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

Dette arbeidet ble finansiert av NIH-tilskudd HD043680, MH106392, DA013137 og NS100624.

Alle dyreprotokoller ble gjennomgått og godkjent av Animal Care and Use Committee ved University of South Carolina (føderalt forsikringsnummer: D16-00028). Seks voksne menn F344/N rotte ble paret plassert i et kontrollert miljø under en 12/12 lys: mørk syklus med ad libitum tilgang til mat og vann. Alle dyrene ble tatt vare på ved hjelp av retningslinjer fastsatt av National Institutes of Health i Guide for care and use of Laboratory Animals.
1. Virusemballasje i 293 FT-celler
<ol><li>K...

<ol>
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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. 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I denne protokollen etablerte vi en EcoHIV-indusert HIV-infeksjonsmodell hos rotter. Spesielt beskrev vi en bilateral stereotaxisk injeksjon av EcoHIV i cortex som med hell induserte aktiv HIV-infeksjon i rottehjernen 7 dager etter injeksjon. Futhermore, vi viser at EcoHIV infeksjon hos rotter kan være et godt biologisk system for å studere viktige aspekter av HAND. Åtte uker etter EcoHIV-infeksjon viste rotter betydelige nevrokognitive svekkelser, som inkluderte endringene i tidsbehandling og synaptisk dysfunksjon ho...

Biologiske systemer har forbedret vår forståelse av HIV-1 assosierte nevrokognitive lidelser (HAND) og deres underliggende nevrale mekanismer2. Å bestemme hvilket biologisk system som passer best for en gitt studie, er ofte avhengig av spørsmålet om interesse2. Begrensningen i utvalget av vertsdyrmodeller utfordrer studier av HIV-1 sykdomsutvikling. For å undersøke HIV-1 viral replikasjon og patogenese opprettet Potash et al.3 en musemodell av aktiv HIV-1-infeksjon, og erstattet kodingsområdet for HIV-overflatekonvolutt glykoprotein, gp120, med økotropisk MLV gp80, noe som førte til vellykket viral replikasjon hos mus4. Etter hale vene injeksjoner i chimeric HIV (EcoHIV) mus, mange egenskaper ble observert som ligner de av HIV-1 seropositive individer (f.eks. infiserte lymfocytter og makrofager, rettet mot antivirale immunresponser, og betennelse3,5,6).
Selv om mus og rotter begge er medlemmer av Muridae, kan grunnleggende artsforskjeller påvirke deres egnethet for spesifikke eksperimentelle spørsmål7. Derfor vil utvidelsen av EcoHIV-infeksjonsmodellen til rotter (ofte brukt i studier av narkotikamisbruk og nevrokognitive lidelser) være fordelaktig i studiet av nevroHIV. For eksempel gjør deres større størrelse jugularkateterimplantasjon for selvadbemanningsprosedyrer for legemidler mer praktisk8. Selvadberettigede teknikker for narkotika hos rotter har blitt brukt til å evaluere motivasjon i HIV-19. Videre ble mange nevrokognitive / atferdsoppgaver opprinnelig designet for rotter10. Her rapporterer vi bruken av stereotaksiske injeksjoner av EcoHIV hos rotter for å utvide EcoHIV-infeksjonsmodellen og har råd til en nøkkelmulighet til å ta opp nye spørsmål relatert til neuroHIV og HAND.

<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

Det har blitt godt studert at EcoHIV-infisert musemodell er av betydelig nytte i å undersøke HIV-assosierte nevrologiske komplikasjoner. Etablering av EcoHIV-infisert rottemodell for studier av narkotikamisbruk og nevrokognitive lidelser, ville være gunstig i studiet av nevroHIV og HIV-1 assosierte nevrokognitive lidelser (HAND). I den nåværende studien demonstrerer vi vellykket opprettelse av en rottemodell av aktiv HIV-infeksjon ved hjelp av chimerisk HIV (EcoHIV). For det første ble lentiviral konstruksjon av EcoHIV pakket i dyrkede 293 FT-celler i 48 timer. Deretter ble det betingede mediet konsentrert og titert. Deretter utførte vi bilaterale stereotaksiske injeksjoner av EcoHIV-EGFP i F344 / N rotte hjernevev. En uke etter infeksjon ble EGFP fluorescenssignaler oppdaget i det infiserte hjernevevet, noe som indikerer at EcoHIV med hell induserer en aktiv HIV-infeksjon hos rotter. I tillegg ble immunostaining for den mikrogliale cellemarkøren, Iba1, utført. Resultatene indikerte at mikroglia var den dominerende celletypen som inneholdt EcoHIV. Videre viste EcoHIV-rotter endringer i tidsbehandling, en potensiell underliggende nevrobehavioral mekanisme for HAND samt synaptisk dysfunksjon åtte uker etter infeksjon. Samlet utvider den nåværende studien EcoHIV-modellen for HIV-1-infeksjon til rotten som tilbyr et verdifullt biologisk system for å studere HIV-1 virusreservoarer i hjernen, samt HÅND og tilhørende komorbiditeter som narkotikamisbruk.

