This work was funded by NIH grants DA059310, DA058586, AG082539, and GM109091. We appreciate the generous gift of the EcoHIV-NL4-3-EGFP from Dr. Potash of the Icahn School of Medicine at Mount Sinai.

The Animal Care and Use Committee at the University of South Carolina approved all animal protocols (federal assurance number: D16-00028). All experiments strictly followed the guidelines established by the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals. Tmem119-EGFP knock-in mice (30 days old, male, 23-26 g body weight) were obtained from a commercial source and group-housed in AAALAC-accredited facilities. All animals were housed under a 12/12 h light-dark cycle with free access to food and water. The details of the animals, reagents, and equipment used in this study are listed in the Table of Materials.
1. EcoHIV-mScarlet packaging in 293FT cells
<ol>
	<li>Incubate the 293 FT cells in a gelatin-precoated 75 cm2 flask. Keep cells growing to 30% confluent at transfection.</li>
	<li>Perform the transfection of plasmid DNA (15 &#181;g) of EcoHIV-mScarlet (Supplementary File 1) using Lipofectamine 3000 (22.5 &#181;L) following the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Culture the cells in DMEM medium with 10% FBS serum for 3 days at 37 &#176;C.</li>
	<li>Collect the conditional medium with viral suspension. Centrifuge at 500 &#215; g for 10 min at 4 &#176;C. Transfer the supernatant with a 10 mL pipette to a sterile 50 mL tube.</li>
	<li>Add a certain amount of Lenti-x concentrator (1:3 ratio) into the viral mixture (for example, 8 mL of concentrator with 24 mL of viral mixture). Gently invert the tube five times.</li>
	<li>Keep the virus-Lenti concentrator mixture at 4 &#176;C for 2 days. Centrifuge at 1,500 &#215; g, 45 min, 4 &#176;C. Carefully remove supernatant as much as possible using a pipette.</li>
	<li>Re-suspend the pellet with prechilled 200 &#181;L of 100 mM PBS. Store the virus at -80 &#176;C. 
	NOTE: The details of EcoHIV-mScarlet packaging in 293FT cells were described in our previous study16. Do not vortex or introduce air bubbles in the viral solution.</li>
</ol>
2. EcoHIV-mScarlet infection in primary rat brain cells
<ol>
	<li>Perform the primary brain cell isolation from rat fetuses (18 days) following the previously published report16.</li>
	<li>Transfer dissociated cells to poly-L-Lysine pre-coated 12-well plates with glass inserts containing 1 mL of DMEM/F12 medium plus 10% FBS. Replace medium the next day for Neurobasal medium with B27 supplement.</li>
	<li>Culture primary fetal brain cells in a 5% CO2 incubator for 3 weeks.</li>
	<li>Add EcoHIV-mScarlet (60 &#181;L, 1.26 &#215; 106 TU/mL) into the culture medium. Incubate the cultured brain cells with EcoHIV-mScarlet for 6 days.</li>
	<li>Fix the cells with 4% PFA and perform immunostaining on infected brain cells using specific primary antibodies (CD11b/c, Iba1).</li>
	<li>Obtain images using 40&#215; objective under a confocal microscopy system.</li>
</ol>
3. EcoHIV-mScarlet virus infection in primary glia cells from adult mice
<ol>
	<li>Anesthetize adult mice with 5% sevoflurane for 5 min (following institutionally approved protocols). Sterilize the skin of the head with 70% EtOH.</li>
	<li>After confirming the mouse is no longer responsive to noxious stimuli, use a sterilized sharpened scissor to perform decapitation. Transfer the head to a new Petri dish filled with 5 mL of HBSS.</li>
	<li>Open the scalp and transfer brain tissue into another Petri dish containing 5 mL of sterilized HBSS. Peel off the meninges and transfer the frontal cortex into 2 mL of HBSS.</li>
	<li>Add 20 &#181;L of 0.25% Trypsin-EDTA into the mixture. Incubate for 15 min at room temperature; gently swirl the tube every few minutes.</li>
