eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Adult Intracerebral Inoculation of Zika Virus
<ol>
	<li>Inject adult mice intraperitoneally with&#160;ketamine hydrochloride (80-100 mg/kg) and xylazine (5-10 mg/kg)&#160;diluted in sterile saline to induce anesthesia. Inject buprenorphine-SR (0.5-1.0mg/kg) subcutaneously to induce analgesia.&#160;Toe/tail pinch the mouse to ensure its anesthetic state, and subsequently mount it on the stereotaxic instrument (with a heating pad) to immobilize the head during surgery.&#160;</li>
	<li>Add ophthalmic ointment to the eyes to&#160;prevent the eyes of the mouse from drying out during surgery. Shave the scalp starting behind the eyes to the beginning of the ears. Sterilize the exposed skin with iodine and 70% ethanol (EtOH) three times.&#160;Alternate the iodine and alcohol wipes, wipe in a circular motion away from the surgical site.</li>
	<li>Re-check the anesthetic depth prior to incising the skin. Using a scalpel, make a 0.5 cm incision along the medial sagittal line of the head in the sterilized location, exposing&#160;bregma. Using a rolling motion with a cotton-tipped applicator, clear the surface of the skull of meningeal tissues, while simultaneously pushing the skin away from the injection sites.</li>
	<li>Starting from&#160;bregma, align the surgical drill bit and identify the injection sites using the following coordinates: anteroposterior (AP): -0.5 mm, mediolateral (ML): +/-1.5 mm. Using the control foot pedal, drill into skull slowly as to only drill bone until a hole has been cleared for the needle.</li>
	<li>Replace the drill with the microinjector pump and gas-tight syringe (10 &#181;L volume) on the stereotaxic. Leaving a small air bubble between the saline solution and the virus, draw ~ 4-5 &#181;L of virus. Determine the device type code for the specific syringe volume (ie, D for 10 &#956;L volume syringe). Lower the needle to dorsoventral (DV): -1.5 mm from the brain surface. To inject 1 &#181;L of virus, program a rate of 10 nL/s, injecting approximately 1 &#181;L over 1.5 min.</li>
	<li>When the injection finishes, wait 30 s, and remove the needle in 0.5 mm increments, waiting 30 s after each turn. This reduces backflow and leakage of the injected virus.</li>
	<li>When the injection(s) are finished, use forceps to loosen the skin and recover the exposed skull. Suture the scalp back together using 4.0 sutures and&#160;remove the mouse from the stereotaxic instrument.&#160;Place the mouse in a cage partially resting on a heating pad to allow the mouse to escape to a non-heated area if needed and monitor while they recover (1 - 2 h) from anesthesia.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Flexible Drive Shaft Drill Hanging Motor</td>
			<td>Leica</td>
			<td>39416001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Stereotax</td>
			<td>Kopf</td>
			<td>04557R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro4 Microsyringe Pump Controller</td>
			<td>WPI</td>
			<td>SYS-MICRO4</td>
			<td></td>
		</tr>
		<tr>
			<td>UMP3 UltraMicroPump</td>
			<td>WPI</td>
			<td>UMP3</td>
			<td></td>
		</tr>
		<tr>
			<td>Modulamp</td>
			<td>Schott</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-lock tubing (19-gauge)</td>
			<td>Hamilton</td>
			<td>90619</td>
			<td></td>
		</tr>
		<tr>
			<td>Melting Point Capillary</td>
			<td>Kimble</td>
			<td>34500-99</td>
			<td>Glass needle</td>
		</tr>
		<tr>
			<td>10 &#181;L, Model 1701 RN SYR, Small Removable NDL, 26s ga, 2 in, point style 2</td>
			<td>Hamilton</td>
			<td>80030</td>
			<td>for neonate/adult</td>
		</tr>
		<tr>
			<td>4-0 Ethilon Nylon Sutures</td>
			<td>Ethicon</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>VWR</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Grinder</td>
			<td>Narishige</td>
			<td>EG-44</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing a model of zika virus-induced neurodegeneration in an adult mouse

Source: Herrlinger, S. A. et al. <a href="https://app.jove.com/v/56486">Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult.</a> J. Vis. Exp. (2018)
The video demonstrates intracerebral injection of Zika virus suspension in an adult mouse to induce neurodegeneration. An anesthetized mouse is secured in a stereotaxic frame, and a hole is drilled into the exposed skull for injection. A suspension of the Zika virus is injected into the brain tissue. The viral infection causes inflammation, leading to neurodegeneration.

Establishing a Model of Zika Virus-Induced Neurodegeneration in an Adult Mouse

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Adult ...

Take an anesthetized mouse secured in a stereotaxic frame. A heating pad maintains its body temperature.
Shave and sterilize the scalp, make a midline incision to expose the bregma, a landmark on the skull to identify the injection location, and drill a small hole.
Inject a Zika virus suspension into the brain tissue. Slowly withdraw the needle to prevent backflow.
Suture the scalp and transfer the mouse to a cage for recovery.
The virus reaches the subventricular zone, an area rich in neural progenitor cells, or NPCs, and glial cells.
The virus binds to NPC receptors and is endocytosed. Viral replication triggers a cellular stress response, inducing apoptosis.
Microglia, the resident immune cells, engulf the virus and produce oxidative bursts that damage surrounding tissue. They also release cytokines and neurotoxic factors, which recruit cytotoxic T-cells to kill the virus-infected cells.
The resulting depletion of NPCs and glial cells reflects virus-induced neurodegeneration.

establishing-model-zika-virus-induced-neurodegeneration-an-adult

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Infectious Diseases

21 visualizações. Fonte: Herrlinger, S. A. et al. Estabelecendo modelos de camundongos para distúrbios neurológicos induzidos pelo vírus Zika usando estratégias de injeção intracerebral: embrionária, neonatal e adulta. J. Vis. Exp. (2018) O vídeo demonstra a injeção intracerebral da suspensão do zika vírus em um camundongo adulto para induzir a neurodegeneração. Um camundongo anestesiado é preso em uma estrutura estereotáxica e um orifício é perfurado no crânio exposto para injeção. Uma susp...

Vídeo: Estabelecendo um modelo de neurodegeneração induzida pelo vírus Zika em um camundongo adulto - Conceito

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Infecção por vírus Zika do cérebro Fetal humano culta células-tronco neurais para análise de Immunocytochemical

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

JoVE Journal

Biologia do Desenvolvimento

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Avaliação das respostas de células T Zika vírus específicos em órgãos Immunoprivileged de infectados Ifnar1- / - ratos

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Imunologia e Infecção

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

Isolamento e quantificação do vírus Zika de múltiplos órgãos em um mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Establishing a Model of Zika Virus-Induced Neurodegeneration in an Adult Mouse

Transcrição

Establishing a Model of Zika Virus-Induced Neurodegeneration in an Adult Mouse