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

This work was funded by NIH grants HD043680, MH106392, DA013137, and NS100624.

All animal protocols were reviewed and approved by the Animal Care and Use Committee at the University of South Carolina (federal assurance number: D16-00028). Six adult male F344/N rat was pair housed in a controlled environment under a 12/12 light: dark cycle with ad libitum access to food and water. All animals were cared for using guidelines established by the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals.
1. Virus packaging in 293 FT cells
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In this protocol, we established an EcoHIV-induced HIV infection model in rats. Specifically, we described a bilateral stereotaxic injection of EcoHIV into the cortex which successfully induced active HIV infection in the rat brain 7 days post-injection. Futhermore, we demonstrate that EcoHIV infection in rats could be a good biological system to study key aspects of HAND. Eight weeks post- EcoHIV infection, rats exhibited significant neurocognitive impairments, which included the alterations in temporal processing and s...

Biological systems have enhanced our understanding of HIV-1 associated neurocognitive disorders (HAND) and their underlying neural mechanisms2. Determining which biological system is most appropriate for any given study is often dependent upon the question of interest2. The limitation of the range of host animal models challenges studies of HIV-1 disease development. To investigate HIV-1 viral replication and pathogenesis, Potash et al.3 created a mouse model of active HIV-1 infection, replacing the coding region of HIV surface envelope glycoprotein, gp120, with ecotropic MLV gp80, which led to successful viral replication in mice4. After tail vein injections in chimeric HIV (EcoHIV) mice, many characteristics were observed resembling those of HIV-1 seropositive individuals (e.g., infected lymphocytes and macrophages, targeted for antiviral immune responses, and inflammation3,5,6).
Although mice and rats are both members of the Muridae, fundamental species differences may influence their suitability for specific experimental questions7. Therefore, the extension of the EcoHIV infection model to rats (commonly used in studies of drug abuse and neurocognitive disorders) would be advantageous in the study of neuroHIV. For example, their larger size makes jugular catheter implantation for drug self-administration procedures more practical8. Drug self-administration techniques in rats have been utilized to evaluate motivation in HIV-19. Furthermore, many neurocognitive/behavioral tasks were initially designed for rats10. Here, we report the utilization of stereotaxic injections of EcoHIV in rats to extend the EcoHIV infection model and afford a key opportunity to address novel questions related to neuroHIV and 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

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

The conditioned medium was collected from lentivirus of EcoHIV-EGFP infected 293FT cells. Next, it was concentrated and titered, then stereotaxically injected into the brain (cortical region) of F344/N rats. Seven days post-injection, rats were sacrificed and images were taken from coronal brain slices ranging from bregma 5.64 mm to bregma -4.68 mm. In Figure 1A, there are significant EcoHIV-EGFP signals throughout the brain, especially in the cortex and the hippocampal dentate gyrus. Furthe...

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

A Rat Model of EcoHIV Brain Infection

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

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

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