	<li>Transfer dissociated cells to a poly-L-Lysine pre-coated 75 cm2 flask containing 10 mL of DMEM/F12 medium and 10% FBS.</li>
	<li>Culture cells at 37 &#176;C, 5% CO2 incubator, until 90% confluency. Digest brain cells with 2 mL of 0.25% Trypsin-EDTA.</li>
	<li>Subculture brain cells into 35 mm glass-bottom dishes containing 5 mL of DMEM/F12 growth medium till 80% confluency.</li>
	<li>Add EcoHIV-mScarlet (8 &#181;L, 1.26 &#215; 106 TU/mL) into the culture medium. Incubate mouse glial cells for 2 days.</li>
	<li>Check red (mScarlet) fluorescence signals daily under a fluorescent microscope.</li>
</ol>
4. EcoHIV-mScarlet virus retroorbital injection into Tmem119-EGFP mice
<ol>
	<li>Use 3% sevoflurane to anesthetize the Tmem119-EGFP mice (both male and female mice at 30 days of age) until they are no longer responsive to noxious stimuli.</li>
	<li>Secure the mice in a lateral position with the injection eye facing up and respiration through the nose cone, which is linked to an anesthesia system. Use an appropriate nose cone size to provide continuous anesthesia.</li>
	<li>Thaw the EcoHIV-mScarlet on ice. Fill the viral solution into an intraocular injector syringe with a 33 G blunt needle.</li>
	<li>Place the mouse&#160;in the right lateral recumbency and keep its head facing to the left. Identify the location of the medial canthus as the injection site.</li>
	<li>After proptosing the eye, slowly and gently insert a needle (45-degree angle) into the medial canthus of the eye. Carefully insert the needle forward into the vessels behind the eyeball (retro-orbital sinus).</li>
	<li>Gently inject 6.5 &#181;L of EcoHIV-mScarlet (1.26 &#215; 106 TU/mL, bilateral-eye inoculation) into the retro-orbital sinus. Carefully remove the needle from the retro-orbital sinus and gently apply pressure to the eyelids to provide hemostasis.</li>
	<li>Apply lubricant to the eye to prevent the cornea from drying or becoming injured.</li>
	<li>Allow the mice to recover in a recovery chamber with a heating pad until it has woken up. 
	NOTE: The needle bevel should not be positioned too deep so that the arteries are not ruptured or the bones fractured. Viral infusion time is dependent upon multiple factors (e.g., injection volume, titer, animal size). For EcoHIV viral infusion, significant expression has been observed one week after retro-orbital injections11,16,17.</li>
</ol>
5. Visualization of brain tissue slices
<ol>
	<li>Deeply anesthetize mice using 5% sevoflurane. Move on to step 5.2 when the mice show no response to noxious stimuli and reflexes are absent.</li>
	<li>Keep the mice in a supine position inside a chemical fume hood.</li>
	<li>Open the skin along the thoracic midline. Separate the diaphragm and open the chest with scissors.</li>
	<li>Insert a 22 G1 1/2 needle into the left ventricle. Open the right atrium with scissors.</li>
	<li>Perfuse 50 mL of pre-chilled 100 mM PBS. Perfuse 100 mL of pre-chilled 4% paraformaldehyde buffer16.</li>
	<li>Remove the entire mouse brain16.</li>
	<li>Fix overnight with 4% paraformaldehyde.</li>
	<li>Secure the brain tissue using tissue adhesive on the metal platform of the Vibratome. Cut 50 &#181;m thickness of coronal sections with carbon steel blades.</li>
	<li>Place the brain slices onto glass slides using a brush. Immediately add 0.1 mL of the antifade mounting medium to each section.</li>
	<li>Place a 22 mm x 50 mm coverslip over brain sections. Dry the super-frost slides in the dark for 1 day.</li>
	<li>Configure the confocal microscope system to a magnification of 60&#215; (A/1.4, oil) and set a Z-plane interval of 0.15 &#181;m, with a pinhole size of 30 &#181;m and a back-projected pinhole radius of 167 nm.</li>
	<li>Use the 488 nm and 594 nm wavelengths to acquire multi-channel images of brain regions of interest.</li>
</ol>

