Identification of EcoHIV-Infected Cells in Microglia-Manipulated Transgenic Mice

This protocol describes how the combination of EcoHIV infection with Tmem119-EGFP mice offers a valuable biological system for investigating microglial alterations and viral reservoirs in rodent models of HIV-associated neurocognitive disorders. Combined antiretroviral therapy has dramatically improved the quality of life of people who live with HIV. To understand how HIV impacts the central nervous system, a reliable and feasible model of HIV is necessary.

Previously, a novel biological system using chimeric HIV, EcoHIV in the colation was developed in rat model to investigate neurocognitive impairment and synaptic dysfunction. A significant challenge remains in clarifying the neuroanatomic distribution of EcoHIV, particularly its differential expression in various cell types in the brain. In the current study, EcoHIV with mScarlet fluorescence labeling was modified and retro-orbitally injected into Tmem119-EGFP knock-in mice to determine if microglia are the major cell type responsible for viral expression and the reservoir of HIV in the brain.

To begin, transfer the dissociated fetal rat brain cells to a poly-L-lysine pre-coated 12-well plate with glass inserts containing one milliliter of DMEM-F12 plus 10%FBS. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide. The next day, replace the medium with a Neurobasal medium supplemented with B27 and continue culturing cells at 37 degrees Celsius with 5%carbon dioxide for three weeks.

Then add 60 microliters of EcoHIV-mScarlet into the brain cell culture and incubate for six days. After incubation, fix the cells with 4%paraformaldehyde. Perform immunostaining on infected brain cells using specific primary antibodies and image them on a confocal microscope under a 40x objective.

Microglia were identified as the major infected cell type in rat brain cell cultures, as evidenced by colocalization of mScarlet with CD11b/c and Iba1 markers. To begin, open the scalp of a decapitated adult mouse and transfer the brain tissue into another Petri dish containing five milliliters of sterilized HBSS. Peel off the meninges and transfer the frontal cortex into two milliliters of HBSS.

Next, add 20 microliters of 0.25%trypsin-EDTA into the mixture. Incubate for 15 minutes at room temperature, swirling the tube every few minutes. Transfer the dissociated cells to a poly-L-lysine pre-coated 75 square centimeter flask containing 10 milliliters of DMEM-F12 medium and 10%FBS.

Culture cells in a 37 degrees Celsius and 5%carbon dioxide incubator until they reach 90%confluency. Next, digest the brain cells with two milliliters of 0.25%trypsin-EDTA. Subculture the digested brain cells into a 35-millimeter glass bottom dish containing five milliliters of DMEM-F12 growth medium until they reach 80%confluency.

Then add eight microliters of EcoHIV-mScarlet into the mouse glial cells and incubate for two days. Adult mouse primary glial cells were successfully infected by EcoHIV-mScarlet after two days of treatment. To begin, secure the anesthetized Tmem119-EGFP mouse in a lateral position.

Thaw the EcoHIV-mScarlet on ice. Fill the viral solution into an intraocular injector syringe with a 33-gauge blunt needle. Place the mouse in the right lateral recumbency with the head facing left.

Identify the injection area around the eye region. Slowly and gently insert the needle into the medial canthus of the eye. Then advance the needle into the vessels behind the eyeball.

Inject 6.5 microliters of EcoHIV-mScarlet into the retro-orbital sinus. After injection, carefully remove the needle. Place the anesthetized mouse in a supine position inside a chemical fume hood.

Open the skin along the thoracic midline. Then separate the diaphragm and open the chest with scissors. Now insert a 22-gauge one-and-half needle into the left ventricle.

Open the right atrium with scissors. Perfuse the right atrium with 50 milliliters of pre-chilled 100 millimolar PBS, then perfuse with 100 milliliters of pre-chilled 4%paraformaldehyde. Remove the entire mouse brain.

After fixing the brain in 4%paraformaldehyde, secure the brain tissue using tissue adhesive on the metal platform of the Vibratome. Cut 50-micrometer thick coronal sections with carbon steel blades. Next, place the brain slices onto glass slides using a brush.

Immediately add 0.1 milliliters of anti-fade mounting medium to each section. Place a 22 by 50-millimeter cover slip over the brain sections and dry the Superfrost slides in the dark for one day. The next day, use a confocal microscope with 488 nanometers and 594 nanometers wavelengths to acquire multi-channel images of the brain region of interest.

EcoHIV-mScarlet signals were primarily observed in microglial cells labeled with EGFP in the Tmem119-EGFP mouse brain. No significant neuronal infection was detected in cultured rat brain cells treated with EcoHIV-mScarlet.