generation of a murine model with a central nervous system infection

Generation of a Murine Model with a Central Nervous System Infection

Before initiating the infection procedure, dilute non-adapted dengue virus 2 stock to a 1 times 10 to the fifth plaque forming units per 40 microliters of RPMI 1640 medium concentration. Next, load 10.3 milliliter syringe equipped with a 30-gauge needle with 10 microliters of diluted virus and 10.3 milliliters syringe equipped with a 30-gauge needle with 30 microliters of virus per animal.
For intracerebral delivery of the virus, press the oracle between the thumb and index finger to gently restrain a seven-day-old suckling Institute of Cancer Research Mouse in a prone position, and inject the 10 microliter volume of diluted virus into the lambda area at the intersection of the sagittal and lambdoid suture. After the intracerebral injection, use the thumb and index finger to hold the mouse in a supine position and gently intraperitoneally, inject the 30-microliter volume of diluted virus into the Murine abdomen.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Infection Procedure
<ol>
	<li>Prepare nonadapted DENV2 (strain PL046) stocks9 (originally obtained from the Centers for Disease Control in Taiwan, ranging from 2.5 x 107 to 1 x 109 PFU/mL).</li>
	<li>Dilute the virus stock to 1 x 106 PFU with Roswell Park Memorial Institute (RPMI) 1640 medium to a total volume of 40 &#956;L.</li>
	<li>Fill one 0.3 mL syringe fitted with a 30 G needle with 10 &#956;L (2.5 x 105 PFU) of diluted virus and fill another 0.3 mL syringe fitted with a 30 G needle with 30 &#956;L (7.5 x 105 PFU) of diluted virus.</li>
	<li>Hold the 7-day-old ICR (Institute of Cancer Research) suckling mouse and conduct the following steps.
	<ol>
		<li>For the intracerebral injection, hold the mouse in a prone position by pressing the auricle between the index finger and thumb, and intracerebrally inject 10 &#956;L of diluted virus into the lambda area, the point at the intersection of the sagittal and lambdoid suture.</li>
		<li>After the intracerebral injection, hold the mouse in a supine position using the index finger and thumb, and gently inject 30 &#956;L of diluted virus intraperitoneally into the murine abdomen. 
		NOTE: To avoid cannibalism, 75% alcohol is used to create a temporary loss of the mother&#39;s olfactory sensation. Additionally, covering the litter with their mother&#39;s stool and urine is suggested.</li>
	</ol>
	</li>
	<li>Lay the suckling mice back in their cages and wait for 5 min to check their safety poststimulation with the avidity of mice, including walking and mother milk sucking. 
	NOTE: Most often, mice demonstrate normal activity poststimulation, and there do not seem to be any harmful effects following the technical challenge.</li>
	<li>Evaluate the daily progress of the mice in terms of body weight (by using a microgram balance), acute viral encephalitis-like disease (by disease scoring as described in section 2), and survival rate.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Roswell Park Memorial Institute 1640 medium (RPMI)</td>
			<td>Gibco</td>
			<td>11875-085</td>
			<td>Diluting virus</td>
		</tr>
		<tr>
			<td>0.3-mL Insulin Syringe</td>
			<td>BD Ultra-Fine&#173;II</td>
			<td>328838</td>
			<td>Intracerebral and intraperitoneal injection</td>
		</tr>
		<tr>
			<td>Microbalance</td>
			<td>METTLER TOLEDO&#39;s LabX</td>
			<td>AL104</td>
			<td>Weight mouse</td>
		</tr>
		<tr>
			<td>Non-adapted DENV2 (strain PL046)</td>
			<td>The Centers for Disease Control of Taiwan</td>
			<td>-</td>
			<td>Infect mouse</td>
		</tr>
		<tr>
			<td>Institute of Cancer Research (ICR) suckling mouse</td>
			<td>BioLASCO Taiwan Co., Ltd</td>
			<td>-</td>
			<td>Our murine model</td>
		</tr>
	</tbody>
</table>

Source: Shen, T., et al. <a href="https://app.jove.com/v/59132">A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease.</a> J. Vis. Exp. (2019)
This video demonstrates the generation of a mouse model with a CNS infection. A suckling pup is injected with the virus both intracranially and intraperitoneally. The virus spreads, infecting the CNS causing neuronal cell death and inflammation. The pup is assessed for symptoms of infection as the disease progresses.

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

Begin with two syringes filled with different volumes of media containing the viruses targeted for central nervous system (CNS) infection.
Keep the mouse pup in a prone position. Disinfect the head and inject the smaller volume of the virus solution intracerebrally through the lambda region.
This allows the viruses to bypass the blood-brain barrier and directly reach the brain tissue.
Next, hold the mouse pup in a supine position. Disinfect and inject the larger volume of the virus solution into the abdomen.
This facilitates the systemic spread of the virus through the blood vessels, infecting other parts of the CNS.
The injected viruses enter neuronal cells and use host machinery to replicate, leading to cell death.
The viruses also trigger the immune system, causing CNS inflammation and establishing a CNS infection model.

10 ビュー. 中枢神経系感染症を発症したマウスモデルの生成

ビデオ: 中枢神経系感染症を発症したマウスモデルの生成 - 実験

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

感染マウス感染モデルにおける髄膜炎の連鎖球菌性バイオ フィルムの役割の調査のための頭蓋内クモ膜下ルート

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

JoVE Journal

免疫・感染学

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.

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).

エコHIV脳感染症のラットモデル

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

神経科学

In Vivo Mouse Model of Spinal Implant Infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

脊椎インプラント感染のin vivoマウスモデル

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

Generation of a Murine Model with a Central Nervous System Infection

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Generation of a Murine Model with a Central Nervous System Infection