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...

<ol>
	<li>Dreher, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+Disease+and+Outcome+in+Children+with+Symptomatic+Congenital+Cytomegalovirus+Infection.">Spectrum of Disease and Outcome in Children with Symptomatic Congenital Cytomegalovirus Infection.</a> J of Pediatr. 164 (4), 855-859 (2014).</li><li>Lanzieri, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+outcomes+of+children+with+symptomatic+congenital+cytomegalovirus+disease.">Long-term outcomes of children with symptomatic congenital cytomegalovirus disease.</a> J of Perinatol. 37 (7), 875-880 (2017).</li><li>Naseer, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+WDR62+mutation+causes+primary+microcephaly+in+a+large+consanguineous+Saudi+family.">A novel WDR62 mutation causes primary microcephaly in a large consanguineous Saudi family.</a> Ann Saudi Med. 37 (2), 148-153 (2017).</li><li>Abuelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcephaly+Syndromes.">Microcephaly Syndromes.</a> Semin Pediatr Neurol. 14 (3), 118-127 (2007).</li><li>Nicholas, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WDR62+is+associated+with+the+spindle+pole+and+is+mutated+in+human+microcephaly.">WDR62 is associated with the spindle pole and is mutated in human microcephaly.</a> Nat Genet. 42 (11), 1010-1014 (2010).</li><li>Pulvers, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+mouse+Aspm+(abnormal+spindle-like+microcephaly+associated)+cause+not+only+microcephaly+but+also+major+defects+in+the+germline.">Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline.</a> Proc Natl Acad Sci U S A. 107 (38), 16595-16600 (2010).</li><li>Shao, Q., Herrlinger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+infection+disrupts+neurovascular+development+and+results+in+postnatal+microcephaly+with+brain+damage.">Zika virus infection disrupts neurovascular development and results in postnatal microcephaly with brain damage.</a> Development. 143 (22), 4127-4136 (2016).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Disrupts+Neural+Progenitor+Development+and+Leads+to+Microcephaly+in+Mice.">Zika Virus Disrupts Neural Progenitor Development and Leads to Microcephaly in Mice.</a> Cell Stem Cell. 19 (5), 672 (2016).</li><li>Miki, T., Fukui, Y., Takeuchi, Y., Itoh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+study+of+the+effects+of+prenatal+X-irradiation+on+the+development+of+cerebral+cortex+in+rats.">A quantitative study of the effects of prenatal X-irradiation on the development of cerebral cortex in rats.</a> Neurosci Res. 23, 241-247 (1995).</li><li>Cragan, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-based+microcephaly+surveillance+in+the+United+States,+2009+to+2013:+An+analysis+of+potential+sources+of+variation.">Population-based microcephaly surveillance in the United States, 2009 to 2013: An analysis of potential sources of variation.</a> Birth Defects Res Part A Clin Mol Teratol. 106 (11), 972-982 (2016).</li><li>Cragan, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baseline+Prevalence+of+Birth+Defects+Associated+with+Congenital+Zika+Virus.">Baseline Prevalence of Birth Defects Associated with Congenital Zika Virus.</a> MMWR Morb Mortal Wkly Rep. 66 (8), 219-220 (2017).</li><li>Mlakar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Associated+with+Microcephaly.">Zika Virus Associated with Microcephaly.</a> N Engl J Med. 374 (10), 951-958 (2016).</li><li>Jaenisch, T., Rosenberger, D., Brito, C., Brady, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+microcephaly+after+Zika+virus+infection+in+Brazil,+2015+to+2016.">Risk of microcephaly after Zika virus infection in Brazil, 2015 to 2016.</a> Bull World Health Organ. 95 (3), 191-198 (2017).</li><li>Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+developmental+time+across+mammalian+species.">Translating developmental time across mammalian species.</a> Neuroscience. 105 (1), 7-17 (2001).</li><li>Driggers, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Infection+with+Prolonged+Maternal+Viremia+and+Fetal+Brain+Abnormalities.">Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain Abnormalities.</a> N Engl J Med. 374 (22), 2142-2151 (2016).</li><li>Semple, B. D., Blomgren, K., Gimlin, K., Ferriero, D. M., Noble-Haeusslein, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+development+in+rodents+and+humans:+Identifying+benchmarks+of+maturation+and+vulnerability+to+injury+across+species.">Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species.</a> Prog Neurobiol. 106, 1-16 (2013).