Det betingede mediet ble samlet inn fra lentivirus av EcoHIV-EGFP-infiserte 293FT-celler. Deretter ble den konsentrert og titerert, deretter stereotaxically injisert i hjernen (kortikale regionen) av F344 / N rotter. Syv dager etter injeksjon ble rotter ofret og bilder ble tatt fra koronal hjerneskiver fra bregma 5,64 mm til bregma -4,68 mm. I figur 1Aer det betydelige EcoHIV-EGFP-signaler i hele hjernen, spesielt i cortex og hippocampal dentate gyrus. Videre ga dobbeltmerking med Iba1 og Ec...

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

A Rat Model of EcoHIV Brain Infection

Her presenterer vi en protokoll for å etablere en ny rottemodell av aktiv HIV-infeksjon ved hjelp av chimerisk HIV (EcoHIV), som er avgjørende for å styrke vår forståelse av HIV-1-virusreservoarer i hjernen og tilby et system for å studere HIV-assosierte nevrokognitive lidelser og tilhørende komorbiditeter (dvs. narkotikamisbruk).

En rottemodell av EcoHIV hjerneinfeksjon

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

Ved hjelp av rotter kan denne protokollen brukes til å etablere en ny modell for aktiv HIV-infeksjon ved hjelp av chimerisk HIV. med rotter, kan etableringen av EcoHIV-infeksjonsmodellen for studier av narkotikamisbruk og nevrokognitive lidelser være gunstig for studiet av nevro HIV og HIV-1 assosierte nevrokognitive lidelser. Demonstrerende prosedyre vil være Hailong Li, en forskningsmedarbeider fra laboratoriet mitt.For pakking av viruset i 293 T-celler, frø fem ganger 10 til femte 293 T-celler per milliliter DMEM supplert med 10% FBS i hver av de to gelatinbelagte 75 kvadratcentimeter kolber og inkubere cellene ved 37 grader Celsius til kulturene når 50% samløp. På dagen for transfeksjonen fortynner du 22,5 mikroliter transfeksjonsreagens i 750 mikroliter medium per kolbe i et 1,5 milliliter mikrosentermikrosenterrør og virveler løsningen i tre sekunder. Fortynn 20 mikrogram av det chimeriske EcoHIV plasmid-DNA-et i 750 mikroliter medium i et andre mikrocentrifugerør på 1,5 milliliter per kolbe med grundig blanding.Kombiner det fortynnede DNA-et med det fortynnede transfeksjonsreagenset med skånsom blanding og inkuber den resulterende løsningen i 15 minutter ved romtemperatur. På slutten av inkubasjonen legger du til hver virusfjæring til 10 milliliter førkrigs DMEM-medium og legger til hver virussupplert løsning i en kolbe på 293 T-celler. Etter to dager med cellekultur, kan du samle supernatanten fra kolber i et enkelt 50 milliliter konisk rør for sentrifugering og overføre den avklarte supernatanten til et nytt 50 milliliterrør.Tilsett åtte milliliter Lenti-X konsentrator til den klargjorte supernatanten og bland med mild inversjon. Etter en to-dagers inkubasjon ved fire grader Celsius, sentrifuger blandingen og fjern forsiktig supernatanten, og resuspender deretter pellet forsiktig med 100 mikroliter 100 millimolar PBS og bruk et P24 ELISA-sett for å titere viruskonsentrasjonen. For EcoHIV-EGFP injeksjon, etter å ha bekreftet mangel på respons på pedalrefleks, barber håret fra hjerneregionen og steriliser den eksponerte huden to ganger med 70% etanol og en klorhexidinbasert skrubb.Sikre rotten i en utsatt posisjon i et stereotaxisk apparat og gjør et fem til seks centimeter snitt gjennom huden langs hodebunnen midtlinjen. Merk en boreposisjon på 0,8 millimeter lateral og en 1,2 millimeter rostral til bregma og bor et hull med en diameter på 0,4 millimeter ved hver skalleposisjon. Legg 1,04 ganger 10 til den sjette transduksjonsenheten per milliliter titert EcoHIV Lentivirus-oppløsning i en 10 mikroliter injeksjonssprøyte og fest sprøyten til det stereotaktiske apparatet.Flytt nålen nær overflaten av ett borehull og sett nålen 2,5 milliliter inn i hullet. Tilsett en mikroliter virusløsning med en hastighet på 0,2 mikroliter virus per minutt. Når alt viruset er injisert, la nålen inne i injeksjonsområdet i fem minutter før du sakte trekker nålen tilbake til den er utenfor rotteskallen.Lukk hudsnittet med en 4-0 silketråd sutur og steriliser løsningen med 70% etanol, og plasser deretter rotten i et gjenopprettingskammer med en varmepute med overvåking til heftelse. En til åtte uker etter viral infusjon, etter å ha bekreftet mangel på respons på pedalrefleks, fest rotten i en liggende stilling inne i en avtrekkshette og brenn huden langs thoracic midline. Klipp membranen for å åpne thoracic hulrommet og sett inn en 20 gauge med 25 millimeter nål i venstre ventrikel.