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Neuroscience

A Rat Model of EcoHIV Brain Infection

Published: January 21st, 2021

DOI:

10.3791/62137

1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

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. 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.

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 su....

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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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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.......

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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.......

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

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Name Company Catalog Number Comments
293FT cells ThermoFisher Scientific R70007
Antibiotic-Antimycotic solution Cellgro 30004CI 100X
Corning BioCoatGelatin 75cm² Rectangular Canted Neck Cell Culture Flask with Vented Cap Life Technologies 354488
Corning DMEM with L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate Life Technologies 10013CV
Cover glass VWR 637-137
drill
Dumont #5 Forceps World Precision Instruments 14095
Dumont #7 Forceps World Precision Instruments 14097
Eppendorf Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes Life Technologies 22600028
Ethicon Vicryl Plus Antibacterial, 4-0 Polyglactin 910 Suture, 27in. FS-2 Med Vet International VCP422H
Hamilton syringe Hamilton 1701
Invitrogen Lipofectamine 3000 Transfection Reagent Life Technologies L3000015
Iris Forceps World Precision Instruments 15914
Iris Scissors World Precision Instruments 500216
Lentivirus-Associated p24 ELISA Kit Cell Biolabs, inc. VPK-107-5
Lenti-X Concentrator Takara PT4421-2
Opti-MEM I Reduced Serum Medium Life Technologies 11058021
Paraformaldehyde Sigma-Aldrich 158127-500G
Paraformaldehyde Sigma P6148
ProLong Gold Fisher Scientific P36930
Sevoflurane Merritt Veterinary Supply 347075
stereotaxic apparatus Kopf Instruments Model 900
SuperFrost Plus Slides Fisher Scientific 12-550-154%
Vannas Scissors World Precision Instruments 500086

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