropositieve personen (bijv. geïnfecteerde lymfocyten en macrofagen, gericht op antivirale immuunresponsen, en ontsteking3,5,6).
Hoewel muizen en ratten beide lid zijn van de Muridae, kunnen fundamentele soortenverschillen hun geschiktheid voor specifieke experimentele vragenbeïnvloeden 7. Daarom zou de uitbreiding van het EcoHIV-infectiemodel tot ratten (vaak gebruikt in studies naar drugsmisbruik en neurocognitieve aandoeningen) voordelig zijn in de studie van neuroHIV. Hun grotere formaat maakt bijvoorbeeld de implantatie van de halskatheter voor zelftoedieningsprocedures van geneesmiddelen praktischer8. Zelftoedieningstechnieken voor geneesmiddelen bij ratten zijn gebruiktom de motivatie bij HIV-19 te evalueren . Bovendien werden veel neurocognitieve / gedragstaken aanvankelijk ontworpen voor ratten10. Hier rapporteren we het gebruik van stereotaxic injecties van EcoHIV bij ratten om het EcoHIV-infectiemodel uit te breiden en een belangrijke kans te bieden om nieuwe vragen met betrekking tot neuroHIV en HAND te beantwoorden.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293FT cells</td>
			<td>ThermoFisher Scientific</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Cellgro</td>
			<td>30004CI</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Corning BioCoatGelatin 75cm&#178; Rectangular Canted Neck Cell Culture Flask with Vented Cap</td>
			<td>Life Technologies</td>
			<td>354488</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning DMEM with L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate</td>
			<td>Life Technologies</td>
			<td>10013CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>637-137</td>
			<td></td>
		</tr>
		<tr>
			<td>drill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14097</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes</td>
			<td>Life Technologies</td>
			<td>22600028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethicon Vicryl Plus Antibacterial, 4-0 Polyglactin 910 Suture, 27in. FS-2</td>
			<td>Med Vet International</td>
			<td>VCP422H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Lipofectamine 3000 Transfection Reagent</td>
			<td>Life Technologies</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Forceps</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>World Precision Instruments</td>
			<td>500216</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus-Associated p24 ELISA Kit</td>
			<td>Cell Biolabs, inc.</td>
			<td>VPK-107-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Concentrator</td>
			<td>Takara</td>
			<td>PT4421-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Fisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotaxic apparatus</td>
			<td>Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat model of ecohiv brain infection

Het is goed bestudeerd dat het ecoHIV geïnfecteerde muismodel van aanzienlijk nut is bij het onderzoeken van hiv-geassocieerde neurologische complicaties. De vaststelling van het ecoHIV-geïnfecteerde rattenmodel voor studies naar drugsmisbruik en neurocognitieve aandoeningen zou gunstig zijn bij de studie van neuroHIV- en HIV-1-geassocieerde neurocognitieve aandoeningen (HAND). In deze studie tonen we de succesvolle creatie van een rattenmodel van actieve hiv-infectie met chimeric HIV (EcoHIV). Ten eerste werd de lentivirale constructie van EcoHIV gedurende 48 uur verpakt in gekweekte cellen van 293 FT. Vervolgens werd het voorwaardelijke medium geconcentreerd en getiterd. Vervolgens voerden we bilaterale stereotaxic injecties uit van de EcoHIV-EGFP in F344/N rattenhersenweefsel. Een week na infectie werden EGFP-fluorescentiesignalen gedetecteerd in het geïnfecteerde hersenweefsel, wat aangeeft dat EcoHIV met succes een actieve HIV-infectie bij ratten induceert. Bovendien werd immunostaining uitgevoerd voor de microgliale celmarker, Iba1. De resultaten gaven aan dat microglia het overheersende celtype was dat EcoHIV herbergde. Bovendien vertoonden EcoHIV-ratten veranderingen in de temporele verwerking, een potentieel onderliggend neurobehavioral mechanisme van HAND en synaptische disfunctie acht weken na infectie. Gezamenlijk breidt de huidige studie het EcoHIV-model van HIV-1-infectie uit naar de rat die een waardevol biologisch systeem biedt om hiv-1 virale reservoirs in de hersenen te bestuderen, evenals HAND en bijbehorende comorbiditeiten zoals drugsmisbruik.

Het geconditioneerde medium werd verzameld uit lentivirus van EcoHIV-EGFP geïnfecteerde 293FT cellen. Vervolgens werd het geconcentreerd en getiterd, waarna stereotaxisch werd geïnjecteerd in de hersenen (corticale regio) van F344 / N-ratten. Zeven dagen na de injectie werden ratten opgeofferd en werden beelden genomen van coronale hersenschijfjes variërend van bregma 5,64 mm tot bregma -4,68 mm. In figuur 1Azijn er significante EcoHIV-EGFP-signalen in de hersenen, vooral in de cortex en ...

