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

Detta arbete finansierades av NIH-bidrag HD043680, MH106392, DA013137 och NS100624.

Alla djurprotokoll granskades och godkändes av Animal Care and Use Committee vid University of South Carolina (federalt försäkringsnummer: D16-00028). Sex vuxna manliga F344/N råtta parades i en kontrollerad miljö under en 12/12 ljus: mörk cykel med ad libitum tillgång till mat och vatten. Alla djur togs om hand med hjälp av riktlinjer som fastställts av National Institutes of Health i guiden för vård och användning av laboratoriedjur.
1. Virusförpackning i 293 FT-celler
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<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 detta protokoll etablerade vi en EcoHIV-inducerad HIV-infektionsmodell hos råttor. Specifikt beskrev vi en bilateral stereotaxic injektion av EcoHIV i cortex som framgångsrikt inducerad aktiv HIV-infektion i råtta hjärnan 7 dagar efter injektion. Dessutom visar vi att EcoHIV-infektion hos råttor kan vara ett bra biologiskt system för att studera viktiga aspekter av HAND. Åtta veckor efter EcoHIV infektion uppvisade råttor betydande neurokognitiv försämringar, som inkluderade ändringarna i tidsbehandling och ...

Biologiska system har förbättrat vår förståelse av HIV-1 associerade neurokognitiva störningar (HAND) och deras underliggande neurala mekanismer2. Att avgöra vilket biologiskt system som är lämpligast för en viss studie beror ofta på frågan om intresse2. Begränsningen av utbudet av värddjursmodeller utmanar studier av hiv-1-sjukdomsutveckling. För att undersöka HIV-1 viral replikering och patogenes skapade Potash et al.3 en musmodell av aktiv HIV-1-infektion, som ersatte kodningsregionen för HIV-ytkuvertglykoprotein, gp120, med ekotropisk MLV gp80, vilket ledde till framgångsrik viral replikering hos möss4. Efter svansven injektioner i chimeric HIV (EcoHIV) möss observerades många egenskaper som liknar hiv-1 seropositiva individer (t.ex. infekterade lymfocyter och makrofager, riktade mot antivirala immunsvar och inflammation3,5,6).
Även om möss och råttor båda är medlemmar i Muridae, kan fundamentala artskillnader påverka deras lämplighet för specifika experimentella frågor7. Därför skulle förlängningen av EcoHIV-infektionsmodellen till råttor (som vanligtvis används i studier av narkotikamissbruk och neurokognitiva störningar) vara fördelaktig i studien av neuroHIV. Till exempel gör deras större storlek halsterkateterimplantation för drog självadministration förfaranden mer praktiska8. Drog självadministration tekniker hos råttor har använts för att utvärderamotivation i HIV-19 . Dessutom var många neurokognitiva / beteendemässiga uppgifter ursprungligen utformade för råttor10. Här rapporterar vi användningen av stereotaxiska injektioner av EcoHIV hos råttor för att utöka EcoHIV-infektionsmodellen och ge en viktig möjlighet att ta itu med nya frågor relaterade till neuroHIV och HAND.

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	<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 studerats väl att ecoHIV infekterade mus modell är av betydande nytta för att undersöka HIV associerade neurologiska komplikationer. Upprättandet av EcoHIV infekterad råtta modell för studier av narkotikamissbruk och neurokognitiva störningar, skulle vara fördelaktigt i studien av neuroHIV och HIV-1 associerade neurokognitiva störningar (HAND). I den aktuella studien visar vi att en råttmodell av aktiv HIV-infektion med hjälp av chimerisk HIV (EcoHIV) har skapats framgångsrikt. Först förpackades den lentivirala konstruktionen av EcoHIV i odlade 293 FT-celler i 48 timmar. Sedan koncentrerades det villkorliga mediet och titererades. Därefter utförde vi bilaterala stereotaxic injektioner av EcoHIV-EGFP i F344/N råtta hjärnvävnad. En vecka efter infektion upptäcktes EGFP fluorescenssignaler i den infekterade hjärnvävnaden, vilket indikerar att EcoHIV framgångsrikt inducerar en aktiv HIV-infektion hos råttor. Dessutom utfördes immunostaining för microglial cell markör, Iba1. Resultaten visade att mikroglia var den dominerande celltypen som hyser EcoHIV. Dessutom uppvisade EcoHIV råttor förändringar i tidsmässiga bearbetning, en potentiell underliggande neurobehavioral mekanism av HAND samt synaptisk dysfunktion åtta veckor efter infektion. Sammantaget utvidgar den nuvarande studien EcoHIV-modellen av HIV-1-infektion till att erbjuda ett värdefullt biologiskt system för att studera HIV-1-virusreservoarer i hjärnan samt HAND och tillhörande komorbiditeter som drogmissbruk.

