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.&#160; &#160;&#160;
1. Animal Model 
<ol>
	<li>Intercross hemizygous Tg(Gfap-luc+/-) mice (transgenic mice expressing firefly luciferase) with hemizygous Tg(M83+/-) mice (transgenic mice expressing human alpha-synuclein with the familial A53T mutation)&#160; to generate hemizygous bigenic Tg(M83+/-:Gfap-luc+/-) mice.</li>
	<li>Genotype the progeny with real-time polyemerase chain reaction (PCR) for the presence of the transgene encoding human alpha-synuclein and with standard PCR for firefly luciferase.</li>
	<li>Inoculate the animals at an age of six-to-eight weeks.</li>
</ol>
2. Inoculum Preparation 
<ol>
	<li>Prepare monomeric recombinant human alpha-synuclein with the A53T mutation in Tris-buffered saline (TBS) buffer containing 150 mM NaCl in 20 mM Tris-HCl (pH 7.2).</li>
	<li>Prepare fibrillar alpha-synuclein for inoculations by agitating 3 &#181;g/&#181;L of monomeric recombinant human alpha-synuclein in an orbital shaker at 800 rpm and 37 &#176;C for 5 days.</li>
	<li>Dilute fibril assemblies in phosphate-buffered saline (PBS) to reach a final concentration of 1 &#181;g/&#181;L for intraperitoneal inoculations.</li>
	<li>Fragment the alpha-synuclein fibrils with a rod sonicator for 1 min (40 pulses of 0.5 s duration with a one-second pause between each pulse and an amplitude set to 50%) on ice. To avoid contamination by cross-seeding, use clean sonication probes to prepare different inoculum.</li>
</ol>
3. Intraperitoneal Injections 
<ol>
	<li>For intraperitoneal injections, narcotize the animals shortly in an anesthesia chamber by inhalation with isoflurane/oxygen using a flow rate of 2 L/min and the vaporizer set to 2%. Pinch the animal&#39;s toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia.</li>
	<li>Fill a 27 G disposable hypodermic syringe with 50 &#181;L of sonicated alpha-synuclein fibrils or PBS.</li>
	<li>Directly inject into the peritoneum of the mouse and avoid penetrating the small intestine or cecum located behind the abdominal wall by holding the animal in a dorsal position with the head facing away from the investigator and downward at approximately 45&#176;. Monitor animals until they have recovered from the anesthesia.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Invitrogen</td>
			<td>14190169</td>
		</tr>
		<tr>
			<td>27-gauge syringe</td>
			<td>VWR</td>
			<td>613-4900</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>PZN 4831850</td>
		</tr>
		<tr>
			<td>Sonopuls Mini20 sonicator</td>
			<td>Bandelin</td>
			<td>3648</td>
		</tr>
		<tr>
			<td>Tg(M83+/-) mice or B6;C3-Tg(PrnpSNCA*A53T)83Vle/J mice</td>
			<td>The Jackson Laboratory</td>
			<td>4479</td>
		</tr>
	</tbody>
</table>

induction of neuroinflammation in mice via injection of alpha-synuclein fibrils

Source: Breid, S. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55503">Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils.</a> J. Vis. Exp. (2017).
This video demonstrates the injection of alpha-synuclein fibrils into a mouse to induce neuroinflammation. The fibrils are injected intraperitoneally into a transgenic mouse expressing mutated alpha-synuclein proteins. Upon reaching the central nervous system, the fibrils enter neurons and induce aggregation of the mutated proteins. The aggregates spread to neighboring neurons, microglia, and astrocytes. Recognition of the aggregates triggers microglia to induce neuroinflammation, damaging the neurons.

Induction of Neuroinflammation in Mice via Injection of Alpha-Synuclein Fibrils

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160; ...

Position a transgenic mouse in a dorsal position. The mouse expresses a mutated form of the alpha-synuclein protein, which is prone to aggregation.
Take a suspension of alpha-synuclein fibrils, aggregates of misfolded alpha-synuclein protein.
Inject the fibrils into the peritoneal cavity to allow their systemic distribution.
Once in the bloodstream, the fibrils reach the central nervous system and bind to endothelial cells of the blood-brain barrier.
The fibrils are transported via vesicles across the cells and enter the nervous tissue. They then invade neurons through endocytosis and promote aggregation of the mutated alpha-synuclein proteins.
These aggregates are released by exocytosis and spread to neighboring neurons, microglia, and astrocytes.
Recognition of the aggregates triggers microglia to release reactive oxygen species, which damages the tissue, and cytokines, which recruit additional immune cells. This results in neuroinflammation that leads to neural cell death.
Allow the mouse to recover to assess alpha-synuclein-mediated neuroinflammation.

induction-neuroinflammation-mice-via-injection-alpha-synuclein

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

40 次观看. 来源:Breid， S. et al.转基因小鼠外周接种 α-突触核蛋白原纤维后神经炎症的生物发光成像。J. Vis. Exp.（2017 年）。 该视频演示了将 α-突触核蛋白原纤维注射到小鼠体内以诱导神经炎症。将原纤维腹膜内注射到表达突变的 α-突触核蛋白的转基因小鼠中。到达中枢神经系统后，原纤维进入神经元并诱导突变蛋白质的聚集。聚集体扩散到邻近的神经元、小胶质细胞和星形胶质细胞。...

视频: 通过注射 α-突触核蛋白原纤维诱导小鼠神经炎症 - 概念

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

科研

JoVE Journal

神经科学

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

从单体和体内使用的Α-syr核 in 预成型纤维的产生

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Induction of Neuroinflammation in Mice via Injection of Alpha-Synuclein Fibrils

成績單

Induction of Neuroinflammation in Mice via Injection of Alpha-Synuclein Fibrils