Name: a rat model of ecohiv brain infection
Uploaded: 2021-01-21T14:30:00.000+00:00
Duration: 8 min 48 s
Description: Watch this Scientific Journal Video about A Rat Model of EcoHIV Brain Infection at JoVE.com

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 synaptic dysfunction in MSNs of the NAc. Given the importance of animal models for the study of neuroHIV and HAND2, the development of a new biological system may be advantageous for addressing novel questions within the field. Potash et al.3 first reported the use of EcoHIV to induce active HIV-1 infection in mice. Specifically, mice were inoculated with a 0.1 mL of solution of EcoHIV virus through tail vein injections3. Six weeks after infection, the HIV-1 viral DNA was detected in spleen cells and lymphocytes. Additionally, one inoculation of EcoHIV injection was sufficient to induce infection in more than 75% of the mice. Utilization of bilateral stereotaxic injections, as in this study, successfully infected 100% of the rats (n = 6 or n = 4, respectively) evidenced by the detection of EcoHIV-EGFP in the brain seven days post-injection.
Previous studies have shown that infection by EcoHIV in the mouse strongly implicated that brain microglia are highly susceptible to EcoHIV virus infection3. In this study, combined with Iba1 (a microglial cell marker) immunostaining, a significant co-localization of EGFP signal with Iba1+ cells was observed, strongly suggesting that microglia were the major cell type for EcoHIV infection in rat brain. Observations of significant EcoHIV infection in microglia are consistent with data in EcoHIV infected mice6, as well as HIV-1 seropositive individuals15 and other biological systems commonly utilized to model HIV16,17. We also performed retro-orbital injection of EcoHIV-EGFP into F344/N rats and the data also indicated high expression of EcoHIV-EGFP in both the cortex and hippocampal dentate gyrus after only seven days (data not shown). In contrast, I.P. injection of EcoHIV in F344/N rats led to undetectable viral expression in the rat brain, inspite of high doses of EcoHIV lentivirus.
Regarding this protocol, researchers should ensure that the conditioned medium, including the EcoHIV lentivirus packaging in 293 FT cells, is concentrated and titered before using for stereotaxic injection. These steps are critical to ensuring consistent and replicable results across experiments. Furthermore, we also found that EcoHIV infection was propagated from stereotaxic injection in brain to spleen tissue at 8 weeks after injection. Meanwhile, the temporal processing deficits were observed in EcoHIV-infected rats as early as 14 days and maintained through 8 weeks post-infection (the current experimental terminal time, Figure 2). Compared to the generalized EcoHIV infection model in mice, the more specific regional stereotaxic injection of EcoHIV produced efficient HIV infection in brain areas and produced a temporal processing deficit in rats, which is key for the study of HIV associated neurocognitive dysfunction.
Limitations providing further opportunities for the current protocol include the identification of other cell types such as neurons and astrocytes, a time-course study of changes in EcoHIV-EGFP expression after infection to indicate viral replication and latency in brain, a longitudinal study to address the potential impacts of HIV associated neurocognitive deficits, and evaluating if the stereotaxic injection of EcoHIV evades the immune system further spreading viral expression throughout the body. Additionally, this should be tested on other rat strains to confirm the generalizability of EcoHIV infection in rats.

