an intracerebroventricular injection to analyze murine cytomegalovirus distribution in a neonatal mouse brain

An Intracerebroventricular Injection to Analyze Murine Cytomegalovirus Distribution in a Neonatal Mouse Brain

Begin by sterilizing a 10-microliter syringe and a 27-gauge needle with 70% alcohol. Then, load 5 microliters of murine cytomegalovirus injection solution or fluorescent microbeads into the needle by carefully pulling the plunger of the syringe.
After anesthetizing and immobilizing a P0.5 neonatal mouse by placing it on ice for three to four minutes, use the toe-pinch response method to determine the depth of anesthesia. Mark the injection site on the anesthetized mouse with a nontoxic laboratory pen at a location approximately 0.7 to 1.0 millimeters lateral to the sagittal suture, and 0.7 to 1.0 millimeters caudal from the neonatal bregma. This diagram shows the site in more detail.
For reference, mark 2 millimeters from the tip of the needle with a nontoxic marker. Next, insert the needle 2 millimeters deep, perpendicular to the skull surface at the marked injection site. Then, slowly inject 5 microliters of murine cytomegalovirus into the lateral ventricle without opening the scalp.
In another group of mice, inject a 5-microliter solution containing fluorescent microbeads by the same method. After injection, allow the needle to remain in place for 10 to 20 seconds to prevent backflow. Then, slowly remove the needle. Allow the injected animals to recover for 5 to 10 minutes in a warm container until movement and general responsiveness are restored.

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Universidad San Sebastian

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JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Infectious Diseases

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1. ICV Injection of MCMV and Fluorescent Microbeads into Neonatal Mice
<ol>
	<li>Maintain normal pregnant ICR mice in a temperature-controlled facility under a 12 hr light/dark cycle. The neonates are designated as P 0.5 on the day of birth.</li>
	<li>Sterilize a 10-&#181;l syringe and a 27 G needle with 70% alcohol.</li>
	<li>Load the injection solution (5 &#181;l) containing MCMV or fluorescent microbeads into the needle by carefully pulling the plunger of the syringe.</li>
	<li>Restrain neonatal mouse (P 0.5) by putting the mouse on ice for 3 - 4 min. Once the animal is under anesthesia, use the toe-pinch response method to determine the depth of anesthesia.</li>
	<li>Mark the injection site with a non-toxic laboratory pen at a location approximately 0.7 - 1.0 mm lateral to the sagittal suture and 0.7 - 1.0 mm caudal from the neonatal bregma (Figure 1A).</li>
	<li>Insert the needle 2 mm deep, perpendicular to the skull surface at the marked injection site. For reference, mark 2 mm from the tip of needle with a non-toxic maker.</li>
	<li>Slowly inject 5 &#181;l of MCMV (approximately 5 &#215; 105 PFU) into the lateral ventricle without opening the scalp (Figure 1B).</li>
	<li>In another group of mice, inject a 5 &#181;l solution containing fluorescent microbeads (0.1 - 0.3 &#181;m, approximately 5.75 &#215; 108 particles) by the same method.</li>
	<li>Slowly remove the needle 10 - 20 sec after discontinuing the plunger movement to prevent backflow.</li>
	<li>To recover, keep the mice for 5 - 10 min in a warm container until movement and general responsiveness are restored.</li>
	<li>Harvest the brains at a range of time points (3, 12, 24, 48, and 72 hr) post-injection.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SPHERO TM Fluorescent Polystyrene Nile Red 0.04-0.06</td>
			<td>Spherotech, Inc.</td>
			<td>FP-00556-2</td>
			<td></td>
		</tr>
		<tr>
			<td>SPHERO TM Fluorescent Polystyrene Nile Red 0.1-0.3</td>
			<td>Spherotech, Inc.</td>
			<td>FP-0256-2</td>
			<td></td>
		</tr>
		<tr>
			<td>SPHERO TM Fluorescent Polystyrene Nile Red 1.7-2.2</td>
			<td>Spherotech, inc.</td>
			<td>FP-2056-2</td>
			<td></td>
		</tr>
		<tr>
			<td>10% mouse serum</td>
			<td>DAKO</td>
			<td>X0910</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mouse</td>
			<td>SLC, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ICR mouse</td>
			<td>SLC, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Modified Microliter Syringes (7000 Series)</td>
			<td>Hamilton company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35-gauge needle</td>
			<td>Saito Medical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pEGFP-N1 vector</td>
			<td>Clontech</td>
			<td>#6085-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Kawasaki, H., et al. <a href="https://app.jove.com/v/54164">Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain.</a> J. Vis. Exp. (2016).
This video demonstrates the intracerebroventricular injection process for studying murine cytomegalovirus (MCMV) distribution in the neonatal mouse brain. MCMV, engineered to express an enhanced green fluorescent protein (EGFP) for visibility, is injected into the lateral ventricle of a mouse pup&#39;s brain. The injected virus circulates through the cerebrospinal fluid, infecting cells in the periventricular region, including the marginal zone, choroid plexus, and subventricular zone.

<img alt="Figure 1" src="/files/ftp_upload/22973/22973_Figure1.jpg" /> 
Figure 1: Injection Sites of the Neonatal Mouse. (A, B) ICV injection of 5 &#181;l MCMV (approximately 5 &#215; 105 PFU) or of 5 &#181;l microbeads (approximately 5.75 &#215; 109 particles). (A) The injection site is at the midpoint between the ear and eye (at a location approximately 0.7 - 1.0 mm lateral to the sagittal suture and 0.7 - 1.0 mm caudal from t...

1. ICV Injection of MCMV and Fluorescent Microbeads into Neonatal Mice

	Maintain normal pregnant ICR mice in a temperature-controlled facility under a 12 ...

Take a suspension of murine cytomegalovirus, genetically engineered to express an enhanced green fluorescent protein, allowing for visualization.
Load the suspension into a syringe with an attached needle.
Next, anesthetize a mouse pup by placing it on ice.
Identify the injection site on the pup&#39;s head.
Insert the needle perpendicular to the skull surface until it reaches the lateral ventricle, a cerebrospinal fluid, or CSF filled cavity in the brain.
Inject the suspension into the lateral ventricle.
Allow the pup to recover.
The injected virus circulates through the ventricular CSF, reaching the ependymal cells of the marginal area and the epithelial cells of the choroid plexus.
The virus attaches to surface receptors on these cells, internalizes, undergoes replication, and spreads from cell to cell, establishing an acute infection in the choroid plexus, the marginal area, and the subventricular zone.

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Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

科研

JoVE Journal

医学

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

小鼠用树脂叶毒素和疼痛测试进行脑血管内治疗

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

神经科学

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

通过脑室内注射血红蛋白模拟新生儿脑室内出血

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

An Intracerebroventricular Injection to Analyze Murine Cytomegalovirus Distribution in a Neonatal Mouse Brain

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An Intracerebroventricular Injection to Analyze Murine Cytomegalovirus Distribution in a Neonatal Mouse Brain