EcoHIV Infection in Tmem119-EGFP Mice for Studying Microglial Alterations

<ol><li>Vigorito, M., Lashomb, A. L., Chang, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+learning+and+memory+in+HIV-1+transgenic+rats%5BTitle%5D)+AND+%22J+Neuroimmune+Pharmacol%22%5BJournal%5D)">Spatial learning and memory in HIV-1 transgenic rats.</a> J Neuroimmune Pharmacol. 2, 319-328 (2007).</li>
<li>Moran, L. M., Booze, R. M., Mactutus, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Time+and+time+again%3A+Temporal+processing+demands+implicate+perceptual+and+gating+deficits+in+the+HIV-1+transgenic+rat%5BTitle%5D)+AND+%22J.+Neuroimmune+Pharmacol%22%5BJournal%5D)">Time and time again: Temporal processing demands implicate perceptual and gating deficits in the HIV-1 transgenic rat.</a> J. Neuroimmune Pharmacol. 8 (4), 988-997 (2013).</li>
<li>Repunte-Canonigo,, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+changes+consistent+with+neuroAIDS+and+impaired+working+memory+in+HIV-1+transgenic+rats%5BTitle%5D)+AND+%22Mol+Neurodegener%22%5BJournal%5D)">Gene expression changes consistent with neuroAIDS and impaired working memory in HIV-1 transgenic rats.</a> Mol Neurodegener. 9, 26(2014).</li>
<li>Reid, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+HIV-1+transgenic+rat+that+develops+HIV-related+pathology+and+immunologic+dysfunction%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction.</a> Proc Natl Acad Sci USA. 98 (16), 9271-9276 (2001).</li>
<li>McLaurin, K. A., Li, H., Booze, R. M., Mactutus, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+timing%3A+NeuroHIV+progression+in+the+post-cART+era%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Disruption of timing: NeuroHIV progression in the post-cART era.</a> Sci Rep. 9, 827(2019).</li>
<li>Roscoe, R. F., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-1+transgenic+female+rat%3A+Synaptodendritic+alterations+of+medium+spiny+neurons+in+the+nucleus+accumbens%5BTitle%5D)+AND+%22J+Neuroimmune+Pharmacol%22%5BJournal%5D)">HIV-1 transgenic female rat: Synaptodendritic alterations of medium spiny neurons in the nucleus accumbens.</a> J Neuroimmune Pharmacol. 9 (5), 642-653 (2014).</li>
<li>Denton, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+monoaminergic+and+histaminergic+circuit+dysregulation+following+long-term+HIV-1+protein+exposure%5BTitle%5D)+AND+%22J+NeuroVirol%22%5BJournal%5D)">Selective monoaminergic and histaminergic circuit dysregulation following long-term HIV-1 protein exposure.</a> J NeuroVirol. 25 (4), 540-550 (2019).</li>
<li>Peng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+HIV-1+transgenic+rat+as+a+model+for+HIV-1+infected+individuals+on+HAART%5BTitle%5D)+AND+%22J+Neuroimmunol%22%5BJournal%5D)">The HIV-1 transgenic rat as a model for HIV-1 infected individuals on HAART.</a> J Neuroimmunol. 218 (1-2), 94-101 (2010).</li>
<li>Abbondanzo, S. J., Chang, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-1+transgenic+rats+display+alterations+in+immunophenotype+and+cellular+responses+associated+with+aging%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">HIV-1 transgenic rats display alterations in immunophenotype and cellular responses associated with aging.</a> PLoS ONE. 9, e105256(2014).</li>