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Infects+Neural+Progenitors+in+the+Adult+Mouse+Brain+and+Alters+Proliferation.">Zika Virus Infects Neural Progenitors in the Adult Mouse Brain and Alters Proliferation.</a> Cell Stem Cell. 19 (5), 593-598 (2016).</li><li>Vermillion, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrauterine+Zika+virus+infection+of+pregnant+immunocompetent+mice+models+transplacental+transmission+and+adverse+perinatal+outcomes.">Intrauterine Zika virus infection of pregnant immunocompetent mice models transplacental transmission and adverse perinatal outcomes.</a> Nat. Commun. 8, 14575 (2017).</li><li>Goodfellow, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Induced+Mortality+and+Microcephaly+in+Chicken+Embryos.">Zika Virus Induced Mortality and Microcephaly in Chicken Embryos.</a> Stem Cells Dev. 25 (22), 1-27 (2016).</li><li>Dick, G. W. A., Kitchen, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+(I).+Isolations+and+serological+specificity.">Zika Virus (I). Isolations and serological specificity.</a> Trans R Soc Trop Med Hyg. 46 (5), 509-520 (1952).</li><li>Shao, Q., Herrlinger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+African+Zika+virus+MR-766+is+more+virulent+and+causes+more+severe+brain+damage+than+current+Asian+lineage+and+Dengue+virus.">The African Zika virus MR-766 is more virulent and causes more severe brain damage than current Asian lineage and Dengue virus.</a> Development. , (2017).</li></ol>

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 ...

The overall goal of the surgical methods presented here is to establish in vivo models of intracranial Zika virus infection. This method can help to address the key question in the field, such as pathology and the mechanism of Zika virus infection in the brain at both early and the later developmental stage as well as adult stage. The main advantage of this technique is that the direction infection of the brain can easily be achieved.The implications of this technique extend toward therapies or diagnoses of Zika virus-induced microcephaly or other neuro-developmental disorders. Although this method can provide insight into Zika virus pathogenesis, it can also be applied to study other flavivirus infection in the brain, such as dengue virus. Just prior to surgery, load the Zika virus injection mix into a prepared glass injection needle by first assembling the gas-tight 50-microliter injection syringe Luer-lock attachment and tubing.Saturate the assembled syringe and tubing with mineral oil. Once saturated, attach the needle and draw approximately six to seven microliters of virus into the needle. After anesthetizing a dam pregnant with E14.5 embryos, test the depth of anesthesia by pinching the toe or tail.When a surgical plane of anesthesia is achieved, insure that the dam is laying supine on a heating pad. Shave the surface of the abdomen and sterilize with iodine and alcohol three times. Drape the skin surrounding the surgical site with sterile cloths to avoid contamination of the incision and the instruments.Pinch and lift the skin of the abdomen with forceps, then use sterile scissors to make a 1 to 1 1/2-centimeter cut in the lower abdomen at the medial-sagittal line and expose the embryos. Next, cut a slit in the middle of a small sterile gauze and place over the surgical opening. Hydrate the opening and gauze with sterile saline.Then, pull the embryos through the slit to rest on the gauze, taking care to not remove more than four or five embryos at once. Hydrate the embryos with sterile saline prior to and throughout the inoculation to ensure they do not dry out. Illuminate the embryo to be injected with a lamp to visualize the head and skull sutures, then gently place a spatula under the head of the embryo to be injected and manipulate the embryo until the head is pushed up directly against the uterine wall.Hold the embryo in place with the non-dominant hand. Use the syringe to inject Zika virus or control medium into the lateral ventricles of the embryo brain. For the E11.5 stage intrauterine injection, use a one-milliliter syringe and a 27 gauge needle to inject Zika virus or a control medium into the intrauterine space.To improve retention of the pregnancy, avoid injecting the two embryos next to the ovaries and the two embryos next to the upper vagina. If performing an intrauterine injection, inject into the intrauterine space or into the placenta. Place the embryos back into the pregnant dam and fill the abdominal cavity with approximately 0.5 milliliters of sterile saline.Suture both the abdominal peritoneal muscle and external skin layer with 4-0 sterile sutures. Keep the dam on a heating