Watch this Scientific Journal Video about A Rat Model of EcoHIV Brain Infection at JoVE.com

A Rat Model of EcoHIV Brain Infection

Hier presenteren we een protocol om een nieuw rattenmodel van actieve hiv-infectie met chimerische hiv (EcoHIV) vast te stellen, wat van cruciaal belang is voor het verbeteren van ons begrip van hiv-1 virale reservoirs in de hersenen en het aanbieden van een systeem om hiv-geassocieerde neurocognitieve aandoeningen en bijbehorende comorbiditeiten (d.w.z. drugsmisbruik) te bestuderen.

Een rattenmodel van EcoHIV-herseninfectie

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Met behulp van ratten kan dit protocol worden gebruikt om een nieuw model van actieve hiv-infectie met chimerische hiv vast te stellen. met ratten kan de vaststelling van het EcoHIV-infectiemodel voor studies naar drugsmisbruik en neurocognitieve aandoeningen gunstig zijn voor de studie van neuro-hiv en hiv-1 geassocieerde neurocognitieve aandoeningen. De demonstratieprocedure is Hailong Li, een onderzoeksmedewerker van mijn laboratorium.Voor het verpakken van het virus in 293 T-cellen, zaad vijf keer 10 tot de vijfde 293 T-cellen per milliliter DMEM aangevuld met 10% FBS in elk van de twee met gelatine bedekte kolven van 75 vierkante centimeter en incubeer de cellen bij 37 graden Celsius totdat de culturen 50% samenvloeiing bereiken. Verdun op de dag van de transfectie 22,5 microliter transfectiereagens in 750 microliter medium per kolf in een microcentrifugebuis van 1,5 milliliter en draai de oplossing gedurende drie seconden. Verdun 20 microgram van het chimerische EcoHIV plasmide DNA in 750 microliter medium in een tweede microcentrifugebuis van 1,5 milliliter per kolf met grondige menging.Combineer het verdunde DNA met het verdunde transfectiereagens met een zachte menging en incubeer de resulterende oplossing gedurende 15 minuten bij kamertemperatuur. Voeg aan het einde van de incubatie elke virussuspensie toe aan 10 milliliter dmem-medium vóórwarm en voeg elke virusoplossing toe aan één kolf van 293 T-cellen. Na twee dagen celkweek, pool het supernatant uit de kolven in een enkele 50 milliliter conische buis voor centrifugeren en breng het geklaarde supernatant over naar een nieuwe buis van 50 milliliter.Voeg acht milliliter Lenti-X Concentrator toe aan het geklaarde supernatant en meng met zachte inversie. Na een incubatie van twee dagen bij vier graden Celsius, centrifugeer het mengsel en verwijder voorzichtig het supernatant, resuspend de pellet vervolgens voorzichtig met 100 microliter van 100 millimolar PBS en gebruik een P24 ELISA-kit om de virusconcentratie te titeren. Voor EcoHIV-EGFP-injectie, na het bevestigen van een gebrek aan reactie op pedaalreflex, scheert u het haar uit het hersengebied en steriliseert u de blootgestelde huid twee keer met 70% ethanol en een scrub op basis van chloorhexidine.Zet de rat in een gevoelige positie in een stereotaxisch apparaat en maak een incisie van vijf tot zes centimeter door de huid langs de hoofdhuidmiddenlijn. Markeer één boorpositie op 0,8 millimeter zijdelings en één 1,2 millimeter rostral naar bregma en boor een gat met een diameter van 0,4 millimeter bij elke schedelpositie. Laad 1,04 maal 10 tot de zesde transductie-eenheden per milliliter getiterde EcoHIV Lentivirus-oplossing in een injectiespuit met 10 microliter en bevestig de spuit aan het stereotaxic-apparaat.Beweeg de naald dicht bij het oppervlak van één boorgat en steek de naald 2,5 milliliter in het gat. Infuseer één microliter virusoplossing met een snelheid van 0,2 microliter virus per minuut. Wanneer al het virus is geïnjecteerd, laat u de naald vijf minuten in het injectiegebied staan voordat u de naald langzaam terugtrekt totdat deze zich buiten de schedel van de rat bevindt.Sluit de huidincisie met een 4-0 zijden draad hechtdraad en steriliseer de oplossing met 70% ethanol en plaats de rat vervolgens in een herstelkamer met een verwarmingskussen met bewaking tot ligfiets. Een tot acht weken na virale infusie, na het bevestigen van een gebrek aan reactie op pedaalreflex, fixeer de rat in een liggende positie in een zuurkast en inciseer de huid langs de thoracale middellijn. Snijd het membraan om de borstholte te openen en steek een naald van 20 bij 25 millimeter in de linkerventrikel.Open onmiddellijk het rechter atrium met een schaar en doordrenkt 50 milliliter voorgekoelde 100 millimolar PBS met een debiet van vijf milliliter per minuut. Wanneer alle PBS is geleverd, perfuseren met 100 milliliter koude 4%paraformaldehyde. Wanneer al het fixatief is geleverd, verwijdert u de hersenen uit de schedel en bevestigt u het weefsel 's nachts in verse 4%paraformaldehyde.Plaats het monster de volgende ochtend in 40 milliliter 30% sacharose in 100 millimol PBS in een buis van 50 milliliter gedurende ongeveer drie dagen totdat de hersenen zich op de bodem van de buis nestelen voordat het hersenweefsel twee minuten in methyl butanol wordt bevroren bij min 80 graden Celsius. Gebruik na het invriezen een minus 20 graden Celsius cryostaat en een fijne kwast om 50 micron dikke coronale secties te verkrijgen. Wanneer alle secties zijn verkregen, dek de monsters af met 300 microliter anti-fade medium en monteer ze met 22 coverslips van 50 milliliter.Wanneer het medium is opgedroogd, beeldt u de secties in door confocale microscopie. Zeven dagen na lentivirus injectie, rat coronale hersenen slice beelden onthullen een significante aanwezigheid van EcoHIV-EGFP signalen in het hersenweefsel, vooral in de cortex en de hippocampale dentate gyrus. Dubbele etikettering met en Iba1- en EcoHIV-EGFP-sondes leveren sterk bewijs dat microglia het overheersende celtype zijn dat EcoHIV-expressie in de hersenen herbergt.Acht weken na infectie vertonen ecoHIV geïnjecteerde dieren een relatieve ongevoeligheid voor de manipulatie van het interstimulusinterval, zoals blijkt uit een relatief vlakkere intervalfunctie in vergelijking met zoutoplossingscontroles. Bovendien vertonen ecoHIV geïnjecteerde ratten diepgaande veranderingen in hun dendritische wervelkolommorfologie, zoals blijkt uit een verhoogde relatieve frequentie van kortere dendritische stekels met verhoogde hoofd- en nekdiameters ten opzichte van controledieren. De concentratie en titratie van het conditiemedium vóór stereotaxic injectie is van cruciaal belang om de consistente en reproduceerbare resultaten in experimenten te garanderen.Deze specifieke regionale stereotaxic injectie van EcoHIV zorgt voor een efficiënte HIV-infectie in de hersenen en produceert tijdelijke verwerkingstekorten bij ratten. Het gebruik van stereotaxic HIV-injecties bij ratten om het EcoHIV-infectiemodel uit te breiden, biedt een belangrijke kans om nieuwe vragen met betrekking tot neuro-hiv bij de hand te beantwoorden.

