A Rat Model of EcoHIV Brain Infection

Summary

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).

Abstract

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.

Introduction

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....

Protocol

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

1. Culture the 293 FT cells (5×10⁵/mL) in gelatin coated 75 cm² flasks with DMEM plus 10% FBS¹¹. Gr....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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