Det konditionerade mediet samlades in från lentivirus av EcoHIV-EGFP infekterade 293FT celler. Därefter koncentrerades och titererades det, sedan stereotaxiskt injiceras i hjärnan (när regionen) av F344/N råttor. Sju dagar efter injektion offrades råttor och bilder togs från koronar hjärnskivor som sträcker sig från bregma 5,64 mm till bregma -4,68 mm. I figur 1Afinns det signifikanta EcoHIV-EGFP-signaler i hela hjärnan, särskilt i cortex och hippocampal dentate gyrus. Dessutom g...

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

A Rat Model of EcoHIV Brain Infection

Här presenterar vi ett protokoll för att etablera en ny råttmodell av aktiv HIV-infektion med hjälp av chimerisk HIV (EcoHIV), vilket är avgörande för att öka vår förståelse av HIV-1-virusreservoarer i hjärnan och erbjuda ett system för att studera HIV-associerade neurokognitiva störningar och tillhörande komorbiditet (dvs. drogmissbruk).

En råttmodell av EcoHIV hjärninfektion

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

Med hjälp av råttor kan detta protokoll användas för att etablera en ny modell för aktiv HIV-infektion med hjälp av chimerisk HIV. med råttor kan inrättandet av EcoHIV-infektionsmodellen för studier av narkotikamissbruk och neurokognitiva störningar vara fördelaktigt för studier av neuro HIV och HIV-1 associerade neurokognitiva störningar. Hailong Li, en forskare från mitt laboratorium, kommer att demonstrera.För förpackning av viruset till 293 T-celler, frö fem gånger 10 till femte 293 T-celler per milliliter DMEM kompletterat med 10%FBS i var och en av de två gelatinbelagda 75 kvadratcentimeterkolvarna och inkubera cellerna vid 37 grader Celsius tills kulturerna når 50% beredskap. På transfektens dag späd 22,5 mikroliter transfection reagens i 750 mikroliter medium per kolv i ett 1,5 milliliter mikrocentrifugrör och virvellösningen i tre sekunder. Späd 20 mikrogram av det chimeriska EcoHIV-plasmid-DNA:t i 750 mikroliter medium på en sekund 1,5 milliliter mikrocentrifugrör per kolv med noggrann blandning.Kombinera det utspädda DNA:t med det utspädda transfectionreagenset med skonsam blandning och inkubera den resulterande lösningen i 15 minuter vid rumstemperatur. I slutet av inkubationen, tillsätt varje virusupphängning till 10 milliliter dmem-medium förewarm och tillsätt varje virus kompletterad lösning till en kolv med 293 T-celler. Efter två dagars cellkultur, slå samman supernaten från flaskorna i ett enda koniskt rör på 50 milliliter för centrifugering och överför den klarlagda supernaten till ett nytt 50 milliliterrör.Tillsätt åtta milliliter Lenti-X-koncentrator till den klarlagda supernaten och blanda med mild inversion. Efter en två dagars inkubation vid fyra grader Celsius, centrifug blanda och försiktigt ta bort supernatanten, sätt sedan försiktigt tillbaka pelleten med 100 mikroliter på 100 millimolar PBS och använd ett P24 ELISA-kit för att titer viruskoncentrationen. För EcoHIV-EGFP injektion, efter att ha bekräftat brist på svar på pedal reflex, raka håret från hjärnregionen och sterilisera den exponerade huden två gånger med 70% etanol och en klorhexidin-baserad skrubb.Säkra råttan i ett benägenläge i en stereotaxisk apparat och gör ett fem till sex centimeter snitt genom huden längs hårbottens mittlinje. Markera ett borrläge vid 0,8 millimeters sidoläge och en 1,2 millimeters rostral till knäform och borra ett hål med diametern 0,4 millimeter vid varje skalleposition. Ladda 1,04 gånger 10 till den sjätte transduktionsenheterna per milliliter titererad EcoHIV Lentivirus-lösning i en 10 mikroliterinsprutningsspruta och fäst sprutan på stereotaxiska apparaten.Flytta nålen nära ytan på ett borrhål och för in nålen 2,5 milliliter i hålet. Ingjuta en mikroliter viruslösning med en hastighet av 0,2 mikroliter virus per minut. När allt virus har injicerats, lämna nålen inne i injektionsområdet i fem minuter innan du långsamt drar tillbaka nålen tills den är utanför råttskalle.Stäng hudsnittet med en 4-0 silketråd sutur och sterilisera lösningen med 70% etanol, placera sedan råttan i en återhämtningskammare med en värmeplatta med övervakning tills den är avslappning. En till åtta veckor efter viral infusion, efter att ha bekräftat brist på svar på pedalreflex, fixera råttan i ett supin läge inuti en rökhuva och luta huden längs bröstmittlinjen. Skär membranet för att öppna brösthålan och sätt in en 20 gauge med 25 millimeters nål i vänster ventrikel.