<li>Potash, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+for+study+of+systemic+HIV-1+infection%2C+antiviral+immune+responses%2C+and+neuroinvasiveness%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness.</a> Proc Natl Acad Sci USA. 102 (10), 3760-3765 (2005).</li>
<li>Li, H., McLaurin, K. A., Illenberger, J. M., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+HIV-1+expression%3A+Role+in+HIV-1+associated+neurocognitive+disorders%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Microglial HIV-1 expression: Role in HIV-1 associated neurocognitive disorders.</a> Viruses. 13 (5), 924(2021).</li>
<li>Kaiser, T., Feng, G. Tmem119-EGFP and Tmem119-CreERT2 transgenic mice for labeling and manipulating microglia. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKaiser%2C+T+%22eNeuro%22">eNeuro</a>. 6 (4), (2019).</li>
<li>Bennett, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+tools+for+studying+microglia+in+the+mouse+and+human+CNS%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci.+USA%22%5BJournal%5D)">New tools for studying microglia in the mouse and human CNS.</a> Proc Natl Acad Sci. USA. 113 (12), E1738-E1746 (2016).</li>
<li>Satoh, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TMEM119+marks+a+subset+of+microglia+in+the+human+brain%5BTitle%5D)+AND+%22Neuropathology%22%5BJournal%5D)">TMEM119 marks a subset of microglia in the human brain.</a> Neuropathology. 36 (1), 39-49 (2016).</li>
<li>Li, H., Aksenova, M., Bertrand, S. J., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+filamentous+actin+%28F-actin%29+puncta+in+rat+cortical+neurons%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Quantification of filamentous actin (F-actin) puncta in rat cortical neurons.</a> J Vis Exp. (108), e53697(2016).</li>
<li>Li, H., McLaurin, K. A., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rat+model+of+EcoHIV+brain+infection%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">A rat model of EcoHIV brain infection.</a> J Vis Exp. (167), e62137(2021).</li>
<li>Alfar, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+optimizing+production+and+quality+control+of+infective+EcoHIV+virions%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol for optimizing production and quality control of infective EcoHIV virions.</a> STAR Protoc. 4 (3), 102368(2023).</li>
<li>Malatesta, P., Hartfuss, E., Götz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+radial+glial+cells+by+fluorescent-activated+cell+sorting+reveals+a+neuronal+lineage%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage.</a> Development. 127 (24), 5253-5263 (2000).</li>
<li>Parpura, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glutamate-mediated+astrocyte-neuron+signaling%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Glutamate-mediated astrocyte-neuron signaling.</a> Nature. 369 (6483), 744-747 (1994).</li>
<li>Wahl, A., Al-Harthi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV+infection+of+non-classical+cells+in+the+brain%5BTitle%5D)+AND+%22Retrovirology%22%5BJournal%5D)">HIV infection of non-classical cells in the brain.</a> Retrovirology. 20 (1), 1(2023).</li>
