Using rats, this protocol can be used to establish a new model of active HIV infection using chimeric HIV. with rats, the establishment of the EcoHIV infection model for studies of drug abuse and neurocognitive disorders could be beneficial to the study of neuro HIV and HIV-1 associated neurocognitive disorders. Demonstrating procedure will be Hailong Li, a research associate from my laboratory.
For packaging of the virus into 293 T-cells, seed five times 10 to the fifth 293 T-cells per milliliter of DMEM supplemented with 10%FBS into each of the two gelatin-coated 75 square centimeter flasks and incubate the cells at 37 degrees Celsius until the cultures reach 50%confluency. On the day of the transfection, dilute 22.5 microliters of transfection reagent in 750 microliters of medium per flask in a 1.5 milliliter microcentrifuge tube and vortex the solution for three seconds. Dilute 20 micrograms of the chimeric EcoHIV plasmid DNA in 750 microliters of medium in a second 1.5 milliliter microcentrifuge tube per flask with thorough mixing.
Combine the diluted DNA with the diluted transfection reagent with gentle mixing and incubate the resulting solution for 15 minutes at room temperature. At the end of the incubation, add each virus suspension to 10 milliliters of prewarm DMEM medium and add each virus supplemented solution to one flask of 293 T-cells. After two days of cell culture, pool the supernatant from the flasks in a single 50 milliliter conical tube for centrifugation and transfer the clarified supernatant to a new 50 milliliter tube.
Add eight milliliters of Lenti-X Concentrator to the clarified supernatant and mix with gentle inversion. After a two-day incubation at four degrees Celsius, centrifuge the mixture and carefully remove the supernatant, then gently resuspend the pellet with 100 microliters of 100 millimolar PBS and use a P24 ELISA kit to titer the virus concentration. For EcoHIV-EGFP injection, after confirming a lack of response to pedal reflex, shave the hair from the brain region and sterilize the exposed skin two times with 70%ethanol and a chlorhexidine-based scrub.
Secure the rat in a prone position in a stereotaxic apparatus and make a five to six centimeter incision through the skin along the scalp midline. Mark one drilling position at 0.8 millimeters lateral and one 1.2 millimeters rostral to bregma and drill a 0.4 millimeter diameter hole at each skull position. Load 1.04 times 10 to the sixth transduction units per milliliter of titered EcoHIV Lentivirus solution into a 10 microliter injection syringe and secure the syringe to the stereotaxic apparatus.
Move the needle close to the surface of one drilling hole and insert the needle 2.5 milliliters into the hole. Infuse one microliter of virus solution at a rate of 0.2 microliters of virus per minute. When all of the virus has been injected, leave the needle inside the injection area for five minutes before slowly retracting the needle until it is outside of the rat skull.
Close the skin incision with a 4-0 silk thread suture and sterilize the solution with 70%ethanol, then place the rat in a recovery chamber with a heating pad with monitoring until recumbency. One to eight weeks after viral infusion, after confirming a lack of response to pedal reflex, fix the rat in a supine position inside a fume hood and incise the skin along the thoracic midline. Cut the diaphragm to open the thoracic cavity and insert a 20 gauge by 25 millimeter needle into the left ventricle.
Immediately open the right atrium with scissors and perfuse 50 milliliters of pre-chilled 100 millimolar PBS at a five milliliters per minute flow rate. When all of the PBS has been delivered, perfuse with 100 milliliters of cold 4%paraformaldehyde. When all of the fixative has been delivered, remove the brain from the skull and fix the tissue overnight in fresh 4%paraformaldehyde.
The next morning, place the sample in 40 milliliters of 30%sucrose in 100 millimolar PBS in a 50 milliliter tube for about three days until the brain settles to the bottom of the tube before snap freezing the brain tissue in methyl butanol for two minutes at minus 80 degrees Celsius. After freezing, use a minus 20 degree Celsius cryostat and a fine paintbrush to acquire 50 micron thick coronal sections. When all of the sections have been obtained, cover the samples with 300 microliters of anti-fade medium and mount them with 22 50-milliliter coverslips.
When the medium has dried, image the sections by confocal microscopy. Seven days after Lentivirus injection, rat coronal brain slice images reveal a significant presence of EcoHIV-EGFP signals throughout the brain tissue, especially within the cortex and the hippocampal dentate gyrus. Dual labeling with and Iba1 and EcoHIV-EGFP probes provide strong evidence that microglia are the predominant cell type harboring EcoHIV expression in the brain.
Eight weeks post-infection, EcoHIV injected animals exhibit a relative insensitivity to the manipulation of interstimulus interval as evidenced by a relatively flatter interval function compared to saline controls. In addition, EcoHIV injected rats display profound alterations in their dendritic spine morphology as evidenced by an increased relative frequency of shorter dendritic spines with increased head and neck diameters relative to control animals. The concentration and titrating of the condition medium before stereotaxic injection is critical to ensuring the consistent and the replicable results across experiments.
This specific regional stereotaxic injection of EcoHIV provides an efficient HIV infection within the brain and produces temporal processing deficits in rats. The utilization of stereotaxic HIV injections in rats to extend the EcoHIV infection model affords a key opportunity for addressing novel questions related to neuro HIV at hand.