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. Furthermore, dual-labeling with Iba1and EcoHIV-EGFP probes provided strong evidence that microglia were the predominant cell type harboring EcoHIV expression in the brain (Figure 1B). Notably, the distribution pattern of EcoHIV-EGFP is consistent with the relative regional concentrations of microglia in the rat brain (i.e., cortex and dentate gyrus of the hippocampus).
In a subsequent study, we validated the utility of EcoHIV infection in rats to model key aspects of HAND. Using the protocol detailed above, F344/N rats were stereotaxically injected with either EcoHIV-EGFP or saline. First, eight weeks post-infection, temporal processing, a potential elemental dimension of HAND12, was evaluated using visual gap prepulse inhibition (Figure 2). EcoHIV animals exhibited a relative insensitivity to the manipulation of interstimulus interval (ISI), evidenced by a relatively flatter ISI function compared to saline controls. Specifically, significant differences in the slope of the ISI function from the 50 ms ISI to the 200 ms ISI were observed (Semilog Line-X is Log, Y is Linear, R2s &#8805; 0.99; F(1,2)=642.9, p&#8804;0.001). Second, ballistic labeling was used to investigate the impact of EcoHIV-EGFP injections on the morphology of dendritic spines in medium spiny neurons (MSN) of the nucleus accumbens (NAc; Figure 3); parameters which can be utilized to draw inferences about synaptic function13. EcoHIV rats displayed profound alterations in dendritic spine morphology, evidenced by an increased relative frequency of shorter dendritic spines (Genotype x Bin Interaction, F(16, 218) = 4.3, p &#8804; 0.001) with an increased head diameter (Genotype x Bin Interaction, F(12, 96) = 18.7, p &#8804; 0.001) and increased neck diameter (Genotype x Bin Interaction, F(15, 120) = 16.3, p &#8804; 0.001) relative to control animals. Detailed methodology for the assessment of temporal processing14 and ballistic labeling13 have been previously reported.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62137/62137fig01.jpg" /> 
Figure 1. The EcoHIV-EGFP infected cells distributed in rat brain. (A) The representative confocal images (20x) of EcoHIV-EGFP expression in hippocampal dentate gyrus or cortex regions at 7 days after injection. (B) The representative confocal images (60X) of co-localization of Iba1 immunostaining with EcoHIV-EGFP infected cells at 7 days after injection. <a href="https://www.jove.com/files/ftp_upload/62137/62137fig01large.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/62137/62137fig02.jpg" /> 
Figure 2. EcoHIV infection induced prominent neurocognitive deficits in temporal processing. Visual gap prepulse inhibition was conducted eight weeks after stereotaxic injections of either EcoHIV or saline. EcoHIV infection induced prominent alterations in temporal processing evidenced by the relative insensitivity to the manipulation of interstimulus interval relative to control rats. Detailed methodology described in McLaurin et al.13. <a href="https://www.jove.com/files/ftp_upload/62137/62137fig02large.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/62137/62137fig03.jpg" /> 
Figure 3. Infectivity with EcoHIV-EGFP altered the morphological parameters of dendritic spines, supporting profound synaptic dysfunction. EcoHIV rats displayed profound alterations in dendritic spine morphology, evidenced by an increased relative frequency of shorter dendritic spines (A) with an increased head diameter (B) and increased neck diameter (C) relative to control animals. <a href="https://www.jove.com/files/ftp_upload/62137/62137fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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JoVE Journal

Neuroscience

3.1K Views.  University of South Carolina. Met behulp van ratten kan dit protocol worden gebruikt om een nieuw model van actieve hiv-infectie met chimerische hiv vast te stellen. met ratten kan de vaststelling van het EcoHIV-infectiemodel voor studies naar drugsmisbruik en neurocognitieve aandoeningen gunstig zijn voor de studie van neuro-hiv en hiv-1 geassocieerde neurocognitieve aandoeningen. De demonstratieprocedure is Hailong Li, een onderzoeksmedewerker van mijn laboratorium.Voor het verpakken van het virus in 293 T-cellen...

Video: A Rat Model of EcoHIV Brain Infection

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biology

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Immunology and Infection

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Medicine

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop epilepsy as a consequence, with rates being significantly higher in less economically developed countries. This work provides an overview of modeling epilepsy of infectious etiology and using it as a platform for novel antiseizure compound testing. A protocol of epilepsy induction by non-stereotactic intracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice is presented, which replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. The clinical evaluation of mice during encephalitis to monitor seizure activity and detect the potential antiseizure effects of novel compounds is described. Furthermore, histopathological consequences of viral encephalitis and seizures such as hippocampal damage and neuroinflammation are shown, as well as long-term consequences such as spontaneous epileptic seizures. The TMEV model is one of the first translational, infection-driven, experimental platforms to allow for the investigation of the mechanisms of epilepsy development as a consequence of CNS infection. Thus, it also serves to identify potential therapeutic targets and compounds for patients at risk of developing epilepsy following a CNS infection.

Intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. This paper describes the virus infection, symptoms, and histopathology of the TMEV model.

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop ...

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/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.

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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