<li>Pandey, H. S., Seth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Friends+turn+foe-astrocytes+contribute+to+neuronal+damage+in+NeuroAIDS%5BTitle%5D)+AND+%22J.+Mol.+Neurosci%22%5BJournal%5D)">Friends turn foe-astrocytes contribute to neuronal damage in NeuroAIDS.</a> J. Mol. Neurosci. 69 (2), 286-297 (2019).</li>
<li>Minagar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+macrophage%2Fmicroglia+and+astrocytes+in+the+pathogenesis+of+three+neurologic+disorders%3A+HIV-associated+dementia%2C+Alzheimer%27s+disease%2C+and+multiple+sclerosis%5BTitle%5D)+AND+%22J+Neurol+Sci%22%5BJournal%5D)">The role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: HIV-associated dementia, Alzheimer's disease, and multiple sclerosis.</a> J Neurol Sci. 202 (1-2), 13-23 (2002).</li>
<li>Borrajo, A., Spuch, C., Penedo, M. A., Olivares, J. M., Agís-Balboa, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Important+role+of+microglia+in+HIV-1+associated+neurocognitive+disorders+and+the+molecular+pathways+implicated+in+its+pathogenesis%5BTitle%5D)+AND+%22Ann+Med%22%5BJournal%5D)">Important role of microglia in HIV-1 associated neurocognitive disorders and the molecular pathways implicated in its pathogenesis.</a> Ann Med. 53 (1), 43-69 (2021).</li>
<li>González-Scarano, F., Martín-García, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neuropathogenesis+of+AIDS%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">The neuropathogenesis of AIDS.</a> Nat Rev Immunol. 5 (1), 69-81 (2005).</li>
<li>Li, H., McLaurin, K. A., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+proliferation+underlies+synaptic+dysfunction+in+the+prefrontal+cortex%3A+Implications+for+the+pathogenesis+of+HIV-1-associated+neurocognitive+and+affective+alterations%5BTitle%5D)+AND+%22J.+Neurovirol%22%5BJournal%5D)">Microglia proliferation underlies synaptic dysfunction in the prefrontal cortex: Implications for the pathogenesis of HIV-1-associated neurocognitive and affective alterations.</a> J. Neurovirol. 29 (4), 460-471 (2023).</li>
<li>Kim, B. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EcoHIV+infection+of+primary+murine+brain+cell+cultures+to+model+HIV+replication+and+neuropathogenesis%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">EcoHIV infection of primary murine brain cell cultures to model HIV replication and neuropathogenesis.</a> Viruses. 16 (5), 693(2024).</li>
<li>Réu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+lifespan+and+turnover+of+microglia+in+the+human+brain%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">The lifespan and turnover of microglia in the human brain.</a> Cell Rep. 20 (4), 779-784 (2017).</li>
<li>Jones, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+of+glial+cell+modulators+in+the+management+of+substance+use+disorders%5BTitle%5D)+AND+%22CNS+Drugs%22%5BJournal%5D)">Potential of glial cell modulators in the management of substance use disorders.</a> CNS Drugs. 34 (7), 697-722 (2020).</li>
<li>Kaalund, S. S., Johansen, A., Fabricius, K., Pakkenberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Untreated+patients+dying+with+aids+have+loss+of+neocortical+neurons+and+glial+cells%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">Untreated patients dying with aids have loss of neocortical neurons and glial cells.</a> Front Neurosci. 13, 1398(2020).</li>
</ol>

