Name: establishing mouse models for zika virus-induced neurological disorders using intracerebral injection strategies: embryonic, neonatal, and adult
Uploaded: 2018-04-26T13:00:00
Description: Watch this Scientific Journal Video about Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult at JoVE.com

The authors would like to acknowledge Dr. Abdellatif Benraiss at the University of Rochester for his mentorship and discussions relevant to learning the adult surgical and neonate techniques. The authors would also like to acknowledge Dr. James Lauderdale at UGA for the use of his stereotaxic equipment and discussions related to setting up the methodology for this technique, and the Advancement for Research College Scientists (ARCS) foundation for their support and our NIH support (NINDS grants R01NS096176-02, R01NS097231-01, &#38; F99NS105187-01).

All animal use protocols follow the animal care guidelines of the University of Southern California and the University of Georgia.&#160;Euthanasia methods for pregnant dams and adults are performed according to approved protocols: carbon dioxide asphyxiation, followed by cervical dislocation as a secondary method to ensure euthanasia. Neonatal pups are euthanized by decapitation.
Caution: The following protocol involves handling a pathogenic virus. Proper precaution should be taken while handl...

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Described here is a method for intracerebral inoculation of the ZIKV at embryonic, neonatal, and adult stages for the investigation of ZIKV-induced damages in brain development. While straightforward, there are a few considerations that investigators should take to ensure the quality of the study and the safety of those involved.
DENV is closely related to ZIKV in the flavivirus genus. DENV has not been causally linked with pediatric brain disorders in humans. DENV2 can successfully infect and...

Microcephaly is a condition resulting from defective brain development characterized by smaller than average head size in newborns. Children with microcephaly exhibit a range of symptoms which can include developmental delay, seizure, intellectual disability, hearing loss, vision problems, and problems with movement and balance, among others, depending on the severity of the disease and cause1,2,3. This condition is multifactorial in nature, with genetic, infectious agent, and environmental factors linked to causing microcephaly4,5,6,7,8,9. Prior to the 2015 - 2016 ZIKV outbreak, 8 children out of 10,000 births were diagnosed with microcephaly in the United States according to the CDC10. On February 1st of 2016 the World Health Organization declared the Zika virus a Public Health Emergency of International Concern due to the alarming increase in microcephaly diagnoses associated with ZIKV infection in mothers11,12. A recent study from the CDC on ZIKV cases in the United States suggests that maternal ZIKV infection results in a 20-fold increased risk for a child to develop microcephaly compared to uninfected individuals, and 4% of ZIKV infected mothers from the USA have resulted in children with microcephaly11. The rate of microcephaly-associated birth defects during pregnancy from ZIKV infection in Brazil have been reported to have affected up to 17% of babies in infected mothers, indicating that other factors in Latin America may be contributing to the increased risk13. While we know that the ZIKV can cause microcephaly and pathogenesis in the neural progenitor cell (NPC) population7,8,14, the complete pathogenesis of ZIKV in the developing brain remains elusive. It is important to develop animal models to further investigate the disease mechanisms underlying the brain abnormalities associated with ZIKV infection.
To directly study the effect that the ZIKV has on brain development, we first developed a mouse model using intracerebral inoculation of embryonic day 14.5 (E14.5) brain with ZIKV7. This stage was chosen as it is considered representative of the end of the first trimester in human gestation14. Pups can survive up to postnatal day 5 (P5) with this embryonic intracerebral injection method (~ 1 &#181;L of 1.7 x 106 tissue culture infective dose (TCID50/mL)). These postnatal pups exhibit a range of phenotypes similarly observed in infected human infants including enlarged ventricles, neuronal loss, axonal rarefaction, astrogliosis, and microglial activation12,15. A newborn mouse brain is relatively immature, akin to the developmental stage of the human brain at mid-gestation16, and mouse brain development includes a major postnatal component. To study later gestation stage infections, a method for postnatal infection is also described. Neonates infected with ZIKV at P1 are able to survive up to 13 days post-injection. Blood-born adult stage infection has been described in the mouse previously17 but requires the use of the interferon (IFN) regulatory factor (IRF) transcription factors IRF-3, -5, -7 triple knockout strain. This protocol describes a method of inoculating ZIKV intraventricularly to circumvent disabling the antiviral response of the murine model in the adult. While this circumvents the murine immune system, this route of injection does not directly mimic the typical route of infection. To address this discrepancy directly, the experimenter can perform an intrauterine infection of ZIKV instead of the intracranial route. Adopted from previous work18, we have briefly described this technique in this embryonic infection protocol.
The Zika virus strains implemented with this technique include the Mexican isolate MEX1-447,19 and the African isolate MR-766 isolated in 194720. Zika MEX1-44 was isolated in Chiapas, Mexico in January of 2016 from an infected Aedes aegypti mosquito. We obtained this virus with permission through the University of Texas Medical Branch at Galveston (UTMB). In addition, the Dengue virus serotype 2 (DENV2) was inoculated using this technique in a comparison study. DENV2, strain S16803 (sequence GenBank GU289914), was isolated from a patient sample from Thailand in 1974 and passaged in C6/36 cells. The virus was passaged twice in Vero cells by the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) before mouse injections. This demonstrates that this technique works equally well for diverse strains of ZIKV and other flaviviruses that may have an impact on brain development.

<table>
	<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>Fluoro-Max: Red Fluorescent Microspheres</td>
			<td>Thermo Scientific</td>
			<td>R25</td>
			<td>No dilution; Use for practice injections</td>
		</tr>
		<tr>
			<td>10 &#181;L, Model 1701 LT SYR</td>
			<td>Hamilton</td>
			<td>80001</td>
			<td>for embryonic inoculation</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>
		<tr>
			<td>Fastgreen FCF Dye</td>
			<td>Sigma</td>
			<td>F7252</td>
			<td>inject with 0.5% Dye</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flavivirus group antigen antibody</td>
			<td>Millipore</td>
			<td>MAB10216</td>
			<td>ms IgG2a 1:400 (Figure 2, Figure 3)</td>
		</tr>
		<tr>
			<td>Pax6</td>
			<td>DBHB</td>
			<td>Pax6-s</td>
			<td>ms IgG1 1:20</td>
		</tr>
	</tbody>
</table>

establishing mouse models for zika virus-induced neurological disorders using intracerebral injection strategies: embryonic, neonatal, and adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Representative images of our injection methods for the ZIKV inoculation of embryonic brain are shown in diagrams depicting intracerebral injections (Figure 1A) and intrauterine and intraplacental injections (Figure 1B), illustrating the way the pregnant dam and embryos should be viewed and oriented for surgery (embryonic inoculation protocol). Figure 2A exhibits ZIKV (MEX1-44) infection (immunostaine...

Watch this Scientific Journal Video about Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult at JoVE.com

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

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