Öppna omedelbart rätt atrium med sax och granska 50 milliliter förkyld 100 millimolar PBS med en flödeshastighet på fem milliliter per minut. När all PBS har levererats, granska med 100 milliliter kall 4%paraformaldehyd. När alla fixativ har levererats, ta bort hjärnan från skallen och fixa vävnaden över natten i färsk 4% paraformaldehyd.Nästa morgon, placera provet i 40 milliliter 30% sackaros i 100 millimolar PBS i ett 50 milliliter rör i ungefär tre dagar tills hjärnan sätter sig till botten av röret innan snäpp fryser hjärnvävnaden i metyl butanol i två minuter vid minus 80 grader Celsius. Efter frysning, använd en minus 20 grader Celsius cryostat och en fin pensel för att förvärva 50 mikron tjocka koronarsektioner. När alla sektioner har erhållits, täck proverna med 300 mikroliter anti-blekmedel och montera dem med 22 50 milliliter täckglas.När mediet har torkat, avbilda sektionerna med konfokal mikroskopi. Sju dagar efter Lentivirus injektion, råtta koronal hjärnskiva bilder avslöjar en betydande närvaro av EcoHIV-EGFP signaler i hela hjärnvävnaden, särskilt inom cortex och hippocampal dentate gyrus. Dubbel märkning med och Iba1 och EcoHIV-EGFP sonder ger starka bevis för att mikroglia är den dominerande celltypen som hyser EcoHIV uttryck i hjärnan.Åtta veckor efter infektion uppvisar EcoHIV injicerade djur en relativ okänslighet för manipulering av interstimulus intervall som framgår av en relativt plattare intervall funktion jämfört med saltlösning kontroller. Dessutom uppvisar EcoHIV injicerade råttor djupgående förändringar i deras dendritiska ryggradsmorfologi, vilket framgår av en ökad relativ frekvens av kortare dendritiska ryggrader med ökade huvud- och halsdiametrar i förhållande till kontrolldjur. Koncentrationen och titrering av tillståndsmediet före stereotaxisk injektion är avgörande för att säkerställa konsekventa och reproducerbara resultat över experiment.Denna specifika regionala stereotaxiska injektion av EcoHIV ger en effektiv HIV-infektion i hjärnan och producerar temporala bearbetning underskott hos råttor. Användningen av stereotaxiska HIV-injektioner hos råttor för att utöka EcoHIV-infektionsmodellen ger en viktig möjlighet att ta itu med nya frågor relaterade till neuro HIV till hands.

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 Leverans till Postnatal råtthjärna av icke-ventrikulär Plasmid Injection och elektroporation

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.

Stereotaktisk kirurgi för excitotoxisk skada av specifika områden i hjärnan i vuxen råtta

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 råtta modell av Mild intrauterin Hypoperfusion med Microcoil stenos

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.

En In Vivo -bedömning av blod - hjärnbarriären störningar i en råtta modell av ischemisk Stroke

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 råtta modell av Central trötthet använder en modifierad metoden med flera plattform

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 metod att studera primära händelser i Neurodegeneration använder Ex Vivo råtta hjärnan skivor

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 bänktop till den platsspecifika blodhjärnbarriäröppningen med fokuserat ultraljud i en råttmodell

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 hydrofobisk vävnadsrensningsmetod för råtta hjärnvävnad

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.

Induktion och bedömning av Levodopa-inducerade dyskinesier i en råttmodell av Parkinsons sjukdom

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 och beteendebedömningar i en mTBI Rat-modell

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.

Författare i fokus: Främja forskning om traumatisk hjärnskada - en sluten modell för korrekt replikering och snabb bedömning 

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.

Råttmodell av lindrig traumatisk skada med stängt huvud och dess validering

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