None of the authors have conflicts of interest to declare.

In the present study, it was found that (1) the novel EcoHIV-mScarlet successfully infected primary rat brain cells in vitro; (2) the triple labeling of mScarlet, CD11b/c, and Iba1 identified microglia as the predominant cell type for this EcoHIV expression in rat brain cells in vitro; (3) primary mouse brain cells from adults in vitro further evidence the EcoHIV-mScarlet infection; (4) the EcoHIV-mScarlet distribution in the Tmem119-EGFP knock-in mouse line demonstrated a microglia-specific distribution pattern of EcoHIV infection.
Emerging studies suggested that various types of brain cells identified within the central nervous system (neurons, astrocytes, microglia, oligodendrocytes, etc.) exhibit functional and transcriptomic alteration during HIV and HIV-associated neurocognitive disorders18,19. For example, astrocytes contribute 30%-70% of the brain and perform surveillance to maintain brain homeostasis20. Astrocytes also modulate immune function and regulate the secretion of multi-cytokines and chemokines, especially in the situation of brain inflammation and neurodegeneration in HIV21,22. HIV infection of microglia not only results in a continuous release of viral proteins and proinflammatory cytokines and chemokines, but also provides predominant sources for HIV viral reservoirs23,24,25,26. Furthermore, activated microglia contribute a critical immunological function in the CNS; however, prolonged activation may also exacerbate neurodegeneration in HIV progression10,27. Oligodendrocytes also play an important function, including releasing several neurotrophins (such as nerve growth factor, brain-derived neurotrophic factor, etc.)28. A previous study found that the number of oligodendrocytes significantly decreased in the brains of AIDS patients, which may indicate direct damage to oligodendrocytes from HIV viral proteins29. Therefore, a specific type of cell-manipulated infection model of HIV should provide fundamental means of identifying the differential functions of various brain cells following infection. In the current study, a biological system was developed to mimic features of HIV-1 by chimeric HIV (EcoHIV) inoculation. This HIV inoculation was also combined with the Tmem119-EGFP knock-in mouse line to generate and validate a microglia-manipulated rodent model of HIV.
However, the limitations of the present study must be recognized. There were a few Iba1/CD11b/c negative cells that presented mScarlet fluorescent signals in vitro. Other types of brain cells, such as brain macrophages or pericytes, may be involved in EcoHIV infection, or alternatively, the cell culture environment may promote aberrant infection patterns relative to in vivo inoculation. Future, whole animal studies should further clarify the function of mScarlet+ microglia in the process of EcoHIV infection, and further define the regional distribution of mScarlet+EcoHIV microglia in the brain. Additionally, neurocognitive alterations occurring as a result of microglial infections may also be addressed in this rodent model. Collectively, the EcoHIV-mScarlet inoculation of Tmem119-EGFP knock-in mice provides a novel model and research strategy for investigating the microglial-driven mechanisms of HIV-associated neurocognitive disorders.

Despite the profound benefits derived from antiretroviral therapy, people living with HIV (PLWH) still experience neurocognitive disorders. To better understand the neuronal mechanisms of HIV-associated neurocognitive disorder (HAND), there is a critical need for HIV models to further elucidate the specific cell type involvement in NeuroHIV.
The HIV-1 transgenic rat, which has constitutive exposure to HIV-1 viral proteins, is a popular rodent model used to investigate neurocognitive disorders1,2,3,4and neuroanatomical alterations5,6,7 associated with HAND. The functional deletion of the gag and pol domains prevents viral replication, rendering the HIV-1 transgenic rat noninfectious8,9. Recently, a chimeric HIV (EcoHIV) infection model in mice was initially reported by Potash et al.10 and later extended to rats, which may be advantageous for further studies on HAND and substance use disorders11. In this novel biological system, systemic HIV-1 infection was observed along with many clinical features of HIV-1 in humans, including lymphocyte and macrophage involvement, antiviral immune responses, neuroinvasiveness, and brain inflammation.
Microglia play a critical role as specialized brain-resident macrophages in maintaining brain function and homeostasis. To distinguish microglia from closely related cell types (e.g., blood monocytes, perivascular macrophages, meningeal macrophages), the current study used the Tmem119-EGFP knock-in mouse line. Previous studies have reported that transmembrane protein 119 (Tmem119) exhibits an exclusively microglia-specific expression pattern in rodent and human brain tissue12,13,14,15. The EGFP signal in Tmem119-EGFP knock-in mice was observed throughout the brain and localized specifically to microglial cells.
In the present study, Tmem119-EGFP knock-in mice were inoculated with the EcoHIV-mScarlet virus, and cells positive for EcoHIV in the central nervous system were identified. Here, we present a protocol for EcoHIV-mScarlet inoculation in Tmem119-EGFP knock-in mice, providing a reliable model for therapeutically targeting microglial alterations in HIV.