Åpne umiddelbart riktig atrium med saks og perfuse 50 milliliter forhåndskjølt 100 millimolar PBS med en strømningshastighet på fem milliliter per minutt. Når hele PBS er levert, parfymerer du med 100 milliliter kald 4% paraformaldehyd. Når alt fikseringsmiddelet er levert, fjern hjernen fra skallen og fest vevet over natten i frisk 4% paraformaldehyd.Neste morgen, plasser prøven i 40 milliliter 30% sukrose i 100 millimolar PBS i et 50 milliliterrør i omtrent tre dager til hjernen legger seg til bunnen av røret før snap fryser hjernevevet i metyl butanol i to minutter ved minus 80 grader Celsius. Etter frysing, bruk en minus 20 grader Celsius kryostat og en fin pensel for å skaffe 50 mikron tykke koronal seksjoner. Når alle seksjonene er oppnådd, dekk prøvene med 300 mikroliter anti-fade medium og monter dem med 22 50 milliliter deksler.Når mediet har tørket, bilde seksjonene ved konfikal mikroskopi. Syv dager etter Lentivirus injeksjon, rotte coronal hjerne skiver bilder avslører en betydelig tilstedeværelse av EcoHIV-EGFP signaler gjennom hjernevevet, spesielt innenfor cortex og hippocampal dentate gyrus. Dobbel merking med og Iba1- og EcoHIV-EGFP-sonder gir sterke bevis på at mikroglia er den dominerende celletypen som skjuler EcoHIV-uttrykket i hjernen.Åtte uker etter infeksjon viser EcoHIV injiserte dyr en relativ ufølsomhet overfor manipulering av interstimulusintervall som det fremgår av en relativt flatere intervallfunksjon sammenlignet med saltvannskontroller. I tillegg viser EcoHIV injiserte rotter dype endringer i deres dendritiske ryggmorfologi som det fremgår av en økt relativ frekvens av kortere dendrittiske spines med økte hode- og nakkediametre i forhold til kontrolldyr. Konsentrasjonen og titreringen av tilstandsmediet før stereotaxisk injeksjon er avgjørende for å sikre konsistente og replikerbare resultater på tvers av eksperimenter.Denne spesifikke regionale stereotoksiske injeksjonen av EcoHIV gir en effektiv HIV-infeksjon i hjernen og produserer tidsmessige behandlingsunderskudd hos rotter. Utnyttelsen av stereotaksiske HIV-injeksjoner hos rotter for å utvide EcoHIV-infeksjonsmodellen gir en nøkkelmulighet til å ta opp nye spørsmål knyttet til nevro HIV for hånden.

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.

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.

Stereotaxic Kirurgi for Excitotoxic lesjon i spesifikke hjerneområder i 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.

En rotte modell av Mild Intrauterine Hypoperfusion med 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.

Bedømme i Vivo blod - hjerne barrieren avbrudd i en rotte modell av iskemiske hjerneslag

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).

En rotte modell av sentral Fatigue bruker en modifisert flere plattform-metoden

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.

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, ...

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.

En benchtop tilnærming til plasseringen spesifikke blod hjernebarriere åpning ved hjelp av fokusert ultralyd i en rotte modell

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.

En hydrofob vevsryddingsmetode for rottehjernevev

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.

Induksjon og vurdering av Levodopa-indusert dyskinesi i en rottemodell av Parkinsons sykdom

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

Validering av skaderespons og atferdsvurderinger i en mTBI rottemodell

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.

Forfatter Spotlight: Fremme forskning på traumatisk hjerneskade - En lukket hodemodell for nøyaktig replikasjon og rask vurdering 

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.

Rottemodell av mildt traumatisk skade med lukket hode og validering

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