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 arbejde blev finansieret af NIH tilskud HD043680, MH106392, DA013137, og NS100624.

Alle dyreprotokoller blev gennemgået og godkendt af Animal Care and Use Committee ved University of South Carolina (føderalt sikkerhedsnummer: D16-00028). Seks voksne mandlige F344/N rotte blev parret opstaldet i et kontrolleret miljø under en 12/12 lys: mørk cyklus med ad libitum adgang til mad og vand. Alle dyr blev passet ved hjælp af retningslinjer, der er fastlagt af National Institutes of Health i vejledningen for pleje og brug af forsøgsdyr.
1. Virusemballage i 293 FT-celler
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<ol>
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(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 protokol etablerede vi en EcoHIV-induceret HIV-infektionsmodel hos rotter. Specifikt beskrev vi en bilateral stereotaxisk injektion af EcoHIV i cortex, som med succes inducerede aktiv HIV-infektion i rottehjernen 7 dage efter injektionen. Derudover viser vi, at EcoHIV-infektion hos rotter kan være et godt biologisk system til at studere centrale aspekter af HÅND. Otte uger efter EcoHIV-infektion udviste rotter betydelige neurokognitive svækkelser, som omfattede ændringer i tidsmæssig behandling og synaptisk ...

Biologiske systemer har forbedret vores forståelse af HIV-1 associerede neurokognitive lidelser (HAND) og deres underliggende neurale mekanismer2. Det er ofte afhænger af spørgsmålet om interesse2, at det afgøres, hvilket biologisk system der er bedst egnet til en given undersøgelse . Begrænsningen af rækken af værtsdyrmodeller udfordrer undersøgelser af hiv-1-sygdomsudvikling. For at undersøge HIV-1 viral replikation og patogenese skabte Potash et al.3 en musemodel af aktiv HIV-1-infektion og erstattede kodningsområdet for HIV-overfladekonvolut glycoprotein, gp120, med ecotropisk MLV gp80, hvilket førte til vellykket viral replikation hos mus4. Efter hale vene injektioner i kimære HIV (EcoHIV) mus, mange egenskaber blev observeret ligner dem af HIV-1 seropositive individer (f.eks inficerede lymfocytter og makrofager, målrettet mod antivirale immunrespons, og inflammation3,5,6).
Selvom mus og rotter begge er medlemmer af Muridae, kan grundlæggende artsforskelle påvirke deres egnethed til specifikke eksperimentelle spørgsmål7. Derfor ville udvidelsen af EcoHIV-infektionsmodellen til rotter (almindeligvis anvendt i undersøgelser af stofmisbrug og neurokognitive lidelser) være fordelagtig i undersøgelsen af neuroHIV. For eksempel gør deres større størrelse jugularkateterimplantation til medicinsk selvadministrationsprocedurer mere praktisk8. Drug self-administration teknikker hos rotter er blevet udnyttet tilat evaluere motivation i HIV-19 . Desuden blev mange neurokognitive / adfærdsmæssige opgaver oprindeligt designet til rotter10. Her rapporterer vi udnyttelsen af stereotaxiske injektioner af EcoHIV hos rotter for at udvide EcoHIV-infektionsmodellen og give en vigtig mulighed for at løse nye spørgsmål relateret til neuroHIV og 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 er blevet godt undersøgt, at EcoHIV inficeret mus model er af betydelig nytte i at undersøge HIV associerede neurologiske komplikationer. Etablering af EcoHIV-inficeret rottemodel til undersøgelser af stofmisbrug og neurokognitive lidelser ville være gavnlig i undersøgelsen af neuroHIV og HIV-1 tilknyttede neurokognitive lidelser (HAND). I denne undersøgelse demonstrerer vi den vellykkede oprettelse af en rottemodel af aktiv hiv-infektion ved hjælp af kimær HIV (EcoHIV). For det første blev den lentivirale konstruktion af EcoHIV pakket i dyrkede 293 FT-celler i 48 timer. Derefter var det betingede medium koncentreret og titered. Dernæst udførte vi bilaterale stereotaxiske injektioner af EcoHIV-EGFP i F344/N rottehjernevæv. En uge efter infektion blev EGFP-fluorescenssignaler påvist i det inficerede hjernevæv, hvilket indikerer, at EcoHIV med succes fremkalder en aktiv HIV-infektion hos rotter. Derudover blev immunostaining til mikroglial cellemarkøren, Iba1, udført. Resultaterne viste, at microglia var den fremherskende celletype, der husede EcoHIV. Desuden udviste EcoHIV-rotter ændringer i tidsmæssig behandling, en potentiel underliggende neuroadfærdsmekanisme af HÅND samt synaptisk dysfunktion otte uger efter infektion. Kollektivt, denne undersøgelse udvider EcoHIV model af HIV-1 infektion til rotte tilbyder et værdifuldt biologisk system til at studere HIV-1 viral reservoirer i hjernen samt hånd og tilhørende comorbiditeter såsom stofmisbrug.