<table><tbody><tr><td>293FT cells</td><td>ThermoFisher Scientific</td><td>R70007</td><td></td></tr><tr><td>33 G, Small Hub RN Needle, (Point Style: 3, Needle Length: 19.25 mm)</td><td>Hamilton</td><td>7803-05</td><td></td></tr><tr><td>Antibiotic-Antimycotic solution</td><td>Cellgro</td><td>30004CI</td><td>100x</td></tr><tr><td>C57BL/6-Tmem119em2Gfng/J</td><td>The Jackson Laboratory</td><td>Strain #:031823</td><td></td></tr><tr><td>Corning BioCoat Gelatin 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&amp;nbsp;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>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>EndoFree Plasmid Maxi Kit (10)</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>Eppendorf&amp;nbsp;Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes</td><td>Life Technologies</td><td>22600028</td><td></td></tr><tr><td>Feather Double Edge Carbon Steel Blades</td><td>Ted Pella, inc.</td><td>121-9</td><td></td></tr><tr><td>Fisher BioReagents&amp;nbsp;Microbiology Media: LB Broth, Miller</td><td>Fisher Scientific</td><td>BP1426-500</td><td></td></tr><tr><td>Human recombinant anti-CD11b antibody</td><td>Miltenyi Biotec</td><td>130-120-214</td><td>1:50 dilution</td></tr><tr><td>Intraocular Injector Syringe (6.5 &amp;micro;L), Removable Needle</td><td>Hamilton</td><td>6609071-01</td><td></td></tr><tr><td>Invitrogen&amp;nbsp;Lipofectamine 3000 Transfection Reagent&amp;nbsp;</td><td>Life Technologies</td><td>L3000015</td><td></td></tr><tr><td>Invitrogen&amp;nbsp;One Shot TOP10 Electrocomp&amp;nbsp;E. coli</td><td>Fisher Scientific</td><td>C404052</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&amp;nbsp;</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>PELCO easiSlicer&amp;nbsp;Vibratory Tissue Slicer</td><td>Ted Pella, inc.</td><td>11000</td><td></td></tr><tr><td>PELCO&amp;nbsp;Pro CA44 Tissue Adhesive</td><td>Ted Pella, inc.</td><td>10033</td><td></td></tr><tr><td>PELCO&amp;nbsp;Pro Specimen Retrievers</td><td>Ted Pella, inc.</td><td>101-33</td><td></td></tr><tr><td>ProLong Gold</td><td>Fisher Scientific</td><td>P36930</td><td></td></tr><tr><td>Rabbit monoclonal anti-Iba1 antibody</td><td>Abcam</td><td>ab178847</td><td>1:500 dilution</td></tr><tr><td>RN Compression Fitting 1 mm</td><td>Hamilton</td><td>55750-01</td><td></td></tr><tr><td>Sevoflurane</td><td>Merritt Veterinary Supply</td><td>347075</td><td></td></tr><tr><td>Sprague Dawley pregnant rat</td><td>Inotiv</td><td></td><td></td></tr><tr><td>SuperFrost Plus Slides</td><td>Fisher Scientific</td><td>12-550-154%&amp;nbsp;</td><td></td></tr><tr><td>To-Pro-3</td><td>ThermoFisher Scientific</td><td>T3605</td><td>Nucleus staining kit</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>ThermoFisher Scientific</td><td>25200-056</td><td></td></tr><tr><td>Vannas Scissors</td><td>World Precision Instruments</td><td>500086</td><td></td></tr></tbody></table>

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.