Research

JoVE Journal

Neuroscience

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Gene Levering aan Postnatale Rat Brain door niet-ventriculaire Plasmide-injectie en elektroporatie

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Stereotaxische Chirurgie voor excitotoxische laesie van specifieke hersengebieden in de Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Een Model van de Rat van milde uteriene hypoperfusie met Microcoil stenose

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

In Vivo eenbeoordeling van de verstoring van de bloed - hersen barrière in een Rat Model van ischemische beroerte

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

Een Rat Model van Centrale vermoeidheid met een gewijzigde methode met verscheidene Platform

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

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

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

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

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Een alternatieve benadering te bestuderen van primaire gebeurtenissen in neurodegeneratie Ex Vivo Rat hersenen segmenten gebruiken

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

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Een benchtop benadering van de locatie specifieke bloed hersenbarrière opening met behulp van gerichte echografie in een rat model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Een hydrofobe weefselopruimingsmethode voor rattenhersenweefsel

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson&#039;s Disease

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Inductie en beoordeling van Levodopa-geïnduceerde dyskinesieën in een rattenmodel van de ziekte van Parkinson

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Validatie van letselrespons en gedragsbeoordelingen in een mTBI-rattenmodel

Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

Auteur in de schijnwerpers: Onderzoek naar traumatisch hersenletsel bevorderen - een gesloten model voor nauwkeurige replicatie en snelle beoordeling 

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

Rattenmodel van licht traumatisch letsel met gesloten hoofd en de validatie ervan

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful ...