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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;&#160;&#160;&#160;
C57BL/6 (H-2b) female mice, 8 weeks of age, were purchased and C57BL/6 AQP4-/- mice were provided by A. Verkman.
1. Immunization of Mice with AQP4 Peptides
<ol>
	<li>Prepare complete Freund&#39;s adjuvant (CFA) working stock.&#8203;
	<ol>
		<li>Finely grind a desiccated preparation of Mycobacterium tuberculosis (H37Ra) using a mortar and pestle.</li>
		<li>Add ground H37Ra to incomplete Freund&#39;s adjuvant to a final concentration of 4 mg/mL. CFA can be stored at 4 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Dissolve lyophilized antigen (Ag) AQP4 peptide in phosphate buffered saline (PBS) to a final concentration of 1 mg/mL. Maintain at 4 &#176;C or on ice.</li>
	<li>Prepare the assembly of 2 luer-lock syringes, joined with a stopcock, containing the CFA/peptide emulsion.
	<ol>
		<li>Attach a glass luer-lock syringe without plunger to a 3-way nylon stopcock with 2 male luer-lock connections. Close the stopcock by switching the lever towards the syringe. Position the open end of the syringe up, allowing the syringe to act as a test tube. Place this in a tube rack for added stability.</li>
		<li>Vortex the CFA working stock and the Ag solution. Add a 1:1 volume of peptide / CFA to the open syringe. 200 &#181;L of CFA/Ag (4 x 50 &#181;L) is required per mouse. 
		NOTE: Typically, about 1 mL of preparation is lost in the stopcock, which will reduce the amount available for immunization. Therefore, it is important to incorporate this into the calculation of total volume required. 
		Example: For immunization of 5 mice: (200 &#181;L/animal x 5 animals) + 1 mL extra volume = 2 mL total CFA/Ag required.</li>
		<li>Insert the glass plunger into the syringe and while holding it in place, invert the syringe so that the stopcock is oriented upward. Switch the stopcock lever to the unused female connection. Carefully apply pressure to the glass plunger in order to remove excess air from the syringe. The liquid fills the second male luer-lock connection of the stopcock.</li>
		<li>Attach a second glass syringe (plunger fully inserted) to the second male luer-lock connection of the stopcock. This should be a closed system of 2 glass syringes connected with a stopcock containing as little excess air as possible.</li>
		<li>To create an emulsion, mix by pushing the plungers to pass the liquid alternately between the 2 syringes for about 2 min. Chill the syringes on ice for about 10 min. Repeat the chilling and mixing until there is a clear change in the viscosity of the preparation, resulting in a very stiff, white emulsion. Refrigerate the syringes containing the emulsion at 4 &#176;C or maintain on ice.</li>
	</ol>
	</li>
	<li>Transfer all of the emulsion to one glass luer-lock syringe, remove the empty glass syringe and replace it with a 1 mL luer-lock syringe for injection. Fill the new syringe with emulsion, remove it from the stopcock and attach a needle (25G x 5/8).</li>
	<li>&#34;Water-test&#34; the emulsion for consistency. Expel a drop of the emulsion into a small dish of water. The emulsion should not disperse, indicating that it is suitable for injection.
	<ol>
		<li>If the emulsion disperses, it is not ready for injection; transfer the liquid back into the glass syringes and continue to mix and then chill again until the emulsion is stiff.</li>
		<li>Test the emulsion again until the proper consistency has been achieved.</li>
	</ol>
	</li>
	<li>Inject donor AQP4-/- mice subcutaneously (s.c.) with emulsion containing the AQP4 peptide. Each mouse receives 4 x 50 &#181;L s.c. injections (200 &#181;L total (100 &#181;g peptide)). The 4 sites include both sides of the lower abdomen, which drain into the inguinal lymph nodes (LN), and both sides of the chest wall medial to each armpit, which drain into axillary LN. The adoptive transfer will require 1-2 immunized donor mice per recipient mouse.</li>
</ol>
2. T Cell Culture and Proinflammatory T cell Polarization
<ol>
	<li>10-12 days after immunization, collect LN from the mice.
	<ol>
		<li>In an ice tray, prepare 60 mm Petri dishes fitted with a cell strainer (mesh size 70 &#181;m) and containing chilled Roswell Park Memorial Institute (RPMI) media with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin-100 &#181;g/mL streptomycin (pen-strep) (RFPS).</li>
		<li>Euthanize mice by exposure to CO2, followed by cervical dislocation. Immerse mice in 70% ethanol and then pin each footpad to a dissection board.</li>
		<li>Cut a midline skin incision from the groin to the neck and down each leg. Pull the skin away from the peritoneum and pin it tautly to the board. Dissect inguinal and axillary LN and place into the Petri dish containing RFPS, on ice.</li>
		<li>For adoptive transfer experiments, combine LN from all animals within the same immunization group. For flow cytometric analyses of V&#946; usage, separate LN from individual animals.</li>
	</ol>
	</li>
	<li>Place the cell strainer containing the LN onto a 50-mL centrifuge tube containing RFPS. Use the flat end of a sterile 5 mL syringe plunger to press the LN cells through the strainer, washing with 20-30 mL of RFPS to facilitate recovery of the cells. Maintain LN cells on ice throughout the processing until time for culture.</li>
	<li>Wash twice, centrifuging at 393 x g for 5 min. After the second wash, resuspend the LN cells in T cell media (TCM). TCM contains RPMI with 10% heat-inactivated FBS, 292 &#181;g/mL L-glutamine, 110 &#181;g/mL sodium pyruvate, and 55 &#181;M 2-mercaptoethanol and pen-strep. Typically, a volume of 5 mL TCM per donor mouse is used.</li>
	<li>Count the LN cells. Expected yield is approximately 3-6 x 107 LN cells per immunized mouse donor.</li>
	<li>Set up polarizing cultures for adoptive transfer.
	<ol>
		<li>Prepare a Th17 polarizing culture of 5 x 106 LN cells per mL with 10 &#181;g/mL Ag, 20 ng/mL recombinant mouse interleukin (IL)-23 and 10 ng/mL recombinant mouse IL-6 in TCM.</li>
		<li>Alternatively, for Th1 polarization, prepare a culture of 5 x 106 LN cells per mL with 10 &#181;g/mL Ag and 10 ng/mL recombinant mouse IL-12 in TCM.</li>
		<li>Add 2 mL (1 x 107 cells) per well of the polarizing culture mixture into a 12-well plate. The LN cells from 2-4 donor mice will fill one 12-well plate. Maintain cultures in a humidified incubator at 37 &#176;C with 5% CO2 for 72 h.</li>
		<li>Visually check the LN cells in culture for activation at 48 h and 72 h. Activated cells will form extensive clusters and the T cells will increase in size, becoming more pleomorphic. The increase in metabolism will cause a lowering of the pH in the media, changing from peach to orange. If the pH is low enough to turn the media yellow, add 1 mL of fresh TCM to prevent toxicity to the cells in the remaining 24 h.</li>
		<li>Proceed for adoptive transfer.</li>
	</ol>
	</li>
</ol>
3. Adoptive Transfer of Pathogenic AQP4-specific T cells
NOTE: This step follows from Section 2.5: T cell culture and proinflammatory T cell polarization.
<ol>
	<li>Recover polarized cells from 12-well plates by pipetting up and down to dislodge, and transferring them to a 50 mL tube. Maintain on ice until injections.</li>
	<li>Count the cells, wash them twice in cold PBS, and prepare a suspension of 1 x 108 cells/mL in PBS. Generally, inject cells within the hour.</li>
	<li>Gently mix the cells by swirling them and transfer them to a 1 mL syringe at the time of injection. Administer 200 &#181;L of the cells (2 x 107) intravenously (i.v.) to each mouse, through the tail vein.</li>
	<li>Inject each recipient mouse i.p. with 200 ng of B. pertussis toxin diluted in 200 &#181;L of PBS that day and 2 days later. Monitor mice daily for signs of clinical disease.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>M. tuberculosis H37Ra</td>
			<td>BD Difco</td>
			<td>231141</td>
			<td>Dessicated, killed M. tuberculosis</td>
		</tr>
		<tr>
			<td>Incomplete Freund&#39;s Adjuvant</td>
			<td>BD Difco</td>
			<td>263910</td>
			<td></td>
		</tr>
		<tr>
			<td>AQP4 peptide p135-153</td>
			<td>Genemed</td>
			<td>Custom Synthesis</td>
			<td>Peptide sequence: LVTPPSVVGGLGVTMVHGN</td>
		</tr>
		<tr>
			<td>AQP4 peptide p201-220</td>
			<td>Genemed</td>
			<td>Custom Synthesis</td>
			<td>Peptide sequence: HLFAINYTGASMNPARSFGP</td>
		</tr>
		<tr>
			<td>MOG peptide p35-55</td>
			<td>Genemed / Auspep</td>
			<td>Custom Synthesis</td>
			<td>Peptide sequence: MEVGWYRSPFSRVVHLYRNGK</td>
		</tr>
		<tr>
			<td>3-way Stopcock</td>
			<td>Kimble</td>
			<td>420163-4503</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum (Characterized)</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30071</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-23</td>
			<td>R&#38;D Systems (BioTechne)</td>
			<td>1887-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-6</td>
			<td>R&#38;D Systems (BioTechne)</td>
			<td>406-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-12</td>
			<td>R&#38;D Systems (BioTechne)</td>
			<td>419-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Pertussis Toxin from B. pertussis</td>
			<td>List Biological Laboratories</td>
			<td>181</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable-Flow Peristaltic Pump</td>
			<td>Fisher Scientific</td>
			<td>13-876-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

inducing paralysis in a mouse model via transfer of aquaporin-4-specific th17 cells

Source: Sagan, S. A. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56185">Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4.</a> J. Vis. Exp. (2017)
This video demonstrates the procedure of inducing paralysis by transferring activated Th17 cells targeting aquaporin-4 into a mouse model. In the central nervous system, the activated Th17 cells interact with aquaporin-4-expressing astrocytes, triggering an immune response that leads to tail and limb paralysis in the mouse.

Inducing Paralysis in a Mouse Model via Transfer of Aquaporin-4-Specific Th17 Cells

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

Begin with a syringe containing T-helper or Th17 cells capable of recognizing an aquaporin-4 protein of astrocytes in the CNS, including the spinal cord, which plays a role in signal transmission to the brain.
Inject these Th17 cells into the tail vein of a restrained mouse. The cells enter the bloodstream and circulate throughout the body.
Simultaneously, intraperitoneally administer a bacterial toxin derived from the Bordetella pertussis. 
The toxin disturbs the endothelial barrier of blood vessels and allows Th17 cells to infiltrate the spinal cord.
The infiltrated Th17 cells interact with the astrocytes expressing the aquaporin-4 and become activated.&#160;
These activated cells release pro-inflammatory cytokines, which causes the infiltration of mononuclear immune cells, including macrophages.
Macrophages release inflammatory molecules that damage the myelin sheath of the surrounding nerve fibers.
This demyelination disrupts nerve signal transmission, leading to tail stiffness and restricted hind limb movement, indicating the onset of paralysis.

inducing-paralysis-mouse-model-via-transfer-aquaporin-4-specific-th17

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neuromuscular Disorders

40 ビュー. 出典: Sagan, S. A. et al., Induction of paralysis and visual system injury in mice by T cells Specific for Neuromyelitis Autoantigen Aquaporin-4. J. Vis. Exp. (2017年) このビデオでは、アクアポリン-4を標的とする活性化Th17細胞をマウスモデルに移すことにより、麻痺を誘発する手順を示しています。中枢神経系では、活性化されたTh17細胞がアクアポリン-4を発現するアストロサイトと相互作用し、免疫応答を?...

ビデオ: アクアポリン-4特異的Th17細胞の移植によるマウスモデルにおける麻痺の誘導 - 概念

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

マウスおよび種々の組織における免疫細胞の疾患に依存する分布の評価における実験的自己免疫性脳脊髄炎の誘導

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

JoVE Journal

免疫・感染学

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

細胞ベースのアッセイによるひと血清抗アクアポリン 4 免疫グロブリン G の検出

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

クリア脳におけるアストロサイトとニューロンの空間的相互作用の解明

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

Inducing Paralysis in a Mouse Model via Transfer of Aquaporin-4-Specific Th17 Cells

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Inducing Paralysis in a Mouse Model via Transfer of Aquaporin-4-Specific Th17 Cells