Det betingede medium blev indsamlet fra lentivirus af EcoHIV-EGFP inficerede 293FT celler. Dernæst var det koncentreret og titered, så stereotaxically injiceres i hjernen (kortikale region) af F344/N rotter. Syv dage efter injektionen blev rotter ofret, og der blev taget billeder fra koronar hjerneskiver fra bregma 5,64 mm til bregma -4,68 mm. I figur 1Aer der betydelige EcoHIV-EGFP-signaler i hele hjernen, især i cortex og hippocampal dentate gyrus. Desuden gav dobbeltmærkning med Iba1-...

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

A Rat Model of EcoHIV Brain Infection

Her præsenterer vi en protokol til at etablere en ny rottemodel for aktiv hiv-infektion ved hjælp af kimær HIV (EcoHIV), som er afgørende for at forbedre vores forståelse af HIV-1 virale reservoirer i hjernen og tilbyde et system til at studere HIV-associerede neurokognitive lidelser og tilknyttede comorbiditeter (dvs. stofmisbrug).

En rottemodel af EcoHIV-hjerneinfektion

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

Ved hjælp af rotter kan denne protokol bruges til at etablere en ny model for aktiv HIV-infektion ved hjælp af kimær HIV. med rotter kan etableringen af EcoHIV-infektionsmodellen for undersøgelser af stofmisbrug og neurokognitive lidelser være gavnlig for undersøgelsen af neuro HIV og HIV-1 tilknyttede neurokognitive lidelser. Demonstration procedure vil blive Hailong Li, en forskningsmedarbejder fra mit laboratorium.Til emballering af virus i 293 T-celler, frø fem gange 10 til den femte 293 T-celler pr milliliter DMEM suppleret med 10% FBS i hver af de to gelatine-belagt 75 kvadratcentimeter kolber og inkubere cellerne ved 37 grader Celsius, indtil kulturerne når 50% sammenløb. På dagen for transfektet fortyndes 22,5 mikroliter transfekturereagens i 750 mikroliter medium pr. kolbe i et 1,5 milliliter mikrocentrifugerør og vortex opløsningen i tre sekunder. Fortynd 20 mikrogram af det kimære EcoHIV plasmid DNA i 750 mikroliter medium i et andet 1,5 milliliter mikrocentrifugerør pr. kolbe med grundig blanding.Kombiner det fortyndede DNA med det fortyndede transfektreagens med blid blanding, og inkuber den resulterende opløsning i 15 minutter ved stuetemperatur. Ved afslutningen af inkubationen tilsættes hver virussuspension til 10 milliliter DMEM-medium før krigen, og hver virus suppleret opløsning til en kolbe på 293 T-celler. Efter to dages cellekultur samles supernatanten fra kolberne i et enkelt 50 milliliter konisk rør til centrifugering og overfører det afklarede supernatant til et nyt 50 milliliter rør.Tilsæt otte milliliter Lenti-X Concentrator til den afklarede supernatant og bland med blid inversion. Efter en to-dages inkubation ved fire grader Celsius, centrifuge blandingen og forsigtigt fjerne supernatant, derefter forsigtigt genbruge pellet med 100 mikroliter af 100 millimolar PBS og bruge en P24 ELISA kit til titer virus koncentrationen. For EcoHIV-EGFP injektion, efter at have bekræftet en manglende reaktion på pedal refleks, barbere håret fra hjernen regionen og sterilisere den udsatte hud to gange med 70% ethanol og en chlorhexidin-baserede krat.Fastgør rotten i en udsat position i et stereotaxisk apparat og lav et fem til seks centimeter snit gennem huden langs hovedbundens midterlinje. Marker en boreposition ved 0,8 millimeter lateral og en 1,2 millimeter rostral til bregma og bor et hul med en diameter på 0,4 millimeter ved hver kranieposition. 1,04 gange 10 indlæses i den sjette transduktionsenhed pr. milliliter titered EcoHIV Lentivirus-opløsning i en 10 mikroliter injektionssprøjte, og fastgør sprøjten til det stereotaxiske apparat.Flyt nålen tæt på overfladen af et borehul og sæt nålen 2,5 milliliter ind i hullet. Infuse en mikroliter virusopløsning med en hastighed på 0,2 mikroliter virus i minuttet. Når al virus er blevet injiceret, skal du lade nålen inde i injektionsområdet i fem minutter, før du langsomt trækker nålen tilbage, indtil den er uden for rottekranken.Luk hudens snit med en 4-0 silketråd sutur og steriliser opløsningen med 70% ethanol, og placer derefter rotten i et genopretningskammer med en varmepude med overvågning indtil recumbency. En til otte uger efter viral infusion, efter at have bekræftet en manglende reaktion på pedalrefleks, fastgør rotten i liggende stilling inde i en røghætte og hæld huden langs brystmidten. Skær mellemgulvet for at åbne brysthulen og indsæt en 20 gauge med 25 millimeter nål i venstre hjertekammer.