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
<ol>
	<li>Culture the 293 FT cells (5&#215;105/mL) in gelatin coated 75 cm2 flasks with DMEM plus 10% FBS11. Grow cells at 37 &#176;C to be 50% confluent at transfection.</li>
	<li>Dilute 22.5 &#181;L of transfection reagent (e.g., Lipofectamine 3000) in 750 &#181;L of medium (e.g., Opti-MEM) in a 1.5 mL micro-centrifuge tube and vortex for 3 s.</li>
	<li>Dilute 20 &#181;g of EcoHIV plasmid DNA in 750 &#181;L of medium in a 1.5 mL micro-centrifuge tube and mix well.</li>
	<li>Add diluted DNA into the tube of diluted transfection reagent and mix gently. Incubate for 15 min at room temperature.</li>
	<li>Add the mixture to 10 mL of prewarmed DMEM medium in 75 cm2 flask. Incubate cells for 2 days at 37 &#176;C.</li>
	<li>Harvest and combine 24 mL of conditional medium from the two flasks that include the packaged lentivirus.</li>
	<li>Centrifuge all 24 mL of conditional medium at 500 x g for 10 minutes at 4 &#176;C. Transfer the clarified supernatant to a sterile 50 mL tube.</li>
	<li>Combine 8 mL of Lenti-x concentrator with 24 mL of clarified supernatant (1:3 ratio). Mix by gentle inversion. Incubate mixture at 4 &#176;C for two days.</li>
	<li>Centrifuge mixture at 1,500 x g for 45 minutes at 4 &#176;C. Carefully remove the supernatant.</li>
	<li>Gently re-suspend the pellet with 100 &#181;L of 100 mM PBS.</li>
	<li>Titer virus concentration with a p24 ELISA kit.</li>
</ol>
2. EcoHIV-EGFP virus stereotaxic surgeries
<ol>
	<li>Anesthetize rats using 3% sevoflurane. Proceed to step 2.2 when the rats are not responsive to noxious stimuli and reflexes are absent.</li>
	<li>Shave the hairs from the brain region and sterilize the skin twice with 70% ethanol and a chlorhexidine-based scrub. Secure the rat in a prone position in the stereotaxic apparatus.</li>
	<li>Make an incision (5-6 cm) through the skin along the scalp midline.</li>
	<li>Mark two drilling positions at 0.8 mm lateral, 1.2 mm rostral to bregma. Drill a hole (diameter 0.4 mm) in each skull position.</li>
	<li>Fill titered EcoHIV lentivirus solution (1.04 &#215; 106 TU/mL) in a 10 &#181;L injection syringe. Secure the syringe to the stereotaxic apparatus.</li>
	<li>Move down the needle close to the surface of drilling hole. Measure and move 2.5 mm in depth.</li>
	<li>Infuse 1 &#181;L of virus solution at a rate of 0.2 &#181;L/min. Keep the needle inside of the injection area for 5 min. Slowly move the needle up until it is outside of the rat skull.</li>
	<li>Suture the skin with a 4-0 silk thread.</li>
	<li>Sterilize the incision with 70% ethanol once. Subcutaneously inject butorphenol (Dorolex, 0.1 mg/kg body weight).</li>
	<li>Transfer the rat to a recovery chamber with a heating pad until it wakes up.</li>
</ol>
3. Visualization of brain sections
NOTE: Wait one to eight weeks after EcoHIV viral infusion.
<ol>
	<li>Anesthetize the rat with 5% sevoflurane. Continue to step 3.2 when the rats are not responsive to noxious stimuli and reflexes are absent.</li>
	<li>Fix the rat in a supine position inside a fume hood.</li>
	<li>Incise the skin along the thoracic midline. Cut the diaphragm and open the thoracic cavity.</li>
	<li>Insert a 20 G &#215; 25 mm needle into the left ventricle.</li>
	<li>Immediately open the right atrium with scissors.</li>
	<li>Perfuse 50 mL of prechilled 100 mM PBS at a rate of 5 mL/min.</li>
	<li>Perfuse 100 mL of cold 4% paraformaldehyde at a rate of 5 mL/min.</li>
	<li>Decapitate the rat, open the scalp and remove the brain.</li>
	<li>Postfix overnight with 4% paraformaldehyde.</li>
	<li>Transfer the brain to 40 mL of 30% sucrose in 100 mM PBS in 50 mL tube until the brain floats down to the bottom (about 3 days).</li>
	<li>Snap freeze the brain in methylbutanol for 2 min at - 80 &#176;C.</li>
	<li>Secure the brain tissue on a metal platform inside a -20 &#176;C cryostat.</li>
	<li>Cut 50 &#181;m thick coronal sections using the cryostat.</li>
	<li>Transfer the brain slices onto glass slides with a fine brush.</li>
	<li>Mount sections in 0.3 mL of antifade medium and cover with 22 mm 50 mm coverslips.</li>
	<li>Keep the glass slides in the dark at room temperature till dry.</li>
	<li>Image the targeted neurons with a confocal microscope using Z-stack based on brain region boundaries and morphological characteristics of neurons. 
	NOTE: The confocal microscope settings used were: magnification of 60 X (A/1.4, oil), and a Z-plane interval of 0.15 &#181;m (pinhole size 30 &#181;m; back-projected pinhole radius 167 nm) using a wavelength of 488 nm.</li>
</ol>

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None of the authors have conflicts of interest to declare.

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><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&amp;sup2; 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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Neuroscience

3.1K Views.  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).

Video: A Rat Model of EcoHIV Brain Infection

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Immunology and Infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

EcoHIV Infection in Tmem119-EGFP Mice for Studying Microglial Alterations

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

Combined antiretroviral therapy (cART) has dramatically improved the quality of life for people living with HIV (PLWH). However, over 4 million PLWH are over the age of fifty and experience accompanying HIV-associated neurocognitive disorders (HAND). 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) inoculation was developed in a rat model to investigate neurocognitive impairments and synaptic dysfunction. Nevertheless, a significant challenge remains in clarifying EcoHIV's neuroanatomical distribution, 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 (which express enhanced green fluorescence protein primarily in microglia) to determine if microglia are the major cell type responsible for viral expression and reservoirs of HIV in the brain. The current data show that: (1) in vitro, EcoHIV-mScarlet fluorescence signals were predominantly localized in microglia-like cells among primary rodent brain cells; (2) in vivo, injection of EcoHIV-mScarlet into Tmem119-EGFP mice induced significant HIV expression in the mouse brain. The co-localization of mScarlet and EGFP signals suggests that microglia are the main cell type harboring HIV in the brain. Overall, EcoHIV in rodents offers a valuable biological system to study microglial alterations, viral reservoirs in the brain, and the neurological mechanisms of HIV-associated neurocognitive disorders.

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 (cART) has dramatically improved the quality of life for people living with HIV (PLWH). However, over 4 million PLWH are ...

Establishing an Active HIV Infection in a Rat Model

Source: Li, H., McLaurin, et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62137">A Rat Model of EcoHIV Brain Infection.</a> J. Vis. Exp. (2021).
This video demonstrates the procedure of injecting chimeric HIV viruses into the cortex of a rat&#39;s brain, where the virus RNA integrates into the host genome, replicates, and spreads to establish an active HIV infection in the experimental model.

Ustalenie aktywnego zakażenia wirusem HIV w modelu szczurzym

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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A Rat Model of EcoHIV Brain Infection

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Humanized NOD/SCID/IL2rγ^null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

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

Ustalenie aktywnego zakażenia wirusem HIV w modelu szczurzym