A fragment of mScarlet (1858 bp) containing enzyme sites of &#34;Cla1&#34; at the 3&#39; end and &#34;Not1&#34; at the 5&#39; end was inserted into pNL4-3-EcoHIV lentiviral vector (Figure 1). To validate the expression of EcoHIV-mScarlet, primary brain cells isolated from the cortex of rat E18 embryos were treated with EcoHIV-mScarlet (60 &#181;L, 1.26 &#215; 106 TU/mL) for 6 days in vitro. The data in Figure 2 showed that red fluorescent signals of mScarlet were mainly localized in glial types of cells based on the different cell morphology. Furthermore, CD11b/c and Iba1 (cell markers for microglia) labeling showed that mScarlet signals were co-localized with CD11b/c + and/or Iba1 + cells. The data indicated that microglia were the major cell type of EcoHIV-mScarlet distribution in vitro. There was no significant neuronal infection in the cultured cells (Supplementary Figure 1). 
Next, the infection of EcoHIV-mScarlet was tested on adult mouse primary mixed glial cells. To do so, mixed glia cells were first isolated and purified from adult mice (2 months) and infected with EcoHIV-mScarlet (8 &#181;L, 1.26 &#215; 106 TU/mL) for 2 days. The images in Figure 3 showed that EcoHIV-mScarlet successfully infected adult mouse glia.
To further address the distribution pattern of EcoHIV-mScarlet in the mouse brain, specifically for identifying the infected cell type, EcoHIV-mScarlet was retro-orbital injected into Tmem119-EGFP knock-in mouse line in which microglia were specifically labeled with EGFP signals without any other macrophage types of conflicts5. The results in Figure 4 (also in Supplementary Figure 2) show that mScarlet red fluorescence signals were observed in EGFP-positive cells, suggesting microglia as the major cell type of EcoHIV expression in mouse brains.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67150/67150fig01.jpg" /> 
Figure 1: The vector map of EcoHIV-NL-4-3-mScarlet. <a href="https://www.jove.com/files/ftp_upload/67150/67150fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67150/67150fig02.jpg" /> 
Figure 2: The EcoHIV-mScarlet infection in primary rat brain cells. (A,E) Representative images of CD11b/c staining at primary brain cells. The brain cells were isolated from E18 rat embryos and infected with the EcoHIV-mScarlet virus for 6 days. (B,F) Representative images of mScarlet fluorescent signals from in vitro brain cells. (C,G) Representative images of Iba1 staining at primary brain cells. (D,H) Merged images of triple labeling of CD11b/c, mScarlet, and Iba1. Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67150/67150fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67150/67150fig03.jpg" /> 
Figure 3: EcoHIV-mScarlet infection in primary mouse mixed glial cells. (A,B) Representative confocal images of mScarlet distribution in vitro. The primary mixed glia cells were isolated from adult C57BL6 mice (2-month-old) and cultured for 2 weeks before viral infection. The EcoHIV-mScarlet was added into the culture medium for two days and captured the images under the 60&#215; objective of the Confocal microscope. Scale bar: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67150/67150fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67150/67150fig04.jpg" /> 
Figure 4: EcoHIV-mScarlet distribution in Tmem119-EGFP knock-in mouse line. (A,B) Merged images of mScarlet/EGFP/ToPro3 signals in brain sections. The yellow frame indicates the target area of (B). Scale bar: 300 &#181;m. (C) Representative image of mScarlet distribution in the external plexiform layer of the olfactory region of Tmem119-EGFP knock-in mouse line. (D) Representative image of EGFP distribution. The fluorescent signals were localized in microglia cells in the Tmem119-EGFP knock-in mouse line. (E) Representative image of TO-PRO-3 nucleus staining. Scale bars: 300 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67150/67150fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: MAP2 and MOG staining of primary rat brain cells infected with EcoHIV-mScarlet virus. Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67150/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Confocal images of EcoHIV-mScarlet infection in Tmem119-EGFP knock-in mouse line. Scale bars: 75 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67150/Supplementary Figure 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 1: EcoHIV-mScarlet plasmid DNA sequence. <a href="https://www.jove.com/files/ftp_upload/67150/Supplementary data - EcoHIV-mScarlet plasmid DNA sequence.zip" target="_blank">Please click here to download this File.</a>

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

identification-ecohiv-infected-cells-microglia-manipulated-transgenic

Research

JoVE Journal

Neuroscience

1.2K Views.  University of South Carolina. 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.

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

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 ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Immunology and Infection

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Medicine

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 ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

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.

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. ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

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

Podsumowanie

Przeglądaj więcej filmów

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

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

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

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

A Rat Model of EcoHIV Brain Infection

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route