pad and monitor while recovering from anesthesia. Thaw the virus on ice, then load the syringe by filling it with saline to reduce the dead volume in the syringe.Draw up to 0.75 microliters of air to separate the saline solution from the injection material. To set up a warmed, humidified recovery chamber, place a closable container with a sterile gauze on a heat box set at 37 degrees Celsius and soak with saline. After setting up the microinjector pump and controller, load the injection syringe with virus and sterilize the pump head mount of the surgical setup with 70%ethanol.Then, after cryo-anesthetizing the neonatal pups for five minutes, ensure sufficient anesthesia for injection by the lack of response to a tail or toe pinch. Place the pup on the head mount. Sterilize the surface of the head with iodine then ethanol, and then wipe dry with an alcohol wipe.Mark lambda with a black marker, then measure the distance from lambda to the edge of the eye and, using stereotaxic coordinates, calculate the 2/5 distance from lambda to the eye. Next, use a 26 gauge needle to carefully and superficially puncture the scalp and skull at the injection location and create an opening for the injection needle. Now lower the injection needle into the puncture site and, once the needle has just breached the skin, calculate a one-millimeter depth for injection into the lateral ventricle using the stereotaxic instrument.Once the needle is lowered to the injection depth, inject one microliter of virus at a rate of 10 nanoliters per second. Wait 30 seconds after injection, and then retract the needle in 0.5-millimeter increments by rotating the screw dorsally, waiting 30 seconds after each increment to reduce leakage of injected virus. After finishing the injection, transfer the pup to the pre-warmed, humidified chamber and monitor until recovered.Immobilize the head of an anesthetized adult mouse in the stereotaxic instrument and place a heating pad under the mouse. After a toe or tail pinch to insure a surgical plane of anesthesia, shave the scalp starting behind the eyes to the beginning of the ears. Sterilize the exposed skin with iodine and 70%ethanol.After making a 0.5-centimeter incision along the medial-sagittal line of the head in the sterilized location to expose bregma, clear the surface of the skull of meningeal tissues while simultaneously pushing the skin away from the injection sites. Align the surgical drill bit with bregma, and then identify the injection site by the stereotaxic coordinates. Using the control foot pedal, slowly drill into the skull until the hole is clear for the needle.Replace the drill bit with the microinjector pump and gas-tight 10-microliter syringe. Leave a small air bubble between the saline solution and the virus and draw up four to five microliters of virus. Lower the needle to 1.5 millimeters below the brain surface from the brain surface and inject one microliter of virus at a rate of 10 nanoliters per second, injecting the one microliter over 1 1/2 minutes.After removing the needle from the brain in increments as before, use forceps to loosen the skin and re-cover the exposed skull. Once the scalp has been sutured with 4-0 sutures, remove the mouse from the stereotax and monitor on a heating pad until recovered. Neuro-progenitor cells are immuno-labeled with Pax6, seen in red, and Zika virus via flavivirus antigens, seen in green, four days after injection at E18.5.The embryonic brain is infected with the MEX1-44 strain of Zika virus across all layers of the developing neocortex. The TCID50 assay using tissue from post-natal day three brain tissue inoculated at E14.5 shows that the Zika virus can efficiently replicate and grow in developing brains. This image shows MEX1-44 Zika virus infection at P13 cerebral cortex after P0 injection.Zika is detected using antibody against flavivirus group antigen seen in red. These images show the fluorescent beads that were used to practice injection location and lateral ventricle injection success in adults. While attempting this procedure, it's important to remember to keep track of the embryos injected, have appropriate controls, and keep the surgical wounds small.Following this procedure, other methods like intraperitoneal or arterial injection can be performed to answer the additional question like how Zika virus cross the placenta. After watching this video, you should have a good understanding of how to generate the in vivo mouse model of Zika virus infection in the brain.

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Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...