Åbn straks højre atrium med en saks og perfuse 50 milliliter forkølet 100 millimolar PBS ved en fem milliliter per minut flow hastighed. Når alle PBS er blevet leveret, perfuse med 100 milliliter kold 4%paraformaldehyd. Når alle de fikseringsmiddel er blevet leveret, fjerne hjernen fra kraniet og fastsætte væv natten over i frisk 4%paraformaldehyd.Den næste morgen, placere prøven i 40 milliliter af 30% saccharose i 100 millimolar PBS i en 50 milliliter rør i cirka tre dage, indtil hjernen sætter sig til bunden af røret, før snap frysning hjernevævet i methyl butanol i to minutter på minus 80 grader Celsius. Efter frysning skal du bruge en minus 20 grader Celsius kryostat og en fin pensel til at erhverve 50 mikron tykke koronarsektioner. Når alle sektionerne er opnået, skal prøverne dækkes med 300 mikroliter anti-fade medium og monteres med 22 50-milliliter coverslips.Når mediet er tørret, skal du afbilde sektionerne ved konfokal mikroskopi. Syv dage efter Lentivirus injektion, rotte koronar hjerne skive billeder afslører en betydelig tilstedeværelse af EcoHIV-EGFP signaler i hele hjernevævet, især inden for cortex og hippocampal dentate gyrus. Dobbelt mærkning med og Iba1 og EcoHIV-EGFP sonder giver stærke beviser for, at microglia er den fremherskende celletype huser EcoHIV udtryk i hjernen.Otte uger efter infektion udviser EcoHIV injicerede dyr en relativ ufølsomhed over for manipulation af interstimulusintervallet, som det fremgår af en relativt fladere intervalfunktion sammenlignet med saltvandskontroller. Derudover udviser EcoHIV injicerede rotter dybtgående ændringer i deres dendritiske rygsøjlemorfologi som det fremgår af en øget relativ hyppighed af kortere dendritiske rygsøjler med øgede hoved- og nakkediametre i forhold til kontroldyr. Koncentrationen og titreringen af tilstandsmediet før stereotaxisk injektion er afgørende for at sikre de ensartede og replikerbare resultater på tværs af eksperimenter.Denne specifikke regionale stereoskatiske injektion af EcoHIV giver en effektiv HIV-infektion i hjernen og producerer tidsmæssige behandling underskud hos rotter. Udnyttelsen af stereotaxiske hiv-injektioner hos rotter for at udvide EcoHIV-infektionsmodellen giver en vigtig mulighed for at løse nye spørgsmål relateret til neuro HIV ved 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.

Stereotaktisk kirurgi for excitotoksiske læsion af specifikke områder i hjernen i den voksne rotte

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 Model af Mild intrauterin 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.

En In Vivo evaluering af blod - hjerne barrieren forstyrrelser i en rotte Model af iskæmisk slagtilfælde

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 Model af Central træthed ved hjælp af en modificeret flere Platform metode

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 metode til at studere primære begivenheder i Neurodegeneration ved hjælp af 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 tilgang til placeringsspecifik blodhjernebarriereåbning ved hjælp af fokuseret ultralyd i en rottemodel

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 vævsclearingmetode til rottehjernevæv

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 og vurdering af Levodopa-induceret dyskinesier i en rottemodel af Parkinsons sygdom

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

Validering af skadesrespons og adfærdsvurderinger i en mTBI rottemodel

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 af forskning i traumatisk hjerneskade - En lukket hovedmodel til nøjagtig replikation og hurtig 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.

Rottemodel af mild traumatisk skade med lukket hoved og dens validering

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